PHOTODYNAMIC THERAPY OF CHEMICALLY- AND ULTRAVIOLET B RADIATION-INDUCED MURINE SKIN PAPILLOMAS BY...

Phorochemistry and Photobiology Vol. 56, No. 1, pp. 42-50, 1992 Printed in Great Britain. All rights reserved

0031 -8655/92 $05 .00+0.00 Copyright @ 1992 Pergamon Press Ltd

PHOTODYNAMIC THERAPY OF CHEMICALLY- AND ULTRAVIOLET B RADIATION-INDUCED MURINE SKIN

PAPILLOMAS BY CHLOROALUMINUM PHTHALOCYANINE TETRASULFONATE

RAJESH AGARWAL, MOHAMMAD ATHAR, CRAIG A. ELMETS, DAVID R. BICKERS and HASAN MUKHTAR*

Department of Dermatology, Skin Diseases Research Center, University Hospitals of Cleveland, Case Western Reserve University and Department of Veterans Affairs Medical Center, Cleveland,

OH 44106, USA

(Received 8 Augusr 1991; accepted 4 December 1991)

Abstract-Photodynamic therapy (PDT) of cancer combines irradiation of tumors with visible light following selective uptake of the photosensitizer by the tumor cells. PhotofrinR-I1 (Pf-11) is the only photosensitizer which is in clinical use in PDT, whereas chloroaluminum phthalocyanine tetrasulfonate (AIPcTS) has also shown promise in preclinical studies. In most such studies, the effectiveness of the photosensitizers has been assessed in implanted tumor model systems rather than in model systems where tumors are allowed to grow in their own connective tissue matrix. In this study the pharmacokinetics, tumor ablation capability and cutaneous photosensitization response of AlPcTS have been assessed in mice bearing chemically- and ultraviolet B radiation (UVB)-induced benign skin papillomas. When tumor-bearing animals were injected intraperitoneally with AlPcTS (5 mg/kg body wt), maximum tumor:normal skin ratio of 2.4 was observed at 48 h, at which time the mice were irradiated within the absorption spectrum of the photosensitizer. In tumor ablation studies with SENCAR mice bearing chemically-induced skin tumors, AlPcTS resulted in greater than 80% ablation in tumor volume at 20 days post-irradiation. In cutaneous photosensitization response, AlPcTS produced only transient effects (no effect after 24 h) in SENCAR mice. Pharmacokinetics data, tumor ablation effects and cutaneous photosensitization response of AlPcTS were comparable in SKH-1 hairless mice bearing UVB-induced skin tumors. Our data indicate that AlPcTS produces significant photodynamic effects towards the ablation of murine skin tumors, and that it does not produce prolonged cutaneous photosensitivity.

INTRODUCTION

Photodynamic therapy (PDT)t, which defines the use of photosensitizing chemicals combined with light, is a modality for the treatment of malignant solid tumors that has been increasingly studied in recent years (Dougherty, 1987; Gomer et al., 1989 and references therein). Porphyrins, especially PhotofrinR-I1 (Pf-II), are the major class of chemicals that have been assessed for this purpose (Dougherty, 1981, 1984; Moan, 1986; Gomer et al., 1988, 1989). However, due to certain drawbacks, specifically significantly high and extended skin phototoxicity (Gomer et al., 1984; Tralau et al. , 1989), associated with the use of porphyrins in the PDT of cancer, there has been a need to develop better photosensitizers. One such class of photosensitizers, the phthalocyanines, is being extensively studied as an alternative to porphyrins in the PDT of cancer (Ben-Hur and Rosenthal, 1985a; Ben- Hur, 1987). Using in vitro cellular systems, metal-

'To whom correspondence should be addressed at Depart- ment of Veterans Affairs Medical Center.

tA bbreviarions: AIPcTS, chloroaluminum phthalocyanine tetrasulfonate; DMBA, 7,12-dimethylbenz(a)anthracene; i.p., intraperitoneal; PDT, photodynamic therapy; Pf-11, PhotofrinR-11; TPA, 12-0-tetradecanoylphorbol-13-acetate; UVB, ultraviolet B radiation.

