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Aluminium phthalocyanine mediated photodynamic therapy in experimental malignant glioma
Aluminium phthalocyanine mediated photodynamic therapy in experimental malignant glioma S S Stylli eSc* J S Hill PhD* W H Sawyer PhDt A H K a y e MD FRACS* The Melbourne Neuroscience Centre*, Departments of Neurosurgery and Surgery, Royal Melbourne Hospital, and Department of Biocbemistry~, University of Melbourne, Parkville*
T h e efficacT o f aluminium tetrasulfonated phthalocyanine (A1SPc) as a photosensitizer f o r p h o t o d y n a m i c therapy (PDT) was investigated in the C6 glioma m o d e l in Wistar rats. T h e d e p t h and extent o f t u m o u r necrosis was d e p e n d e n t on b o t h the dosage o f intravenously (IV) administered AISPc, and the dose and time post-sensitization o f 675 n m light administration. T h e r e was no effect on t u m o u r or n o r m a l brain if the sensitizer dose was less than 0.5 m g / k g . Selective necrosis of t u m o u r was evident 6 hours post-sensitization at an A1SPc dose up to 1 m g / k g and fight doses u p to 200 J / cm 2. Necrosis occurred in n o r m a l brain at higher doses o f light and sensitizer. T h e r e was no photosensitizer effect in animals treated 24 hours post AISPc administration. T h e ability o f A1SPc to act as a selective photosensitizer causing photonecrosis provides a basis f o r the generation o f m o d i f i e d A1SPc species as future agents in the P D T o f brain tumours, especially as these sensitizers absorb light at a higher wavelength than those that are currently available. Journal of Clinical Neuroscience 1995, 2 (2) :146-151
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Keywords: Brain tumour, Glioma, Photodynamic therapy, Aluminium tetrasulfonated phthalocyanine
Introduction Cerebral tumours are responsible for approximately 2% of all cancer deaths and the high grade cerebral glioma, glioblastoma m u l t i f o r m e (GBM), is the most c o m m o n cerebral t u m o u r in adults. Conventional therapies for the treatment of malignant gliomas have only limited success. T h e best available p r o t o c o l , using surgery, r a d i a t i o n therapy and systemic c h e m o t h e r a p y result in a median survival time of less than one year for GBM. 1'e'3 Most t r e a t m e n t failures occur because of local r e c u r r e n c e of the t u m o u r indicating that a m o r e aggressive local therapy would be desirable for the t r e a t m e n t of gliomas. 4'5'6'7'8 Photodynamic therapy (PDT) is a t r e a t m e n t modality that involves the selective uptake of a photosensitizer by the t u m o u r followed by irradiation of the t u m o u r with light that will activate the sensitizer and cause selective t u m o u r death. PDT utilising the sensitizers h a e m a t o p o r p h y r i n d e r i v a t i v e ( H p D ) or its m o r e e n r i c h e d commercial form, Photofrin, has b e e n used in a n u m b e r o f trials in t h e t r e a t m e n t o f p a t i e n t s w i t h b r a i n tumours, 4'5'6'7'swith over 120 patients being treated in the Royal M e l b o u r n e Hospital study. 4'5 Despite showing clinical efficacy,4'5 H p D and Photofrin possess m a n y characteristics which m a k e t h e m less than ideal p h o t o s e n s i t i z e r s . In the synthesis o f H p D a n d
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Photofrin, a hydrolysis mixture of acetic and sulphuric acid is used to treat haematoporphyrin, resulting in a large n u m b e r o f o l i g o m e r i c , m o n o m e r i c e t h e r a n d ester c o m p o n e n t s . ° N u m e r o u s attempts have b e e n m a d e to d e t e r m i n e the 'active' c o m p o u n d in H p D , and several candidates have b e e n nominated. 8'1°'11'12T h e composition of H p D may also vary f r o m b a t c h to b a t c h ultimately altering its biological efficacy) 3 Additional disadvantages are that H p D absorbs poorly in the region of light (600700 nm) desirable for optimal tissue penetration and that patients remain photosensitive for several weeks due to retention of appreciable a m o u n t s of dye in the skin) 4 These constraints have encouraged the search for alternative photosensitizers for clinical use. O n e potential g r o u p of c o m p o u n d s are the phthalocyanines, which have b e e n suggested as possible candidates f o r r e p l a c i n g H p D in P D T p r o t o c o l s . 15 T h e y a r e characterised by strong light absorption in the 670-700 n m range and are easily synthesised in a p u r e form. In this study, the efficacy of A1SPc as a p o t e n t i a l photosensitizer in the PDT t r e a t m e n t of the C6 glioma in the Wistar rat has b e e n investigated and the appropriate dose of sensitizer and light dosimetry which caused destruction of t u m o u r with sparing of n o r m a l brain was established.
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Materials and m e t h o d s Chemicals A1SPc was p u r c h a s e d in p o w d e r f o r m in l g quantities ( P o r p h y r i n P r o d u c t s , U t a h , USA). T h e m o l e c u l a r structure of the tetrasulfonated c o m p o u n d is shown in F i g u r e 1, a l t h o u g h h i g h p e r f o r m a n c e liquid c h r o m a t o g r a p h y of this p r o p r i e t r y MSPc showed it to be a m i x t u r e consisting of the mono-, di-, tri- a n d tetrasulfonated forms (unpublished results). O n receipt, it was stored dessicated and shielded f r o m light at -20°C until subsequent use. Immediately prior to use, itwas suspended in isotonic saline a n d as a precaution was stored in the dark at 4°C.
intracranial gliomas. In brief, the h e a d was shaved, a 1.5 cm midline scalp incision was made, and a b u r r hole was placed just in front of the coronal suture, 3 m m to the left of the midline. T h e C6 glioma cells were suspended in a solution o f 2X RPMI 1640 containing 1% Sea plaque agarose (FMC Corp, Rockland, Maine) at a density of 105 cells per 25 gl. This volume was injected using a H a m i l t o n Microlitre syringe with a 27 g a u g e disposable n e e d l e covered by a plastic sleeve to a d e p t h of 2 m m f r o m the outer table of the bone. T h e needle was withdrawn 30 seconds after injection of the suspension, and the hole was sealed with b o n e wax. T h e w o u n d was then closed with clips. T h e o p e r a t i o n was p e r f o r m e d u s i n g an operating microscope.
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Photodynamic therapy PDT was administered to animals on the 10th day following tumour cell implantation. T h e animals were sensitized with AISPc via intravenous (IV) tail vein injection either 6 hours or 24 hours prior to the PDT p r o c e d u r e as described.
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Fig. 1 Chemical structure of AISPc.
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Cells T h e C6 glioma cell line was obtained f r o m the American Type Culture Collection, and the cells grown at 37°C in a humidified a t m o s p h e r e containing 5 % CO z in RPMI 1640 m e d i u m s u p p l e m e n t e d with 5% n e w b o r n calf s e r u m (Commonwealth Serum Laboratories, Parkville, Australia).
Animals and tumour implantation All in vivo a n i m a l p r o c e d u r e s w e r e p e r f o r m e d in accordance with r e q u i r e m e n t s established by the Royal Melbourne Hospital Institutional Animal Ethics Committee.
