Studies on the photodynamic effect of haematoporphyrin derivative

Journal of Photochemistry and Photobiology, B : Biology, 6 (1990) 297-308

297

STUDIES ON THE PHOTODYNAMIC EFFECT OF HAEMATOPORPHYRIN DERIVATIVE* M . KARAIVANOVAt Research Institute of Pharmacology and Pharmacy, Bulgarian Medical Academy, Department of Oncopharmacology, Sofia 1000 (Bulgaria) S . KARANOV National Ontological Centre, Bulgarian Medical Academy, Clinic of Surgery, Department of Abdominal Surgery, Sofia 1156 (Bulgaria)

M . SHOPOVA, E . KAISHEVA and M. PEEVA Institute of Organic Chemistry, Bulgarian Academy of Sciences, Department of PDT, Sofia 1113 (Bulgaria) H . GETOV and H . PROKOPANOV National Ontological Centre, Bulgarian Medical Academy, Clinic of Surgery, Department of Pathology, Sofia 1156 (Bulgaria) (Received October 5, 1989 ; accepted December 15, 1989)

Haematoporphyrin derivative, photodynamic therapy, mouse adenocarcinoma .

Keywords.

Summary A case-control photodynamic therapy (PDT) was studied on adenocarcinoma (AC755) in BDF1 mice . Haematoporphyrin derivative (HPD ; Porphyrin Products, U .S .A.) and a Bulgarian HPD were used as photosensitizers at doses of 10 mg kg - ' . An argon dye laser system with Aim =630 nm (400 mW cm - ') was used for PDT with a total light dose of 400 J cm -2 . The therapeutic effect was assessed by the changes in tumour dimensions, the size of photonecrosis and the mean survival time of the animals . Histologic and ultrastructural studies were carried out . No significant difference was recorded between the antitumour effects of the two photosensitizers . Best results were obtained in small tumours (less than 10 mm) with incision of covering skin . Results are discussed in an attempt to advocate an optimal regimen for PDT in experimental tumours .

*Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989 . Author to whom correspondence should be addressed .

Elsevier Sequoia/Printed in The Netherlands

298 1 . Introduction Photodynamic therapy (PDT) is a new method for the treatment of solid malignant tumours - superficial or located within the caval organs . The most common photosensitizer used is haematoporphyrin derivative (HPD) . A number of experimental and clinical investigations have been devoted to the photodynamic effect of HPD [1-51 and to the mechanisms of PDTinduced tumour destruction [6, 7] . We carried out comparative studies concerning the photodynamic effect of HPD on transplanted mouse tumour adenocarcinoma 755 (AC755) .

2 . Materials and methods 2 .1 . Synthesis of haematoporphyrin derivative (HPD) Haematoporphyrin dihydrochloride (0 .345 mg) was dissolved in 7 ml of a mixture of glacial acetic acid and sulphuric acid in the ratio 19 :1 . The mixture was stirred for 60 min at room temperature and then left for 24 h . The pH of the reaction mixture was adjusted to 5 .3 by adding 700 mi of 3% CH 3 000Na and 0.280 ml of 50% NaOH . The mixture was then left for 24 h at room temperature . The precipitated material was separated by vacuum filtration through a glass microfibre filter and washed with bidistilled water until the salts were removed and a pH of about 6 .0 was reached . The HPD thus obtained was subjected to lyophilization . 2 .2. Tumour model The tumour model used was adenocarcinoma of the mammary gland AC 755 transplanted in adult female mice BDF 1 . The tumour line was maintained by serial transplants of tenfold diluted cell suspensions on the left flank of the animals . Parts of the animals were inoculated subcutaneously (s .c .) with 106 cells per 0 .5 ml, while other mice received 2 X 10 6 cells . No cases of spontaneous regression were observed during the serial transplants . 2.3 . Photodynamic therapy The light source used was a laser system consisting of an argon ion laser (Spectra Physics No . 171) and a dye-pumped laser (Spectra Physics No . 375) with 632 nm wavelength emission (Kiton Red dye) . The opticalfibre system (Quentron, Austria) had a normal cross-section end piece and a diffuser allowing interstitial irradiation . The photosensitizers (Bulgarian HPD and HPD supplied by Porphyrin Products, U .S .A .) were administered intraperitoneally (i .p .), after alkaline hydrolysis and pH adjustment to 7 .2, at a dose of 10 mg kg - ' body weight . After 48 h, the animals were irradiated with a total light dose of 400 J cm - Z . The mice were anaesthetized with a 0 .2% solution of Thiopental (0 .2-0 .3 ml per mouse, applied i .p.) and their skin was depilated . The animals were divided into the following groups (12 mice per group) .

