transdermal delivery of nonsteroidal anti - inflammatory drugs is a topical treatment routinely used in physiotherapy to reduce pain and inflammation in musculoskeletal disorders.14 in order to weaken the skin barrier and to achieve a clinically effective drug concentration in the target tissues , various transdermal enhancement techniques , such as sonophoresis , iontophoresis , and occlusion , are used.511 however , little information is available concerning the efficacy of the treatment protocol and the penetration profiles of the substances used in the physiotherapy setting . indeed , most clinical studies have complex treatment protocols , and are unable to relate clinical outcome with drug penetration . a recent meta - analysis ( 2012 ) suggested a better clinical outcome for iontophoretically delivered substances.12 despite methodological flaws ( eg , combined therapies , lack of control for passive diffusion and occlusion ) , definite conclusions regarding the possible penetration enhancement of electrically assisted delivery can not be inferred.12 the building up of a stratum corneum ( sc ) reservoir is an important component of the pharmacodynamics of topically applied substances . rougier et al used the reservoir effect of the sc after 30 minutes of application to predict the total amount absorbed.13 moreover , the human sc has the capacity to store topically applied substances through a prolonged time.14 the existence of a reservoir within the sc has been documented for several topically applied solutes such as corticosteroids,1418 caffeine,19,20 nicotine,21 diclofenac ( df),22 and chemical ultraviolet filters.23 roberts et al emphasized that reservoir properties can also be attributed to the viable epidermis and dermis . specifically , df - binding capacities in dermal tissue have been reported.24 lambrecht et al estimated df skin bioavailability by quantifying the reduction of methyl nicotinate ( mn)-induced vasodilatation using chromametry and laser doppler flowmetry . they detected a significant reduction in mn response 90 minutes after different application modalities of df . open application resulted in a 32% reduction in mn response , application under occlusion accounted for a reduction of 66% , and the iontophoretic application resulted in a 65% reduction.11 at that time , one could not discriminate between the occlusive and electrically assisted delivered df . to obtain more insight into the pharmacodynamics and reservoir properties of df , the aim of the present study was to evaluate the bioavailability in the skin of df at longer time intervals after df application using different application modalities . therefore , three different forms of application ( passive , occlusive , iontophoretic ) were examined in order to detect the anti - inflammatory effect and thereby get a better understanding of the skin - penetrating and reservoir - building behavior of df as a function of time . mn responses were quantified by skin colorimetry at different time points ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) after initial df application . five different groups , ( group 1 , n=13 [ 1.5 hours ] ; group 2 , n=12 [ 6 hours ] ; group 3 , n=12 [ 24 , 32 hours ] ; group 4 , n=14 [ 48 , 72 hours ] and group 5 , n=9 [ 96 , 120 hours ] ) of healthy volunteers ( n=60 , 23 male and 37 female ) not treated with any drugs participated in this study . approval by the ethical committee of the vrije universiteit brussel was obtained for this study ( 2002/136 ) . before testing , all subjects were informed of the research protocol and signed an informed consent . during the duration of the experiments , the volunteers were asked to maintain their daily activities , but to abstain from swimming and extensive showering . the measurements were performed under standardized temperature ( 20c2c ) and relative humidity ( 45%5% ) conditions . before every df application and each measurement session , the volunteers participated in a 30-minute acclimatization period . for each time interval tested , five randomized circular skin areas ( 7 cm ) were demarcated on the subjects volar forearms , for the following treatment and/or control modes : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , 3 ) iontophoretic application , 4 ) standard mn response , and 5 ) untreated skin side . this meant that either five skin areas for groups 1 and 2 or ten skin areas for groups 3 , 4 , and 5 ( testing two time intervals ) were randomly demarcated on both arms . this procedure enabled a single application of mn on every skin site at different time intervals , avoiding the occurrence of tachyphylaxis . df skin bioavailability was assessed by quantifying the reduction of the mn response , as proposed by lambrecht et al.11 topical application of mn provokes an increase of local cutaneous blood flow ( erythema).25 when df is present in the skin , the nicotinate response is depressed in a concentration - dependent way.26 treffel and gabard demonstrated a good correlation between the in vivo inhibition of