Photodiagnosis and Photodynamic Therapy (2006) 3, 190—201

Influence of formulation factors on methyl-ALA-induced protoporphyrin IX accumulation in vivo Ryan F. Donnelly PhD a,b,∗, Petras Juzenas b,c, Paul A. McCarron a, Li-Wei Ma b, A. David Woolfson a, Johan Moan b,d a

School of Pharmacy, Queens University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK b Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310, Oslo, Norway c Fellow of the Norwegian Cancer Society, P.O. Box 4 Sentrum 0101, Oslo, Norway d Institute of Physics, University of Oslo, P.O. Box 1048 Blindern 0316, Oslo, Norway Available online 3 May 2006

KEYWORDS 5-Aminolevulinic acid; Drug delivery; Bioadhesion; Patches; Fluorescence

Summary Photodynamic therapy (PDT) is a medical treatment by which a combination of a photosensitising drug and visible light cause the destruction of selected cells. Thick lesions, such as nodular basal cell carcinomas (BCCs), or lesions with overlying keratinous debris, are reported as being difficult to eradicate using 5aminolevulinic acid-based photodynamic therapy (ALA-PDT). Such treatment failures have been attributed to the shallow penetration of water-soluble drugs like ALA. In addition, the current scarcity of sophisticated drug delivery research centered on PDT applications has meant that accurate comparison of similar clinical studies is difficult. This paper investigates, for the first time, novel drug delivery systems for controlled drug delivery of methyl-ALA (M-ALA). Pressure sensitive adhesive (PSA) and bioadhesive patches containing defined M-ALA loadings and a standard cream containing equivalent amounts of drug were applied to the skin of mice for defined periods of time and the fluorescence of the protoporphyrin IX (PpIX) induced measured over 24 h. Of major importance, the PSA patches containing low drug loadings induced high PpIX levels, which were limited to the site of application, after only 1 h applications. Such systems have the potential to improve selectivity of PpIX accumulation, increase simplicity of treatment and, due to the low drug loadings required, reduce costs of clinical PDT. PSA patches would be most suitable for application

Abbreviations: AKs, actinic keratoses; ALA, 5-aminolevulinic acid; BCCs, basal cell carcinomas; CCR, complete clearance rate; PDT, photodynamic therapy; PMVEMA, poly(methylvinylether/maleic anhydride); PpIX, protoporphyrin IX; PSA, pressure sensitive adhesive; TPM, tripropyleneglycol methyl ether ∗ Corresponding author. Tel.: +44 2890 972 251; fax: +44 2890 247 794. E-mail address: [email protected] (R.F. Donnelly). 1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2006.03.007

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to areas of dry skin, while bioadhesive patches would be suitable for moist areas, such as the mouth or lower female reproductive tract and have been shown here to induce significant PpIX production at the site of application after 4 h applications of patches containing high drug loadings. © 2006 Elsevier B.V. All rights reserved.

Introduction Photodynamic therapy (PDT), based on topical application of 5-aminolevulinic acid (ALA), has been widely reported to give high clearance rates when used in the treatment of superficial skin lesions, such as superficial basal cell carcinoma [1—4], Bowen’s disease [5—7] and actinic keratosis [8—10]. Excellent tissue preservation and lack of scarring are noted advantages over conventional surgical treatment options for such lesions. However, thicker lesions, such as nodular basal cell carcinomas (BCCs), or lesions with overlying keratinous debris, are reported as being difficult to eradicate using ALA-PDT [11,12]. Such treatment failures have been attributed to the shallow penetration of water-soluble drugs like ALA [2]. Consequently, more lipophilic ALA derivatives have been produced with the aim of improving the penetration and subsequent treatment success rates. The most commonly investigated ALA derivative is its methyl ester (M-ALA). High clearance rates of nodular basal cell carcinomas have been reported after topical application of M-ALA [12]. ALA and M-ALA are typically delivered to surface lesions using topically applied creams, which are covered with occlusive dressings to aid retention at the site, enhance drug absorption and provide protection from light for the accumulating protoporphyrin IX (PpIX) [13—15]. However, great variability in the amount of creams applied per unit area has been reported [2,6,7,10,16—19]. Application of occlusive dressings over the applied creams leads to smudging and spreading of the cream away from the site of application in an irreproducible fashion, adding further uncertainty to the technique. Consequently, comparison of the results of different studies is difficult. Clearly there is a need for a unit dosage form for use in PDT based on the topical application of ALA or its derivatives. Use of such a system would eliminate the interclinician variability seen at present and allow accurate critical comparisons of different studies to be made. An ideal dosage form would be self-adhesive and backed with an occlusive and opaque material, thereby negating the need for a covering dressing and simplifying and standardizing treatment. We have previously described a bioadhesive patch, backed with an occlusive layer of opaque