299 (1) Animals were irradiated on the tenth day after tumour transplantation (10 6 cells per mouse), when the mean tumour diameter was 9 .0 nun, using a normal cross-section end piece of the optical fibre . (2) Mice were treated under the same conditions as group 1, but had a mean tumour diameter of 8 .4 mm. (3) Animals were irradiated 11 days after tumour transplantation (2 x 106 cells per mouse), and had a mean tumour diameter of 13 .2 mm . (4) Animals were irradiated under conditions similar to group 3, but a diffuser end piece of the optical fibre was used allowing interstitial (intratumoural) irradiation . In this case the mean tumour diameter was 15 .3 mm . (5) Two groups of control animals were used : (i) with tumours after transplantation of 10 6 cells per mouse, without irradiation and without sensitizer, and (ii) with tumors after transplantation of 2 X 10 8 cells per mouse, without irradiation and without sensitizer .

2 .4. Assessment criteria On the fourth day after irradiation half of the animals (six of each group) were killed by cervical dislocation . Tumour preparations were weighed and fixed in 10% formalin . The material was treated using routine histological techniques for microscopic investigations: determination of the degree of induced necrosis and morphological characterization of the tumour tissue . Parts of the tumours were subjected to double fixation with 3% glutaraldehyde and 1% osmium tetroxide, dehydrated with alcohol and propylene oxide at increasing concentrations, and mixed with Durcupan for electron microscope observations (using a Hitachi 11E electron microscope) . The remaining animals were left for further observations : presence of tumours ; tumour diameters (a and b representing the long and short axes of the tumour parallel to the surface) ; survival time . The results were statistically processed using Student's t test .

3 . Results

3 .1 . Changes in tumour diameter alter PDT Figure 1 presents the results of the photodynamic treatment of AC755 with the two porphyrin products as reflected by the changes in tumour diameter with time . As can be seen from Fig . 1A, photodynamic treatment administered on the tenth day after transplantation (when the tumours had mean diameters of up to 10 mm) caused an immediate abrupt decrease in tumour mass (group 1 (treated with the imported derivative) and group 2 (treated with our product)) . On the fourth day after PDT the mean tumour diameters were reduced by 50% and these dimensions were preserved almost unchanged during the next 3 days . After that (up to day 24) a certain increase in tumour size was observed (tumour diameters reaching 8 .2 mm and 4 .2 mm for the first and second groups respectively), but the tumours were much smaller than those in the control group (31 .2 mm) . The observed

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(B) Fig. 1 . Tumour growth curves of adenocarcinoma 755 after PDT . Groups of six mice transplanted with 106 cells per mouse (A) and 2 x 106 cells per mouse (B) were treated (except for the controls) with HPD and irradiated with 400 J cm -2 at 630 nm 48 h later. - - -, controls; --0--, 10 mg kg - ' HPD (Porphyrin Products, USA) ; - •- , 10 mg kg - ' HPD (Bulgarian product) .

tumour growth was peripheral - the tumours were flat and elliptical in shape with a central necrotic region and a condensed peripheral area . When tumours with larger diameters (13 .2 nun) were irradiated (Fig . 113) similar changes in the growth curve were observed : a 50% decrease in

301

tumour size within 4 days after irradiation and a slow peripheral growth thereafter.

3.2. Survival time assay Following the lethality of the animals with time (Table 1), a statistically reliable (for P<0 .05) increase in the survival time was established for the PDT-treated mice of groups 1 and 2 compared with the untreated control animals (the mean survival time of the control group was 32 .5 days) . In the group of animals with larger tumour size (group 3) the difference between the survival times of the treated and untreated (control II) animals was not reliable .

