Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 143-148
PHOTODYNAMIC THERAPY WITH ENDOGENOUS PROTOPORPHYRIN IX: B A S I C P R I N C I P L E S A N D P R E S E N T CLINICAL EXPERIENCE* J. c. KENNEDY* Departments of Oncology and Pathology, Queen's University, Kingston, Ontario KTL 3N5 (Canada)
R. H. POTTIER Department of Urology, Queen's University, Kingston, Ontario KTL 3N5 (Canada) and Department of Chemist~j and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario KTK 5LO (Canada)
D. C. PROSS Departments of Oncology and Medicine, Queen's University, Kingston, Ontario KTL 3N5 (Canada)
(Received October 5, 1989; accepted January 10, 1990)
K e y w o r d s . P h o t o d y n a m i c t h e r a p y , p r o t o p o r p h y r i n IX, b a s a l cell c a r c i n o m a ,
s q u a m o u s cell c a r c i n o m a .
Summary 5-Aminolaevulinic acid (ALA) is a p r e c u r s o r o f p r o t o p o r p h y r i n IX ( P p IX) in the b i o s y n t h e t i c p a t h w a y f o r h a e m . Certain t y p e s of cells h a v e a large c a p a c i t y to s y n t h e s i z e P p IX w h e n e x p o s e d to an a d e q u a t e c o n c e n t r a t i o n o f e x o g e n o u s ALA. Since the c o n v e r s i o n of P p IX into h a e m is relatively slow, s u c h cells t e n d to a c c u m u l a t e p h o t o s e n s i t i z i n g c o n c e n t r a t i o n s o f P p IX. P p IX p h o t o s e n s i t i z a t i o n c a n b e i n d u c e d in cells o f t h e e p i d e r m i s a n d its a p p e n d a g e s , b u t n o t in the dermis. M o r e o v e r , since ALA in a q u e o u s solution p a s s e s readily t h r o u g h a b n o r m a l keratin, b u t n o t t h r o u g h n o r m a l keratin, the t o p i c a l a p p l i c a t i o n of ALA in a q u e o u s s o l u t i o n to actinic k e r a t o s e s or superficial b a s a l cell or s q u a m o u s cell c a r c i n o m a s i n d u c e s P p IX p h o t o sensitization t h a t is r e s t r i c t e d p r i m a r i l y to the a b n o r m a l epithelium. S u b s e q u e n t e x p o s u r e to p h o t o a c t i v a t i n g light selectively d e s t r o y s s u c h lesions. In o u r o n g o i n g clinical trial o f A L A - i n d u c e d P p IX p h o t o d y n a m i c t h e r a p y , the r e s p o n s e r a t e for b a s a l cell c a r c i n o m a s following a single t r e a t m e n t h a s b e e n 9 0 % c o m p l e t e r e s p o n s e a n d 7.5% partial r e s p o n s e f o r the first 80 lesions t r e a t e d . *Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989. tAuthor to whom correspondence should be addressed.
© Elsevier Sequoia/Printed in The Netherlands
144 The cosmetic results have been excellent, and patient acceptance has been very good.
1. I n t r o d u c t i o n Standard forms of photodynamic therapy (PDT) involve the administration of an exogenous photosensitizer. If such a photosensitizer accumulates preferentially within malignant tissues, then subsequent exposure of selected tissue volumes to an adequate dose of photoactivating light may cause destruction of the malignant tissues without producing serious damage to adjacent normal tissues. The tissue photosensitizers that are in routine clinical use at present are haematoporphyrin derivative (HPD) (a complex mixture of various porphyrin monomers, dimers and polymers) and Photofrin II (a semi-purified proprietary preparation of HPD). Both show a clinically useful degree of specificity for many types of malignant tissue, although they also accumulate in liver, spleen, kidneys, skin, embryonic tissue and fresh scar tissue. Unfortunately, the HPD or Photofrin II that accumulates in the skin leads to clinically significant photosensitization that persists for at least 2 weeks, and sometimes for several months. During that time, the patients are at risk of serious accidental phototoxic reactions. This hazard is the major reason why PDT is not used much more widely. Consequently, there is a great need for new tissue photosensitizers that combine acceptably low toxicity and clinically useful tissue specificity with much more rapid clearance from the skin.
