Biochimica et Biophysica Acta 1514 (2001) 191^205

New, highly e¤cient formulation of diclofenac for the topical,
transdermal administration in ultradeformable drug carriers,
a; b;1
Gregor Cevc *, Gabriele Blume
Medizinische Biophysik, Technische Universita«t Mu«nchen, Ismaningerstrasse 22, D-81675 Munich, Germany
IDEA AG, Frankfurter Ring 193a, D-80807 Munich, Germany

Received 14 September 1999; received in revised form 6 June 2001; accepted 20 June 2001


Transfenac, a lotion-like formulation of diclofenac, is described. It consists of pharmaceutically acceptable ingredients and
mediates the agent transport through intact skin and into the target tissues. Therapeutically meaningful drug concentrations
in the target tissue are reached even when the administered drug dose in Transfenac is below 0.5 mg/kg body weight.
Ultradeformable agent carriers, called Transfersomes, form the basis of Transfenac. These Transfersomes are proposed to
cross the skin spontaneously under the influence of transepidermal water activity gradient (see [Biochim. Biophys. Acta 1104
(1992) 226]). Diclofenac association with ultradeformable carriers permits it to have a longer effect and to reach 10-times
higher concentrations in the tissues under the skin in comparison with the drug from a commercial hydrogel. For example,
Transfenac achieves intramuscular agent concentrations between 0.5 and 2 Wg/g and 2 and 20 Wg/g at t = 12 h, depending on
the tissue depth, when it is administered in the dose range 0.25^2 mg/kg of rat body weight. A much higher drug
concentration in a hydrogel (1.25^10 mg/kg body weight) creates the drug level of only 6 0.5 Wg/g in the muscle. The drug
concentration in the rat patella for these two types of formulation is between 1 Wg/g and 5 Wg/g or 0.4 Wg/g, respectively. The
relative advantage of diclofenac delivery by means of ultradeformable carriers increases with the treated muscle thickness and
with decreasing drug dose, as seen in mice, rats and pigs; this can be explained by assuming that the drug associated with
carriers is cleared less efficiently by the dermal capillary plexus. In pigs it suffices to use 0.3 mg of diclofenac in highly
deformable vesicles per kg body weight, spread over an area of 25 cm2 , to ensure therapeutic drug concentration in a 5-cm
thick muscle specimen, collected under the agent application site. When the drug is used in a hydrogel at 8 times higher dose,
the average intramuscular concentration is at least three times lower and subtherapeutic. This suggests that diclofenac in
Transfersomes has the potential to replace combined oral/topical diclofenac administration in humans. ß 2001 Published
by Elsevier Science B.V.

Keywords: Lipid vesicles; Drug delivery; NSAID; Targeted application ; Biodistribution ; Pharmacokinetics ; Phospholipid bilayer

1. Introduction

Diclofenac is one of the most potent and commer-
* Corresponding author. Fax: +49-89-4140-4980. cially successful non-steroidal anti-in£ammatory
E-mail address: (G. Cevc). agents [1,2]. It has an annual turnover of over 1 bil-
Address during the study. lion US dollars, approximately 15% of which is

0005-2736 / 01 / $ ^ see front matter ß 2001 Published by Elsevier Science B.V.
PII: S 0 0 0 5 - 2 7 3 6 ( 0 1 ) 0 0 3 6 9 - 8

BBAMEM 78137 31-8-01

G. The `moisture seeking' (hydrotaxis) such a goal can only be achieved by delivering diclo. Transfenac. time of approximately 1 h [1]. is a trademark of IDEA AG. resulting from the drug binding to certain (e. The primary site of such barrier [8^10]. dration gradient. drug concentrations in the target tissues. this formulation has vesicles. is controllable by formulation/administration param- ous applications of special diclofenac formulations eters and provides a good basis for the topical as well were introduced. eral skin permeation enhancers are used to overcome The nearly perfect drug absorption in the gastro.g. phospholipid vesicles (liposomes) [1.. but this level circulation leaves too little drug in the target organs is not maintained long. so-called administered diclofenac is therefore poorly tolerated Transfersomes3 for the non-invasive delivery of phar- and causes stomach ulcerations. the skin barrier [6]. inability of conventional liposomes to cross the skin prostaglandin) receptors. with a terminal elimination half. owing to the amphipathic character of the from the epicutaneously applied Voltaren Emulgel is weakly acidic drug. 1 cm under its administra. Cevc. The other topical diclofenac lates in the urine. The best known is Voltaren Emul. gel. In Voltaren diclofenac concentration in the plasma. or tissue to Emulgel. The depth of diclofenac permeation below the skin 2 3 Voltaren Emulgel is the trademark of Novartis. hence promises to become the remedy of ¢rst choice this medication is typically combined with oral Vol. for ous tissue: the drug spill-over into the blood the daily dose between 100 and 150 mg. the di¡u- intestinal tract is partly o¡set by the ¢rst-pass hepat. for the treatment of diseases of super¢cial tissues. The probable reason for this is the icity. These ad. The remainder ¢nally accumu. We argue that a single admin- reached nearly 15% of the total market to date. biologically available. These ultradeformable It would be desirable to reach the therapeutic drug drug carriers trespass the intact skin spontaneously. As istration of the lotion-like Transfenac formulation on Voltaren gel normally falls short of its therapeutic the normal skin creates therapeutically meaningful dose by the factor of 10. as systemic therapy [16^18]. taren to ensure good therapeutic e¡ects. transcutaneous hy- agent concentrations as low as possible. Transfenac tion site on the skin (see [3^5] for further discussion). ministered drug across the skin barrier. Obviously. formulations are less e¡ective by the factor of 2 [7]. Approximately 30% of the for adequate stand-alone therapy [4]. orally administered diclofenac is excreted in the It is believed that only around 10% of diclofenac bile. Here we report some of our results obtained using ministration. This corresponds to a therapeutic skin permeability is also an obstacle. This notwithstanding. but previously overlooked. `carrying body'. of approximately 0. concentration in the target tissue [3. of Transfersomes [13] permits the carrier to bring fenac into the body via the route other than the more than 50% [14^16. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 achieved with the topical remedies.192 G. adverse action is the gastrointestinal tract [1].19] of the epicutaneously ad- mouth. that would be in the therapeutic concentration range istration is not the insu¤cient bioavailability but across the skin.2 developed speci¢cally for the epicutaneous ad. The process For these reasons. topical injections or epicutane. Orally We have introduced special drug carriers.5 Wg/g tissue [2].12] without solv- verse e¡ects are mainly due to the poor agent specif. In an attempt to transport an amount of the drug The chief disadvantage of oral diclofenac admin. Although the drug penetration depth of the formulation of diclofenac in ultradeformable Emulgel is merely 3^4 mm [4]. ing the problem. BBAMEM 78137 31-8-01 . Around 25 mg of is limited by the relatively good drug solubility in the drug in a body ensure good antiphlogistic and aqueous systems ( s 1%) and fast clearance. Transfersome. maceuticals across the skin. diclofenac was incorporated into rather the serious side e¡ects of the drug.4] while simulta. such as muscles or joints. sive £ow of the drug monomers across the skin can- ic biotransformation of V50%. Low analgesic e¡ects. The peak plasma not match the drug elimination from the subcutane- concentration of diclofenac is close to 1 Wg/ml. diethylammonium salt of the drug and sev- be treated. probably under the in£uence of the naturally occur- neously keeping the systemic and gastrointestinal ring.