3.3. Tumour regressions The two types of HPD showed a pronounced photodestructive effect in AC755 which was expressed by tumour regression (Table 2) . Thus when the photosensitizers were administered to animals with tumour diameters of

TABLE I Survival time of mice with AC755 after PDT Group

Mean tumour diameter on day of irradiation

MST±SD' (days)

Mortality (days)

(mm) 1 2 Control 1

9 .0 8 .4 14 .2

39 .6±2 .8 39 .0±1 .9 32 .5±2 .3

30-44 35-44 24-38

3 Control 11

13 .2 18 .5

37 .6±3 .6 30 .5±3 .5

28-44 22-34

'MST, mean survival time; SD, standard deviation. Groups of six mice were treated i .p. with HPD (Porphyrin Products, U.S-k.) (1) and HPD (Bulgarian product) (2 and 3) and irradiated with 400 J cm -2 48 h later . The animals were transplanted with 10 6 tumour cells per mouse (groups 1, 2 and control I) or 2 x 10 6 cells per mouse (group 3 and control II) . TABLE 2 Tumour regression after PDT Group

1 2 3

Tumour regression (96) Day 4

Day 7

Day 14

50 .0 66 .6 16 .6

33 .3 50.0 0

16 .6 33 .3 0

Animals were transplanted and treated as indicated in Table 1 . Spontaneous tumour regression was not observed in the control group .



302 up to 10 mm (groups I and 2), full regression was observed in 50 9/6-66 .6% of the cases . In animals with larger tumours (group 3), regression was observed only in 16 .6% of the cases .

3 .4. Necrosis region Figure 2 shows the degree of induced necrosis after PDT . Through histological investigations the highest degree of necrosis (up to 6096-70% necrotic tissue) was found in irradiated tumours of small size (groups 1 and 2) . The diameters of the necrotic regions correspond, in principle, to the results of the histological investigations . However, in larger tumours, induced necrotic regions with relatively large diameters were found (about 5 mm) which constitute only about 55% of the total tumour mass . The presence of necrotic tissue was also noted in the control (about 15% for C-Il) .

3 .5. PDT using interstitial irradiation Table 3 presents the therapeutic results obtained after PDT using interstitial irradiation and i .p . administration of Bulgarian HPD ; 80% necrosis was achieved . The tumours were soft and easily injured by palpation . A decrease in the mean survival time was found : 28 .7 days compared with 30 .5 days for the untreated control animals .

3.6 . Morphological evaluation of treated tumour tissues Two zones of tumour damage (necrotic and hyperaemic zones), were determined histologically . In early tumour treatment (mean tumour diameter, 8 .4 mm), a moderate photocytotoxic effect was observed on the fourth day Ib)

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Fig. 2 . Degree of necrosis in AC755 induced after PDT (irradiation with 400 j CM-2 at 630 nm 48 h after application of HPD) : 1, with HPD (Porphyrin Products, USA .) ; 2 and 3, with HPD (Bulgarian product) ; K, and K,,, controls without treatment. (A) Necrotic region (nun) determined macroscopically on the basis of the mean diameter of the superficial necrotic areas ; (B) percent necrotic tissue (in relation to tumour mass) determined by light microscopic investigation .

303 TABLE 3 PDT of AC755 with interstitial irradiation Group

Mean tumour diameter on day of irradiation

Necrosis on day 4

MST±SD

(%)

(days)

80 30

28 .7 ± 4 .5 30 .5±3 .5

(mm) 4 Control II

15 .3 18 .5

Transplantation with 2 x 10 6 cells per mouse . Total light dose of irradiation, 400 J cm - ' ; drug dose, 10 mg kg - ' body weight of Bulgarian HPD .

after PDT with Bulgarian HPD . The necrotic region (approximately 70% of the tumour mass) was surrounded by oedemic tissue with moderate inflammatory infiltration . Most of the tumour vessels were intact, some of them exhibiting extensive blood stasis . The endothelial cells in some of the vessels were inflated and exhibited slight degenerative alterations (Fig . 3) . The results of the treatment with Porphyrin Product's HPD (group 1) were essentially similar . In tumours with a mean diameter of 13 .2 mm, we observed larger necrotic zones and a considerable number of non-viable cells . In 55% of the tumour cells the presence of pyknotic nuclei, karyorexis and karyolysis was established (Fig. 4(A)) . A hyperaemic-haemorrhagic zone was outlined around the necrotic area (Fig. 4(B)) . Although there were haemorrhages in the necrotic region, most of the vessels were intact and congested with red blood cells . More rarely we observed capillaries with pronounced endothelial cell destruction and surrounded by oedemic tissues with inflammatory infiltration . The electron microscope investigations provided data on the changes in the ultrastructure of the tumours . Intercellular spaces were widened to various degrees, whereby some of the cells were singly located . Specialized intercellular contacts were considerably reduced in number, and even missing in some fields (Fig. 5(A)) . As to the cytological characteristics, nuclei were found to be poorly sensitive to PDT : their chromatin was more condensed, both centrally and marginally, in comparison with the untreated control tumours, whereas the nuclear membrane was smooth or undulated . Predominantly single nucleoli were observed, centrally located, of compact (homogeneous) type . The cytoplasm of these cells was poor in organelles and inclusions . Only single sacs of granular endoplasmic reticulum were found and single mitochondria often degeneratively changed . Numerous free ribosomes, vacuoles, lysosomes, free membrane structures and single myelin structures were also noted (Fig . 5(B)) . 4 . Discussion Comparative studies of the two types of HPD show that these derivatives, when applied at a dose of 10 mg kg - ' 48 h prior to irradiation, are