green PVC, and containing a defined ALA loading. We have used this system in successful PDT of various superficial vulval neoplasias and dysplasias [20—23]. Moreover, we have recently developed strategies for enhanced PDT of thick lesions [24,25]. This paper combines these two research themes and, therefore, describes the formulation and in vivo investigation of two novel M-ALA-containing patches which may be used to enhance PDT of thick lesions.

Materials and methods Chemicals Gantrez® AN-139, a copolymer of methylvinylether and maleic anhydride (PMVE/MA), was provided by ISP Co. Ltd., Guildford, UK. Tripropyleneglycol methyl ether (DowanolTM TPM) and 5aminolevulinic acid methyl ester (hydrochloride salt) were purchased from Sigma Aldrich, Dorset, UK. Plastisol® medical grade poly(vinyl chloride) emulsion, containing diethylphthalate as plasticiser, was provided by BASF Coatings Ltd., Clwyd, UK. Unguentum Merck® was obtained from Merck, Darmstadt, Germany. Duro-Tak® 2074 was provided by National Starch and Chemical Company, Berkeley, CA, USA.

Patch and cream manufacture Bioadhesive patches evaluated in this study were prepared by a conventional casting technique [26] using a gel containing 20% (w/w) PMVE/MA and 10% (w/w) TPM. PMVE/MA was added to ice-cooled water (reagent grade 1), stirred vigorously and heated to 95 ◦ C until a clear solution was formed. Upon cooling, the required amount of tripropyleneglycol methyl ether was added and the casting blend adjusted to a final weight with water. Appropriate amounts of M-ALA were dissolved directly into defined volumes of this aqueous blend immediately prior to casting. It was decided to load each square centimetre of the patch with an equivalent dose of M-ALA to that contained in the amounts of proprietary creams typically applied per square centimetre to neoplastic lesions.

192 A cream (Unguentum Merck® ) was applied, in the thickness used clinically, to each of 25 cm2 , ruled out on the back of a gloved hand. Each square centimetre was individually cleared of cream using a microspatula and each aliquot of cream weighed. As Metvix® cream, the most commonly used M-ALA product in Europe, contains 16% (w/w) M-ALA, the mean M-ALA dose per square centimetre was determined by calculation. This drug loading was then used as a starting point in the patch design process. Patches containing M-ALA loadings of 30.4 mg cm−2 were prepared initially and other loadings, above and below this original level, were then tested. Given the instability of M-ALA at elevated pH [27], no adjustment was required, and the pH of the cast gels was allowed to remain close to 2. Bioadhesive films were prepared by slowly casting the drug-loaded gel into a pre-levelled mould (internal dimensions 30 mm by 50 mm), lined with a release liner to facilitate film removal. This was placed in a constant air flow at 25 ◦ C for 24 h. Pressure sensitive adhesive (PSA) films, containing the same M-ALA loadings as the bioadhesive films, were prepared by mixing M-ALA with DuroTak® 2074 (25% (w/w)) in ethyl acetate. The resulting gels were then cast as above and allowed to dry over a period of 30 min in a fume cupboard. PVC backing films were prepared using a forced smear technique, where uncured polymer was knife-drawn over a glass surface to produce a film of approximately 100 ␮m thick. This was cured at 160 ◦ C, removed and applied to the exposed surface of the dry bioadhesive or PSA film, using gentle pressure to affirm attachment. Finished patches were removed by simply peeling the release liner, with attached film, off the base of the mould. Creams containing M-ALA were prepared in such a way that application of a defined weight to a defined area of skin would deliver the same amount of drug as the patches. Creams were produced by simply mixing M-ALA crystals directly into Unguentum Merck® . All formulations were used immediately after preparation.