Intracranial implantation o f C6 glioma in Wistar rats A d u l t Wistar rats w e i g h i n g b e t w e e n 200 a n d 400 g, o b t a i n e d f r o m the j o i n t animal facility of the Ludwig Institute for Cancer Research (Melbourne Tumour Biology B r a n c h ) a n d D e p a r t m e n t o f Surgery, Royal Melbourne Hospital, were used for C6 glioma intracranial implantation. T h e rats were anaesthetised using methoxyflurane inhalation followed by administration of chloral hydrate (0.1 m l / 1 0 g b o d y w e i g h t in a solution containing 3.6 g / 1 0 0 ml water (wt/vol)) by intraperitoneal injection. The m e t h o d developed by Kaye et aP 6was used to establish
A 2 cm midline incision was m a d e to expose the skull and allow the p e r i c r a n i u m to be reflected. A craniotomy of 45 m m long and 3-4 m m wide was p e r f o r m e d with a high s p e e d d e n t a l drill ( F a r o Pty Ltd, Italy). T h e e x a c t dimensions of the craniotomy were r e c o r d e d to be used later in calculating the laser light dose. T h e a n t e r i o r margin of the craniotomywas placed 1 m m in front of the coronal suture and medially e x t e n d e d to within 1 m m of the midline.
Tumour bearing animals An incision was m a d e as before, but incorporating the previous b u r r hole after reflection of the pericranium. A craniotomy was p e r f o r m e d as in the n o r m a l rats over the C6 t u m o u r injection site to ensure the exposure was over the surface of the tumour. PDT o f rats P D T was a d m i n i s t e r e d 6 or 24 h o u r s a f t e r the IV administration of A1SPc. Light of 675 n m was g e n e r a t e d u s i n g an A r g o n - - i o n p u m p e d K i t o n r e d dye l a s e r p r o d u c i n g a b r o a d b a n d (615--695 nm) output (SpectraPhysics, Model 164-05 Argon Ion Laser, Model 375 dye laser, Mountain View, California). A birefringent grating was u s e d to t u n e the o u t p u t to 675 + 2 n m a n d the wavelength o u t p u t verified using a slit m o n o c h r o m e t e r . A 600 g m diameter flat cut quartz fibre was used to deliver the light. T h e light output was measured by placing the tip of the fibre in a photoelectric cell (Spectra Physics Model 404, Spectra Physics, Bayswater, Australia) a n d calculating the light dose relative to the craniotomy area. T h e fibre tip was held at a distance of 3 to 4 m m over the craniotomy so that red light evenly covered the exposed tissue.
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T h e laser power at the fibre tip varied f r o m 50 to 500 m W and the dose of light that was delivered varied f r o m 0 to 800 J / c m 2. T h e surface of the brain was kept cool by irrigation with isotonic saline at r o o m temperature, which has b e e n shown in previous studies to p r e v e n t hypert h e r m i a of the irradiation surface and underlying tissue. 6 On completion of the irradiation, a single layer of Surgicel (Johnson and Johnson, N o r t h Ryde, Australia) was used to cover the craniotomy site, and the incision closed with w o u n d clips.
Quantification of photodynamic effect T h e a n i m a l s were culled 5 days after laser treatment. The b r a i n s were r e m o v e d f i x e d in 10% f o r m a l d e h y d e , sectioned through the area of irradiation, and stained with haematoxylin and eosin. T h e extent of n o r m a l brain or t u m o u r necrosis was m e a s u r e d on serial sections using a graticule m i c r o m e t e r (Leitz, Wetzler, West Germany) in a Nikon Diaphot microscope.
g r e a t e r t h a n 50 J / c m 2, necrosis of n o r m a l brain was observed, with the d e p t h a n d e x t e n t o f this necrosis d e p e n d e n t on the light dose administered.
Effect of PDT a n d AISPc o n intracerebral tumours
W h e n C6 t u m o u r cells were implanted 2 m m deep f r o m the outer b o n e margin, intracerebral tumours grew up to the surface of the brain at 10 days post inoculation. This allowed for the surface of the t u m o u r to be irradiated with red laser light after the craniotomy was p e r f o r m e d . T h e depths of PDT mediated m m o u r kill 6 hours post administration of doses of A1SPc f r o m 0-5 m g / k g , and of 675 n m light f r o m 0-800 J / c m 2 at 6 hours, are shown in Figure 3.
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Fig. 2 AISPc mediated PDT on Wistar rat normal brain. Nont u m o u r bearing Wistar rats were administered intravenously (IV) w i t h AISPc at doses of 0.5 (n), 1.0 (s) or 5.0 (u) mg/kg. PDT w i t h 675 nm light on normal brain was carried out 6 hours post AISPc administration. Values shown are the mean from 5 animals per group, w i t h the standard error being equal or less than 10% of the mean value shown.
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T h e effect of light dosage on n o r m a l brain in animals sensitized with A1SPc, but without i m p l a n t e d tumour, was studied to d e t e r m i n e the dosimetry limits of selective kill. T h e PDT effect on n o r m a l brain was studied following A1SPc doses of either 0, 0.5, 1 or 5 m g / k g , and light doses up to 800 J / c m 2. Necrosis of the n o r m a l brain was not evident following light doses up to 800 J / c m 2, both in animals sensitized at a dose o f 0.5 m g / k g a n d n o n sensitized animals. Following administration of AlSPc at a dose of 1 m g / k g , n o r m a l brain necrosis was not evident up to a light dose of 2 0 0 J / c m 2, but was a p p a r e n t at higher light doses (Fig. 2). At a dose of 5 m g / k g and a light dose
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Fig. 3 AISPc mediated PDT against C6 glioma in Wistar rats. 1 x 10 s C6 glioma cells were inoculated intracerebrally into Wistar rats on day 0. AISPc was administered intravenously (IV) on day 10, at doses of 0.5 (n), 1.0 (s) or 5.0 (u) mg/kg. PDT w i t h 675 nm light on t u m o u r was carried out 6 hours post AISPc administration. Values shown are the mean from 5 animals per group with the standard error being equal or less than 10% of the mean value shown.
T h e major finding was that following administration of MSPc at a dose of 5 m g / k g and 1 0 0 J / c m 2 of 675 n m light, there was variable animal death, with 5 out of 9 animals surviving the 5 day post laser irradiation period. Animals that were not able to move or feed were sacrificed. Mso, sensitized animals treated at a light dose of 900 J / cm 2 were sacrificed within 24 hours of t r e a t m e n t as they were suffering f r o m neurological symptoms, which were n o t a p p a r e n t in n o n - s e n s i t i z e d c o n t r o l a n i m a l s administered the same light dose. Wide spread cerebral o e d e m a was evident in these animals. Based on these results, animals administered 5 m g / k g MSPc were not treated at light doses higher than 100 J / c m 2. T h e r e was no t u m o u r necrosis in a n i m a l s which received laser t r e a t m e n t alone up to light doses of 800 J / c m 2, or laser t r e a t m e n t following sensitization at an A1SPc dose of 0.5 mg/kg.
April 1995
Aluminium phthalocyanine PDT
Laboratory investigations Fig. 5b
Following a sensitizer dose of I m g / k g , and light doses up to 200 J / c m 2, selective kill of the C6 rat glioma with sparing of n o r m a l brain was evident (Fig. 4). T h e d e p t h of necrosis was similar in rats treated with light doses between 200 and 8 0 0 J / e r a 2. At the highest selective laser dose of 200 J / c m 2, the m e a n d e p t h of t u m o u r necrosis was 3.4 m m , with 4.9 m m b e i n g the deepest d e p t h of necrosis, and in 2 out of 5 animals the d e p t h of necrosis was greater than 3.5 m m .
Haematoxylin and eosin section of Wistar rat normal brain. The animal was previously administered intravenously (IV) with 1.0 mg/kg and treated with 675 nm light 6 hours post AISPc administration. The light dose given was 800 J/cm2.
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Fig. 6 Haematoxylin and eosin section of C6 tumour inoculated in Wistar rat brain. The animal was previously administered intravenously (IV) 1.0 mg/kg and treated with 675 nm light 6 hours post AISPc administration. The area aof necrosis is shown by arrows (x 10).
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Fig. 4 Comparison of AISPc mediated PDT of normal brain (s) and C6 tumour (n) at 1.0 mg/kg, taken from Figures 2 and 4. The arrow depicts the point at which selective tumour necrosis ceased (200 J/cm2).