lophthalocyanines have been shown to be efficient photodynamic sensitizers (Ben-Hur and Rosenthal, 1985b; Langlois et al., 1986; Brasseur et al., 1987a, 1988). In general, phthalocyanines are non-toxic and quite resistant to chemical and photochemical degradation (Chan et al., 1986). Phthalocyanines absorb at longer wavelengths (600-700) than do porphyrins, and thereby possess improved tissue penetration (Chan er al., 1986; Reddi et af., 1987). The type of metal ion chelated within a phthalocyanine has a considerable impact on the tumor retention efficiency of the photosensitizer (Rousseau et al., 1983; 1985; Rosenthal, 1991 and references therein).

Among various phthalocyanines, chloroaluminum phthalocyanine tetrasulfonate (AIPcTS) has been the most widely studied photosensitizer for the PDT of tumors (Rosenthal, 1991). Several laboratories have assessed the cellular distribution (Tralau et al., 1987a; Berg et al., 1989; Chan et al., 1989) and in vitro photodynamic effects (Ben-Hur et al., 1987; Paquette ef al., 1988; Ramakrishnan et al., 1988) of AlPcTS. In addition, studies have been conducted in vivo which show that AlPcTS is preferentially localized in tumor tissues and following exposure to red light (600-700) evokes tissue necrosis (Chan et al., 1987, 1988; Reddi et al., 1987; Tralau et al . , 1987b).

43

44 RAIESH AGARWAL er al.

The majority of preclinical da t a o n the efficacy of the photosensitizers in PDT of tumors have been generated using transplanted tumors which are not growing in their native connective tissue matrix, a situation that does not mimic human conditions. I t would be preferable therefore to evaluate photosensitizers for the PDT of tumors using model systems in which tumors grow in their own connective tissue matrix. While limited studies have been conducted to evaluate the PDT effects of hematoporphyrin derivative in laryngeal and other papillomas (Shikowitz et al., 1986, 1988; Abramson et al., 1988), no study has been d o n e t o show the PDT effects of any photosensitizer utilizing skin tumors. Tumors induced in murine skin by chemical carcino- gens as well as by ultraviolet B radiation (UVB) are widely employed for the study of environmental carcinogenesis. By manipulating these tumor induction protocols, it is possible t o alter the number, size, and types of tumors such as papillomas and/or carcinomas that can be induced in murine skin (Agarwal and Mukhtar , 1991). In the present study, we investigated the photodynamic effects of AlPcTS in chemically- and UVB-induced skin tumors in SENCAR and SKH-1 hairless mice, respectively.

MATERIAIA AND METHODS

Chemicals. AlPcTS was purchased from Porphyrin Products, Logan, UT. 7,12-Dimethylbenz(a)anthracene (DMBA) was purchased from Aldrich Chemical Co., Mil- waukee, WI and 12-0-tetradecanoylphorbol-13-acetate (TPA) from Sigma Chemical Co., S t Louis, MO. All other chemicals used in the study were obtained in the purest form commercially available.

Animals and induction of benign papillomas on mouse skin. As described by Mukhtar er al. (1988). for chemically-induced tumors on SENCAR mouse skin, 6-week- old female SENCAR mice obtained from the National Cancer Institute-Frederick Cancer Research Facility, Bethesda, MD were used, and the dorsal skin of each mouse was shaved with electric clippers 2 days prior to the beginning of the experiment. Only those mice in the resting phase of the hair cycle were used and NairH depila- tory was applied to remove any unshaved hairs left on the dorsal skin of the animals. Skin tumors were induced chemically by a standard two-stage initiation-promotion protocol (Hennings er ul . , 1983; Agarwal and Mukhtar, 1991). Tumor initiation was accomplished by a single top- ical application of 5 kg of DMBA in 0.2 mL of acetone under subdued light. One week following initiation, the mice were treated topically with twice weekly applications of 1 pg TPA in 0.2 mL acetone as the tumor promotor. Using this protocol, at 12 weeks on test, 100% of the animals developed tumors; of which about 30% mice had an average of 4-5 tumors per mouse of 5-8 mm dia and 2-5 mm thickness. These tumors were at identical stage of dysplasia and were used for the present study, whereas the remaining 70% of the mice exhibited larger or lower tumor burden and therefore were not utilized in this study. At this stage of tumor induction protocol, the animals selected for the PDT study were withdrawn from the TPA treatment. It is known that when TPA treatment is stopped in these animals bearing papillomas, the growth and/or regression of the tumors become negligible, if any, at least up to 20 weeks (Aldaz ef nl., 1991). Of the tumor-bearing animals selected to assess the pharmacokinetics and the photodynamic effects of AIPcTS, few were randomly sacri-