the mn - induced inflammation and the in vitro determination ( chromatographic analysis ) of nonsteroidal anti - inflammatory drug - concentration levels in the sc.27 lambrecht et al evaluated the inhibition of mn - induced erythema with both laser doppler flowmetry and the colorimetric method , which resulted in equivalent conclusions . therefore it was decided that the chromametric evaluation be used.11,28 in the present study , the inhibition of the mn response with sum of the a * parameter ( a * ) was evaluated up to 50 minutes post - mn application , making the protocol more feasible and bearable for the subjects . skin - surface colorimetric measurements were performed with a minolta cr 200 chromameter operating in the international commission on illumination l*a*b * color space . for the quantification of skin - surface color and erythema , the a * parameter is especially useful , as it represents the chromaticity between red / magenta and green ( negative values indicate green , while positive values indicate magenta).29,30 chromameter measurements were carried out before df application , prior to mn application , and every 5 minutes until 50 minutes post - mn application for the different reservoir - estimation time points . since different groups of subjects were involved , an untreated skin area was included in order to be able to correct for blank values . for treatment modes 1 , 2 , and 3 , a commercially available 1% df ( voltaren emulgel ; novartis pharma , bern , switzerland ) formulation ( 12 mg ) the mn test was applied using paper - filter disks that were saturated in a 0.005 m aqueous mn solution and applied on the circular demarcated skin areas for 30 seconds . after removal of the filter disk , excess solution was gently wiped away using a tissue paper . df was applied for 20 minutes under three different conditions : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , and 3 ) iontophoretic application ( direct current , 0.2 ma / cm , cathode ) . to reproduce physiotherapeutic conditions , two marked skin areas were not treated with df : the application side for the standard mn - induced response and the blank untreated skin side . the bioavailability of df in the sc under the three conditions was assessed by quantification of mn induced erythema at 1.5 , 6 , 24 , 48 , 72 , 96 , and 120 hours post - df application . to estimate the presence of df in the skin , mn responses at the different time periods following initial df application ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) were compared . the a * corrected for blank values ( untreated skin ) up to 50 minutes post - mn application was used as an indicator for the magnitude of the mn response . smirnov goodness - of - fit test . at the different time periods following initial df application , the skin response to the different application modalities statistical significance between any application mode and the standard mn response was used as an indication for the presence of df in the skin at that particular time point following initial df application . five different groups , ( group 1 , n=13 [ 1.5 hours ] ; group 2 , n=12 [ 6 hours ] ; group 3 , n=12 [ 24 , 32 hours ] ; group 4 , n=14 [ 48 , 72 hours ] and group 5 , n=9 [ 96 , 120 hours ] ) of healthy volunteers ( n=60 , 23 male and 37 female ) not treated with any drugs participated in this study . approval by the ethical committee of the vrije universiteit brussel was obtained for this study ( 2002/136 ) . before testing , all subjects were informed of the research protocol and signed an informed consent . during the duration of the experiments , the volunteers were asked to maintain their daily activities , but to abstain from swimming and extensive showering . the measurements were performed under standardized temperature ( 20c2c ) and relative humidity ( 45%5% ) conditions . before every df application and each measurement session , the volunteers participated in a 30-minute acclimatization period . for each time interval tested , five randomized circular skin areas ( 7 cm ) were demarcated on the subjects volar forearms , for the following treatment and/or control modes : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , 3 ) iontophoretic application , 4 ) standard mn response , and 5 ) untreated skin side . this meant that either five skin areas for groups 1 and 2 or ten skin areas for groups 3 , 4 , and 5 ( testing two time intervals ) were randomly demarcated on both arms . this procedure enabled a single application of mn on every skin site at different time intervals , avoiding the occurrence of tachyphylaxis . df skin bioavailability was assessed by quantifying the reduction of the mn response , as proposed by lambrecht et al.11 topical application of mn provokes an increase of local cutaneous blood flow ( erythema).25 when df is present in the skin , the nicotinate response is depressed in a concentration - dependent way.26 treffel and gabard demonstrated a good correlation between the in vivo inhibition of the mn - induced