D e l a y e d e f f e c t o f PDT a n d AISPc o n n o r m a l
brain and intracerebral t u m o u r s T h e d e p t h a n d e x t e n t o f n e c r o s i s f o l l o w i n g AISPc m e d i a t e d PDT of n o r m a l brain and C6 t u m o u r was also investigated at 24 h o u r s post sensitization. A1SPc was administered at either 0.5 m g / k g or 1.0 m g / k g and 675 n m light at doses up to 8 0 0 J / c m 2. No evidence of n o r m a l brain necrosis in sensitized animals was evident even at the highest light dose of 8 0 0 J / c m 2 (Fig. 5a). In contrast, there was minimal t u m o u r necrosis at an A1SPc dose of 1.0 m g / k g and 800 J / c m 2 of light (Fig. 5b) to the same dose regime 6 hours post A1SPc administration (Fig. 6) which resulted in a m e a n d e p t h of necrosis of 3.5 m m . Fig. 5a Haematoxylin and eosin section of Wistar rat normal brain. The animal was previously administered intravenously (IV) with 1.0 mg/kg and treated with 675 nm light 24 hours post AISPc administration. The light dose given was 800 J/cm2.
Discussion Most cerebral gliomas r e c u r locally after the p r e s e n t c o n v e n t i o n a l t h e r a p i e s o f surgery, r a d i o t h e r a p y a n d c h e m o t h e r a p y indicating a failure to control the local tumour. 1,2,S PDT is a targeted adjuvant therapy which has b e e n shown to aid local t u m o u r control 4,5 and has b e e n the subject of several clinical trials, 4-8 some of which have d e m o n s t r a t e d it to be clinically effective. 4,5 We have previously shown that A1SPc administered via the 1V route into CBA mice bearing the intracranial C6 glioma was selectively retained by the tumour, with p e a k uptake at 6 hours post-sensitization37 The data r e p o r t e d here indicate that selective kill of a cerebral glioma with sparing of the n o r m a l brain can be achieved with A1SPc m e d i a t e d PDT. Selective t u m o u r destruction occurred at an A1SPc dose of 1 m g / k g and at light doses less than 200 J / c m 2 administered 6 hours post-sensitization. Increasing the light dose f r o m 2 0 0 J / c m 2 to 8 0 0 J / c m z (in 2 0 0 J / c m -9 increments) did not significantly increase the d e p t h of t u m o u r necrosis but did result in d a m a g e to the n o r m a l brain. T h e depths of t u m o u r kill r e p o r t e d here are similar to those previously r e p o r t e d by Kaye and Morstyn using H p D in doses between 5 to 20 m g / k g . 16 The timing of the PDT p r o c e d u r e was f o u n d to be critical since t u m o u r bearing and n o n - t u m o u r bearing rats injected with 0.5 m g / k g or 1.0 m g / k g A1SPc, a n d treated with 0 J / c m 2 to 8 0 0 J / c m 2 24 hours postA1SPc administration did not show any evidence of n o r m a l brain or t u m o u r necrosis, a result in accord with the p h a r m a c o k i n e t i c data. 17 These results are in contrast to a r e p o r t using the VMDk m u r i n e glioma model, where mice were treated with A1SPc m e d i a t e d PDT 18 a n d n o n - t u m o u r bearing animals treated at 48 hours post-sensitization with 0.5 m g / k g or 5.0 m g / k g . These workers f o u n d that PDT administered 48 hours
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following a dose of 0.5 m g / k g resulted in all animals exposed to 2 0 0 J / c m 2 light dying within 24 hours of light exposure. Our results show that if PDT was administered 6 hours post A1SPc dosage, the time of maximal sensitizer uptake, 17no deaths occurred immediately postoperatively or in the 5 day postoperative period at light and sensitizer doses o f 50 J / c m 2 and 5 m g / k g A1SPc respectively. However, using a dose regime of 5 m g / k g A1SPc and 100 J / c m 2, there was variable death of the animals, suggesting the threshold light dose is between 50 and 100 J / c m 2. Animals treated at 24 hours with either 0.5 m g / k g or 1.0 m g / k g showed minimal evidence of either normal brain or t u m o u r necrosis. The differences in the results reported here and those previously published, 18 may well r e f l e c t e i t h e r v a r i a t i o n s in the t u m o u r m o d e l s or differences in the composition of the sensitizer prep a r a t i o n . Variations in t u r n o u t m o d e l s have b e e n recognised, with conflicting reports regarding HpD peak u p t a k e times in d i f f e r e n t brain t u m o u r models. 19'2° Boggan et al 2° observed a patchy uptake of HpD into the 9L gliosarcoma rat model with only 33-44% of the t u m o u r area being fluorescent, and the maximal fluorescence not apparent until 24 hours after the administration of HpD. After IV administration of HpD into mice bearing the C6 glioma, patchy uptake was evident at 4 hours, and maximal and uniform fluorescence was present t h r o u g h o u t the t u r n o u t at 6 h o u r s , with a d e c l i n e in f l u o r e s c e n c e beginning at 24 hours. Kaye et aP 9 have also shown that the C6 glioma cell line in vitro is more sensitive to kill by light after exposure to HpD than V79 fibroblast cells. Differences in cellular uptake of the a m o u n t of HpD or its various components by cells of different origin may also explain the discrepancies in uptake in different in vivo models. Variations between the C6 and 9L models may also be critical since Boggan et a121 have suggested that in the 9L m o d e l h e t e r o g e n e i t y a m o n g t u r n o u t capillaries, some of which were highly permeable and some of which maintained characteristics of the normal blood-brain barrier, caused the patchy uptake. In addition, it has also been observed that protein b o u n d drugs and high molecular weight aggregates such as HpD diffuse only minimal distances from permeable capillaries. 22 A similar situation may apply to the phthalocyanines as we have shown that they can associate with serum proteins, particularly lipoproteins, which may affect the degree of t u m o u r uptake. Variations in the degrees of sulfonation in a phthalocyanine mixture, such as that used in this PDT study, will affect the h y d r o p h o b i c p r o p e r t i e s o f the c o m p o u n d with hydrophilicity increasing with increasing sulfonation. We have shown that the pharmacokinetics of uptake and distribution in the glioma cells in vitro and the C6 model in vivo, are d e p e n d e n t on the degree of sulfonation) 7 This variation in time to peak uptake may explain the variations between the results reported here, and those of Sandeman et al. 18 Thus further PDT studies utilising the purified sulfonated forms ofA1SPc (mono-, di-, tri- and tetra-) are n e e d e d to d e t e r m i n e b o t h the optimal timing of PDT, and the degree of sulfonation required for maximal t u m o u r kill. Few studies addressing these issues have b e e n reported, although Brasseur et
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a123,24have compared the photodynamic activity of various p h t h a l o c y a n i n e s with t h a t o f H p D in t r e a t i n g a subcutaneously implanted EMT-6 m a m m a r y t u m o u r in mice. The aluminium and gallium chelated complexes of the phthalocyanines were as tumouricidal as HpD, while disulfonated phthalocyanine has b e e n shown to elicit superior tumouricidal activity to an A1SPc mixture, various s u l f o n a t e d gallium p h t h a l o c y a n i n e c o m p l e x e s a n d HpD.2S, 24 T h e disulfonated aluminium phthalocyanine has also been shown to be superior to HpD as a PDT sensitizer in mice bearing a MS-2 fibrosarcoma or B16 melanoma. 25 With light penetration being significantly reduced in the pigmented m e l a n o m a , it is possible that localisation of the disulfonate form to a critical subcellular target may have resulted in greater tumouricidal effects. Whilst h y p e r t h e r m i a has b e e n r e g a r d e d by some investigators as a major c o m p o n e n t of PDT, 26'27'28this study a n d p r e v i o u s studies u s i n g H p D have s h o w n t h a t hyperthermia does not occur if the site is irrigated with isotonic saline, and that PDT's efficacy is i n d e p e n d e n t of h y p e r t h e r m i a . T h e critical t u m o u r targets o f H p D sensitized PDT are yet to be definitively d e s c r i b e d , although evidence from some studies suggests that since the observed depth of t u m o u r damage 16 is beyond the proposed depth of penetration of light, 29,3° it is probable that vascular d a m a g e with s u b s e q u e n t d e e p e r tissue infarction may be a major basis for some of the t u m o u r destruction, a6,31 As A1SPc is comprised o f a mixture of the various sulfonate species (J Bommer, Porphyrin Products, personal communication, Stylli et al, unpublished observations), it is possible that the a m o u n t of the more active disulfonate c o m p o n e n t is proportionately low. Thus a description of the t u m o u r distribution of the various sufonate species comprising A1SPc is n e e d e d to d e t e r m i n e the critical targets for this sensitizer. The results presented in this study show that A1SPc possesses characteristics that may allow it to be considered for Phase I clinical trials of PDT. A1SPc shows selective t u m o u r uptake, and mediates selective photonecrosis of implanted glioma. Further studies examining the efficacy o f the s e p a r a t e s u l f o n a t e d f o r m s o f A1SPc w a r r a n t a d d i t i o n a l e v a l u a t i o n a n d are c u r r e n t l y u n d e r investigation.