Animals Female Balb/c athymic nude mice were obtained from Bomholt Gaard (Ry, Denmark). At the start of the experiments, the mice were 7—8 weeks old, with an average body weight of 20—25 g. Three mice were housed per cage with autoclaved filter covers in a room with subdued light at constant temperature (24—26 ◦ C) and humidity (30—50%). Food and bedding were sterilised and the mice were given tap water ad libitum in sterilised bottles.

R.F. Donnelly et al. The mice were divided into three main groups, one to have creams applied, one the bioadhesive patches and the other the PSA patches. All animal experiments were conducted according to the policy of the Federation of European Laboratory Animal Science Associations (FELASA) and The European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, with the implementation of the principle of the 3Rs (replacement, reduction, refinement). The animal experiments were performed under a Project (No. 108) approved by the Animal Department of the Norwegian Radium Hospital.

ALA application Approximately 190 mg of the freshly prepared cream was applied on a single spot of 1 cm2 area on the right flank of each mouse in the first group. The cream was then covered with an occlusive dressing (OpSite Flexigrid® , Smith & Nephew Medical Ltd., Hull, UK). An area (1 cm2 ) of either bioadhesive or PSA patch was applied on a single spot on the right flank of each mouse in the second and third groups. Approximately 10 ␮l of water was applied to the skin prior to bioadhesive patch application to initiate adhesion. For application of the cream, it was necessary to anaesthetise the animals. This was done using a subcutaneous injection of Hyponorm® /Dormicum® (50/50 mix, approximately 4 ml kg−1 body weight). This was unnecessary for patch application. However, to ensure a fair comparison, all animals were anaesthetised. The animals woke up within 30 min and appeared normally active for the duration of the experiment. Creams, occlusive dressings and patches were removed from the animals after a period of 1 or 4 h, to mimic application times used clinically, and the area washed with warm water and blotted dry with tissue paper.

Fluorescence measurements Fluorescence was measured by means of a Perkin Elmer LS50B luminescence spectrometer (Norwalk, CT). The measurements were carried out using a standard fibre optic probe connected to the spectrometer. The fluorescence excitation wavelength was set at 407 nm, corresponding to the maximum of the Soret band of PpIX in cells. The emission wavelength was scanned from 620 to 650 nm. The excitation and emission slits were set at 10 and 15 nm, respectively. Scattered excitation light was removed from the detected light with a 515 nm cut-off filter. Skin autofluorescence was subtracted from all fluorescence spectra.

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Imaging The fluorescence of the mice was visualised by a Kodak digital camera (DCS 720×, Eastman Kodak Co., Rochester, NY). Images in room light and with the excitation light (TLD 18W/08, Philips, The Netherlands; emission 350—400 nm, max 368 nm) were captured using camera settings f 2.8, 1/40, ISO1000. The fluorescence images were recorded using a longpass cut-off 630 nm filter in front of the camera objective and camera settings f 2.8, 1/60, ISO3200.

Statistical analysis Comparison between cream and patch formulations, in terms of PpIX fluorescence, were made using the Mann—Whitney U test. In all cases p < 0.05 denoted significance.

Results Table 1 shows the mean (±S.D.) weights of cream applied per square centimetre. Table 1 also shows the mean M-ALA dose available per square centimetre. Given that the proprietary Metvix® cream contains 16% (w/w) M-ALA and that it was found that approximately 190 mg of cream was applied per square centimetre, an M-ALA loading of 30.4 mg cm−2 was used as a starting point for patch design. M-ALA loadings of 4.0, 15.2, 30.4 and 40.0 mg cm−2 were all investigated with respect to the influence of different M-ALA contents on protoporphyrin IX production in murine skin in vivo. Formed bioadhesive films were completely clear in all cases, with no evidence of crystallization. However, PSA films containing 30.4 and 40.0 mg cm−2 M-ALA were opaque and light microscopy revealed the presence of crystalline material throughout the matrix. Fig. 1 shows the influence of formulation factors and drug loading on PpIX accumulation at the site of application in murine skin after 1 h applications. Fig. 2 shows the influence of these two Table 1 Outcome of experiment to determine approximate M-ALA loadings for bioadhesive and PSA patches (mean ± S.D., n = 25) Mean mass of cream applied (g cm−2 ) (±S.D.) M-ALA delivered by the 16% (w/w) proprietary cream (approximate amount) (mg cm−2 )

