Photodynamic Therapy of Malignant Tumours

1175 past can now be attributed to its latency and to the fact that viral " rescue " has not heretofore been attempted. supported in part by Deutsche ForschungsAZ 270/8, Stiftung Volkswagenwerk and Forschungsmittel des Landes Niedersachsen. This work was gemeinschaft, Requests for reprints should be addressed to V. t. M., Institut Virologie, Universitat Wurzburgj Versbacher, Landstrasse 7, Wurzburg, Federal Republic of Germany. fur REFERENCES Hayne, A. L., Slotowski, E. L. Am. J. Dis. Child. 1947, 73, 554. Ehrengut, W. Arch. ges. Virusforsch. 1965, 16, 311. Holliday, P. B., Jr. J. Pediat. 1950, 36, 185. Gibbs, F. A., Gibbs, E. L., Carpenter, P. R., Spies, H. W. J. Am. med. Ass. 1959, 171, 1050. 5. Pampiglione, G. Br. med. J. 1964, ii, 1296. 6. Scott, T. F. Med. Clins N. Am. 1967, 51, 701. 7. McLean, D. M., Best, J. M., Smith, P. A., Larke, R. P. B., McNaughton, G. A. Can. med. Ass. J. 1966, 94, 905. 8. Müller, D., Meulen, V. ter. Acta neuropath. 1969, 12, 227. 9. Meulen, V. ter, Enders-Ruckle, G., Müller, D., Joppich, G. ibid. 1. 2. 3. 4. p. 244. 10. Katz, M., Oyanagi, S., Koprowski, H., Nature 1969, 222, 888. 11. 12. 13. 14. 15. Enders-Ruckle, G. Personal communication. Meulen, V. ter, Katz, M., Käckell, Y. M., Barbanti-Brodano, G., Koprowski, H., Lennette, E. H. J. infect. Dis. 1972, 126, 11. Walthard, K. M. Z. ges. Neurol. Psychiat. 1930, 124, 176. Meulen, V. ter, Katz, M., Müller, D. Curr. Topics Microbiol. Immun. 1972, 57, 1. Drzeniek, R., Rott, R. Int. Archs Allergy, 1969, 36, 146. Preliminary Communications PHOTODYNAMIC THERAPY OF MALIGNANT TUMOURS STEVEN G. GRANELLI SURL NIELSEN RICHARD JAENICKE IVAN DIAMOND ANTONY F. MCDONAGH CHARLES B. WILSON Departments of Neurology, Pediatrics, Neurosurgery, Medicine, and Pathology, University of California, San Francisco, U.S.A. Summary Porphyrins are powerful photodynamic agents which sensitise cells so that damaged when exposed to light. Malignant tumours take up and retain hæmatoporphyrin to a greater extent than does normal tissue. This study is a test of the idea that hæmatoporphyrin can serve as a selective photosensitising agent to destroy tumour cells by exposure to visible light. The administration of hæmatoporphyrin followed by light therapy proved lethal to glioma cells in culture and produced massive destruction of porphyrin-containing gliomas transplanted subcutaneously in rats. Treatment with light or hæmatoporphyrin individually was without effect. Photodynamic therapy offers a new approach to the treatment of brain tumours and other neoplasms resistant to existing forms of therapy. they are INTRODUCTION often treated by extensive effort in many laboratories to find an effective radiosensitising agent.1 Many neoplasms take up and retain BECAUSE malignant tumours are X-ray irradiation, there has been an hsematoporphyrin preferentially 2-4 and Schwartz et al. suggested that porphyrins might be therapeutically useful as radiosensitising agents for cancer therapy." However, injection of porphyrins into patients with malignant tumours did not appear to potentiate the effects of X-ray irradiation. Porphyrins are powerful photodynamic agents which can sensitise biological preparations so that they are severely damaged when exposed to visible or near-ultraviolet light These photo-oxidation reactions appear to involve the production of electronically excited metastable molecular oxygen (singlet oxygen) as a reactive and highly toxic intermediate. 9, 10 It seemed reasonable to expect that accumulation of hxmatoporphyrin in a malignant tumour would result in specific sensitisation of the tumour so that it could be destroyed by visible light. The results of this study demonstrate that a combination of hxmatoporphyrin and light treatment is lethal to tumour cells in culture and in laboratory animals. MATERIALS AND METHODS Commercial haematoporphyrin (free base, Sigma Chemical Co., St. Louis, Mo.) was used without purification; thinlayer chromatography in two systems showed it to contain one major component. Haematoporphyrin was dissolved in saline containing 20 mM NaOH and the solution neutralised to pH 7-4 with HCl. The tumour model used was a glioma induced by methylnitrosourea in an inbred rat strain. 11I In-vitro experiments were carried out with actively growing cultures of the rat glioma maintained in Eagle’s basal medium supplemented with 10% fetal-calf serum. An inoculum of 4 x 104 cells was allowed to equilibrate in culture tubes for 48 hours. At this time haematoporphyrin (1O-ClO-5M) was added to duplicate culture tubes and controls received neutralised saline. The cultures were maintained in either dark or lighted incubators. Cell death was determined by staining with trypan-blue. In-vivo studies were performed with 20 Fisher 344 (150-170 g.) bearing tumours produced by imsubcutaneously in the right flank. Nineteen to twenty-one days after implantation 20 tumour-bearing rats were used for study. 