Acknowledgements These investigations were p e r f o r m e d with s u p p o r t of grants f r o m National H e a l t h and Medical Research Council, Anti-Cancer Council of Victoria, Stroke Research Foundation, Victor Hurley Trust and the Slezak Trust. Received 2 August 1994 Accepted for publication 15 November 1994
Correspondence and offprint requests: S S Stylli The Melbourne Neuroscience Centre Royal Melbourne Hospital, Universityof Melbourne, Victoria 3050 Australia Tel: 61 3 342 7616 Fax: 61 3 347 8332
April 1995
Aluminium phthalocyanine PDT
Laboratory investigations
References 1. Walker MD, Alexander E Jr, Hunt WC et al. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial. J Neurosurg 1978;49:333-43. 2. Walker MD, Green SB, Byar DP et al. Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. New EnglJ Med 1980;303:1323-29. 3. Wilson CB. Brain tumours. New Engl. J. Med 1979;300:1469-71. 4. KayeAH, Morstyn G, Brownbill D. Adjuvant high dose photoradiation therapy in the treatment of cerebral glioma. A phase I-II study. J Neurosurg 1987;67:520-25. 5. KayeAH, HillJS. Photodynamic therapy of cerebral tumours. Neurosurgery Quarterly 1992;1:233-58. 6. Laws ER, Cortese DA, KinseyJH, Boggan RT, Anderson RE. Photoradiation therapy in the treatment of malignant brain tumours. Phase I (feasibility study). Neurosurgery 1981;9:672-78. 7. McCulloch GAJ, ForbesJJ, Lee See K, Cowled PA,Jacka FJ, Ward AD. Phototherapy of malignant brain tumours. In: Porphyrin Localization and Treatment of Tumours, 1984;709. 8. Perria C, Capuzzo T, Cavagnaro G, et al. First attempts at the photodynamic treatment of human gliomas. J Neurosurg Sci 1980;24:119-29. 9. Lipson RL, Baldes EJ, Olsen AM. The use of a derivative of hematoporphyrin in tumor detection. J Natl Cancer Inst 1961;26:1-11. 10. Berenbaum MC, Bonnet R, Scourides PA. In vivo biological activity of the components of haematoporphyrin derivative. BrJ Cancer 1982;47:571-81. 11. Dougherty TJ, Weishaupt KR. Structure and properties of the active fraction of hematoporphyrin derivative. Photochem Photobiol 1983;37:815-21. 12. Kessel D, Cheng M-L. Biological and biophysical properties of the tumor-localizing component of hematoporphyrin derivative. Cancer Res 1985;45:3053-58. 13. Dougherty TJ, Boyle DG, Weishaupt BA et al. Photoradiation therapy--clinical and drug advances. In: Porphyrin Photosensitization,1983:3-18. 14. Dougherty TJ, Lawrence G, KaufmanJH, Boyle D, Weishaupt KR, Goldfarb A. Photoradiation in the treatment of recurrent breast carcinoma. J Natl Cancer Inst 1979;62:231-37. 15. Rosenthal I. Yearly Review: Phthalocyanines as photodynamic sensitizers. Photochem Photobiol 1991;53:859-70. 16. Kaye AH, Morstyn G. Photoradiation therapy causing selective tumor kill in a rat glioma model. Neurosurgery 1987;20:408-15. 17. Stylli SS, HillJS, Sawyer WH, Kaye AH. The use of phthalocyanines as potential photosensitizers in the
18.
19. 20.
21.
22. 23.
24.
25.
26. 27. 28.
29. 30. 31.
treatment of central nervous system neoplasms. J Clinical Neuroscience 1995;(in press). Sandeman DR, Bradford R, Buxton P, Bown SG, Thomas DGT. Short communication : Selective necrosis of malignant gliomas in mice using photodynamic therapy. BrJ Cancer 1987;55:647-49. KayeAH, Morstyn G, Ashcroft RC. Uptake and retention of hematoporphyrin derivative in an in vivo/in vitro model of cerebral glioma. Neurosurgery 1985;17:883-90. BogganJE, Walker R, Edwards MSB, BorcichJK, Davis RL, Koonce M, Berns MW. Distribution of hematoporphyrin derivative in the rat 9L gliosarcoma brain tumor analysed by digital video fluorescence microscopy. J Neurosurg 1984;61:1113-19. BogganJE, Bolger C, Edwards MSB. Effect of hematoporphyrin derivative photoradiation therapy on survival in the rat 9L gliosarcoma brain tumor model. J Neurosurg 1985;63:917-21. Levin VA, Freeman-Dove M, Landahl HD. Permeability characteristics of brain adjacent to tumors in rats. Arch Neurol 1975;32:785-91. Brasseur N, Ali H, Langlois R, Wagner JR, RousseauJ. van LierJE. Biological activities of phthalocyanines V. Photodynamic therapy of EMT-6 mammary tumors in mice with sulfonated phthalocyanines. Photochem Photobiol 1987;45:581-86. Brasseur N, Ali H, Langlois R, van LierJE. Biological activities of phthalocyanines--IX. Photosensitization of V79 chinese hamster cells and EMT-6 mouse mammary tumor by selectively sulfonated zinc phthalocyanines. Photochem Photbiol 1988;47:705-11. Canti G, Franco P, Marelli O, Cubeddu R, Taroni P, Ramponi R. Comparative study of the therapeutic effect of photoactivated hematoporphyrin derivative and aluminium disulfonated phthalocyanines on tumor bearing mice. Cancer Lett 1990;53:123-27. KinseyJH, Cortese DA, Neel HB. Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983;43:1562-67. Cheng MK, McKeanJ, Boisvert D, Tulip T, Mielke BW. Effect of photoradiation therapy on normal rat brain. Neurosurgery 1984;15:804-10. Cheng MK, McKeanJ, Mielke B, Tulip J, Boisvert D. Photoradiation therapy of 9L- gliosarcoma in rats: Hematoporphyrin derivative (types I and II) followed by laser energy.J Neurooncol 1985;3:217-28. Svaasand LO, Ellingson R. Optical properties of human brain. Photochem Photobiol 1983;38:292-99. Svaasand LO, Ellingson R. Optical penetration in human intracranial tumors. Photochem Photobiol 1985;41:73-6. Bugelski PG, Poster CW, Dougherty TJ. Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res 1981;41: 4606-12.