0.19 ± 0.05 30.40 ± 0.01

Figure 1 Influence of drug loading on PpIX accumulation in normal murine skin at the site of application following 1 h application of bioadhesive patches (A), cream (B) and PSA patches (C). Fluorescence was measured in each case before application; creams and patches were then applied for 1 h and then removed, and further measurements taken up to 24 h. Results are plotted as mean ± S.D. (n = 3).

factors on the maximum PpIX fluorescence generated at the site of application in each case. For each formulation examined, the peak fluorescence was observed between 3 and 5 h, and the skin fluorescence returned to background levels within 24 h. For bioadhesive patches, the peak fluorescence increased significantly as drug loading increased, with the transition in loading from 30.4 to 40.0 mg cm−2 causing a particularly profound (p = 0.0495) increase. For creams, the peak

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Figure 2 Influence of drug loading and formulation type on the maximum PpIX fluorescence at the site of application in normal murine skin following 1 h applications. Results are plotted as mean ± S.D. (n = 3). The drug loading for creams is expressed in terms of the amount of drug delivered per square centimetre by 190 mg of cream applied to this area, rather than the percent (w/w) loading, so as to allow direct comparison with patches.

fluorescence increased significantly as the drug loading was increased from 2.1 to 8.0% (w/w) (p = 0.0495). Further increasing the drug loading had no significant effect on peak fluorescence. In all cases, the peak fluorescence generated by the cream was significantly greater than that generated by the corresponding bioadhesive patch. For PSA patches, the peak fluorescence increased non-significantly (p = 0.2752) as the drug loading was raised from 4.0 to 15.2 mg cm−2 . The peak fluorescence then fell significantly (p = 0.0495) as the drug loading was increased from 15.2 to 30.4 mg cm−2 . There was no significant difference (p = 0.5127) in the peak fluorescence generated by 1 h applications of PSA patches containing 30.4 or 40.0 mg M-ALA cm−2 . The PSA patches containing 4.0 and 15.2 mg M-ALA cm−2 generated significantly higher peak fluorescences than any of the other formulations investigated, irrespective of greater loadings in creams and bioadhesive patches. Fig. 3 shows the influence of formulation factors and drug loading on PpIX accumulation at the opposite flank in murine skin after 1 h applications. Fig. 4 shows the influence of these two factors on the maximum PpIX fluorescence generated at the opposite flank in each case. For PSA patches and bioadhesive patches, the PpIX fluorescence at the opposite flanks of the mice did not increase significantly above background during the study period. However, for the creams, the PpIX fluorescence peaked between 3 and 4 h in each case. As drug loading increased from 8 to 21% (w/w), the peak fluorescence increased significantly (p = 0.0495), with

Figure 3 Influence of drug loading on PpIX accumulation in normal murine skin on the opposite flank following 1 h application of bioadhesive patches (A), creams (B) and PSA patches (C). Fluorescence was measured in each case before application; creams and patches were then applied for 1 h and then removed, and further measurements taken up to 24 h. Results are plotted as mean ± S.D. (n = 3).

a 1 h application of the 21% (w/w) cream producing a mean peak fluorescence of 34.4 a.u. on the opposite flank of the mice. Fig. 5 shows the influence of formulation factors and drug loading on PpIX accumulation at the site of application in murine skin after 4 h applications. Fig. 6 shows the influence of these two factors on the maximum PpIX fluorescence generated at the site of application in each case.

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Figure 4 Influence of drug loading and formulation type on the maximum PpIX fluorescence at the opposite flank in normal murine skin following 1 h applications. Results are plotted as mean ± S.D. (n = 3). The drug loading for creams is expressed in terms of the amount of drug delivered per square centimetre by 190 mg of cream applied to this area, rather than the percent (w/w) loading, so as to allow direct comparison with patches.