ficed for histopathological evaluation of the tumors. These tumors were verified as benign papillomas.

As described earlier by Das ef al. (1985), for UVB- induced tumors on SKH-1 hairless mouse skin, 5-6 week- old female SKH-1 hairless mice obtained from Temple University Health Sciences, Skin and Cancer Hospital, Philadelphia, PA were used. The animals were selected randomly and the dorsal skin was irradiated using a bank of 4 Westinghouse FS-40-T-12 fluorescent sunlamps (National Biological Corp., Twinsburg, OH), which emit- ted ca 80% radiation in the range of 280-340 nm with peak emission at 314 nrn as monitored with a SEE 240 photodetector, 103 filter and 1008 diffuser attached to an IL 700 Research Radiometer (International Light, New- buryport, MA). The mice were exposed 3 times weekly on alternate days for 25 min to UVB at a target skin distance of 23 cm. The UVB dose used was 5 J/m2/s, as measured by a digital radiometer. Using this protocol, tumor development occurred only on the dorsal skin of the animals. Between 24-30 weeks on test, about 40% of the animals exhibited 4-5 tumors per mouse averaging 4-6 mm in dia and 2-4 mm in thickness. These tumors were at identical stage of dysplasia and were used for the present study, whereas the remaining 60% of the animals exhibited either larger or smaller tumors and therefore were not utilized in this study. At this stage of tumor induction protocol by UVB, the animals selected for the PDT study were withdrawn from the UVB exposure. By doing this the tumor growth and/or repression in these animals bearing papillomas become negligible at least for a few weeks (Elmets, 1991). Of the tumor-bearing animals selected to assess the pharmacokinetics and the photodynamic effects of AIPcTS, few were randomly sacrificed for histopathological evaluation of the tumors. These tumors were verified as benign papillomas.

Pharmacokinetics study. Tumor-bearing animals were used for the determination of AlPcTS distribution in tissues including liver, lung, kidney, muscle, normal skin and skin tumors. AlPcTS solution was prepared in 0.1 M saline in the dark at a concentration of 2 mg/mL (wt/vol). The animals were treated with a single intraperitoneal (i-p.) dose of 5 mglkg body wt of AIPcTS. At 6, 12,24,36,48, 60, 72, 84 and 96 h after treatment, the animals were sacrificed by cervical dislocation and the desired tissues were removed. Pieces of tissues were rinsed twice with 0.1 M phosphate buffer, pH 7.2, blotted dry and weighed. To determine the concentration of AIPcTS, tissues from two animals were pooled for each data point prior to extraction of the photosensitizer. For skin tumors, all tumors obtained from two animals were pooled for each data point.

For the determination of AlPcTS concentration, the pieces of washed tissues were digested in 0.1 M sodium hydroxide ( l : l O , wtlvol) at 50°C for 4 h according to the method of Chan et al. (1986). The tissue digests were then centrifuged at 12000 rpm for 20 min, the clear supernatant was aspirated and the fluorescence was determined using 603 nm excitation and 673 nm emission. Standard curve was obtained by adding known concentrations of AlPcTS to corresponding tissue digests prepared from untreated animals. For quenching corrections, the fluorescence of the solution of known concentration of AlPcTS in 0.1 M sodium hydroxide was compared with that observed for the same concentration of AlPcTS added to the corresponding tissue digests prepared from untreated control animals.