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T h e efficacT o f aluminium tetrasulfonated phthalocyanine (A1SPc) as a photosensitizer f o r p h o t o d y n a m i c therapy (PDT) was investigated in the C6 glioma m o d e l in Wistar rats. T h e d e p t h and extent o f t u m o u r necrosis was d e p e n d e n t on b o t h the dosage o f intravenously (IV) administered AISPc, and the dose and time post-sensitization o f 675 n m light administration. T h e r e was no effect on t u m o u r or n o r m a l brain if the sensitizer dose was less than 0.5 m g / k g . Selective necrosis of t u m o u r was evident 6 hours post-sensitization at an A1SPc dose up to 1 m g / k g and fight doses u p to 200 J / cm 2. Necrosis occurred in n o r m a l brain at higher doses o f light and sensitizer. T h e r e was no photosensitizer effect in animals treated 24 hours post AISPc administration. T h e ability o f A1SPc to act as a selective photosensitizer causing photonecrosis provides a basis f o r the generation o f m o d i f i e d A1SPc species as future agents in the P D T o f brain tumours, especially as these sensitizers absorb light at a higher wavelength than those that are currently available. Journal of Clinical Neuroscience 1995, 2 (2) :146-151
© Pearson Professional (Australia) Pry Ltd
Keywords: Brain tumour, Glioma, Photodynamic therapy, Aluminium tetrasulfonated phthalocyanine
Introduction Cerebral tumours are responsible for approximately 2% of all cancer deaths and the high grade cerebral glioma, glioblastoma m u l t i f o r m e (GBM), is the most c o m m o n cerebral t u m o u r in adults. Conventional therapies for the treatment of malignant gliomas have only limited success. T h e best available p r o t o c o l , using surgery, r a d i a t i o n therapy and systemic c h e m o t h e r a p y result in a median survival time of less than one year for GBM. 1'e'3 Most t r e a t m e n t failures occur because of local r e c u r r e n c e of the t u m o u r indicating that a m o r e aggressive local therapy would be desirable for the t r e a t m e n t of gliomas. 4'5'6'7'8 Photodynamic therapy (PDT) is a t r e a t m e n t modality that involves the selective uptake of a photosensitizer by the t u m o u r followed by irradiation of the t u m o u r with light that will activate the sensitizer and cause selective t u m o u r death. PDT utilising the sensitizers h a e m a t o p o r p h y r i n d e r i v a t i v e ( H p D ) or its m o r e e n r i c h e d commercial form, Photofrin, has b e e n used in a n u m b e r o f trials in t h e t r e a t m e n t o f p a t i e n t s w i t h b r a i n tumours, 4'5'6'7'swith over 120 patients being treated in the Royal M e l b o u r n e Hospital study. 4'5 Despite showing clinical efficacy,4'5 H p D and Photofrin possess m a n y characteristics which m a k e t h e m less than ideal p h o t o s e n s i t i z e r s . In the synthesis o f H p D a n d
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Photofrin, a hydrolysis mixture of acetic and sulphuric acid is used to treat haematoporphyrin, resulting in a large n u m b e r o f o l i g o m e r i c , m o n o m e r i c e t h e r a n d ester c o m p o n e n t s . ° N u m e r o u s attempts have b e e n m a d e to d e t e r m i n e the 'active' c o m p o u n d in H p D , and several candidates have b e e n nominated. 8'1°'11'12T h e composition of H p D may also vary f r o m b a t c h to b a t c h ultimately altering its biological efficacy) 3 Additional disadvantages are that H p D absorbs poorly in the region of light (600700 nm) desirable for optimal tissue penetration and that patients remain photosensitive for several weeks due to retention of appreciable a m o u n t s of dye in the skin) 4 These constraints have encouraged the search for alternative photosensitizers for clinical use. O n e potential g r o u p of c o m p o u n d s are the phthalocyanines, which have b e e n suggested as possible candidates f o r r e p l a c i n g H p D in P D T p r o t o c o l s . 15 T h e y a r e characterised by strong light absorption in the 670-700 n m range and are easily synthesised in a p u r e form. In this study, the efficacy of A1SPc as a p o t e n t i a l photosensitizer in the PDT t r e a t m e n t of the C6 glioma in the Wistar rat has b e e n investigated and the appropriate dose of sensitizer and light dosimetry which caused destruction of t u m o u r with sparing of n o r m a l brain was established.
April 1995
Aluminium phthalocyaninePDT
Laboratory investigations
Materials and m e t h o d s Chemicals A1SPc was p u r c h a s e d in p o w d e r f o r m in l g quantities ( P o r p h y r i n P r o d u c t s , U t a h , USA). T h e m o l e c u l a r structure of the tetrasulfonated c o m p o u n d is shown in F i g u r e 1, a l t h o u g h h i g h p e r f o r m a n c e liquid c h r o m a t o g r a p h y of this p r o p r i e t r y MSPc showed it to be a m i x t u r e consisting of the mono-, di-, tri- a n d tetrasulfonated forms (unpublished results). O n receipt, it was stored dessicated and shielded f r o m light at -20°C until subsequent use. Immediately prior to use, itwas suspended in isotonic saline a n d as a precaution was stored in the dark at 4°C.
intracranial gliomas. In brief, the h e a d was shaved, a 1.5 cm midline scalp incision was made, and a b u r r hole was placed just in front of the coronal suture, 3 m m to the left of the midline. T h e C6 glioma cells were suspended in a solution o f 2X RPMI 1640 containing 1% Sea plaque agarose (FMC Corp, Rockland, Maine) at a density of 105 cells per 25 gl. This volume was injected using a H a m i l t o n Microlitre syringe with a 27 g a u g e disposable n e e d l e covered by a plastic sleeve to a d e p t h of 2 m m f r o m the outer table of the bone. T h e needle was withdrawn 30 seconds after injection of the suspension, and the hole was sealed with b o n e wax. T h e w o u n d was then closed with clips. T h e o p e r a t i o n was p e r f o r m e d u s i n g an operating microscope.
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Photodynamic therapy PDT was administered to animals on the 10th day following tumour cell implantation. T h e animals were sensitized with AISPc via intravenous (IV) tail vein injection either 6 hours or 24 hours prior to the PDT p r o c e d u r e as described.
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Fig. 1 Chemical structure of AISPc.
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Cells T h e C6 glioma cell line was obtained f r o m the American Type Culture Collection, and the cells grown at 37°C in a humidified a t m o s p h e r e containing 5 % CO z in RPMI 1640 m e d i u m s u p p l e m e n t e d with 5% n e w b o r n calf s e r u m (Commonwealth Serum Laboratories, Parkville, Australia).
Animals and tumour implantation All in vivo a n i m a l p r o c e d u r e s w e r e p e r f o r m e d in accordance with r e q u i r e m e n t s established by the Royal Melbourne Hospital Institutional Animal Ethics Committee.
Intracranial implantation o f C6 glioma in Wistar rats A d u l t Wistar rats w e i g h i n g b e t w e e n 200 a n d 400 g, o b t a i n e d f r o m the j o i n t animal facility of the Ludwig Institute for Cancer Research (Melbourne Tumour Biology B r a n c h ) a n d D e p a r t m e n t o f Surgery, Royal Melbourne Hospital, were used for C6 glioma intracranial implantation. T h e rats were anaesthetised using methoxyflurane inhalation followed by administration of chloral hydrate (0.1 m l / 1 0 g b o d y w e i g h t in a solution containing 3.6 g / 1 0 0 ml water (wt/vol)) by intraperitoneal injection. The m e t h o d developed by Kaye et aP 6was used to establish
A 2 cm midline incision was m a d e to expose the skull and allow the p e r i c r a n i u m to be reflected. A craniotomy of 45 m m long and 3-4 m m wide was p e r f o r m e d with a high s p e e d d e n t a l drill ( F a r o Pty Ltd, Italy). T h e e x a c t dimensions of the craniotomy were r e c o r d e d to be used later in calculating the laser light dose. T h e a n t e r i o r margin of the craniotomywas placed 1 m m in front of the coronal suture and medially e x t e n d e d to within 1 m m of the midline.