1 MBq/ml). G. 50 WCi/ml). with the stan- dard deviation of size distribution typically around 2. Stressful or painful manipulations were always imately 75% of diclofenac in a typical starting sus. Germany) and 0.1.19]. This ensured that the of 2-propanol and propyleneglycol as the skin per- £exibility of the vesicle membrane was at least 5 times meation enhancers.8 and 7. sonication) was used to diminish the average trimming the hair of mice and rats to the length of vesicle size in original suspension.p. Under the conditions individual applications to prevent suspension drip- chosen. obtained with pH 7.36]. was added to pre-made Transfenac by comparing the rate of enforced vesicle transport or diclofenac in a gel (Voltaren Emulgel) at least across a semi-permeable barrier with the pores v3 3 days before the experiment to the ¢nal concentra- times smaller than the average vesicle diameter. Su¤ciently vigorous stirring Wistar rats.25 ml Rompun (Bayer. and common pigs. The designated application site on the animals back pending on the dosage used. Berlin. but the former compo. Swit- across the skin permeability barrier was thus ful¢lled zerland). a proportion of the drug was thus non-ion. In brief. vesicles with the size in the range 100^ label attached in the upper cyclic part of the drug 200 nm were prepared from soy phosphatidylcholine molecule. [14 C]diclofenac. The animals were or other suitable kind of suspension homogenization prepared for the drug administration by manually (e. The tion of 1. use. partitioning measurements we estimate that approx. 10 mg diclofenac per millilitre of suspension.8 MBq/ml. Leverkusen. displacement glass pipette in the case of Transfer- in di¡erent experiments. ethylamine salt.1 are reported. For refer.. lasting approximately 30 min.1 þ 0. and characterization of Transfenac. Vesicles dimension maximally 2 mm. Germany) marker used. in one arm of the chief requirement for the e¤cacy of drug transfer study.2 MBq/mg (33 WCi/ in detail in [20. with luke warm water.6 mg/ml of diclofenac di- particular formulation type. drug. allowing for su¤cient drying time between brane bound drug is pKa = 6. or Lipoid (Ludwigshafen. of 2 MBq/mg dry mass (1. containing mixture of 6 ml charge as well as to amphipathic character of the 0. Vertical axis label always identi¢es the (from Natterman Phospholipids (Cologne. the liquid was applied in several ence. (In the former case. but here only the data some suspension and with a spatula for hydrogel. Animal experiments 30%. together with an undisclosed amount sition was commonly used. More details on the man. higher than that of standard phosphatidylcholine The radioactive [3 H]diclofenac. Germany). a called Transfersomes [20]. custom made by vesicles in £uid phase. and the drug was administered as described further ufacturing. will be published separately. The drug-to-lipid ratio in the proprietary vehicles.) Oral administration was done through a ¢ne ized and the diclofenac^vesicle association was al. The precise value depended on the pharmacy. De. Cevc. the formulation was prepared by suspending the lipids in an aqueous phase in which Animal experiments were done on NMRI mice. G. the apparent dissociation constant of mem. in pigs the tentative application site was ¢nally brought to the desired value by extruding was washed only. positive The suspension pH value was between 6. This was assessed as described Amersham (speci¢c activity 1. a gift of Novartis (Basel.4. product of Novartis) was purchased from the local tween 1/4 and 1/9. Based on the results of indirect on the skin. was used similarly at speci¢c radioactivity [16. Materials and methods together with the formulation stability data.9% NaCl. rial was applied topically with a precision. 1 ml Ketavet 100 (Parke-Davis. carried out under general injection anaesthesia pension was membrane bound. volumes between 3 or hind extremity was marked with a permanent pen and 75 Wl/cm2 were used. In short. aliquots. the drug was dissolved.1.). The test formulations contained between 4 and Germany) given i. BBAMEM 78137 31-8-01 . It contained 11. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 193 2. used in this study was be.g. the conclusion was made mg) dry mass). Test mate- the suspension through 100^200 nm pore ¢lters. Diclofenac in a hydrogel (Voltaren Emulgel. in the text. but with the Speci¢cally. tube using the same Transfenac that was also used ways appreciable. ping. owing to (partial) (10 Wl/g body weight.