PDT of murine skin rumors. In each experiment, 32 tumor-bearing animals were divided into four groups of eight animals which consisted of: (1) an untreated group; (2) a non-AIPcTS-treated but irradiated group; (3) an AIPcTS-treated but non-irradiated group; and (4) an AlPcTS-treated-irradiated group. The first three groups were used as controls whereas the animals in the fourth group served as the experimental group. All animals were

PDT of murine skin tumors 45

given free access to standard chow and water. AlPcTS (2 mgiml, wt/vol, in 0.1 M saline) was injected i.p. to the animals bearing skin tumors at a dose of 5 mukg body wt. Following injection with AIPcTS, the animals were protected from light except during irradiation. Based upon the tissue distribution studies described above, these animals were irradiated to -675 nm light (Agarwal et al . , 1990) at 48 h after the i.p. administration of AIPcTS, at which time the tumormormal skin ratio of this photosensitizer was found to be maximum (see Results section).

Mice were irradiated in individual cubicles of a plexiglass cage at a tube-to-target distance of 49.5 cm (19.5 inches). The light source used was a 500 W Tungsten Halogen lamp mounted in a reflector housing equipped with a heat absorbing glass plate and covered with a primary red 106 filter sheet (Lee Filters, Andover, U.K.). The spectrum of this light source was monitored in between 2W1000 nm using a 118 m focal length grating monochromator supplied by Oriel. The wavelength dispensed light was detected with a Silicon photodiode placed at the focal plane of the exit slit of the monochromator. Slit widths were set at 0.6 mm giving a 4 nm band pass. The spectrum was found to rise sharply from 600 nm, reaching a broad maximum at -675 nm and falling off gradually to about 25% of the maximum at lo00 nm (data not shown). The power density of the filtered lamp output was measured using a Tektronix 516 radiometer and a 56502 irradiance probe equipped with a filter adapter and 1 inch diameter neutral density filter with a nominal optical density of 3. The power density of this light source at sample distance was found to be 0.1 W/cm2. The total dose of light used for irradiating animals was 200 J/cm*.

Following PDT, each tumor was measured three times per week. Tumor volume was calculated with a calliper according to a hemiellipsoid model [v = 1 (4d3) X (1/2) x ( 4 2 ) X h ; I = length, w = width and h = height] described by Brasseur er al. (1987b). These measurements were continued until the end of the experiment, 20 days post-irradiation.

Cutaneous phorosensirizarion. Untreated control female SENCAR and female SKH-I hairless mice of similar age and from the same lot as used in the pharmacokinetics studies and in the PDT studies but without tumors, were used to assess the effects of AlPcTS on cutaneous photo-

sensitization using ear swelling response as a marker (Haw- kins et al., 1986; Athar er al., 1988). Six animals were used in each group and given free access to standard chow and water. The dose of AlPcTS used for the cutaneous photosensitization study was 5 mukg body wt. The mode of AlPcTS administration, post-irradiation handling of animals, time of irradiation after the administration of photosensitizer, and the light source used were the same as described above under the method of assessing PDT except that the total dose of light used for irradiation was 5 J/cmZ.

Cutaneous photosensitization was determined by com- paring the degree of ear swelling that developed after irradiation of AIPcTS-treated animals (experimental group) with that of untreated animals (control group). Each ear was measured three times with a dial thickness gauge engineer’s micrometer (Mitutoya, Co., Tokyo, Japan) from 2 h post-irradiation up to 144 h (6 days). Increment in ear thickness in the AIPcTS-treated and irradiated group of animals was determined by subtracting the ear thickness of the control animals as described earlier (Hawkins ef al . , 1986).