inflammation and the in vitro determination ( chromatographic analysis ) of nonsteroidal anti - inflammatory drug - concentration levels in the sc.27 lambrecht et al evaluated the inhibition of mn - induced erythema with both laser doppler flowmetry and the colorimetric method , which resulted in equivalent conclusions . therefore it was decided that the chromametric evaluation be used.11,28 in the present study , the inhibition of the mn response with sum of the a * parameter ( a * ) was evaluated up to 50 minutes post - mn application , making the protocol more feasible and bearable for the subjects . skin - surface colorimetric measurements were performed with a minolta cr 200 chromameter operating in the international commission on illumination l*a*b * color space . for the quantification of skin - surface color and erythema , the a * parameter is especially useful , as it represents the chromaticity between red / magenta and green ( negative values indicate green , while positive values indicate magenta).29,30 chromameter measurements were carried out before df application , prior to mn application , and every 5 minutes until 50 minutes post - mn application for the different reservoir - estimation time points . since different groups of subjects were involved , an untreated skin area was included in order to be able to correct for blank values . for treatment modes 1 , 2 , and 3 , a commercially available 1% df ( voltaren emulgel ; novartis pharma , bern , switzerland ) formulation ( 12 mg ) was applied . the mn test was applied using paper - filter disks that were saturated in a 0.005 m aqueous mn solution and applied on the circular demarcated skin areas for 30 seconds . after removal of the filter disk , excess solution was gently wiped away using a tissue paper . df was applied for 20 minutes under three different conditions : 1 ) open application ( without occlusion ) , 2 ) passive diffusion under a semiocclusive humid sponge , and 3 ) iontophoretic application ( direct current , 0.2 ma / cm , cathode ) . to reproduce physiotherapeutic conditions , two marked skin areas were not treated with df : the application side for the standard mn - induced response and the blank untreated skin side . the bioavailability of df in the sc under the three conditions was assessed by quantification of mn induced erythema at 1.5 , 6 , 24 , 48 , 72 , 96 , and 120 hours post - df application . to estimate the presence of df in the skin , mn responses at the different time periods following initial df application ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) were compared . the a * corrected for blank values ( untreated skin ) up to 50 minutes post - mn application was used as an indicator for the magnitude of the mn response . smirnov goodness - of - fit test . at the different time periods following initial df application , the skin response to the different application modalities statistical significance between any application mode and the standard mn response was used as an indication for the presence of df in the skin at that particular time point following initial df application . analysis of the skin color ( a * parameter ) after mn application for the different application modes ( open application , occlusion , and iontophoresis ) and at the different reservoir - estimation times ( 1.5 , 6 , 24 , 32 , 48 , 72 , 96 , and 120 hours ) clearly showed changes in mn response as a function of the application mode and reservoir - estimation time . as an example , kinetics of the skin - color parameters are given in figures 1 and 2 . at 1.5 hours post - df application , a clear inhibition was visible for application under occlusion and under iontophoresis , while open df application did not provoke an inhibition in mn response ( figure 1 ) . at 96 hours post - df application , inhibition of the mn response was no longer perceived ( figure 2 ) . differences between application modes at the different reservoir - estimation times were assessed using the a * values calculated from the different kinetics ( table 1 ) . at 1.5 hours after the initial df application , a significantly decreased response was detected for the occluded and iontophoretically delivered df only . from 6 hours up to 32 hours post - df application , a significantly reduced mn response was detected at all df - pretreated skin sites . at 48 hours post - df application , only the passive and occluded applications resulted in a significant reduction . at 72 hours post - df application , only the response after the passively delivered df remained significant . from 96 hours on , the applied method  inhibition of a physiological reaction ( mn ) due to the presence of the inhibitor ( df ) in the skin reservoir  enabled us to estimate penetration kinetics , df skin - reservoir building , and emptying properties of three different application conditions up to 120 hours after initial df application . in contrast with other reservoir protocols , an active substance - liberating action , such as occlusion or increased hydration , was