Tumour bearing animals An incision was m a d e as before, but incorporating the previous b u r r hole after reflection of the pericranium. A craniotomy was p e r f o r m e d as in the n o r m a l rats over the C6 t u m o u r injection site to ensure the exposure was over the surface of the tumour. PDT o f rats P D T was a d m i n i s t e r e d 6 or 24 h o u r s a f t e r the IV administration of A1SPc. Light of 675 n m was g e n e r a t e d u s i n g an A r g o n - - i o n p u m p e d K i t o n r e d dye l a s e r p r o d u c i n g a b r o a d b a n d (615--695 nm) output (SpectraPhysics, Model 164-05 Argon Ion Laser, Model 375 dye laser, Mountain View, California). A birefringent grating was u s e d to t u n e the o u t p u t to 675 + 2 n m a n d the wavelength o u t p u t verified using a slit m o n o c h r o m e t e r . A 600 g m diameter flat cut quartz fibre was used to deliver the light. T h e light output was measured by placing the tip of the fibre in a photoelectric cell (Spectra Physics Model 404, Spectra Physics, Bayswater, Australia) a n d calculating the light dose relative to the craniotomy area. T h e fibre tip was held at a distance of 3 to 4 m m over the craniotomy so that red light evenly covered the exposed tissue.
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Aluminium phthalocyanine PDT
T h e laser power at the fibre tip varied f r o m 50 to 500 m W and the dose of light that was delivered varied f r o m 0 to 800 J / c m 2. T h e surface of the brain was kept cool by irrigation with isotonic saline at r o o m temperature, which has b e e n shown in previous studies to p r e v e n t hypert h e r m i a of the irradiation surface and underlying tissue. 6 On completion of the irradiation, a single layer of Surgicel (Johnson and Johnson, N o r t h Ryde, Australia) was used to cover the craniotomy site, and the incision closed with w o u n d clips.
Quantification of photodynamic effect T h e a n i m a l s were culled 5 days after laser treatment. The b r a i n s were r e m o v e d f i x e d in 10% f o r m a l d e h y d e , sectioned through the area of irradiation, and stained with haematoxylin and eosin. T h e extent of n o r m a l brain or t u m o u r necrosis was m e a s u r e d on serial sections using a graticule m i c r o m e t e r (Leitz, Wetzler, West Germany) in a Nikon Diaphot microscope.
g r e a t e r t h a n 50 J / c m 2, necrosis of n o r m a l brain was observed, with the d e p t h a n d e x t e n t o f this necrosis d e p e n d e n t on the light dose administered.
Effect of PDT a n d AISPc o n intracerebral tumours
W h e n C6 t u m o u r cells were implanted 2 m m deep f r o m the outer b o n e margin, intracerebral tumours grew up to the surface of the brain at 10 days post inoculation. This allowed for the surface of the t u m o u r to be irradiated with red laser light after the craniotomy was p e r f o r m e d . T h e depths of PDT mediated m m o u r kill 6 hours post administration of doses of A1SPc f r o m 0-5 m g / k g , and of 675 n m light f r o m 0-800 J / c m 2 at 6 hours, are shown in Figure 3.
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Fig. 2 AISPc mediated PDT on Wistar rat normal brain. Nont u m o u r bearing Wistar rats were administered intravenously (IV) w i t h AISPc at doses of 0.5 (n), 1.0 (s) or 5.0 (u) mg/kg. PDT w i t h 675 nm light on normal brain was carried out 6 hours post AISPc administration. Values shown are the mean from 5 animals per group, w i t h the standard error being equal or less than 10% of the mean value shown.
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T h e effect of light dosage on n o r m a l brain in animals sensitized with A1SPc, but without i m p l a n t e d tumour, was studied to d e t e r m i n e the dosimetry limits of selective kill. T h e PDT effect on n o r m a l brain was studied following A1SPc doses of either 0, 0.5, 1 or 5 m g / k g , and light doses up to 800 J / c m 2. Necrosis of the n o r m a l brain was not evident following light doses up to 800 J / c m 2, both in animals sensitized at a dose o f 0.5 m g / k g a n d n o n sensitized animals. Following administration of AlSPc at a dose of 1 m g / k g , n o r m a l brain necrosis was not evident up to a light dose of 2 0 0 J / c m 2, but was a p p a r e n t at higher light doses (Fig. 2). At a dose of 5 m g / k g and a light dose
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Fig. 3 AISPc mediated PDT against C6 glioma in Wistar rats. 1 x 10 s C6 glioma cells were inoculated intracerebrally into Wistar rats on day 0. AISPc was administered intravenously (IV) on day 10, at doses of 0.5 (n), 1.0 (s) or 5.0 (u) mg/kg. PDT w i t h 675 nm light on t u m o u r was carried out 6 hours post AISPc administration. Values shown are the mean from 5 animals per group with the standard error being equal or less than 10% of the mean value shown.
T h e major finding was that following administration of MSPc at a dose of 5 m g / k g and 1 0 0 J / c m 2 of 675 n m light, there was variable animal death, with 5 out of 9 animals surviving the 5 day post laser irradiation period. Animals that were not able to move or feed were sacrificed. Mso, sensitized animals treated at a light dose of 900 J / cm 2 were sacrificed within 24 hours of t r e a t m e n t as they were suffering f r o m neurological symptoms, which were n o t a p p a r e n t in n o n - s e n s i t i z e d c o n t r o l a n i m a l s administered the same light dose. Wide spread cerebral o e d e m a was evident in these animals. Based on these results, animals administered 5 m g / k g MSPc were not treated at light doses higher than 100 J / c m 2. T h e r e was no t u m o u r necrosis in a n i m a l s which received laser t r e a t m e n t alone up to light doses of 800 J / c m 2, or laser t r e a t m e n t following sensitization at an A1SPc dose of 0.5 mg/kg.
April 1995
Aluminium phthalocyanine PDT
Laboratory investigations Fig. 5b
Following a sensitizer dose of I m g / k g , and light doses up to 200 J / c m 2, selective kill of the C6 rat glioma with sparing of n o r m a l brain was evident (Fig. 4). T h e d e p t h of necrosis was similar in rats treated with light doses between 200 and 8 0 0 J / e r a 2. At the highest selective laser dose of 200 J / c m 2, the m e a n d e p t h of t u m o u r necrosis was 3.4 m m , with 4.9 m m b e i n g the deepest d e p t h of necrosis, and in 2 out of 5 animals the d e p t h of necrosis was greater than 3.5 m m .
Haematoxylin and eosin section of Wistar rat normal brain. The animal was previously administered intravenously (IV) with 1.0 mg/kg and treated with 675 nm light 6 hours post AISPc administration. The light dose given was 800 J/cm2.
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Fig. 6 Haematoxylin and eosin section of C6 tumour inoculated in Wistar rat brain. The animal was previously administered intravenously (IV) 1.0 mg/kg and treated with 675 nm light 6 hours post AISPc administration. The area aof necrosis is shown by arrows (x 10).
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Fig. 4 Comparison of AISPc mediated PDT of normal brain (s) and C6 tumour (n) at 1.0 mg/kg, taken from Figures 2 and 4. The arrow depicts the point at which selective tumour necrosis ceased (200 J/cm2).