For each formulation, the peak fluorescence was observed between 5 and 10 h. The skin fluorescence returned to background within 24 h in mice to which bioadhesive patches were applied. The skin fluorescence remained elevated in mice to which PSA patches and creams were applied. In each case, the peak fluorescence was significantly greater after a 4 h application than after a 1 h application. For bioadhesive patches, the peak fluorescence increased significantly as drug loading increased. For creams, the peak fluorescence did not increase significantly as the drug loading was increased from 2.1 to 21.0% (w/w) (p = 0.5127). Apart from the highest drug loadings (p = 0.1266), the peak fluorescence generated by the cream was always significantly greater than that generated by the corresponding bioadhesive patch. For PSA patches, the peak fluorescence did not increase significantly (p = 0.5127) as the drug loading was raised from 4.0 to 15.2 mg cm−2 . The peak fluorescence then fell significantly (p = 0.0495) as the drug loading was increased from 15.2 to 30.4 mg cm−2 , before rising slightly (p = 0.1266) as the loading was increased from 30.4 to 40.0 mg MALA cm−2 . However, it should be noted that the markedly lower peak fluorescences induced by 1 h applications of the latter formulations were not observed after 4 h applications. The PSA patch containing 15.2 mg M-ALA cm−2 generated significantly higher peak fluorescence than any of the other formulations investigated, irrespective of greater loadings in creams, bioadhesive patches or PSA patches.

Figure 5 Influence of drug loading on PpIX accumulation in normal murine skin at the site of application following 4 h application of bioadhesive patches (A), creams (B) and PSA patches (C). Fluorescence was measured in each case before application; creams and patches were then applied for 4 h and then removed, and further measurements taken up to 24 h. Results are plotted as mean ± S.D. (n = 3).

Fig. 7 shows the influence of formulation factors and drug loading on PpIX accumulation at the opposite flank in murine skin after 4 h applications. Fig. 8 shows the influence of these two factors on the maximum PpIX fluorescence generated at the opposite flank in each case. For each formulation, the peak fluorescence at the opposite flank of the mice following a 4 h application was significantly elevated with respect to the peak fluorescence after a 1 h application. Only for bioadhe-

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Figure 6 Influence of drug loading and formulation type on the maximum PpIX fluorescence at the site of application in normal murine skin following 4 h applications. Results are plotted as mean ± S.D. (n = 3). The drug loading for creams is expressed in terms of the amount of drug delivered per square centimetre, rather than the percent (w/w) loading, so as to allow direct comparison with patches.

sive patches containing 40.0 mg cm−2 did the PpIX fluorescence at the opposite flanks of the mice increase appreciably above background during the study period. However, for the creams and PSA patches, the PpIX fluorescence peaked between 6 and 7 h in each case. As drug loading increased from 16 to 21% (w/w), the peak fluorescence increased non-significantly (p = 0.2752), with a 4 h application of the 21% (w/w) cream producing a mean peak fluorescence of 62.2 a.u. on the opposite flank of the mice. The peak fluorescence generated by 4 h applications of the PSA patches generally increased as drug loading increased, with the 30.4 mg cm−2 patch inducing a peak fluorescence of 84.3 a.u. on the opposite flank of the mice. Surprisingly, the peak fluorescence generated at the opposite flank by a 4 h application of the 40 mg cm−2 patch was significantly lower than that induced by the 15.2 and 30.4 mg cm−2 patches. It should be noted that this patch generated high peak fluorescences at the site of application. Fig. 9 (A—C) shows the three formulations in place on the flanks of the mice. Fig. 9 also shows the view through a longpass cut-off 630 nm filter, under UV illumination, of application sites 2 h after removal of bioadhesive patch (D), cream (E) and PSA patch (F) formulations from the skin following 4 h applications. The cream clearly produced a diffuse fluorescence around the application site, while for both types of patch the fluorescence was localised to the application site (demarcated in the figure using a black marker). The fluorescence induced by the PSA patch was extremely intense (Fig. 9F).

Figure 7 Influence of drug loading on PpIX accumulation in normal murine skin on the opposite flank following 4 h application of bioadhesive patches (A), creams (B) and PSA patches (C). Fluorescence was measured in each case before application; creams and patches were then applied for 4 h and then removed, and further measurements taken up to 24 h. Results are plotted as mean ± S.D. (n = 3).