not required.24 this may be a confirmation of the presence of df in the viable tissue ( dermal compound ) , since mn - induced vasodilatation is inhibited immediately.24 the application modality influenced the formation and the emptying of the reservoir . penetration - enhancing factors , such as occlusion and current , induced faster formation and emptying of the reservoir compared to an open ( passive ) df application . the presence of the reservoir for the passive df application after 6 hours only is in contrast with earlier findings from our laboratory.11 lambrecht et al11 presented the data of the mn response as calculated areas under the curve , relating the inhibition of mn response throughout a 65-minute time period . they found a 32% reduction of the mn response 1.5 hours after application , reaching borderline significance ( p=0.04 ) . consequently , the weaker inhibition potential at 1.5 hours post - df application noticed in both studies indicates lower bioavailability at that moment , due to a slower percutaneous penetration compared to the other application modes . according to the literature , the formation of a skin reservoir for topically applied substances is determined by a variety of factors ( eg , lipid / water - solubility , protein - binding capacity , percutaneous absorption , compound concentration , clearance , application time , and application mode).11,1416,23,24 our results support the previous findings of vickers , showing that increased drug diffusivity in the sc provoked by penetration enhancers results in faster formation of the reservoir.14 more specifically , takahashi et al suggested increased diffusivity of df into the sc from vehicles containing urea compared to vehicles without urea . the latter was explained by the hydration - enhancement effect of urea on the sc.31 the results concerning the penetration - enhancement effect of iontophoresis are in line with the results of curdy et al on the in vivo uptake of piroxicam after passive , occlusive , and iontophoretic administration . only after iontophoresis were enhanced drug uptakes found at 30 , 60 , and 125 minutes following the initial application at different depths in the sc . in contrast with our results , curdy et al found no significant difference between passive delivery and the application under occlusion . a possible explanation could be the low lipophilicity of piroxicam resulting in a low passive uptake into the sc.32 fang et al postulated that the route and mechanism for the iontophoretic delivery of df through the skin might be different compared to passive delivery . therefore , the importance of the sc as a rate - limiting barrier is reduced for iontophoretic delivery of df.33 our results indicate that the emptying of the reservoir is influenced by the application mode . after iontophoretic delivery , the df reservoir was present up to 32 hours following initial application . however , after occlusive and passive delivery of df , the reservoir was present up to 48 and 72 hours , respectively . it is well established that diffusivity influences clearance from the reservoir , with faster emptying as a function of increasing diffusivity . increasing sc hydration as well as iontophoresis have been shown to be effective methods for enhancing the percutaneous penetration of df.33 the present data fit in the model proposed by roberts et al with a shorter lag time resulting in a faster reservoir emptying.24 equally , based on the results of rougier et al13 one can assume that the application with the fastest reservoir - buildup conditions may result in a greater delivery of active substances to the viable tissues , which may have an effect on the clinical outcomes of the physiotherapeutic treatment . within the limitations of our experimental design , we can hypothesize that the faster emptying of the reservoir after iontophoretic delivery compared to application under occlusion may be an indication for a higher diffusivity during the current - assisted application , leading to a superior delivery under iontophoretic circumstances . limitations of the present study are the lack of information on absolute df quantities entering the viable skin and the fact that the methods used did not allow differentiation between an epidermal reservoir and a dermal reservoir . further research estimating in vivo tissue concentrations after different modes of application are required for further elaboration of the pharmacodynamics of topical applied substances in physiotherapeutic practice . this study measured the penetration kinetics and reservoir properties of df after a single topical passive , occlusive , and electrical assisted application in a realistic physiotherapeutic setting . the results indicate that the contribution of occlusive and passive penetration in the iontophoretic delivery can be substantial . the prompt inhibition of the vasoactive reaction may be an indication for a dermal df reservoir . the formation and emptying of the reservoir was found to be dependent on the application mode .