0^1. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 The blood samples (V20 Wl) were taken at given kept in separate boxes. ples representative of the depths of 0^0. At the speci¢ed times. `untreated muscle' reference sample. on days 2^6. After killing the animals under anaesthesia. that is. Data analysis 1.1. but always Data analysis was done with the software package covered all the tissue down to the bone. After the addition of 10 ml on the day 1. Wildbad.75^1. The treated skin as well as 80 cm2 . liver and bladder (with the residual urine).2. all probes were neutralized with once or twice daily.5^4 cm2 . following the last drug administration. muscle(s) under the application site were collected 2. Biodistribution or on the hind leg(s).5 cm. On the day 7. all animals many). 1. muscles). On subsequent 5 days. Twenty-four hours all the other organs of interest were excised.1. lications [21]. A otherwise kept under standard conditions (normal 1-cm thick piece of shoulder muscle was taken as the diet. ORIGIN (Microcal. 0. Dreieich. the lations were used on the same leg that was also total radioactivity of samples was determined with treated on days 2^6 with the cold drug. The suspension was left at the site of administration during the entire course of ex. G. somewhat thinner sections were used for the smaller rats.3. corresponding to the treatment 0.1. where stated. Cevc. the biodistribution of diclofenac in the target tissues.2 ml CH3 COOH. the animals were killed periment. Germany).75 cm.2. ples from the sites close to the drug administration the appropriate muscles were sliced in thin pieces by site (typically. They received the drug-loaded lipid suspension from a micropipette on the upper part of the back 2. 12/12 h light/dark regime).3. Patella from the treated knee was also excised. t = 12 h) of the soft-catheter located in one of the dorsal veins in the labelled drug formulation on the ¢rst day of the ex- pigs.25^ 0. NMRI mice Five sequential muscle samples (each V1 cm NMRI mice (8^12 weeks old) were transferred into thick) were collected under the drug administration individual cages on the day of experiment and were site.4 ml H2 O2 and 0. Subsequently. BBAMEM 78137 31-8-01 . Statistical signi¢- cance was checked with Student's t-test and consid- 2. 0.1. were anaesthetized.25 cm and 2. The sam. 2. To get the therapeutically relevant information on periment.2 ml HClO4 at 80³C over. The after the ¢rst drug administration each animal was tissue and organ samples were prepared and used anaesthetized and a 1-cm thick sample was cut from for the diclofenac radioactivity counting as described the treated leg (data not shown). the radioactive formu- Aquasol-2 (NEN-DuPont. Ger. spread without occlusion over 0. They received one (at t = 0) or intervals by tail puncture in rodents and through a two doses (at t = 0 and. then taken care of surgically.25^1. for the highest dose used. super¢cial and deep calf and shank using a pair of sharp anatomical scissors. Wistar rats were treated similarly except in that From the speci¢c radioactivity in these slices the the drug formulation was always administered on drug-related penetration pro¢le of the radioactively hind legs. Pigs ered to be granted at P 6 0. of the femur muscles. spread over an area of up to Other samples included at least two muscle sam- 3 cm2 .5^0. tered on the contralateral leg without occlusion night. All results give the Pigs had a weight of approximately 15 kg and were mean of all measured values þ standard deviation.5 cm for the 500-g rats. the non-labelled drug in ultra- The blood and tissue samples were discoloured deformable vesicles or in a hydrogel was adminis- with 0. The wound was in the following paragraphs and in our previous pub.194 G.25 cm. covering the knee and the proximal part labelled diclofenac was ¢nally determined. Wistar rats down to the bone and sliced to individual samples. 0. killed and used for the measure- ments. Twelve hours a beta-scintillation counter (Berthold.05. The administration site was on one of the by a heart puncture (rodents) or by a lethal dose of hind legs close to the knee and had an area of up to anaesthetic agent (pigs).0 cm. USA). water ad libitum. OR.

3. and 9 mg/kg body weight (left-dashed) of the drug in ultradeformable vesicles. on the hind thigh of mice. and pigs. Cevc. This was done us. by applying a su¤cient amount of vs. drug delivery achieves up to one order of magnitude sible to improve substantially the target-tissue/blood better performance than the use of current commer- drug concentration ratio. thicker tissues.1. therapeutic drug levels in cial drug-in-gel formulations. The proviso was that the drug dose was greater than Biodistribution and some pharmacokinetic param. when the drug concentration in subcutaneous tissue when so desired. Results drug (around 1 Wg/g) in the murine dorsum. 4. (Right) Biodistribution of diclofenac-derived 3 H-radioactivity 12 h after an applica- tion of 2. G. ing mice. hydrogel-based formulation of The localization of diclofenac in the treated tissue diclofenac were studied in detail. In this study the epicutaneously more dramatic (see Section 4). is formulation as well as application-site dependent. and 9 mg/kg body weight (left-dashed) in the com- mercial hydrogel. 0. 1) and carriers. which is already an excessive eters of two Transfersomes-based suspensions.25 mg/kg body weight (right-dashed). 1. (Left) Tissue concentration of diclofenac-derived 3 H-radioactivity at t = 12 h after an epicutaneous application of 2.5 mg/kg body weight. It was pos. Ultradeformable vesicles Several drug doses were tested on the murine hind were found to improve the regio-speci¢city and the legs to support the conjecture. G. Biodistribution the drug per kg body weight in Transfersomes is approx. and agent amount. In the murine Fig. For example. Transfersomes. 4. when the animals are treated with approx. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 195 3. The the drug on intact skin in highly deformable drug di¡erence is greater for lower doses (see Fig.5 Wg/g) whereas the con- radioactivity in mice is illustrated in Fig. rats. centration ratio deep in the hind legs of rats is close Topical use of diclofenac is believed generally to to 10.25 mg/kg body weight (right-dashed).7 Wg/g vs. the relative drug con- centration in the dorsal muscles treated with 9 mg of 3.5 mg/kg body weight (hatched).9 higher than in the case of using gel-based The biodistribution of the [3 H]diclofenac-derived formulation (6. administered Transfenac and Voltaren Emulgel The drug concentration in the blood is approxi- were seen to deliver an appreciable amount of the mately 2^3 times lower at t = 12 h. 2. applied dose is compared (see also Section 4). This is clear especially the systemic blood circulation were also reached. 1. BBAMEM 78137 31-8-01 .6 improve the regio-speci¢city of therapy but is not mg/kg body weight The discrepancy in pigs is even always applicable.5 mg/kg body weight (hatched). of one commercial. 1. The carrier-mediated e¤cacy of agent delivery into the body.