RESULTS

Pharmacokinetics of AlPcTS

As shown in Fig. 1, i.p. administration of AlPcTS (5 mg/kg body wt) to SENCAR (panel A) and SKH- 1 hairless (panel B) mice bearing chemically- and UVB-induced skin tumors, respectively, resulted in distribution of the photosensitizer in each of the tissues studied. The maximum concentration of AlPcTS in liver, kidney, lung, normal skin and rnus- cle was found to be at 24 h in both SENCAR and SKH-1 hairless mice. The AlPcTS concentration in these tissues started declining after 24 h and at 96 h the levels were in the range of 0.3 & 0.1 to 1.2 2 0.2 and 0.55 f 0.1 to 1.5 f 0.1 Fg AlPcTS/g tissue in SENCAR and SKH-1 hairless mice,

12

C - 0 10 E- r a 8 5 3

c.

it 6

$ 3 4 n a 2

0 0 12 24 36 48 60 72 84 96 12 24 36 48 60 72 84 96

Time (hours) Time (hours)

Figure 1. Concentration of AlPcTS in tumor bearing animals. SENCAR mice bearing chemically- induced skin tumors (A) and SKH-1 hairless mice bearing UVB-induced skin tumors (B) were administered an i.p. dose of AlPcTS (5 mgikg body wt) and the concentration of photosensitizer was determined in liver (A) , kidney (A), lung (0). skin tumors (a), normal skin (a) and muscle (0) at increasing times up to 96 h. Each data point represents mean 2 SE of four individual values; the tissues from two animals were pooled for each data point. All (4-5) skin tumors from one animal were pooled and called one tissue. The data for other tissues are shown by solid lines, whereas for skin tumors, a broken line is used for better distinction. For other details, see Materials and Methods.

46 RAJESH AGARWAL el al.

Table 1. Tumor:normal skin ratio of AlPcTS in SENCAR mice bearing chemically- and in SKH-1 hairless mice bearing

UVB-induced skin tumors* ~ ~~ ~

Tumor:normal skin ratio?

Tumor model 1% h 24 h 48 h 72 h 96 h

Chemically-induced tumors 1.4 1.1 2.4 1.7 1.5 UVB-induced tumors 1.4 1 .1 2.5 1.7 1.6

*Skin tumor bearing animals were administered i.p. with AlPcTS (5 mgikg body wt), and were killed at desired time intervals. Normal skin and skin tumors were removed, and the concentration of AlPcTS was determined as described in Materials and Methods.

tData represent mean of four individual values (Fig. 1). For each data point, normal skin and 8-10 skin tumors obtained from two animals were pooled.

respectively. For skin tumors, the maximum AlPcTS concentration (2.6 * 0.4 and 2.5 rt 0.3 pg AIPcTS/g tissue in SENCAR and SKH-1 hairless mice, respectively) was found at 48 h, and after 96 h it declined to 0.9 5: 0.1 pg AIPcTS/g tissue in both SENCAR and SKH-1 hairless mice. As shown in Table 1, when the AlPcTS concentration data were considered in terms of the tumor:normal skin ratio at different time intervals, the maximum tumor to normal skin ratio (2.4 and 2.5 in SENCAR and SKH-1 hairless mice, respectively) was observed at 48 h.

Photodynamic effects

The photodynamic effects of AlPcTS were assessed in tumor-bearing animals injected i.p. with the photosensitizer at a dose of 5 mg/kg body wt and then irradiated to -675 nm light for a total dose of 200 J/cm2 as described in Materials and Methods. As shown in Figs. 2 and 3, only AlPcTS treated and irradiated animals showed significant regression in tumor volume during the first 5 days post-irradiation. In chemically-induced skin tumors (Fig. 2), the initial tumor volume (49.9 ? 4.2 mm3) was reduced to 19.7 3: 3.9 mm3 (P < 0.01, Stud- ent's t-test) in 5 days post-PDT. Similarly, in UVB- induced skin tumors (Fig. 3), the initial tumor volume (41.2 ? 3.9 mni') also reduced to 18.1 ? 3.7 mm3 (P < 0.01, Student's t-test) in 5 days post-PDT. At the termination of the experiment at 20 days post-irradiation, the tumor volumes in AlPcTS treated and irradiated animals were found to be 8.1 f 2.6 mm3 and 5.7 k 2.1 mm3 (P < 0.001, Student's t-test) in case of SENCAR and SKH-1 hairless mice, respectively. In these animals considerable reduction in tumor volumes was observed as early as 2 days post-irradiation, and at 10 days post-irradiation the formation of indurated crusting over the treated sites was evident. In total 84 and 86% tumor necrosis was observed in the chemically- and UVB-induced skin tumors, respect-

50 - 40 - 30 - 20 -

10-

0 . ' . 1 . 1 . 1 . 1 .

