NSC B

Transcription

1 NSC B twwong@mail.ncku.edu.tw

2 The Sequential Studies of Photodynamic Therapy: Development of Optimal Transdermal Delivery Agent and The Fluorescence Monitor Machine NSC B twwong@mail.ncku.edu.tw : (Photodynamic 5-ALA therapy, PDT) Dimethyl sulfoxide (DMSO) 5-Aminolevulinic acid deferoxamin methansulfonic, (5-ALA) (Desferal, CIBA-GEIGY Ltd., Switzerland, Protoporphrin DES) IX (PpIX) 5nm 635nm PpIX 2%5ALA 10%DES %5- ALA 5-ALA PDT 88 PDT Abstract Photodynamic therapy (PDT) began to bloom as a new promising therapy against CCD cancers in recent 10 years. 5- aminolevulinic acid (5-ALA) was one of the most well studied prodrugs, which turns into CCD the photosensitizer protopophrin IX (PpIX) in cancer cell. PpIX, the new second generation photosensitizer emits 635nm red fluorescence under 5nm blue light excitation. The intensity of red 2. fluorescence correlates well with the cellular Spex content of PpIX and thus can be used for SkinSkan (Jobin Yvon Ltd., UK) drug monitoring. Twenty precent 5-ALA cream was often used to treat skin cancers in 2. clinical practice. However, the fluorescence 2

3 intensity monitoring system (around million (1) NT dollars) as well as 5-ALA are quite (non-melanoma skin cancer) expensive. To develop an effective yet inexpensive fluorescence intensity 5-aminolevulinic acid (5-ALA) monitoring system and 5-ALA topical formulation is therefore mandatory. FDA The project composed of two arms: 1. To develop an effective, inexpensive 5-ALA fluorescence monitor device during PDT, 2. To develop a cost effective formulation for % (1) (2,3) transdermal delivery of 5-ALA. By using UV-induced squamous cell (4,5) carcinoma in hairless mice skin model we established in the last NSC project, we found that digital camera could effectively capture 5-ALA fluorescence image for digital analysis. A Protoporphrin IX good correlation between the fluorescence kinetics analyzed with digital images and (PpIX) 5nm 635nm with a commercial machine Spex SkinSkan (Jobin Yvon Ltd., UK). To develop a better formulation for (1,6) transdermal absorption, we combined 5-ALA solution with different concentrations of enhancer (DMSO), elimination inhibitor (DES, deferoxamin methansulfonic, Desferal, CIBA-GEIGY Ltd., Switzerland) and monitoring fluorescence kinetics for 32 hours after 4-hour occlusion. We found that 5 0mJ/cm 2 UVB 2% 5-ALA with 10% DES appeared to be the 62 best formulation for transdermal absorption. In summary, we found that digital (4) 3mm imaging was a cheap and effective way to (4) (T)(Fig 2) monitor fluorescence kinetics during PDT. Two percent 5-ALA with 10% DES provided N 2%5ALA a stronger and longer fluorescence intensity (2% 10%) DMSO in tumor tissue in comparison to other DES combinations. It implied that it could be ( used for clinical studies in stead of % ) SkinSkan ( 5nm ALA which usually used, though it may 0-650nm, peak 635nm) probably unnecessary. %5-ALA ( Keywords: Photodynamic therapy, 5- aminolevulinic acid, UV-induced squamous ) 2%5- cell carcinoma, fluorescence monitor device, ALA+10%DES (Fig 3A-C) transdermal delivery 2%5-ALA 2%5- ALA+10%DES %5-ALA 4 (Photodynamic Kodak DC290 7X therapy, PDT) 1.5 Wood s (Fig 1) Lamp(3-410nm, peak365nm) 3

4 (Fig 2) PC NIH Image 1.61, (Wayne Rasband National (carcinogenesis) Institutes of Health, Bethesda MD, USA) (Fig 4A-C) SkinSkan 2%5-ALA 10%DES NIH Image %5ALA SkinSkan 5-ALA prodrug NIH PpIX ferrochelatase Image in vivo PpIX Fe ++ heme DES PDT Fe ++ ferrochelatase Fe ++ PpIX (1)( 1) 3,4 5 2%5-ALA+10%DES 1. Peng Q, Warloe T, Berg K, et al %5-ALA %5-ALA Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges. Cancer. 79: , (5) PpIX Wong TW, Sheu HM, Lee JYY, Fletcher RJ. Photodynamic therapy for Bowen s isease (squamous cell carcinoma in situ) of ferrochelatase (1) the digit. J Dermatol Surg. (submitted). DES PpIX 3. Jeffes EW, McCullough JL, Weinstein GD, (N) et al. Photodynamic therapy of actinic 3 keratosis with topical 5-aminolevulinic acid. A pilot dose-ranging study. Arch PpIX Dermatol 1997;133: Wong TW, Hsieh WT, Fang YK, Sheen MC. The application of photodynamic %5ALA (3,7) therapy in squamous cell carcinoma: in vivo and in vitro studies. 1999; NSC (Mann-Whitney Test B report. P>0.05) 5. van der Veen N, de Bruijn HS, Berg RJ, Star WM. Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice. Br J Cancer 1996;73: ALA 6. Ackermann G, Abels C, Baumler W, et al. (2,3,7) Simulations on the selectivity of 5-2% 5-ALA % aminolaevulinic acid-induced fluorescence DES in vivo. J Photoch Photobio B 1998;47: Fehr MK, Chapman CF, Krasieva T, et al. PDT Selective photosensitizer distribution in vulvar condyloma acuminatum after topical application of 5-aminolevulinic acid. Am J Obstet Gynecol 1996;174:

5 Glycine + Succinyl-CoA ALA-synthase 5-ALA + Topical 5-ALA administration PBG DMSO PpIX Ferrochelatase Heme DES Fig. 1. Porphyrin metabolism in mammalian cells. Application of ALA bypass the rate determining step of heme on ALA synthase which leads to PpIX accumulation. DMSO enhances topical delivery of ALA theoretically while DES acts as a competitive inhibitor of ferrochelatase (red line represent inhibition). Fig. 2. After 4 hours occlusion with 5-ALA solution, vivid red fluorescence (635nm) could be seen on the UV-induced squamous cell carcinoma of the hairless mouse skin under Wood s lamp (3-410nm, peak 365nm) excitation. 5

6 2 Fluorescence intensity(x3.5x10exp-3ua) Fig. 3A T-2 N-2 Fluorescence intensity 2 0 T-2 N Fig. 4A 2 T-2+10 Fluorescence intensity (x3.5x10exp-3ua) N-2+10 Fluorescence intensity 2 T-2+10 N Fig. 4B Fig. 3B Fluorescence intensity (x3.5x10exp.-3ua) Fig. 3C T- N- Fluorescence intensity T- 2 N Fig. 4C Fig. The fluorescence intensity measured by Skinskan (3A-C) and by digital imaging captured with digital camera (4A-C). Note the similarities between two measurement systems. The scales in Fig3 were proportional reduced in order to compare with Fig 4. T-2, T-2+10, T-: tumor occluded 4 hr with 2% ALA, 2%ALA+10%DES, %ALA respectively; N: normal appearance mice skin. 6