3. The ¢rst set of corresponding biodistribution data is shown in Fig. Transfersomes (full the drug distribution pro¢les in the tissues under symbols.8 Wg/g tissue. Consequently. Voltaren Emulgel in the same dose range only yields values between 0. Inset is used the average drug concentration in the muscle shows the normalized pro¢le to the point where the intramuscu.3 for Trans- fenac and of 1 for Emulgel. 2. Hind extremities are better suited for such studies. 3 which compares [3 H]diclofenac in ultradeformable vesicles. since they can provide sam- ples with a thickness of 5 mm (on a mouse) or up to 25 mm (on a rat). The dorsal musculature of small rodents is typi- cally only 2^3 mm thin. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 hind legs treated with 2. 2. Diclofenac the site of administration. BBAMEM 78137 31-8-01 . [3 H]Diclofenac penetration pro¢le in the rat hind-leg rier-based formulation.3 Wg/g. open columns) on rat hind legs. [3 H]Diclofenac biodistribution after the administration of 2 mg of the drug per kg body weight in highly deformable carriers. much di¡erent from the agent concentration in the Fig. When Voltaren Emulgel concentration in the blood is represented as a dashed line. Mice are therefore unsuit- able for predicting the penetration of diclofenac into more voluminous tissues.4 and 1. which is not lar values become similar to those in the blood. black columns) or in a hydrogel (right panel. Transfersomes (left panel. below the fatty layer is 0. This corresponds to a relative degree of regionalization of 3. but to a di¡erent extent.196 G. Samples were taken 12 h after administering This is also illustrated in Fig. the characteristic of diclofenac bio- distribution in `thick tissues' was studied with the radiolabelled drug formulations on the rat hind legs. left hind leg) or in a hydrogel (open symbols. muscles. Cevc. It demonstrates that the drug-derived radioactivity reaches the super¢cial muscle using a hydrogel as well as using the car- Fig. right leg) on the skin at the dose of 2 mg/kg body weight. G.25^9 mg/kg body weight of Transfenac the drug concentration is close to 5 Wg/g.

For example. (Right) 0. this is seen from the fact that the deep-muscle-to-blood drug concentra- Fig. In the contra- lateral leg treated with Voltaren Emulgel the intra- muscular concentration is around 4 mg/g near the surface and only approximately 0. 4. 0.7 mg/g deep in the muscle.1 mg/cm2 ) or in a hydrogel (open symbols. the average drug concentration in the super¢cial muscle treated with Transfenac is 14^20. which indicates the lack of drug delivery specif- icity. (Left) 0.6 mg/kg in Emulgel on one of the hind legs of four (left) or two (right) test rats. alternatively. deformable vesicles.5 mg/g and approximately half these values for the deeper muscles (data not shown). G. A similar drug distribution pro¢le was ob. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 197 blood. drug doses but Transfenac was again more regio-spe- ci¢c than Voltaren Emulgel (see Fig. 5. This latter value is very close to the cor- responding blood concentration level of V0. G.5 mg to 0.5 mg/kg body weight as Voltaren Emulgel. 4). Diclofenac distribution pro¢le 12 h after an epicutaneous administration of the drug in ultradeformable vesicles (full symbols.25 mg/kg body weight of diclofenac in ultradeform- able vesicles or. with 0.5 mg/ ml. Conversely.2 Wg/g. BBAMEM 78137 31-8-01 .75 mg/kg body weight as 14 C-labelled Transfenac or 1. (Inset) The normalized drug distribution pro¢le expressed as relative di¡er- ence of local and systemic concentrations. 4 stem from measure- ments with rats treated on one hind leg with either 0. the average drug concentration in the muscle below the Transfenac-treated skin is 6. especially deep under the treated skin. Biodistribution of [3 H]diclofenac-derived radioactivity of the drug per kg body weight in a commercial after the topical administration of a small drug amount in very hydrogel. 5 and 7). 4. Transfersomes (black columns) or in a hy- tained under the application site as with the higher drogel (open columns) on the hind legs of Wistar rats. 1 mg/cm2 ). The results illustrated in Fig. A similar trend is observed when the administered drug dose is below 2 mg/kg body weight (Figs.25 mg/kg body weight (labelled with 3 H) in deformable carriers or 0. or more than 8 times higher.6 mg Fig. Cevc.

and there- weight. illustrates this.8 Wg/g) or for the diclofenac level in di¡erence in regio-speci¢city is commented on fur. 6. 4 and 5) causes a 2-fold disparity from Transfersomes is consequently cleared much between drug concentration deep under the skin less e¤ciently by the intradermal capillary plexus al. in lowing more drug to reach the deep subcutaneous favour of the former. 3 and drawn despite the fact that the thickness of and the 5). down-triangles to the deep muscle tissue. site (on average: 20.198 G. (It tissues. deduced from the [3 H]diclofenac-derived radioactivity data. Intramuscular drug concentration. Fig. when Transfenac or Voltaren Emulgel is used. after a single epicutaneous hydrogel is greater deep in the tissue. see Figs. Transfersomes are too large to between 0. Administering diclofenac molecules. Cevc. We therefore infer that the mechanism of drug drug concentration in vehicle was di¡erent for Trans- transport across the skin is di¡erent for the vesicle fenac and Emulgel. taken that the administration in and gel-based diclofenac formulations. see Figs.6 þ 0. stant. 6. in contrast to smaller amount of diclofenac is used. We believe that Similar di¡erences are observed when a much this di¡erence is due to the fact that. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 tion ratio for Transfenac is close to 6 while for the This is also true for the drug concentration in fascia gel-like preparation a value near 1 is found.6 and 29. carrier-mediated delivery. The drug weight (see Figs.) The carrier-mediated diclofenac concentration in the relative advantage of Transfenac over the drug in a skin (726. Up-triangles pertain to the super¢cial. respectively.1 Wg/g). which is with regard to the surface concentration value.5 and 4. the patella near the treated site mediated by Trans- ways the highest at the skin surface. and fairly con- indirectly support such conclusion. Greater divergence is ob. depends strongly on the choice taren Emulgel (see Fig. but the prin.9 Wg/g for Transfenac and Voltaren similar near the skin surface for Transfenac and Vol. which administration of 2 mg diclofenac per kg body shows the results from dose ¢nding study. Emulgel. pears to be similar in either case. therapeutic situation. di¡erence between the drug concentrations in For obvious reasons the drug concentration is al.7 þ 14.25 and 0. This (275. The porcine data discussed later in this text should be noted that there is a large.7 Wg/g). is signi¢cantly higher than that resulting fore presents some gaps.2 Wg/g. of drug vehicle as well. either case was done non-occlusively to mimic the ciple of agent propagation through the muscle ap. 3). The above-mentioned observations suggest that ul- Fig. 2 and The relative drug concentration pro¢le. is 5 Wg/g and 0. The average fenac or Voltaren Emulgel. at two di¡erent times after the administration of [3 H]diclofenac in highly deformable vesicles (full symbols) or in a hydrogel (open symbols). normalized 3).75 mg diclofenac per kg body enter into the cutaneous blood circulation. The drug concentration in the rat patella. G. from a hydrogel administration (87. These conclusions can be served deep in the muscle (see insets to Figs. 2 and 4. BBAMEM 78137 31-8-01 . the super¢cial muscle near the drug administration ther in the text.