D e l a y e d e f f e c t o f PDT a n d AISPc o n n o r m a l
brain and intracerebral t u m o u r s T h e d e p t h a n d e x t e n t o f n e c r o s i s f o l l o w i n g AISPc m e d i a t e d PDT of n o r m a l brain and C6 t u m o u r was also investigated at 24 h o u r s post sensitization. A1SPc was administered at either 0.5 m g / k g or 1.0 m g / k g and 675 n m light at doses up to 8 0 0 J / c m 2. No evidence of n o r m a l brain necrosis in sensitized animals was evident even at the highest light dose of 8 0 0 J / c m 2 (Fig. 5a). In contrast, there was minimal t u m o u r necrosis at an A1SPc dose of 1.0 m g / k g and 800 J / c m 2 of light (Fig. 5b) to the same dose regime 6 hours post A1SPc administration (Fig. 6) which resulted in a m e a n d e p t h of necrosis of 3.5 m m . Fig. 5a Haematoxylin and eosin section of Wistar rat normal brain. The animal was previously administered intravenously (IV) with 1.0 mg/kg and treated with 675 nm light 24 hours post AISPc administration. The light dose given was 800 J/cm2.
Discussion Most cerebral gliomas r e c u r locally after the p r e s e n t c o n v e n t i o n a l t h e r a p i e s o f surgery, r a d i o t h e r a p y a n d c h e m o t h e r a p y indicating a failure to control the local tumour. 1,2,S PDT is a targeted adjuvant therapy which has b e e n shown to aid local t u m o u r control 4,5 and has b e e n the subject of several clinical trials, 4-8 some of which have d e m o n s t r a t e d it to be clinically effective. 4,5 We have previously shown that A1SPc administered via the 1V route into CBA mice bearing the intracranial C6 glioma was selectively retained by the tumour, with p e a k uptake at 6 hours post-sensitization37 The data r e p o r t e d here indicate that selective kill of a cerebral glioma with sparing of the n o r m a l brain can be achieved with A1SPc m e d i a t e d PDT. Selective t u m o u r destruction occurred at an A1SPc dose of 1 m g / k g and at light doses less than 200 J / c m 2 administered 6 hours post-sensitization. Increasing the light dose f r o m 2 0 0 J / c m 2 to 8 0 0 J / c m z (in 2 0 0 J / c m -9 increments) did not significantly increase the d e p t h of t u m o u r necrosis but did result in d a m a g e to the n o r m a l brain. T h e depths of t u m o u r kill r e p o r t e d here are similar to those previously r e p o r t e d by Kaye and Morstyn using H p D in doses between 5 to 20 m g / k g . 16 The timing of the PDT p r o c e d u r e was f o u n d to be critical since t u m o u r bearing and n o n - t u m o u r bearing rats injected with 0.5 m g / k g or 1.0 m g / k g A1SPc, a n d treated with 0 J / c m 2 to 8 0 0 J / c m 2 24 hours postA1SPc administration did not show any evidence of n o r m a l brain or t u m o u r necrosis, a result in accord with the p h a r m a c o k i n e t i c data. 17 These results are in contrast to a r e p o r t using the VMDk m u r i n e glioma model, where mice were treated with A1SPc m e d i a t e d PDT 18 a n d n o n - t u m o u r bearing animals treated at 48 hours post-sensitization with 0.5 m g / k g or 5.0 m g / k g . These workers f o u n d that PDT administered 48 hours
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Laboratory investigations
Aluminium phthalocyanine PDT
following a dose of 0.5 m g / k g resulted in all animals exposed to 2 0 0 J / c m 2 light dying within 24 hours of light exposure. Our results show that if PDT was administered 6 hours post A1SPc dosage, the time of maximal sensitizer uptake, 17no deaths occurred immediately postoperatively or in the 5 day postoperative period at light and sensitizer doses o f 50 J / c m 2 and 5 m g / k g A1SPc respectively. However, using a dose regime of 5 m g / k g A1SPc and 100 J / c m 2, there was variable death of the animals, suggesting the threshold light dose is between 50 and 100 J / c m 2. Animals treated at 24 hours with either 0.5 m g / k g or 1.0 m g / k g showed minimal evidence of either normal brain or t u m o u r necrosis. The differences in the results reported here and those previously published, 18 may well r e f l e c t e i t h e r v a r i a t i o n s in the t u m o u r m o d e l s or differences in the composition of the sensitizer prep a r a t i o n . Variations in t u r n o u t m o d e l s have b e e n recognised, with conflicting reports regarding HpD peak u p t a k e times in d i f f e r e n t brain t u m o u r models. 19'2° Boggan et al 2° observed a patchy uptake of HpD into the 9L gliosarcoma rat model with only 33-44% of the t u m o u r area being fluorescent, and the maximal fluorescence not apparent until 24 hours after the administration of HpD. After IV administration of HpD into mice bearing the C6 glioma, patchy uptake was evident at 4 hours, and maximal and uniform fluorescence was present t h r o u g h o u t the t u r n o u t at 6 h o u r s , with a d e c l i n e in f l u o r e s c e n c e beginning at 24 hours. Kaye et aP 9 have also shown that the C6 glioma cell line in vitro is more sensitive to kill by light after exposure to HpD than V79 fibroblast cells. Differences in cellular uptake of the a m o u n t of HpD or its various components by cells of different origin may also explain the discrepancies in uptake in different in vivo models. Variations between the C6 and 9L models may also be critical since Boggan et a121 have suggested that in the 9L m o d e l h e t e r o g e n e i t y a m o n g t u r n o u t capillaries, some of which were highly permeable and some of which maintained characteristics of the normal blood-brain barrier, caused the patchy uptake. In addition, it has also been observed that protein b o u n d drugs and high molecular weight aggregates such as HpD diffuse only minimal distances from permeable capillaries. 22 A similar situation may apply to the phthalocyanines as we have shown that they can associate with serum proteins, particularly lipoproteins, which may affect the degree of t u m o u r uptake. Variations in the degrees of sulfonation in a phthalocyanine mixture, such as that used in this PDT study, will affect the h y d r o p h o b i c p r o p e r t i e s o f the c o m p o u n d with hydrophilicity increasing with increasing sulfonation. We have shown that the pharmacokinetics of uptake and distribution in the glioma cells in vitro and the C6 model in vivo, are d e p e n d e n t on the degree of sulfonation) 7 This variation in time to peak uptake may explain the variations between the results reported here, and those of Sandeman et al. 18 Thus further PDT studies utilising the purified sulfonated forms ofA1SPc (mono-, di-, tri- and tetra-) are n e e d e d to d e t e r m i n e b o t h the optimal timing of PDT, and the degree of sulfonation required for maximal t u m o u r kill. Few studies addressing these issues have b e e n reported, although Brasseur et
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a123,24have compared the photodynamic activity of various p h t h a l o c y a n i n e s with t h a t o f H p D in t r e a t i n g a subcutaneously implanted EMT-6 m a m m a r y t u m o u r in mice. The aluminium and gallium chelated complexes of the phthalocyanines were as tumouricidal as HpD, while disulfonated phthalocyanine has b e e n shown to elicit superior tumouricidal activity to an A1SPc mixture, various s u l f o n a t e d gallium p h t h a l o c y a n i n e c o m p l e x e s a n d HpD.2S, 24 T h e disulfonated aluminium phthalocyanine has also been shown to be superior to HpD as a PDT sensitizer in mice bearing a MS-2 fibrosarcoma or B16 melanoma. 25 With light penetration being significantly reduced in the pigmented m e l a n o m a , it is possible that localisation of the disulfonate form to a critical subcellular target may have resulted in greater tumouricidal effects. Whilst h y p e r t h e r m i a has b e e n r e g a r d e d by some investigators as a major c o m p o n e n t of PDT, 26'27'28this study a n d p r e v i o u s studies u s i n g H p D have s h o w n t h a t hyperthermia does not occur if the site is irrigated with isotonic saline, and that PDT's efficacy is i n d e p e n d e n t of h y p e r t h e r m i a . T h e critical t u m o u r targets o f H p D sensitized PDT are yet to be definitively d e s c r i b e d , although evidence from some studies suggests that since the observed depth of t u m o u r damage 16 is beyond the proposed depth of penetration of light, 29,3° it is probable that vascular d a m a g e with s u b s e q u e n t d e e p e r tissue infarction may be a major basis for some of the t u m o u r destruction, a6,31 As A1SPc is comprised o f a mixture of the various sulfonate species (J Bommer, Porphyrin Products, personal communication, Stylli et al, unpublished observations), it is possible that the a m o u n t of the more active disulfonate c o m p o n e n t is proportionately low. Thus a description of the t u m o u r distribution of the various sufonate species comprising A1SPc is n e e d e d to d e t e r m i n e the critical targets for this sensitizer. The results presented in this study show that A1SPc possesses characteristics that may allow it to be considered for Phase I clinical trials of PDT. A1SPc shows selective t u m o u r uptake, and mediates selective photonecrosis of implanted glioma. Further studies examining the efficacy o f the s e p a r a t e s u l f o n a t e d f o r m s o f A1SPc w a r r a n t a d d i t i o n a l e v a l u a t i o n a n d are c u r r e n t l y u n d e r investigation.