Discussion Conventional treatments for skin neoplasias include surgical excision and radiotherapy, which are usually successful, with complete clearance rates (CCR) up to 95% on 5 years follow-up reported [28]. However, these treatments are unsuitable for large or multiple lesions and can lead to poor cosmesis [28]. Cryotherapy and curettage are only suitable

Influence of formulation factors on M-ALA-induced PpIX accumulation

Figure 8 Influence of drug loading and formulation type on the maximum PpIX fluorescence at the opposite flank in normal murine skin following 4 h applications. Results are plotted as mean ± S.D. (n = 3). The drug loading for creams is expressed in terms of the amount of drug delivered per square centimetre by 190 mg of cream applied to this area, rather than the percent (w/w) loading, so as to allow direct comparison with patches.

for very superficial tumours and are associated with high rates of recurrence [3,6,13,28]. The most commonly used drug for PDT of skin neoplasias is topically applied ALA. Superficial skin lesions show an excellent response to topical ALA-PDT, but the response of thicker or hyperkeratotic lesions is disappointing. Dijkstra et al. [10] were able to get a complete response in 100% of superficial basal cell carcinomas after a single treatment, while only 50% of nodular BCCs showed a complete response when analysed histologically. Similar results were initially reported by Svanberg et al. [5], however, the complete response rate of nodular BCCs was improved from 64 to 100% after a second treatment. Nodular BCCs greater that 2 mm in depth are not uncommon [1]. Topically applied ALA is unlikely to penetrate the tissue to this depth [29] in conventional application times, which vary between 3 and 6 h. As a result, nodular BCCs are not good candidates for topical ALA-PDT. Lesions that appeared to be cleared may recur, as reported by Calzavara-Pinton [3]. After initial clearance, 33% of nodular BCCs recurred within 19 months. On biopsy, tumour remnants were found in deep dermis and were covered by normal skin. Similarly, Dijkstra et al. [10] achieved only a 50% CCR for hyperkeratotic actinic keratoses (AKs). These results reflect the poor penetration of ALA through skin. Furthermore, it has been suggested that keratotic debris may actually absorb ALA, preventing it from reaching the lesion [19]. Potential strategies for successful PDT of thicker lesions include multiple treatments [3], prolonged

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ALA application times [29], employing penetration enhancers or curettage to allow ALA to penetrate deeper into the dermis within reasonable application times. Another option would be to use ALA esters, which are more lipophilic and should penetrate to greater depths in tissue than their more hydrophilic parent. However, in this case, binding to superficial skin layers may be a problem, possibly even larger than for ALA. Soler et al. [12] report a complete clearance rate of 79% for 350 BCCs, of which 168 were nodular, on 35 months follow-up after curettage followed by treatment with topical M-ALA and red light. In this case, curettage amounted to a simple debulking procedure which removed only visible parts of the tumour using a small surgical curette. Complete removal of the tumour tissue was not attempted and topical M-ALA was applied to the remaining tumour. The most commonly used topical delivery systems for ALA and M-ALA are the proprietary o/w creams Porphin® (20% (w/w) ALA) and Metvix® (16% (w/w) M-ALA), respectively. There is currently little consensus as to the optimum amount of cream required for successful treatment. In fact, the dose of cream applied in clinical studies is rarely reported. Where the dose is given, great variability between studies can be observed. Literature reports have indicated that between 10 and 200 mg of the ALA- or M-ALA-containing vehicle is applied per square centimetre of lesion, giving a 20-fold difference in drug dose [2,6,7,10,16—19]. The use of occlusive dressings to aid retention and enhance absorption can further increase this variability by causing smudging and subsequent spreading of the cream from the site of application. As a result, comparing the outcomes of different studies becomes very difficult. The use of a unit-dose system for topical delivery of ALA or M-ALA would eliminate this inter-clinician variability, enabling comparison of different studies. Moreover, if such a system were self-adhesive and backed with an occlusive material, the need for retentive dressings would be eliminated. This would be of particular benefit in the PDT of oral lesions or those of the lower female reproductive tract, where use of creams and occlusive dressings is impractical. The rapid development of the field of PDT based on ALA and its esters, and the rapid transition from laboratory to clinic have meant that important drug delivery issues have been sidelined [14,15]. In fact, only recently have reports of unit dosage forms containing ALA or M-ALA been published. Bretschko et al. [30] described the formulation of an adhesive patch, loaded with a defined amount of ALA, as a potential dosage form for topical PDT. Crystalline ALA was dispersed in a pressure sensitive adhesive