A twice Fig.088 Wg/g in the patella.5 or 0. respectively. Near the bone.89 measured in the treated muscle is 0. Experimental scattering is quite large but the average concentration measured 12 h after applying 0. An untreated 10^14 for the di¡erent kinds of formulations studied muscle contains 0. 0. The results of our experiments with pigs.021 Wg/g in the untreated The regio-speci¢city also decreases with time until muscle. BBAMEM 78137 31-8-01 . For the hydrogel-based formulation the three increasing doses. the carrier. irrespective of the If the topical administration of diclofenac in a gel is kind of drug formulation (data not shown). 6). The average radioactivity-derived drug concentration in individual pigs tested as described in the text with the drug in daily administration for 6 consecutive days of 0. 7). Similar observations are nac concentration in the target tissue than using the made for the joints near the gel administration site. 4) when the administered Transfenac dose is 0. A 4.5 mg/kg body in a hydrogel yields a drug concentration of 0. 7. ni¢cant level of 1. Cevc. 4) and V1 Wg/g (data not shown). They also carry [3 H]Transfenac application. The muscle-to-liver ratio is typically between 1 and 3 for Voltaren Emulgel and around 6 for Transfenac. G.811 Wg/g in the muscle and to large and at 15 mm in the small rats. between 0. respectively. the following results are obtained.2 mg/kg body weight) of diclofenac 3.1 Wg/g are found. the proximal patella does the treated-muscle/blood ratio falls below the insig. and sometimes even in the patella. followed by a drogel (right panel). Wg/g in the treated and 0.150 mg/kg body weight. G. In the latter case.133 weight In the deeper muscle this advantage is lost.039.5 at t = 24 h. drug content in ultradeformable carriers there is ap- topically administered gel at t = 24 h and even more proximately one order of magnitude higher diclofe- so at t = 12 h (see Fig. Transfersomes (left panel) or in a hy- mg of diclofenac per kg body weight.05) ^ di¡erent from the value of 0.075 ultradeformable vesicles. When the average drug concentration is calculated for a 5-cm thick muscle under the treated site. body weight on the day 1. 0.2 mg/kg The above-mentioned result is not much ^ but sig. that have a skin permeability barrier similar to that of humans [22]. complemented with an oral drug uptake of 1. (data not shown). black single treatment with the detectable radioactive drug. The drug concentration in the `treated patella' is greater than 2 Wg/g tissue (data not shown) and close to 1 Wg/g tissue (Fig.5 at t = 8 h and by a factor of is 2 Wg/g and in the patella 0. 0.8 at t = 12 h when the drug dose is 0. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 199 tradeformable vesicles not only improve the trans. 2U0. Local administration of Trans- fenac gives rise to a therapeutically meaningful drug concentration in the 8-fold muscle-to-blood concentration ratio near the skin is higher dose ( 6 1. single dose of 0. under the drug application site. at a depth of 24 mm in the raises these levels to 0. support the results obtained with rats. not contain any drug within the experimental error. columns) and their proximal patella (open columns) are com- gives concentrations of 0.15 mg/kg body weight at t = 312 h tion site.36 Wg/g (Fig.32 Wg/g while in measured after a single peroral use of diclofenac the untreated one 0. the drug concentration ni¢cantly (P 6 0.25 mg/kg body weight.327 Wg/g in the patella (see Fig. Skin treatment with a a higher payload into the muscles under the applica.163 Wg/g.5^1 mg of diclofenac per kg body weight in a gel is signi¢cantly lower. Treated muscles (5-cm thick sample. the value in the muscle ues by a factor of 17.031 and 0.047 Wg/g for the at t = 12 h.433 Wg/g in the muscle and pared with the untreated muscles (grey columns). Transfenac supersedes in absolute These results indicate that even with a smaller terms the results obtained with the diclofenac-loaded. For a dose of mediated agent accumulation exceeds the serum val. 12 h after the second port of diclofenac through the skin.

treatment of pigs for 6 days is also compatible with The in vivo penetration studies described in this this conjecture (see Fig. Cevc.200 G. according to the manufacturers' (open squares) or through the mouth (crossed diamonds). 8). gives v 10 times higher plasma and 14 times higher urine concentration than the diclofenac gel application on the intact animal skin. Pharmacokinetics h per cm2 . Related lower dose approximately 0.5 Wg/ml over a period of 8 h. been calculated by Roberts and Singh [4] to be be- lease automatically. good performance of the vesicle-based drug formu- tion with a half-time of v4 h (see Fig. Transfenac only needs to be depending on agent's ionization state. the same range as previously published results mea- In topical human therapy the painful regions are sured in di¡erent animal species. often treated two or three times per day with a di- clofenac-loaded hydrogel. closed circles). Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 4. G. t1=2 = 2 h. that is.82U1032 cm/h).5 Wg/h per cm2 . The poor success of such 4.36 Wg/h per cm2 . speci¢cations. The highest reported £ux is conclusion (data not shown). The upper limit estimate is 30 Wg/h per cm2 . The pharmacokinetics of [3 H]diclofenac appearance and chronically with a dose of approximately 2 mg/kg per elimination from rat blood circulation after an epicutaneous ad- day orally. The corresponding blood value falls from 1.57U1034 cm/h) than drug mice treated once or twice daily with a relatively in the protonated form. Results measured with the cell less readily (log P = 3. Oral A single oral diclofenac application at the dose of V5 mg/kg body weight in a guinea-pig model. at low pH (log high dose of diclofenac imply the above-mentioned P = 1. In our experience. 7).09 Wg/h per cm2 and 0. right panel). ultradeformable vesicles to cross the skin and thus to mans. 4.5 to 0.2. in a hydrogel around 1 Wg/ml. Such tered on the skin is cleared from the blood circula.75 mg of drug/kg body weight in highly de- formable carriers (Transfersomes. a single oral application of 9 mg/kg body weight created se- rum drug concentration of approximately 1 Wg/ml at administration of commercial Voltaren Emulgel (see t = 12 h (see Fig. 7). or 30^100 times higher than that gener- ated by a more conventional drug formulation [4]. work suggest that the carrier-mediated £ux of agent through the intact rodent skin in situ is at least 10 Wg/ 3. the drug concentration in the blood is ministration of 0. Transfenac provides a solution The transepidermal £ux of diclofenac in vitro has to this problem as it generates a sustained drug re. tween 0. In humans treated Fig. BBAMEM 78137 31-8-01 .2. Skin palliative therapy must be compensated with the oral uptake of diclofenac. 8. Our data thus fall in Fig. The ionized used once or twice daily on the skin to ensure a diclofenac passes through the rat skin in a Franz- good therapeutic e¡ect. 1. This is lation is in our opinion due to the good capability of longer than the half-time value of Voltaren in hu. In our experiments with mice. The drug from a hydrogel or Transfenac adminis.1. Discussion The relative merits of diclofenac in ultradeform- able vesicles are best judged by comparing the results gathered with Transfenac with those obtained using conventional formulations and/or administrations of the drug. carry agents into the body. ac- cording to the literature [3].

