Aluminium phthalocyanine mediated photodynamic therapy in experimental malignant glioma

S S Styl l i eSc* J S Hill PhD* W H S a w y e r PhDt A H Kaye MD FRACS* The Melbourne Neuroscience Centre*, Departments of Neurosurgery and Surgery, Royal Melbourne Hospital, and Department of Biocbemistry~, University of Melbourne, Parkville*

The efficac T of aluminium tetrasulfonated phthalocyanine (A1SPc) as a photosensi t izer for photodynamic therapy (PDT) was investigated in the C6 glioma mode l in Wistar rats. The depth and extent o f tumour necrosis was dependen t on both the dosage of intravenously (IV) adminis tered AISPc, and the dose and time post-sensitization of 675 n m light administration. There was no effect on tumour or normal brain if the sensitizer dose was less than 0.5 mg/kg . Selective necrosis of tumour was evident 6 hours post-sensitization at an A1SPc dose up to 1 m g / k g and fight doses up to 200 J / cm 2. Necrosis occurred in normal brain at h igher doses of light and sensitizer. There was no photosensi t izer effect in animals t reated 24 hours post AISPc administration. The ability of A1SPc to act as a selective photosensi t izer causing photonecrosis provides a basis for the generat ion of modi f ied A1SPc species as future agents in the PDT of brain tumours, especially as these sensitizers absorb l ight 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

I n t r o d u c t i o n

Cerebral tumours are responsible for approximately 2% of all cancer deaths and the high grade cerebral glioma, glioblastoma mul t i forme (GBM), is the most c o m m o n cerebral tumour in adults. Conventional therapies for the t rea tment of mal ignant gliomas have only limited success. The best available pro tocol , using surgery, rad ia t ion therapy and systemic chemotherapy result in a median survival t ime of less than one year for GBM. 1'e'3 Most t rea tment failures occur because of local recurrence of the tumour indicating that a more aggressive local therapy would be desirable for the t rea tment of gliomas. 4'5'6'7'8

Photodynamic therapy (PDT) is a t rea tment modality that involves the selective uptake of a photosensit izer by the tumour followed by irradiation of the tumour with light that will activate the sensitizer and cause selective t u m o u r death. PDT utilising the sensitizers haemato- p o r p h y r i n der iva t ive ( H p D ) or its m o r e e n r i c h e d commercia l form, Photofrin, has been used in a n u m b e r o f t r ia ls in the t r e a t m e n t o f p a t i e n t s wi th b r a i n tumours, 4'5'6'7's with over 120 patients being treated in the Royal Melbourne Hospital study. 4'5

Despite showing clinical efficacy, 4'5 HpD and Photofrin possess many characteristics which make them less than ideal photosens i t izers . In the synthesis o f H p D and

Photofrin, a hydrolysis mixture of acetic and sulphuric acid is used to treat haematoporphyr in , resulting in a large n u m b e r of o l igomer ic , m o n o m e r i c e t h e r and es ter components . ° Numerous at tempts have been made to de te rmine the 'active' c o m p o u n d in HpD, and several candidates have been nominated. 8'1°'11'12 The composition of H p D may also vary f rom batch to ba tch ult imately altering its biological efficacy) 3 Additional disadvantages are that HpD absorbs poorly in the region of light (600- 700 nm) desirable for opt imal tissue penetra t ion and that patients remain photosensitive for several weeks due to re tent ion of appreciable amounts of dye in the skin) 4 T h e s e cons t r a in t s have e n c o u r a g e d the sea rch fo r alternative photosensitizers for clinical use.

One potential group of compounds are the phthalo- cyanines, which have been suggested as possible candidates fo 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 are characterised by strong light absorption in the 670-700 nm range and are easily synthesised in a pure form. In this study, the efficacy of A1SPc as a potent ia l photo- sensitizer in the PDT t rea tment of the C6 gl ioma in the Wistar rat has been investigated and the appropr ia te dose of sensitizer and light dosimetry which caused destruction of tumour with sparing of normal brain was established.

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Aluminium phthalocyanine PDT Laboratory investigations

Mater ia ls and methods

Chemicals

A1SPc was purchased in powder fo rm in lg quantit ies ( P o r p h y r i n P r o d u c t s , U tah , 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 l i qu id chromatography of this propr ie t ry MSPc showed it to be a mix tu re consisting of the mono- , di-, tri- and tetra- sulfonated forms (unpublished results). On receipt, it was stored dessicated and shielded f rom light at -20°C until subsequent use. Immediately prior to use, itwas suspended in isotonic saline and as a precaut ion was stored in the dark at 4°C.

intracranial gliomas. In brief, the head was shaved, a 1.5 cm midline scalp incision was made, and a bur r hole was placed just in front of the coronal suture, 3 m m to the left of the midline. The C6 gl ioma cells were suspended in a solution of 2X RPMI 1640 conta ining 1% Sea p laque agarose (FMC Corp, Rockland, Maine) at a density of 105 cells per 25 gl. This volume was injected using a Hamil ton Microlitre syringe with a 27 gauge disposable need le covered by a plastic sleeve to a depth of 2 m m from the outer table of the bone. The needle was withdrawn 30 seconds after injection of the suspension, and the hole was sealed with bone wax. The wound 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 us ing an operat ing microscope.