12 animals received 10 mg. hsematoporphyrin (10 mg. per ml.) intraperitoneally and were kept in the dark. Tumours in 8 animals injected with hsmatoporphyrin were exposed to light for a total of 3-5 hours over the next three to five days; 4 animals injected with hsematoporphyrin were not exposed to light. 3 rats were given light treatment alone, and 5 animals were not treated. Changes in tumour volume were estimated by external measurement of tumour diameters with callipers. Formalin-fixed paraffin-embedded tumour sections were stained with haematoxylin and eosin and examined under a light microscope. The light source for in-vitro irradiation, 8 20-watt fluorescent cool-white lamps (’Vitalite’, Duratest Corp., Hillside, New Jersey), delivered approximately 350-450 male rats planting 106 cells footcandles and was kept 1 ft. above the culture tubes in an air-curtain incubator. The temperature of the cultures was maintained at 37 °C. The light source for in-vivo radiation, a 150-watt highintensity light bulb directed through a ’Lucite’ rod, delivered approximately 10,000 footcandles of cool light in a 5 cm. diameter circle. This did not heat the shaved skin overlying the tumour. Treated animals were either restrained without anaathesia or anxsthetised with’Innovar-Vet’ (fentanyl/droperidol). RESULTS Glioma-cell cultures incubated in the dark for 150 minutes, (a) without treatment, (b) with added haemato- 1176 of 0-96 cm. ±0-20 S.D. The estimated average tumour volume was 0-5 c.cm. Light without haematoporphyrin or hEematoporphyrin without light did not affect tumour growth when compared to untreated animals (fig. 2). However, the combination of light and hsematoporphyrin produced a striking regression of tumour size within a few days after the last light treatment. Twenty-eight days after tumour implantation, porphyrin-containing gliomas treated with light had a diameter ranging from 0-5 to 0-65 cm. (volume=0-l c.cm.), while untreated controls and tumours treated with light alone had an average tumour diameter of -LU 4U bU t3U IOU 1U 14U ]bU MINUTES Fig. I-Effect of haematoporphyrm on the viability of glioma cells in culture. Cell death was estimated by staining with trypan-blue. Controls had a 1 % cell-death rate. Each point is the average of 2 cultures. porphyrin (10"Af), or (c) with added neutralised saline, maintained 99% viability. The same viability was found when cultures were exposed to light for 150 minutes without additions or with neutralised saline. However, the combination of hxmatoporphyrin and light proved lethal to glioma cells in vitro (fig. 1). Addition of 10-5M haematoporphyrin and exposure to light led to 100% cell death after 50 minutes, while 10-sM haematoporphyrin and light produced 93% cell death after 120 minutes of light exposure. These results suggested that hasmatoporphyrin might be a selective and lethal sensitising agent if light could be directed into a porphyrin-containing tumour. Twenty to twenty-three days after implantation, flank tumours growing in 14 animals reached a diameter Fig. 3-Effect of light and haematoporphyrin glioma implants in rats. on the growth of Controls were 5 untreated tumour-bearing rats and 3 tumours treated only with light. Control growth-rates are included in fig. 2. 4 animals were treated with hsmatoporphyrin and light. 1-7 cm.:LO-19 S.D. (volume=2-6 c.cm.) (fig. 3). Histo- logical examination of the shrunken tumour nodules removed from 3 animals treated with hxmatoporphyrin and light revealed massive coagulation necrosis with sparing of only a small rim of viable cells along the periphery of tumour most distant from the light source. Tumour-cell kill was estimated to range from 75 % to 95 %. In the 4th animal, cell death was observed only in the lateral third of the tumour mass. Tumour sampled from each control group had the characteristic histological feature of proliferating gliomas. DAYS AFTER TUMOUR INOCULATION Fig. 2-Effect of hseoiatoporphyrin on the growth of glioma implants in rats. Volume was calculated from tumour-diameter measurements. Tumour diameter in the 3 groups increased at a rate of 0-078 cm./day:0’019 S.D. Each point is the average volume JS.D. of 3-5 animals. During the first 30 hours after porphyrin-containing gliomas were treated with light the tumour mass and overlying skin enlarged about 30%, probably due to oedema. In six days a desquamating skin lesion developed at the site of light treatment, but photosensitised skin re-epithelialised in about 5 weeks. Glioma growth arrest was maintained for ten to twenty days after phototherapy. Then tumours began to enlarge slowly as small nodules grew from the deepest surface of the otherwise inactive mass. 1177 MEASUREMENT OF THYROXINE AND TRIIODOTHYRONINE IN HUMAN URINE DISCUSSION This was an exploratory study to test the idea that hxmatoporphyrin could be used to produce photodynamic destruction of malignant tumours byadministering a single dose of hxmatoporphyrin with a variable After animals are injected amount of illumination. with hxmatoporphyrin, fluorescence remains in the R. A. SHAKESPEAR C. W. BURKE T. RUSSELL FRASER Endocrine Unit, Department of Medicine, Royal Postgraduate Medical School, serum up to 3 hours and is found in urine and fasces up to 24 hours. 