Cevc. The corresponding published values super¢cial and close to 0. when a 7. The relative bioavailability of the drug from Six applications of 2U1. with the preparations on the skin in vivo and in vitro.01) Wg/ml were detected in the blood and muscular drug concentration of (0. erts [4]. Singh and Roberts [4] provided the data on the ization of the drug at the site of application on rats permeation of diclofenac from several aqueous drug with slow systemic availability.5 or V1. These application of ultrasound pulsed drug systemic levels authors have estimated the maximum reach of agent could be achieved [12].2% in the tive doses [3].005 Wg/ml. all the drug is re- or in combination with a large amount of hydrogen. Comparable drug concentration is also the drug is used in a hydrogel. The A single application of V10. In a study by a Japanese conventional formulation of this drug is used for the group [11]. around 0.3 mg/ corresponding local drug concentration decrease. G. the [3]. 21 and 5 Wg/ml. tion and biotransformation remains to be investi- tients) was applied on the shaved rat dorsum alone gated we believe that. data not shown). The intramuscular concentration is This is 10 times higher than that of a hydrogel ap. tissue. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 201 Lipid^diclofenac combinations have been tested nated more slowly from the skin than when a more before in the rat model. when the administered dose is close to 1 mg/kg when the drug was applied at the dose of 2. depending on the 4. ac- kg body weight diclofenac. This is carrier-mediated drug concentration in rat skin was 55% and 87% lower than in the plasma.2) Wg/g (1.25 mg/kg. It is also roughly found in the urine of test animals. G. comparable to the value reported by Singh and Rob- gest that the drug delivered by means of ultradeform. application of liposomal diclofenac resulted in local. however. This corresponds to a relative reportedly causes drug concentration in the blood to concentration of approximately 10% in the dermis.12) Wg/ml. over from the contralateral leg treated with Trans- fenac under the same experimental conditions gives fenac is possible. Muscle total lipid concentration used (0. other cases.4.03) Wg/g in the bladder plus residual urine. intradermal drug concentrations between 12 Wg/g and up to 60 Wg/g of the skin tissue. This resulted in crossed the skin.515 Wg of diclofenac per milli- used. Ma¡ei and colleagues also reported that the litre of sample [3]. result in the intramuscular drug concentrations lipid-supplemented formulations over a period of 7 h under the application site of approximately 0. close to a gel on the guinea-pig back.3 Wg/g in rise to a drug concentration of (0. in a commercial cream. In the distal muscle 0. Raising this dose to This documents the compatibility of results between 2 mg/kg body weight increases the intramuscular our and previous studies. We ¢nd that a single application of diclofenac in a cial diclofenac gel used on the murine hind legs hydrogel on the rat hind leg only creates an intra- (0. however.25 þ 0. when a spill- Conversely. Both results sug. body weight (data not shown). near less than 1% in the subcutis.6 mg diclofenac per day in such formulations was rather low.5 þ 0. which con- 726 Wg/g. then similar to that measured in the blood. reach 0. for the three respec.07 Wg/g is found.5-fold lower diclofenac dose was tains approximately 0. reason discussed later. an epicutaneous application of Trans. leased from highly deformable carriers that have ated soya phospholipids (1 mg/cm2 ).37 þ 0.03 þ 0. drug concentration to (0.24 Wg/g [11]. Using a small amount of Transfenac (0. mg/kg body weight) on the skin gives rise to a sig- BBAMEM 78137 31-8-01 . 0. When diclofenac is applied on Wistar rat backs as a 4. While the rate of drug libera- fold the dose normally taken orally by human pa.88 þ 1.3) Wg/g.3. when plication. 15 mg/kg body weight of diclofenac (7. In the experiments reported in this work. ultimately. is approximately onto the shaved but otherwise intact guinea-pig skin 1/10 per millimetre. Blood and urine solution. cording to the same authors [4].02 and 0. respectively [3].25 able vesicles penetrates the skin better and is elimi. and to approximately 0. permeation through the rat skin not to exceed 4 mm. V3. the concentration of this drug deep in the muscle is hence nearly the same as in the plasma.015% in the deep muscle for the urine are approximately 45.1^1 wt%. according to the liter- approximately 5% for the solution and 25% for the ature. In our experiments with the commer.1.