0 5 10 15 20 Time Post irradiation (days)

Figure 2. PDT of skin tumors with AIPcTS. Reduction in tumor volume for SENCAR mice bearing chemically- induced skin tumors; untreated control group (m), non- AIPcTS-treated but irradiated control group (0), AIPcTS- treated but non-irradiated control group (A) and AIPcTS- treated-irradiated experimental group (0). Each data point represents mean 2 SE of 3240 skin tumors obtained from eight mice with 4-5 skin tumors per mouse averaging 5-8 mm in dia and 2-5 mm in thickness. For other details,

see Materials and Methods.

0' " " " . ' . " 0 5 10 15 20

Time Post irradiation (days) Figure 3. PDT of skin tumors with AIPcTS. Reduction in tumor volume for SKH-1 hairless mice bearing UVB- induced skin tumors; untreated control group (W), non- AIPcTS-treated but irradiated control group (0), AIPcTS- treated but non-irradiated control group (A), and AIPcTS- treated-irradiated experimental group (0). Each data point represents mean k SE of 32-40 skin tumors obtained from eight mice with 4-5 skin tumors per mouse averaging 4-6 mm in dia and 2-4 mm in thickness. For other details,

see Materials and Methods.

ively (Table 2). Under similar experimental conditions, in each of the three control groups of animals i.e. untreated, non-AlPcTS treated but irradiated and AlPcTS treated but non-irradiated animals, no reduction in tumor volumes was observed (Figs. 2 and 3, and Table 2) in either tumor model system. Furthermore, spontaneous tumor regression or tumor growth was not observed in any of these animals.


Table 2. Photodynamic effects of AlPcTS in relation to tumor necrosis of chemically- and UVB-induced skin tumors in SEN-

CAR and SKH-1 hairless mice'

System

Chemically-induced tumors No treatment Light alone AlPcTS alone AlPcTS + light

UVB-induced tumors No treatment Light alone AlPcTS alone AIPcTS + light

Tumor volume (mm')t % Necrosis

49.3 f 3.9 50.5 f 4.0 46.2 2 3.0 8.1 f 2.66

40.8 2 3.6 42.7 f 4.1 40.1 2 3.6

5.7 2 2.11 L

86

'The tumor necrosis data represent the final response on day 20 post-irradiation. For details of tumor size, initial tumor volume, tumor type, time of irradiation after i.p. injection of AIPcTS, light dose and source of irradiation. see Materials and Methods.

tData represent mean f SE of 32-40 skin tumors obtained from eight animals with 4-5 tumors per animal.

$No effect. &Significantly lower ( P < 0.001) than the control groups

(Student's [-test).

Cutaneous photosensitization response

To evaluate the potency of AlPcTS as cutaneous photosensitizer, the murine ear swelling response was used as a marker. In initial experiments, the animals were injected with AlPcTS and irradiated with varying doses of light between 1.0 to 20 J/cm2. Optimum ear swelling response occurred at a light dose of 5 J/cm2 (data not shown). Based on this observation, a light-dose of 5 J/cm* was selected for further experiments. As shown in Table 3, AlPcTS showed only transient cutaneous photosensitization response in between 2 and 6 h post-irradiation in both SENCAR and SKH-1 hairless mice. However beyond 24 h post-irradiation, cutaneous photosensitization was not noticed in these animals (Table

Table 3. Cutaneous photosensitization response of AlPcTS in SENCAR and SKH-1 hairlcss mice'

A Ear thickness ( X lo-' mm)i

Time post irradiation (h) SENCAR SKH-1 Hairless

2 6

24 96

144

10 t 1 8 2 0.9 3 t- 0.5 5 2 0.6

ND$ ND ND ND ND ND

~

'SENCAR and SKH-I hairless mice were irradiatcd 48 h after the treatment with AlPcTS (5 mg/kg body wt) using a metal halide lamp equipped with a red filter (== 675 nm). Total light fluence delivered to the animals was 5 J/cmZ. Ear swelling response was determined as described in Materials and Methods.