304

(A)

(B) Fig. 3 . Histological appearance of A0755 (tumour diameter, 8 .4 mm) on the fourth day after PDT (10 mg kg- ' HPD (Bulgarian product), photoirradiation with 400 J CM-2) . (A) Extensive necrosis, inflammatory reaction and oedema surrounding the tumour ; (B) areas of mlcrovasculature congested with extensive blood stasis . Haematoxylin and eosin (H.E.), x 200 .

therapeutically efficient photosensitizers of mouse adenocarcinoma of the mammary gland . The photodynamic effect resulting from sensitization with Bulgarian HPD is equal to that observed after administration of the Porphyrin Product derivative, provided that the same doses and experimental conditions are used. The data are in agreement with clinical PDT results obtained using HPD in recurrent breast carcinoma [8] . Various methods of evaluation have been described in the literature ; these are based on the diameter and/or depth of the induced necrosis [9-111, the tumour weight and volume [12-14], the content of sensitizers in the tumour and normal tissues [15] and the number of "cured" animals [16] .

305

(A)

(B) Fig . 4 . Histological appearance of AC755 (tumour diameter, 13 .2 mm) after PDT (as indicated in Fig . 3) . (A) Extensive necrosis and many nonviable cells throughout the tumour ; (B) hyperaemic-haemorrhagic zone surrounding the necrotic region . Although blood vessel destruction and severe haemorrhage were observed, there were many intact vessels congested with red blood cells H.E ., x 200 .

We used various approaches in an attempt to obtain maximum information about the properties of HPD . On evaluating the effect of PDT as reflected in the dynamics of the mean tumour diameter, we obtained unambiguous data on the decrease in tumour growth with time for both types of photosensitizer . The presence of necrotic tissue in the treated tumours, the changes in their shape (mainly at the expense of the third diameter c) and the predominating peripheral tumour growth indicate that this method does not provide a sufficiently accurate quantitative analysis of the experimental results .

306

(A)

(B) Mg. 5 . Ultrastructural changes in AC755 after PDT (HPD (Bulgarian product), photoirradiation -2 with 400 J cm 48 h later) : (A) changes in structural intercellular interactions ; (B) changes in cytological characteristics, x 6200 .

The data obtained on the survival time of the animals show little differences with respect to the controls . Although this is an important criterion, very close to that clinically employed, the selection of photosensitizers using this evaluation method is still difficult . On assessing the cytotoxic effect of PDT on the basis of the degree of induced necrosis, data were obtained which gave an adequate characterization of the tumour tissue destruction . A sufficient accuracy and an alternative response (presence/absence of tumours) provide an evaluation of the effect of PDT from the number of tumour regressions [1, 16J . Such an approach