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Figure 9 Bioadhesive patch (15.20 mg cm−2 ) (A), cream (21.0% (w/w)) covered with an occlusive dressing (B) and PSA patch (15.20 mg cm−2 ) (C) in place. View through a longpass cut-off 630 nm filter, under UV illumination, of application sites 2 h after removal of bioadhesive patch (D), cream (E) and PSA patch (F) formulations from normal murine skin following 4 h applications.

matrix. In vitro drug release studies demonstrated a 20 h lag-phase before significant amounts of ALA were released from the patch across excised stratum corneum. The reason given for the delay before significant amounts of ALA were released was that the dispersed drug had to dissolve before diffusion could occur. Such a system, therefore, requires moisture to activate release. Moisture, however, compromises the adhesion of PSA devices [31], and

the patch may not stay in place long enough to be clinically effective, especially in wet environments, such as the mouth or the lower female reproductive tract. A similar, ALA-containing, PSA patch was described by Lieb et al. [32]. In this system, based on Eudragit® NE, the release of the dispersed crystalline ALA across excised stratum corneum was enhanced by the incorporation of the plasticiser acetyl tributyl citrate (ATBC) into the formulation.

Influence of formulation factors on M-ALA-induced PpIX accumulation ATBC increased the flexibility of the formed patch, increasing the influx of water. No lag-phase was observed in the drug release profile. However, only 2.5% of the ALA loading was released from the patch across excised stratum corneum and epidermis after 5 h. There was no mention of drug release from a similar system described by Pons et al. [33]. However, pressure sensitive patches containing ALA were used in the successful PDT of actinic keratosis, basal and squamous cell carcinomas. Such systems may be capable of maintaining stability of ALA or its esters on prolonged storage, due to the fact that the drug is in the solid state. However, the poor release and inability to adhere in wet environments may limit their commercial success. An alternative approach to patch production was described by McCarron et al. [20]. The authors described a novel bioadhesive patch cast from an aqueous gel containing poly(methyl vinyl ether/maleic anhydride). In contrast to the pressure sensitive systems described above, ALA was dissolved in the matrix of the patch and no lagtime was observed in the ALA release profile. The patch released approximately 60% of its drug loading across a model membrane over 6 h. The system was subsequently shown to be capable of delivering high levels of ALA down to depths of approximately 2.5 mm in vaginal tissue [34]. The formulation was highly flexible and capable of adhering even in wet environments and was used in the successful PDT of extra-mammary Paget’s disease and lichen sclerosus of the vulva [21,22]. Similar systems were described by Manivasager et al. [35,36] for the delivery of ALA and its methyl ester for the purposes of PDD, although drug release was not evaluated. The mean amount of cream applied per square centimetre used in the present study, 190 mg, falls within the upper reaches of the range detailed above. Knowing the drug loading in the Metvix® cream (16% (w/w) M-ALA), this allowed calculation of the required amount of drug in the patches. A patch containing 30.4 mg cm−2 M-ALA, should contain the same available dose as the respective cream. Drug loadings either side of these were also investigated to reflect the variability in amounts of creams applied clinically. As may be seen from Fig. 2, 1 h applications of the various formulations induced PpIX in murine skin. Increasing drug loading in bioadhesive patches and creams increased the peak PpIX fluorescence at the site of application. Increasing drug loading in PSA patches from 4.0 to 15.2 mg cm−2 significantly increased the peak PpIX fluorescence. However, further increasing the drug loading led to significant and surprising decreases in peak PpIX fluorescence. This is likely to be due to crystallization of