12 Skin photosensitivity may persist for five days,12but hxmatoporphyrin is preferentially retained by malignant tumours for as long as fourteen days. 2,4 For these reasons we chose 24 hours after injection of hasmatoporphyrin as the time to start light treatment to subcutaneous gliomas in rats. Hxmatoporphyrin without light, or exposure to light without hasmatoporphyrin, did not affect glioma viability either in cultures or in animals. However, haematoporphyrin taken up by tumour cells acted as a powerful photodynamic agent that conferred striking photosensitivity. The combination of hxmatoporphyrin and phototherapy was lethal to glioma cells in culture and produced massive destruction of porphyrin-containing tumour cells in rats. Consistent with current concepts it seems likely but not proven that hxmatoporphyrin photosensitisation of tumour cells is mediated through Failure to the production of singlet oxygen. 9, 10 achieve total glioma-cell kill in tumour-bearing animals could be ascribed to inadequate deposition of porphyrin or incomplete delivery of light energy to the entire - mass. Since hxmatoporphyrin is preferentially retained by malignant tissue, 2-4 it is potentially a true sensitising agentwhich may achieve selective photodynamic damage in porphyrin-containing tumour cells with relative sparing of porphyrin-free normal tissues in In particular, photodynamic therapy should vivo. offer a unique approach to the treatment of brain tumours, since (a) hxmatoporphyrin is taken up and retained by intracerebral gliomas but is excluded from normal brain (Granelli and Diamond, unpublished observations) and (b) light penetrates through the intact skull into the brain of large and small animals. 13 Indeed, once the optimal conditions for combining a photosensitising agent with light irradiation are defined, photodynamic therapy may provide a new approach to the management of several human neoplasms resistant to existing forms of treatment. This work was supported by Public Health Service grant CA 13525 and a gift from Phi Beta Psi Sorority. We thank Dr. Rudi Schmid for helpful suggestions. I. D. is the recipient of U.S.P.H.S. research scientist career development award 1-K4-NB23, 131. London W12 0HS Urinary excretion of unconjugated thyroxine (T4) was 2·0 µg. per day in (mean) thirty-six normal subjects. The renal Summary clearance of serum unbound T4 was 26 ml. per minute (mean) in ten subjects. Hydrolysis of conjugated T4 in urine, however, yielded a further 2·8 µg. day (mean) by enzyme hydrolysis or 3·7 µg. per day by acid hydrolysis. Mean figures for percentages of unconjugated and conjugated thyroxine were 39% and 61% of total T4, respectively. The unconjugated fraction should reflect the prevailing serum level of unbound T4, and be a useful thyroid-function test. Urinary immunoassayable triiodothyronine (T3) was 0·8 µg. per day (mean) in thirty-eight normal subjects. This fraction was 52% (mean) of the total T3, which was no more than 1·4 µg. per day (mean), in nine subjects. It is probably close to the amount of unconjugated T3. Renal clearance of serum unbound T3 seemed to be greater than glomerular filtration-rate in some cases, raising the possibility of tubular per excretion of T3. INTRODUCTION MEASURING the urinary excretion of unconjugated thyroxine (T4) and triiodothyronine (T3) might be an easy and useful indirect way of estimating the biologically active, non-protein-bound, levels of T4 and T3 in serum; just as, for example, urinary " free " (unconjugated) cortisol excretion reflects the prevailing level of unbound cortisol in plasma. 1,2 On this hypothesis, two forms of T4 and T3 should be present in urine. First, there would be unconjugated T4-T3 which will have entered the urine by glomerular ultrafiltration of serum non-protein-bound T4 and T3, some being then reabsorbed in the renal tubules. Second, there will be T4 and T3 which has been conjugated with glucuronide or sulphate, either in kidney itself or in liver.3 In addition there will be metabolites of T4 and T3 present. Only the first, unconjugated fraction, may be expected to reflect the serum unbound level. The total excretion of T4 and T3 will depend on liver conjugation, hormone kinetics, Requests for reprints should be addressed to 1. D., Department of Neurology, University of California, San Francisco, California 94122, U.S.A. DR. DIAMOND AND OTHERS: REFERENCES 1. 2. Lancet, Sept. 23, 1972, p. 638. Figge, F. H. J., Weiland, G. S., Manganiello, O. J. Proc. Soc. exp. Biol. Med. 1948, 68, 640. 3. Lipson, R. L., Baldes, E. J., Olsen, A. M. J. natn. Cancer Inst. 1961, 26, 1. 4. Gregorie, H. B., Horger, E. O., Ward, J. L., Green, J. F., Richard, T., Robertson, H. C., Jr., Stevenson, T. B. Ann. Surg. 1968, 167, 820. 5. Schwartz, S., Absolon, K., Vermund, H. J. Lab. clin. Med. 1955, 46, 949. 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