The steady-state nac onto one knee and a placebo gel onto the other measurements with such pad on animals indicated knee of humans [5]. Alternative formulations ten times more e¤ciently than the drug from a hy- drogel administered at an intermediate dose (2 mg/kg Diclofenac to date was chie£y employed orally.36 Wg/g. G. after a single epicutaneous Transfenac blood samples were collected immediately before the application. The data reported by 20:00 h) for 4 consecutive days by eight volunteers Radermacher should hence be compared with the su¡ering from monolateral knee joint e¡usion [25]. after the ninth DHEP plaster application. containing 180 mg DHEP in an auto-adhesive medicated gauze pad of Radermacher and colleagues have applied diclofe. 1. including diclofenac. are thus likely to be due to the short di¡usion dis. Joint incorporated in a plaster [35]. High relative this corresponds to a bioavailability of 2. diclofenac in submicron emulsion proved to be 40% ti¢ed by the results of experiments on pigs. plasma. In such test including humans. steroidal and non-steroidal anti-in- epicutaneously with Transfenac (V2 mg/kg body £ammatory drugs. when the drug. Extrapolation to bigger animal species. Our value exceeds by the factor of 5 the ¢rst dose and 1.35]. Diclofenac gel brings was therefore important and successful commercially the majority of the drug to the depth of merely 3^4 but alternative formulations are now being mm.6. The latter consisted results obtained in murine (see Fig. Cevc.202 G. £ammatory agents [25. is therefore reasonable and is jus. to a total depth of 25 mm. concentrations in the systemically treated (distal) Synovial £uid (SF) from the a¡ected knee joint was knee (0.07) Wg of diclofenac per lowing application or for 12 h the 5th day.75 Wg/g) of collected at the beginning of the study and at 4 h the same test rat. The introduction of Volteren Emulgel Transfenac is used (see Fig.05 þ 0. sought. The formulation of diclofenac in ultradeformable vesicles gets into and across the skin approximately 4. result of our measurements with the rat patella. Diclofenac was detectable at somewhat high- BBAMEM 78137 31-8-01 .5.5^5. Venous gram tissue. 0.8 Wg/ml) [3]. more active than standard Voltaren Emulgel. mainly from the systemic blood supply. Diclofenac hydroxyethylpyrrolidine (DHEP) was 4. were formu- weight) is not speci¢c to this kind of animal: the lated in a submicron emulsion. Plasters were left on the a¡ected area until the fol- which contains (5.09 and 0.1 Wg/ml Similarly favourable results are obtained in pig (see Fig. 3 the drug penetration pro¢le even for the treatment of the well-localized patholog- in the latter situation is seen to extend throughout ical states and despite the systemic side e¡ects of the entire hind leg. 2) hind-leg muscles are semi-quantita. In the treated knee the Transfe. Un- nac-derived drug concentration is also one order of changed diclofenac was measured in plasma and SF magnitude higher than in the blood. The resulting drug accumulation the sustained release (12 h) of the drug [7] with an in the synovial £uid was then concluded to stem estimated absorbed dose of 5^10 mg per application. the speci¢c drug concentration being between the rat model are much closer to therapists desires. trometry technique. 2).5% [7] drug concentrations in the smaller animal joints (3 which is not su¤cient for the successful treatment of Wg/g). standard dimensions (10U15 cm2 ). Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 ni¢cantly lower serum drug level around 0. patella after the topical administration of Transfe- Penetration pro¢les achieved with Transfenac in nac. In Fig. left panel) or of approximately 100 nm large oil droplets [23] and rat (see Fig. This excludes by means of a gas liquid chromatography^mass spec- the possibility of signi¢cant drug in£ux from the sys. Drug accumulation in the hind legs of rats treated For example. 4 and 8 h after the ninth application. 4). Low but durable levels of diclo- temic blood circulation into the treated knee as the fenac were obtained at each collection after dosage in reason for the high drug concentration in the joint. or in the synovial £uid of the repeatedly all kind of diseases sensitive to nonsteroidal antiin- treated human ¢nger and wrist (V0.92 þ 0. body weight). were tested in the somewhat questionable [24] carra- tively similar. geenan-induced paw edema rat model.08) Wg/g or muscle (V0. DHEP plasters were used twice daily (at 08:00 and tances in such tissue specimen.

dyspnoea. facial oedema.8. resulting from the cutaneous other comparable parenteral formulation of such lip- application of diclofenac (Voltaren Emulgel). if any. 20% degradation [29]. 98 adverse drug ance) of aged skin. Phospholipids. are also known demonstrated in a study in which a total of 120 pa. certain phospholipid preparations even seem to trointestinal tract. In two cases a peptic uct. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 203 er concentration in SF at the fourth hour of the should partly be due to the more favourable drug ninth application [25]. are often serious. is unlikely to exceed 1 g/day in most teric-coating diclofenac than in the case of plain di. biodistribution from the carriers that concentrate Impeding the skin barrier increases the propensity the agent in the tissue to be treated. the daily dose of Transfenac for Most commonly gastrointestinal (39%) and cutane. the diclofenac lotion based directly onto a diseased site in a small but su¤cient on ultradeformable vesicles. rheumatic or traumatic conditions. cases. diclofenac should be used pru. that will not exceed the total amount of this drug arrhoea. phospholipid sus- pensions are not detrimental to the skin. trary. Phospholipid suspensions are also reactions to topical nonsteroidal anti-in£ammatory non-irritating to the skin. We therefore expect that the toxicity and the side cian's overall assessment of therapeutic e¤cacy re. For the period improve the hydration (and thus the optical appear- between 1983 and 1991. Transfenac is expected to be a very safe prod- before the onset of bleeding. were randomly the co-applied lipids are likely to minimize the dan- allocated to receive ultrasonic sessions three times ger of allergic contact dermatitis that may be induced weekly for 4 weeks. consequently. This is only 20% of the phospholipid quantity clofenac (P = 0.05). Such an in- 86% of Voltaren Emulgel-treated patients compared crease in the agent safety was already documented with 76% of patients receiving regular gel (P 6 0. 5. Based on these times daily for at least 2 weeks over a large area data. tients. for example.037). Transfenac and carrier safety 4. especially in the gas.7. Eleven general reactions. It is also less than 20% of the natural variability also reported by Zimmerman and colleagues [32]. than the to be superior even to the best commercial diclofenac currently available diclofenac formulations. G. Adverse side e¡ects of the drug When applied on the intact skin. Four cases of upper gastrointes. Cevc. humans should actually be lower: 9 25 mg and prob- ous (20%) adverse events were reported. Transfenac will be applied in a quantity (9 50 mg) such as duodenal ulcer. Tolerability was good or excellent in over 95% of patients in both treatment groups [26]. When used in a BBAMEM 78137 31-8-01 . 4. In our expectation Transfenac. were id. The physi. and the haemor- rhage was massive enough to necessitate blood trans- fusions. Total amount of the phospholip- gastrointestinal to non-gastrointestinal reactions to id placed on the skin in the form of Transfenac. has proven quantity. Transfenac. On the con- The side e¡ects of diclofenac. e¡ects of diclofenac in vivo will be diminished in the vealed that a satisfactory result was achieved in presence of ultradeformable vesicles. ally (9 150 mg/day). Conclusions dently in patients with a history of peptic ulcer [32]. for lidocaine in corresponding carriers [31]. ulcer was identi¢ed retrospectively. for the drug permeation across the skin. when administered In animal experiments. aggravation of used currently parenterally (9 75 mg/injection) or or- bronchospasm. at least up to the degree of agents were reported by the Spanish pharmacovigi. Moreover. of phosphatidylcholine concentration in the plasma Three of the patients had used the medication three for average healthy subjects [27]. The ratio of ably even 9 10 mg. Voltaren Emulgel or regular by topical diclofenac [30]. will cause less side e¡ects. Therefore. due to localized when the latter are surfactant-like [33]. di. This gel for the topical administration. with moderate to severe pain. that is infused daily in the form of Lipofundin or any tinal haemorrhage. lance organization [28]. G. and angioedema were described. gastrointestinal bleeding. to improve the safety of their co-applied agents. diclofenac was higher in the case of slow-release en. This was and especially phosphatidylcholine. gel was used as coupling medium [26].