Fig. 1 Chemical structure

of AISPc.

R J

U N N

R

R S O 2 T E T R A N U L F O N A T E D P H T H A L O C Y A N I N E

Cells

The C6 gl ioma cell line was obtained f rom the American Type Culture Collection, and the cells grown at 37°C in a humidif ied a tmosphere 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% newborn calf s e rum ( C o m m o n w e a l t h S e r u m L a b o r a t o r i e s , Parkv i l l e , Australia).

Animals and tumour implantation

All in vivo a n i m a l p r o c e d u r e s were p e r f o r m e d in accordance with requi rements established by the Royal M e l b o u r n e H o s p i t a l I n s t i t u t i o n a l A n i m a l E th ics Commit tee .

Intracranial implantation of C6 glioma in Wistar rats

Adul t Wistar rats we igh ing be tween 200 and 400 g, ob ta ined f rom the jo in t animal facility of the Ludwig Ins t i tu te for Can ce r Resea rch ( M e l b o u r n e T u m o u r Biology Branch) and D e p a r t m e n t of Surgery, Royal Melbourne Hospital, were used for C6 glioma intracranial implantation. The rats were anaesthetised using metho- xyflurane inhalation followed by administration of chloral hydrate (0.1 ml /10 g bodyweight in a solution containing 3.6 g /100 ml water (wt/vol)) by intraperi toneal injection. The me thod developed by Kaye et aP 6 was used to establish

Photodynamic therapy

PDT was administered to animals on the 10th day following tumour cell implantation. The animals were sensitized with AISPc via intravenous (IV) tail vein injection either 6 hours or 24 hours pr ior to the PDT procedure as described.

Normal animals

A 2 cm midline incision was made to expose the skull and allow the per icranium to be reflected. A craniotomy of 4- 5 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 den t a l dril l (Faro Pty Ltd, I ta ly) . T h e exac t dimensions of the craniotomy were recorded to be used later in calculat ing the laser light dose. The an te r ior margin of the craniotomywas placed 1 m m in f ront of the coronal suture and medially ex tended to within 1 m m of the midline.

Tumour bearing animals

An incision was made as before, but incorporat ing the previous bur r hole after reflection of the pericranium. A craniotomy was p e r f o r m e d as in the normal rats over the C6 tumour injection site to ensure the exposure was over the surface of the tumour.

PDT o f rats

PDT 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 administrat ion of A1SPc. Light of 675 nm was genera ted us ing an A r g o n - - i o n p u m p e d Ki ton r ed dye laser producing a broad band (615--695 nm) output (Spectra- Physics, Model 164-05 Argon Ion Laser, Model 375 dye laser, Mountain View, California). A birefr ingent grating was used to tune the ou tpu t to 675 + 2 n m and the wavelength output verified using a slit monochrometer . A 600 g m diameter flat cut quartz fibre was used to deliver the light. The light output was measured by placing the tip of the fibre in a photoelectr ic cell (Spectra Physics Model 404, Spectra Physics, Bayswater, Australia) and calculating the light dose relative to the craniotomy area. The 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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Laboratory investigations Aluminium phthalocyanine PDT

The laser power at the fibre tip varied f rom 50 to 500 mW and the dose of light that was delivered varied f rom 0 to 800 J / c m 2. The surface of the brain was kept cool by irrigation with isotonic saline at room temperature , which has been shown in previous studies to prevent hyper- thermia of the irradiation surface and underlying tissue. 6 On complet ion of the irradiation, a single layer of Surgicel (Johnson and Johnson, Nor th Ryde, Australia) was used to cover the craniotomy site, and the incision closed with wound clips.

Quantification of photodynamic effect

Thean ima l s were culled 5 days after laser treatment. The b ra ins were r e m o v e d f ixed 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. The extent of normal brain or tumour necrosis was measured on serial sections using a graticule micrometer (Leitz, Wetzler, West Germany) in a Nikon Diaphot microscope.