2. E n d o g e n o u s p h o t o s e n s i t i z e r s The porphyrias are a group of diseases caused by metabolic abnormalities in the biosynthetic pathway for haem. Variegate porphyria is characterized by the accumulation of protoporphyrin IX (Pp IX) in the liver and erythrohepatic porphyria (also known as protoporphyria) is characterized by the accumulation of Pp IX in both liver and erythrocytes [ 1 ]. Pp IX is an efficient photosensitizer, and the particular porphyrias are associated with severe phototoxic skin reactions. Although the concentration of Pp IX in the serum is devated in at least some of the porphyrias , we considered the possibility that the skin photosensitizer was synthesized i n s i tu and that it might be possible to induce the synthesis of photosensitizing concentrations of Pp IX in malignant tissues. Pp IX is the immediate precursor of haem in the biosynthetic pathway for haem. Since haem-containing enzymes are essential for energy metabolism, every nucleated cell in the body must have at least a minimal capacity to synthesize Pp IX. Under normal conditions, haem biosynthesis is regulated so closely that photosensitizing concentrations of Pp IX never accumulate
145 except in a few highly specialized tissues . Little is known about the regulation of haem biosynthesis in tissues other than liver and bone marrow. In the liver, a feedback control mechanism that responds to changes in the concentration of haem regulates the synthesis of 5-aminolaevulinic acid (ALA), an early precursor of both Pp IX and haem . In principle, such feedback control can be bypassed by the provision of an excess of exogenous ALA, and if the rate at which ALA-induced Pp IX is synthesized is greater than the rate at which it is used for the production of haem, then Pp IX should accumulate within the liver. It seemed probable that tissues other than liver might regulate their synthesis of haem in a similar manner, and that therefore it might be possible to induce photosensitizing concentrations of Pp IX in such tissues by providing them with an excess of exogenous ALA. The tissue specificity of such ALA-induced Pp IX would be quite different from that seen with HPD or Photofrin II, since only certain types of cells would have the necessary enzyme profile. Also, the Pp IX would not enter the cells by passage from the extracellular fluid into the plasma membrane, but would be synthesized within the mitochondria of the cells that it would subsequently destroy [2 ]. In 1956, four volunteers who ingested doses of M_A ranging from 12.5 to 35 mg kg -1 body weight as part of a study of porphyrin metabolism developed photosensitization of the skin that persisted for approximately 24 h [3, 4]. This suggested that at least some of the cells in the skin might synthesize and accumulate photosensitizing concentrations of Pp IX if provided with exogenous AIA, and that the photosensitization produced by such a technique would not persist for more than 24 h.
3. A n i m a l e x p e r i m e n t s •Initial experiments demonstrated that the toxicity of exogenous ALA to mice was acceptably low, and that the neurotoxicity that followed the systemic administration of a large dose of ALA was only transient . Of equal importance was the observation that a localized excess of exogenous ALA could induce the localized synthesis of a significant concentration of Pp IX in the skin ; this observation indicates that the Pp IX that accumulates in the skin following the systemic administration of M.A may be synthesized in the skin itself rather than transported to the skin after being synthesized in the liver. Subsequent studies of the dose and time relationships for the ALA-induced synthesis of Pp IX in the skin of mice confirmed these observations . Recently, we have shown that the intensity of the ALA-induced Pp IX fluorescence in various skin structures correlates well with the degree of phototoxic damage that follows exposure of the skin of Pp IX-sensitized mice to a standard dose of photoactivating light . Only certain mouse tissues developed significant Pp IX fluorescence and became photosensitized following exposure to exogenous ALA. In the skin, the epidermal cells and pilosebaceous units (epidermal appendages) did, but the cells of the dermis,
146 the blood vessels and cartilage of the ear did not . In the urinary bladder, the urothelium showed intense fluorescence, whereas the underlying muscle layers showed very little; in the uterus, the endometrium becam e strongly fluorescent, whereas the m yom e t r i um did not . Such tissue specificity would seem to permit the selective destruction of cancers of the urothelium or endometrium without danger of perforating the bladder or uterus.