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M-ALA in PSA patches with loadings greater than 15.2 mg cm−2 . If the drug is in the solid form, it cannot diffuse into skin, and the crystals may actually retard the penetration of the drug dissolved in the matrix by presenting a physical barrier at the release surface of the patch. Indeed, crystals were evident at the surface of the patches containing 30.4 and 40.0 mg M-ALA cm−2 , which felt rough and did not adhere particularly well compared to those with lower loadings. The PSA systems with the lower loadings described here adhered well to murine skin and induced the highest PpIX fluorescence values at the site of application after 1 h applications. This was despite the cream and bioadhesive patch having maximum loadings 10-times greater than the PSA patch with the lowest loading, and may be due to the fact that M-ALA is actually quite hydrophilic (MALA log octanol/water partition coefficient = −0.9; ALA log octanol/water partition coefficient = −1.5) [37], and, hence, should have a significant thermodynamic activity in the hydrophobic PSA matrix leading to enhanced diffusion into skin [38]. Conversely, M-ALA should have a relatively low thermodynamic activity in the water-based bioadhesive patches, leading to reduced M-ALA penetration into skin and the reduced production of PpIX observed. Unguentum Merck® is an o/w emulsion and, consequently should deliver approximately the same amount of M-ALA as the bioadhesive patch, as the drug should all be in the external aqueous phase. However, the cream contains several ingredients, such as, glyceryl monostearate and cetostearyl alcohol, which may act as chemical penetration enhancers. As a result, the penetration of M-ALA into, and through, normal skin is enhanced, leading to the relatively high peak fluorescences observed at the application site. Application of creams for 1 h also led to production of small amounts of PpIX on the opposite flank of the mouse, as shown in Fig. 3. This did not occur with the other formulations and may be due to the presence of the penetration enhancers in the cream formulation. Four-hour applications led to significantly increased peak PpIX fluorescences at the site of application in each case, as may be seen from Fig. 6. Again the PSA patches with the lower loadings produced the highest peak fluorescence values in contrast to other formulations containing greater loadings. The PSA patches with the higher loadings produced significant amounts of PpIX at the site of application after 4 h applications. This may be due to perspiration from the mice dissolving the crystalline M-ALA and allowing its diffusion into skin. Indeed, on removal the formulations appeared relatively clear and appeared

200 smooth, in contrast to the appearance of the same formulations upon removal after 1 h applications. Four-hour applications led to significantly increased peak PpIX fluorescences at the opposite flanks of the mice in each case. The cream and PSA patches produced significant peaks of PpIX fluorescence at the opposite flank. This is in contrast to previously published studies [39,40], and demonstrates that M-ALA can cross the skin barrier and induce PpIX production at distant sites, after prolonged application times of optimal delivery vehicles. From this study it can be seen that PSA patches containing low loadings of M-ALA produce high levels of PpIX at the site of application. If applied for short periods (1 h), very little PpIX is produced at distant sites. Since this type of patch only delivers M-ALA to the exact site of application, in contrast to the commonly used cream formulation (Fig. 9), the efficiency and accuracy of dosing is improved. This type of patch is simple to apply and delivers a defined amount of drug per square centimetre, thus, in theory, improving the simplicity and convenience of dosing by clinicians and allowing accurate comparison of similar clinical studies. Although 4 h applications of the PSA patches induced higher PpIX levels than 1 h applications in each case, the shorter application time allowed PpIX to be localised to the site of application. For this reason, and for the fact that 4 h applications of creams (Fig. 5) of high loadings induces only slightly higher PpIX levels than a 1 h application of PSA patches with low loadings (Fig. 1), it is likely that such PSA systems could have great utility in clinical M-ALA PDT. Given that M-ALA is relatively expensive, the PSAbased approach would also make good economic sense. Stability studies are currently ongoing to investigate the practical viability of this formulation, which can be used on any area of dry skin. Bioadhesive patches would be suitable for moist areas, such as the mouth or lower female reproductive tract and have been shown here to induce significant PpIX production at the site of application after 4 h applications of patches containing high drug loadings.

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Influence of formulation factors on methyl-ALA-induced protoporphyrin IX accumulation in vivo.

Photodynamic therapy (PDT) is a medical treatment by which a combination of a photosensitising drug and visible light cause the destruction of selecte...
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