Acknowledgements These investigations were p e r f o r m e d with s u p p o r t of grants f r o m National H e a l t h and Medical Research Council, Anti-Cancer Council of Victoria, Stroke Research Foundation, Victor Hurley Trust and the Slezak Trust. Received 2 August 1994 Accepted for publication 15 November 1994
Correspondence and offprint requests: S S Stylli The Melbourne Neuroscience Centre Royal Melbourne Hospital, Universityof Melbourne, Victoria 3050 Australia Tel: 61 3 342 7616 Fax: 61 3 347 8332
April 1995
Aluminium phthalocyanine PDT
Laboratory investigations
References 1. Walker MD, Alexander E Jr, Hunt WC et al. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial. J Neurosurg 1978;49:333-43. 2. Walker MD, Green SB, Byar DP et al. Randomized comparisons of radiotherapy and nitrosoureas for the treatment of malignant glioma after surgery. New EnglJ Med 1980;303:1323-29. 3. Wilson CB. Brain tumours. New Engl. J. Med 1979;300:1469-71. 4. KayeAH, Morstyn G, Brownbill D. Adjuvant high dose photoradiation therapy in the treatment of cerebral glioma. A phase I-II study. J Neurosurg 1987;67:520-25. 5. KayeAH, HillJS. Photodynamic therapy of cerebral tumours. Neurosurgery Quarterly 1992;1:233-58. 6. Laws ER, Cortese DA, KinseyJH, Boggan RT, Anderson RE. Photoradiation therapy in the treatment of malignant brain tumours. Phase I (feasibility study). Neurosurgery 1981;9:672-78. 7. McCulloch GAJ, ForbesJJ, Lee See K, Cowled PA,Jacka FJ, Ward AD. Phototherapy of malignant brain tumours. In: Porphyrin Localization and Treatment of Tumours, 1984;709. 8. Perria C, Capuzzo T, Cavagnaro G, et al. First attempts at the photodynamic treatment of human gliomas. J Neurosurg Sci 1980;24:119-29. 9. Lipson RL, Baldes EJ, Olsen AM. The use of a derivative of hematoporphyrin in tumor detection. J Natl Cancer Inst 1961;26:1-11. 10. Berenbaum MC, Bonnet R, Scourides PA. In vivo biological activity of the components of haematoporphyrin derivative. BrJ Cancer 1982;47:571-81. 11. Dougherty TJ, Weishaupt KR. Structure and properties of the active fraction of hematoporphyrin derivative. Photochem Photobiol 1983;37:815-21. 12. Kessel D, Cheng M-L. Biological and biophysical properties of the tumor-localizing component of hematoporphyrin derivative. Cancer Res 1985;45:3053-58. 13. Dougherty TJ, Boyle DG, Weishaupt BA et al. Photoradiation therapy--clinical and drug advances. In: Porphyrin Photosensitization,1983:3-18. 14. Dougherty TJ, Lawrence G, KaufmanJH, Boyle D, Weishaupt KR, Goldfarb A. Photoradiation in the treatment of recurrent breast carcinoma. J Natl Cancer Inst 1979;62:231-37. 15. Rosenthal I. Yearly Review: Phthalocyanines as photodynamic sensitizers. Photochem Photobiol 1991;53:859-70. 16. Kaye AH, Morstyn G. Photoradiation therapy causing selective tumor kill in a rat glioma model. Neurosurgery 1987;20:408-15. 17. Stylli SS, HillJS, Sawyer WH, Kaye AH. The use of phthalocyanines as potential photosensitizers in the
18.
19. 20.
21.
22. 23.
24.
25.
26. 27. 28.
29. 30. 31.
treatment of central nervous system neoplasms. J Clinical Neuroscience 1995;(in press). Sandeman DR, Bradford R, Buxton P, Bown SG, Thomas DGT. Short communication : Selective necrosis of malignant gliomas in mice using photodynamic therapy. BrJ Cancer 1987;55:647-49. KayeAH, Morstyn G, Ashcroft RC. Uptake and retention of hematoporphyrin derivative in an in vivo/in vitro model of cerebral glioma. Neurosurgery 1985;17:883-90. BogganJE, Walker R, Edwards MSB, BorcichJK, Davis RL, Koonce M, Berns MW. Distribution of hematoporphyrin derivative in the rat 9L gliosarcoma brain tumor analysed by digital video fluorescence microscopy. J Neurosurg 1984;61:1113-19. BogganJE, Bolger C, Edwards MSB. Effect of hematoporphyrin derivative photoradiation therapy on survival in the rat 9L gliosarcoma brain tumor model. J Neurosurg 1985;63:917-21. Levin VA, Freeman-Dove M, Landahl HD. Permeability characteristics of brain adjacent to tumors in rats. Arch Neurol 1975;32:785-91. Brasseur N, Ali H, Langlois R, Wagner JR, RousseauJ. van LierJE. Biological activities of phthalocyanines V. Photodynamic therapy of EMT-6 mammary tumors in mice with sulfonated phthalocyanines. Photochem Photobiol 1987;45:581-86. Brasseur N, Ali H, Langlois R, van LierJE. Biological activities of phthalocyanines--IX. Photosensitization of V79 chinese hamster cells and EMT-6 mouse mammary tumor by selectively sulfonated zinc phthalocyanines. Photochem Photbiol 1988;47:705-11. Canti G, Franco P, Marelli O, Cubeddu R, Taroni P, Ramponi R. Comparative study of the therapeutic effect of photoactivated hematoporphyrin derivative and aluminium disulfonated phthalocyanines on tumor bearing mice. Cancer Lett 1990;53:123-27. KinseyJH, Cortese DA, Neel HB. Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983;43:1562-67. Cheng MK, McKeanJ, Boisvert D, Tulip T, Mielke BW. Effect of photoradiation therapy on normal rat brain. Neurosurgery 1984;15:804-10. Cheng MK, McKeanJ, Mielke B, Tulip J, Boisvert D. Photoradiation therapy of 9L- gliosarcoma in rats: Hematoporphyrin derivative (types I and II) followed by laser energy.J Neurooncol 1985;3:217-28. Svaasand LO, Ellingson R. Optical properties of human brain. Photochem Photobiol 1983;38:292-99. Svaasand LO, Ellingson R. Optical penetration in human intracranial tumors. Photochem Photobiol 1985;41:73-6. Bugelski PG, Poster CW, Dougherty TJ. Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res 1981;41: 4606-12.
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