4. Preliminary clinical experiments Since basal cell carcinomas are the most c o m m o n form of cancer among persons of northern E u r o p e a n ancestry and are readily accessible for both treatment and observation, we decided to begin our clinical studies with superficial cancers of the skin. Realizing that the systemic administration of ALA would cause generalized photosensitization of the skin [3, 4], we decided to investigate the effect of topical applications of ALA on bot h normal skin and various types of skin lesions. There was good reason to suspect that at least some types of cells in the skin could be induced to synthesize photosensitizing concentrations of Pp IX if provided with e x o g e n o u s ALA , and we h oped that the topical application of ALA might induce a photosensitization that remained localized to the site of ALA application. Since the systemic administration of ALA did not induce either fluorescence or photosensitization in the dermis of mice , it seem ed likely that any photosensitization induced by the topical application of ALA to human skin would be restricted to the epidermis and the epidermal appendages. Such a tissue-specific photosensitization would be ideal for the treatment of basal cell and squamous cell carcinomas, since they originate by malignant transformation of epidermal cells and therefore might b e c o m e photosensitized while the underlying dermis remained insensitive. Preliminary studies on a male volunteer were encouraging. Saturated aqueous solutions of ALA did not induce detectable Pp IX fluorescence in normal skin located on the underside of the forearm, even after 6 h of exposure. However, the somewhat sun-damaged skin on the cheek, on the back of the hand and on the scalp at the hair-line becam e moderately photosensitized following expos ur e to ALA, with the photosensitization being restricted to zones approximately 1 m m in diameter centred about the hair follicles. The skin in the photosensitized areas eventually peeled as though following a mild sunburn, but in contrast with our experience with mice , the hairs remained firmly rooted. A healing scratch that was covered by a thin layer of normal keratin developed localized Pp IX fluorescence and photosensitization following exposure to ALA. When a solution of ALA was applied for 2 h to areas of skin that included mild actinic keratoses, the actinic keratoses became both fluorescent and photosensitized, but adjacent normal skin did not. Following expos ur e of the photosensitized actinic keratoses to photoactivating light, they oozed serum and then developed thin scabs that eventually fell off to reveal normal skin. There was no
r e c u r r e n c e of the keratoses during the subsequent 18 months. However, neither warts (verruca vulgaris, ver r uca plantaris) nor seborrhoeic keratoses b e came photosensitized following the topical application of ALA.
5. C lin ical t r i a l This is a preliminary r e por t of an ongoing clinical trial of ALA-induced PDT for selected basal cell and squamous cell carcinomas, and for percut aneous nodules of carcinoma of the breast. Single lesions were always biopsied prior to treatment, but only representative lesions were biopsied if the patient had more than two. Standard treatment involved the topical application of 20% ALA dissolved in Glaxal Base, a proprietary oil-in-water emulsion. After 3 - 6 h (to allow penetration of ALA into the tissue and synthesis of Pp IX), the lesions and adjacent normal tissues were e x p o s e d to light from a Kodak slide projector equipped with a 500 W lamp (either Sylvania DEK/DFW or CBA) and a Hoya R-60 long-wave-pass colour glass filter in the slide compartment to eliminate wavelengths lower than approximately 600 nm. As part of the trial, the intensity of the photoactivating light was varied from 150 to 300 mW cm -2, the expos ur e time from 3.5 to 30 rain and the total dose of light from 15 to 150 mWh cm -2. Of the first 80 basal cell carcinomas treated, 72 (90%) showed a complete response when examined 2 - 3 months post-treatment, six (7.5%) showed a partial