15 (1993) 225^234. J.A. but dale). Schreier. In rats. 19 (1993) 89^95. A. Interaction of phosphatidyl- Diclofenac lotion comprising ultradeformable choline liposomes with the human stratum corneum. Zellmer. Sorkin. The e¡ect is more pronounced for therapeutic e¤cacy. This trend seems to increase with tem. E. This is not observed when the drug is Res. Ramachandran. Con- release of diclofenac from the carriers deposited into trolled Release 30 (1994) 1^15. L. Basel. Nishihata. Acta 1237 (1995) 176^182. Radermacher. C. M. G. Assandri. This suggests that the percutaneous transport of diclofenac and in£uence of hydro- simple-to-use. 179^186. Liposomes and niosomes as topical are likely to be due. Antioxidant pro¢le of nimesulide. 268 carrier-associated agent may therefore exceed by (1994) 144^151. A reappraisal of its pharmacodynamic and pharmacokinetic properties and loaded hydrogel. Drug cation site. Todd. T. Botta. Y.A. K.204 G. Flynn. J. conventional diclofenac formulations for the topical Weiner. Drugs Exp. M. 31 (1991) 537^ therapeutic quantity of diclofenac into the treated 541. Pharm. N. Siou¢. Both [8] H. ties. The reach of the after topical application. Res. Lasch. Chem. M. Arzeim. G. agent penetration from the commercial Diclofenac. diclofenac hydroxyethylpyrrolidine plaster. Yamazaki. Clin. Pharmaceutical Press. Clin. Bull. to the sustained drug carriers: dermal and transdermal drug delivery. Pharmacol. Saibene. Transfollicular drug delivery. 1993.S. Giachetti. L. Res. Lustinetz. C. Biophys. The relative advantage of using Transfenac instead [7] A. decreasing number of the drug administrations.C. a single epicutane. Pharmacol. Transfersomes. Die per- etrate deep into the soft tissue under the drug appli. Ma¡ei-Facino. Bio- vesicles. the subcutaneous tissue. Ther. is used. K. Roberts. Strusberg. for their hospitality in animal facili- ously applied diclofenac. combines the safety of the chim. more so with Transfenac than with the diclofenac [2] P. Nakano. the large than for the small animals.. Ultradeformable vesicles even can bring a ma after cutaneous application in in£ammatory and degen- erative joint disease. Scholl. 1993. Lauer. Br. Pharm. M. Tissue React. concentrations of non-steroidal anti-in£ammatory drugs such as sonophoresis [34]. [4] P. L. The relative improvement is berger for their technical support as well as to the a¡ected by the skin/muscle-weight ratio. Diclofenac from ultradeformable vesicles can pen. [12] R. Reynolds (Ed. which is 1/8 sta¡ of Geselschaft fu«r Strahlenforschung in Neu- for mice and makes the murine model suboptimal for Herberg b. Kobayashi. Rat preparations of such drug. Aldini. at least partly. Tomasi. Blume / Biochimica et Biophysica Acta 1514 (2001) 191^205 reasonable dose range in mice. Drugs 35 (1988) 244^285. Pfeil. company brochure. Int. brane model. unless special treatment. Cevc. Lieb. ous application of 2 mg of diclofenac per kg body weight in highly deformable carriers produced at least 4 times higher drug concentration in the treated References muscles than a drug-loaded hydrogel.). Schmid. BBAMEM 78137 31-8-01 . Ciba. S. Diclofenac sodium. J. J. Mac- We believe that such novel formulations will compete ciocchi. K. A. [3] W. more than one order of magnitude the maximum [5] J. A. Jentsch. Schneider.F. Singh.D. Moppert.M. Dr Griebel. Transfenac was Acknowledgements shown to be at least 5 times more potent and signi¢- cantly more site-speci¢c than the `competing' topical We are grateful to Karin Putz and Barbara Scho«n- diclofenac formulations. W.M. [9] S. Exp. 12 (1995) administration with a high or even improved e¤cacy. 35 (1987) 3807^3812. Diclofenac concentration in synovial £uid and plas- loaded gel. Canali. especially to Mrs Mo«llensta«dt and the prediction of therapeutic e¤cacy of epicutane. The latter matches that of the best available oral [11] T. kutane Resorption von Diclofenac. J. J.C. knee with reasonable reproducibility.I. Agent concentration in distal muscles or in serum [1] J. topical Transfenac formulations have genated soya phospholipids. 36 (1986) 1092^1096. the potential to replace oral therapy with diclofenac. J. Forsch. D. Skin permeability and local tissue used in a gel (see Figs. London. Frolich. Kotera. Bouwstra. W. G. The Extra Pharmacopoeia (Martin- is lowered by using a smaller agent amount. J. Carini. [6] Voltaren Emulgel. Riess. indomethacin and successfully with the combined oral/topical treatment diclofenac in phosphatidylcholine liposomes (PCL) as mem- of rheumatoid disease. M. 4^7). M. Local tolerability and of the more conventional diclofenac gel increases in pharmacokinetic pro¢le of a new transdermal delivery sys- the low dose range. [10] A.

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