3 07

is analogous to the criteria used for clinical analysis of the results of PDT [171 . The results of the morphological analysis show the presence of direct photocytotoxicity, changes in some targets of the tumour cell (i .e . membrane, mitochondria, etc .) and changes in the vessels, as indicated by other workers 118-201 . The histologically observed necrosis of the tumour tissue and the high percentage of non-viable cells indicate direct cell damage . The infringement of the blood-vessel walls can only explain, to a certain extent, the histopathological alterations . The relatively high resistance of the vascular walls to the destructive effect of PDT is worth noting . An analysis of the results obtained using the various evaluation techniques emphasizes the need to apply a multiparameter experimental strategy, involving the registration of a large number of parameters (as independent as possible) . The experimental data show that the two HPD samples used are effective photosensitizers for PDT of experimental adenocarcinoma 755 . When applied in combination with laser irradiation, they have a pronounced photodestructive effect, and cause morphological changes and tumour regression in relatively small tumours ; these effects result in an increase in the life of tumour-bearing animals . References 1 T . J . Dougherty, B . Grindey, R . Fiel, K . R . Weishaupt and D . G . Boyle, Photoradiation therapy : cure of animal tumors with hematoporphyrin and light, J . Nall . Cancer Inst ., 55 (1975) 115-121 . 2 T . J . Dougherty, Photosensitizers : therapy and detection of malignant tumors, Photochem . PhotobioL, 45 (1987) 879-890, 3 D . Kessel and T . J. Dougherty, Porphyrin Photosensitization, Plenum, New York, 1983 . 4 G . Jori and C . A. Perria (eds.), Photodynamic Therapy of Tumors and Other Diseases, Libreria Progetto Editore, Padova, 1985 . 5 J. Moan, Porphyrin photosensitization and phototherapy, Photochem . Pholobiol., 43 (1986) 681-690. 6 B . W. Henderson, S . M . Waldow, T . S . Mand, W. R . Potter, P . B . Malone and T. J . Dougherty, Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy, Cancer Res., 45 (1985) 572-576 . 7 G . Moreno, R. H . Pottier and T . G . Truscott, Photosensitization: Molecular, Cellular and Medical Aspects, Springer-Verlag, Berlin, 1988 . 8 T . J . Dougherty, G. Lawrence, J . Kaufman, D . Boyle, K . R . Weishaupt and A . Goldfarb, Photoradiation in the treatment of recurrent breast carcinoma, J. Nall. Cancer Inst ., 62 (1979) 231-237 . 9 M . C . Berenbaum, R . Bonnett and P . A. Scourides, In vivo biological activity of the components of hematoporphyrin derivative, Br. J. Cancer, 45 (1982) 571-575 . 10 J. C . van Gemet, lvl . C. Berenbaum and G . M. Gllsberg, Wavelength and light dose dependence in tumor phototherapy with hematoporphyrin derivative, Br. J. Cancer, 52 (1985) 43-49. 11 C . J. Tralau, A . J . MacRobert, P . D . Coleridge-Smith, H. Barr and S . G . Sown, Photodynamic therapy with phthalocyanine sensitization : quantitative studies in a transplantable rat fibrosareoma, Br. J. Cancer, 55 (1987) 389-395 . 12 M . Shopova, G . Grashev, I . Urumov and K . Idakieva, Comparative investigations of porphyrins as photosensitizers on experimental colon carcinoma, in G . Jori and C. A . Perria (eds .), Photodynamic Therapy of Tumors and Other Diseases, Libreria Progetto Editore, Padova, 1985, pp . 232-234 .

308 13 M. Shopova and G . Grashev, Diagnosis and laser phototherapy of tumors in Bulgaria, 31st Int . Congress of Pure and Applied Chemistry, Sofia, 1987, Bulg. Acad . Set ., Sofia, Bulgaria, 1987, Section 9, pp . 118-132 . 14 V. N . Zalessky, T. Yu. Bass, D . Egorova, K. N. Soloviov and N . Gamaleya, Porphyrinphotosensitized effect of laser radiation on solid Ehrlich carcinoma in mice, Exp. Oncol ., 5 (1983) 70-73 (in Russian) . 15 P . J . Bugeiski, C . W. Porter and T . J. Dougherty, Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse, Cancer Res., 41 (1981) 4606-4612 . 16 D . Kessel, T. J . Dougherty and T . G . Truscott, Photosensitization by diporphyrins joined via methylene bridges, Photochem. PhotobioL, 48 (1988) 741-744 . 17 J . S . McCaughan, P . C . Hawley, B . H . Bethel and J . Walker, Photodynamic therapy of endobronchial malignancies, Cancer, 62 (1988) 691-701 . 18 C . J . Gomer, N . Rucker and A. L . Murphree, Differential cell photosensitivity following porphyrin photodynamic therapy, Cancer Res ., 48 (1988) 4539-4542 . 19 J. R . Evenson and J . Moan, Photodynamic action and chromosomal damage: a comparison of hematoporphyrin derivative (HPD) and light with X-irradiation, Br. J. Cancer, 45 (1982) 456-465 . 20 J. S . Nelson, Li-Huei Liaw, A . Orenstein, W. G . Roberts and M . W. Berns, Mechanism of tumor destruction following photodynamic therapy with hematoporphyrin derivative, chlorin and phthalocyanine, J. NatL Cancer Inst., 80 (1988) 693-699 .