response (area decreased by more than 50%) and two (2.5%) were definite failures. Every lesion that was relatively flat and covered by a rough keratin layer showed a complete response after a single treatment. Two lesions that were elevated more than 1 m m above the skin and completely covered by keratin of approximately normal colour and thickness were definite therapeutic failures. The partial responses o c c u r r e d mainly with lesions that had a central ulcerated area and an elevated rim covered by keratin that a p p e a r e d to be almost normal in colour and thickness. ALA-induced Pp IX PDT was used to treat six lesions having a histological diagnosis of either i n s i t u or early invasive squamous cell carcinoma. All six showed a complete response. However, two squamous cell carcinomas that were elevated approximately 10 m m above the surface of the skin showed only a partial response (more than 50% loss of t u m o u r volume) even after r ep ea t e d weekly treatments. Actinic keratoses r e s p o n d e d well to topical ALA-induced Pp IX PDT. Of the ten lesions treated, nine showed a complete response after a single treatment. Patients with very extensive areas of sun-damaged skin on the face p r es en ted a special problem. We found it advisable to treat only a limited area at a time, in order to minimize the skin reaction. Topical ALA was used to treat four patients with metastatic carcinoma of the breast. Although strong Pp IX fluorescence and photosensitization was induced in p e r cut aneous nodules, there was no effect on subcutaneous nodules or on the more diffuse deposits of malignant cells that characterize
148 the i n f l a m m a t o r y f o r m of b r e a s t c a r c i n o m a . ALA in simple a q u e o u s solution d o e s n o t p e n e t r a t e the keratin o f n o r m a l skin. C o n s e q u e n t l y , even t h o u g h p e r c u t a n e o u s n o d u l e s are s o m e w h a t accessible to t o p i c a l ALA, t h e y are difficult to eradicate b e c a u s e their p e r i p h e r y generally lies b e n e a t h the n o r m a l skin. W e f o u n d topical ALA to be of value in the t r e a t m e n t of c a r c i n o m a o f the b r e a s t only if the p e r c u t a n e o u s n o d u l e s were v e r y small, or if the n o d u l e s w e r e bleeding a n d the s t a n d a r d f o r m s o f t r e a t m e n t for bleeding w e r e either ineffective or inadvisable. In s u m m a r y , topical ALA-induced Pp IX PDT is v e r y effective in the t r e a t m e n t o f s e l e c t e d superficial basal cell c a r c i n o m a s , i n s i t u a n d early invasive s q u a m o u s cell c a r c i n o m a s a n d actinic k e r a t o s e s . It m a y also be of value for controlling the bleeding o f p e r c u t a n e o u s s e c o n d a r i e s o f c a r c i n o m a o f the breast. At p r e s e n t o u r s t a n d a r d t r e a t m e n t p r o t o c o l for the m o r e superficial lesions involves 3 h o f e x p o s u r e to a 20% solution of ALA in Glaxal Base followed b y a d o s e of 30 m W h c m -2 o f p h o t o a c t i v a t i n g light. Thicker lesions are given h i g h e r d o s e s of light, and also l o n g e r t i m e s o f e x p o s u r e to A I ~ . R e s e a r c h n o w in p r o g r e s s is d e s i g n e d to evaluate the clinical potential o f s y s t e m i c a n d topical ALA-induced Pp IX for the d e t e c t i o n and t r e a t m e n t o f b o t h m a l i g n a n t a n d p r e - m a l i g n a n t lesions of the urothelium, e n d o m e t r i u m , cervix, vaginal m u c o s a , oral m u c o s a and r e s p i r a t o r y m u c o s a .
Acknowledgments the and Dr. Dr.
This r e s e a r c h w a s s u p p o r t e d b y the National C a n c e r Institute (Canada), Medical R e s e a r c h Council (Canada) a n d the Ontario C a n c e r T r e a t m e n t R e s e a r c h F o u n d a t i o n . W e are i n d e b t e d to Dr. J. M. Blakeman, FRCP(C), A. K. Wyllie, FRCS(Edin.) a n d FRCS(C), Dr. W. A. Wells, FRCP(C) a n d V. L. Kraus, FRCR(Lond.) for their e n c o u r a g e m e n t and referrals.
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