?Data present mean f SE of three individual values, each comprised of two animals.

$Not detectable.

3). The animals that received irradiation alone or AlPcTS alone or untreated control animals did not develop measurable ear swelling (data not shown).

DISCUSSION

Although PDT was introduced more than 20 years ago, the search for the ideal photosensitizer con- tinues to the present. in a number of recent publications (Nelson et al., 1988; Zhou, 1989; Gibson et al., 1990; McCaughea, 1990; Pandey et al., 1991; Roberts et al., 1991; Rosenthal, 1991) Pf-I1 and AiPcTS have shown promise in this regard. While clinical trials using Pf-I1 are underway, clinical trials of PDT with AlPcTS are about to begin in Israel (Ben-Hur, 1990). Despite considerable advances in the field of PDT of cancer, most published reports have assessed the PDT response in implanted tumors in different organs of experimental animals using various photosensitizers (Nelson et al., 1988; Gomer et al., 1989; Zhou, 1989; Gibson et al., 1990; McCaughea, 1990; Pandey et al., 1991; Roberts et al., 1991; Rosenthal, 1991). In these implanted tumors, although the injected carcinoma cells grow successfully in a short time to form a visible tumor mass (Henderson, 1990 and references therein), they are not in their native connective tissue matrix and therefore do not fully mimic the human situation. Only limited studies have assessed PDT effects of hematoporphyrin derivative in papillomas (Shikowitz et al., 1986; Abramson et al., 1988; Shi- kowitz el al., 1988), and to our knowledge no study has been published where PDT effect of any photosensitizer has been evaluated in murine skin tumors. Since PDT may also find a place in benign papil- lomatous diseases, in this study we employed skin papillomas induced chemically and with UVB in SENCAR or SKH-1 hairless mice, respectively, to study the photodynamic effects of AlPcTS. Since these tumors develop in their own tissue matrix they are much more akin to the in vivo human situation than that of implanted tumor model systems for the PDT of cancer. Our results indicate that AlPcTS, when injected i.p. at a dose of 5 mg/kg body wt followed by irradiation with 4 7 5 nm light at a dose of 200 J/cm*, evokes significant tumor necrosis of both chemically- and UVB-induced skin tumors in SENCAR and SKH-1 hairless mice, respectively.

The data shown in Figs. 2 and 3 in relation to time course of tumor response clearly indicate that within the first 48 h post-irradiation of animals bearing either chemical- or UVB-induced skin tumors and treated with AIPcTS, considerable reduction in tumor volumes occur. However, a significant ablation in tumor volume was apparent from day 3 onward to day 10 post-irradiation. These results, though by and large consistent with other published studies, which, utilizing implanted tumor model systems have shown that within 1 to 2 days after PDT the tumors become flat (Gomer et al., 1989; Rosen-