Results

Effect of PDT and AISPc on normal brain

The effect of light dosage on normal brain in animals sensitized with A1SPc, but without implanted tumour, was studied to de te rmine the dosimetry limits of selective kill. The PDT effect on normal brain was studied following A1SPc doses of ei ther 0, 0.5, 1 or 5 m g / kg , and light doses up to 800 J / c m 2. Necrosis of the normal brain was not evident following light doses up to 800 J / c m 2, both in animals sensi t ized at a dose of 0.5 m g / k g and non- sensitized animals. Following administrat ion of AlSPc at a dose of 1 mg /kg , normal brain necrosis was not evident up to a light dose of 2 0 0 J / c m 2, but was apparen t at higher light doses (Fig. 2). At a dose of 5 m g / k g and a light dose

31 ~ i l l ..................... -I,

3 I'-I~ l i j ..... /

2.5 8 2 /

i • .~ / . . . . . . . . . 0.5 mg/kg

~ , 5 ~ i / ---~ik- ...... 1.0 mg/kg

' ~ - / i ~ $.Omg/kg i' I/ / ~ J

050

0 200 400 600 800 1000

Light dose ( J/cm 2 )

Fig. 2 AISPc mediated PDT on Wistar rat normal brain. Non- tumour bearing Wistar rats were administered intravenously (IV) w i th AISPc at doses of 0.5 (n), 1.0 (s) or 5.0 (u) mg/kg. PDT w i th 675 nm l ight on normal brain was carried out 6 hours post AISPc administration. Values shown are the mean from 5 animals per group, w i th the standard error being equal or less than 10% of the mean value shown.

grea te r than 50 J / c m 2, necrosis of n o r m a l brain was observed, with the dep th and ex ten t o f this necrosis dependen t on the light dose administered.

Effect of PDT and AISPc on intracerebral t u m o u r s

When C6 tumour cells were implanted 2 m m deep f rom the outer bone margin, intracerebral tumours grew up to the surface of the brain at 10 days post inoculation. This allowed for the surface of the tumour to be irradiated with red laser light after the craniotomy was per fo rmed . The depths of PDT media ted m m o u r kill 6 hours post administration of doses of A1SPc f rom 0-5 mg /kg , and of 675 nm light f rom 0-800 J / c m 2 at 6 hours, are shown in Figure 3.

3.5

7 3 v cn

2.5 .o

2

1.5

0.5

I I - ................. -i<- ................. I ................. - i # /

i I

i / /

i/

---II ....... 0.5 mg&g

--,A.- ...... l.Omg&g

- - t - - - - - 5.0 mg&g I

m m

o - - - - - - - - - ---- -" ---

0 200 400 600 800

Light dose ( J /cm 2 )

1 0 0 0

Fig. 3 AISPc mediated PDT against C6 g l ioma in Wistar rats. 1 x 10 s C6 g l ioma cells were inoculated intracerebral ly 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 th 675 nm l ight on t umour 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.

The major finding was that following administrat ion 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 rea tment as they were suffering f rom 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 adminis tered 5 m g / k g MSPc were not treated at light doses higher than 100 J / c m 2. There was no t u m o u r necrosis in an imals which received laser t rea tment alone up to light doses of 800 J / c m 2, or laser t rea tment following sensitization at an A1SPc dose of 0.5 mg /kg .

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Following a sensitizer dose of I mg /kg , and light doses up to 200 J / c m 2, selective kill of the C6 rat gl ioma with sparing of normal brain was evident (Fig. 4). The depth of necrosis was similar in rats t reated with light doses between 200 and 800J / e r a 2. At the highest selective laser dose of 200 J / c m 2, the mean depth of tumour necrosis was 3.4 mm, with 4.9 m m being the deepes t dep th of necrosis, and in 2 out of 5 animals the depth of necrosis was greater than 3.5 mm.

3.

- - l - - - - - C6 mm0ur

1.5

1 al brain

0 200 400 600 800 1000

Light dose ( J/cm 2 )

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 and AISPc on n o r m a l brain and intracerebral tumours

T h e d e p t h a n d e x t e n t o f necros i s fo l lowing AISPc media ted PDT of normal brain and C6 tumour was also invest igated at 24 hours post sensitization. A1SPc was adminis tered at ei ther 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 normal 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 tumour 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 administrat ion (Fig. 6) which resulted in a mean depth of necrosis of 3.5 mm.

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/cm 2.

Fig. 5b 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/cm 2.

Fig. 6 Haematoxylin and eosin section of C6 tumour inoculated in Wistar rat brain. The animal was previously admin- istered intravenously (IV) 1.0 mg/kg and treated with 675 nm light 6 hours post AISPc administra- tion. The area aof necrosis is shown by arrows (x 10).

Discuss ion

Most cerebra l gl iomas recur locally after the p resen t conven t iona l the rap ies of surgery, r a d i o t h e r a p y and chemothe rapy indicating a failure to control the local tumour. 1,2,S PDT is a targeted adjuvant therapy which has been shown to aid local tumour control 4,5 and has been the subject of several clinical trials, 4-8 some of which have demonst ra ted 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 gl ioma was selectively retained by the tumour, with peak uptake at 6 hours post-sensitization37 The data repor ted here indicate that selective kill of a cerebral gl ioma with sparing of the normal brain can be achieved with A1SPc media ted PDT. Selective tumour 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 rom 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 depth of tumour necrosis but did result in damage to the normal brain. The depths of tumour kill repor ted here are similar to those previously repor ted by Kaye and Morstyn using HpD in doses between 5 to 20 mg /kg . 16 The t iming of the PDT procedure was found to be critical since t umour bearing and non- tumour bearing rats injected with 0.5 m g / k g or 1.0 m g / k g A1SPc, and treated with 0 J / c m 2 to 800J / cm 2 24 hours postA1SPc administration did not show any evidence of normal brain or t umour necrosis, a result in accord with the pharmacokinet ic data. 17 These results are in contrast to a repor t using the VMDk mur ine gl ioma model , where mice were t reated with A1SPc media ted PDT 18 and non - tumour bear ing animals t reated at 48 hours post-sensitization with 0.5 m g / k g or 5.0 mg /kg . These workers found that PDT adminis tered 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, 17 no deaths occurred immediately postoperatively or in the 5 day postoperative period at light and sensitizer doses of 50 J / c m 2 and 5 m g / k g A1SPc respectively. However, using a dose regime of 5 mg /kg 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 ei ther 0.5 m g / k g or 1.0 m g /kg showed minimal evidence of ei ther normal brain or tumour necrosis. The differences in the results repor ted here and those previously published, 18 may well r e f l ec t e i t h e r var ia t ions in the t u m o u r mod e l s or differences in the composi t ion of the sensitizer pre- pa ra t i on . Var ia t ions in t u r n o u t mode l s have b e e n recognised, with conflicting reports regarding HpD peak uptake times in d i f fe ren t bra in 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 tumour 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 uni form fluorescence was present th roughout the t u rnou t at 6 hours , with a dec l ine in f l uo re scen ce 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 amount 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 mode l he t e rogene i t y among tu rnou 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 bound 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 tumour 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 roper t i e s o f the compound 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 dependen t on the degree of sulfonation) 7 This variation in time to peak uptake may explain the variations between the results repor ted here, and those of Sandeman et al. 18 Thus fur ther PDT studies utilising the purified sulfonated forms ofA1SPc (mono-, di-, tri- and tetra-) are needed to de te rmine both the optimal timing of PDT, and the degree of sulfonation required for maximal tumour kill. Few studies addressing these issues have been reported, al though Brasseur et

a123,24 have compared the photodynamic activity of various p h t h a l o c y a n i n e s with tha t o f H p D in t r e a t i n g a subcutaneously implanted EMT-6 mammary tumour in mice. The aluminium and gallium chelated complexes of the phthalocyanines were as tumouricidal as HpD, while disulfonated phthalocyanine has been shown to elicit superior tumouricidal activity to an A1SPc mixture, various su l fona ted gal l ium p h t h a l o c y a n i n e c o m p l e x e s and HpD.2S, 24

The 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 penetrat ion being significantly reduced in the pigmented melanoma, 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 been r ega rded by some investigators as a major component of PDT, 26'27'28 this study an d p rev ious s tudies us ing H p D have shown tha t hyper thermia does not occur if the site is irrigated with isotonic saline, and that PDT's efficacy is independen t of h y p e r t h e r m i a . T h e cri t ical t u m o u r targets o f HpD sensi t ized PDT are yet to be defini t ively descr ibed , al though evidence from some studies suggests that since the observed depth of tumour damage 16 is beyond the proposed depth of penetrat ion of light, 29,3° it is probable that vascular damage with subsequent d e e p e r tissue infarction may be a major basis for some of the tumour destruction, a6,31

As A1SPc is comprised of a mixture of the various sulfonate species (J Bommer, Porphyrin Products, personal communication, Stylli et al, unpublished observations), it is possible that the amount of the more active disulfonate componen t is proport ionately low. Thus a description of the tumour distribution of the various sufonate species comprising A1SPc is needed to de te rmine 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 tumour uptake, and mediates selective photonecrosis of implanted glioma. Further studies examining the efficacy of the separa te su l fona ted fo rms of A1SPc war ran t a d d i t i o n a l e v a l u a t i o n an 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 suppor t of grants f rom Nat ional Hea l th 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, University of Melbourne, Victoria 3050 Australia Tel: 61 3 342 7616 Fax: 61 3 347 8332

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Aluminium phthalocyanine PDT Laboratory investigations

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