48 RAJESH AGARWAL el al.

thal, 1991 and references therein), show that highly significant ablation in tumor volumes occurred with in 10 days post-irradiation. It seems logical that such a difference may be due to the: (1) difference in the two tumor model systems; and (2) immunogenetic response if any, associated with such a tumor model system. In the present study, reduction in tumor volumes was observed only in AlPcTS treated and irradiated group of animals, so we are convinced that it was a photodynamic process. It is iinportant to emphasize that when tumor-bearing animals were withdrawn from chemically- and UVB-induced skin tumorigenesis protocols, the tumor volume of skin papillomas remains almost the same for 20 weeks (Aldaz et al., 1991) and for at least a few weeks (Elmets, 1991). respectively. Consistent with these findings, no tumor growth or spontaneous regression of the skin tumors occurred in any of the three control groups of animals (Figs 2 and 3). This observation implies that the tumor necrosis observed in the present study was a photodynamic process. Since a dose of 5 mg/kg body wt of AlPcTS has been used by several workers (Spikes, 1986; Rosenthal, 1991 and references therein) in various experiments utilizing implanted tumor model systems to show the tissue distribution and photodynamic effects of AIPcTS, in the present study we have also selected this dose of the photosensitizer in order to correlate the results of the present study with data reported by others. In other experiments a dose of 1 mg/kg body wt of AlPcTS followed by irradiation to =675 nm light for a total dose of 200 J/cm2 produced no tumor ablation (data not shown). Similarly a light dose of 200 J/cm2 used in the present study was also based on the previous studies (Gomer et al., 1989; Rosenthal, 1991 and references therein). However, it is important to mention here, that since a broad-band light source was used in the present study, the exact amount of effective light dose is not clear. Nevertheless, the photodynamic effects obtained with AlPcTS using skin tumors clearly indicate that this tumor model is useful for evaluating the PDT effects of the photosensitizers. In a study to be published elsewhere (Mukhtar et a!. , 1991), we have shown that Pf-11, when injected at a dose of 5 mg/kg to the same mouse models as used in the present study followed by irradiation for a total light dose of 200 Jlcm*, evoked significant tumor necrosis; however considerable skin photosensitization was also evident in these animals.

The pharmacokinetics data obtained in the present study suggest that the highest concentrations of AlPcTS are found in liver, kidney and lung as compared to skin tumors, normal skin and muscle; however AlPcTS was found to be retained in skin tumors for a longer period than in normal skin, and the rate of clearance of AlPcTS from other organs such as liver, kidney and lung was faster than that from skin tumors and normal skin. These results

support earlier studies in implanted tumors (Tralau et al . , 1987a,b; Lin, 1990) indicating that the uptake of AlPcTS in tumor tissue was higher than in normal tissues, although the levels of the photosensitizer in tumors were less than in the liver, kidney and lung.

Various in vivo models have been developed to study the acute and chronic changes resulted by exogenous and endogenous photosensitizers (Hawk- ins er al . , 1986). Based on prior publications from our laboratory (Elmets and Bowen, 1985; Hawkins et al . , 1986; Athar et al . , 1988), we have employed the ear swelling response as a marker of cutaneous photosensitization. Using this model, the results of the present study support prior observations by several workers (Roberts er al . , 1989; Tralau et al . , 1989, 1990) that AlPcTS produces only transient cutaneous photosensitivity.

In summary, using pharmacokinetics data and photodynamic effects of AlPcTS in chemically- and UVB-induced skin tumors in SENCAR and SKH- 1 hairless mice, respectively, we have shown that such skin tumors are a useful model for the study of the photodynamic effects of the photosensitizers. Further studies are needed to define PDT responses of the photosensitizers in malignant skin tumors.

Acknowledgements-Thanks are due to Ms Sandra Evans for preparing the manuscript. This work is supported by USPHS Grants CA 48763, CA 51802, Po l -CA 48735, P- 30-AR-39750, and by research funds from the Department of Veterans Affairs.

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Abramson, A., M. Waner and J . Brandsma (1988) The clinical treatment of laryngeal papillomas with hematoporphyrin derivative. Arch. Orol. Head Neck Surg. 114, 795-800.

Agarwal, R., M. Athar, D. R. Bickers and H. Mukhtar (1990) Evidence for the involvement of singlet oxygen in the photodestruction by chloroaluminum phthalocyanine tetrasulfonate. Biochem. Biophys. Res. Commun. 173, 34-41.

Agarwal, R. and H. Mukhtar (1991) Cutaneous chemical carcinogenesis. In Pharmacology of the Skin (Edited by H. Mukhtar), pp. 371-387. CRC Press, Boca Raton, FL.

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