Effectiveness of a Lysyl Chlorin P6/Chlorin P6 Mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat1

[CANCER RESEARCH 52, 1235-1239, March I, 1992] Effectiveness of a Lysyl Chlorin p6/Chlorin p6 Mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat1

Michael W. Leach, Robert J. Higgins, James E. Boggan, Shwn-Ji Lee, Susan Autry, and Kevin M. Smith Schools of Veterinary Medicine [M. W. L., R. J. H.J and Medicine [J. E. B., S. A.] and Department of Chemistry [S-J. L., K. M. S.], University of California, Davis, California 95616

ABSTRACT Chlorin photosensitizers are among the new photosensitizers currently being developed and show great promise for use in A new photosensitizer, LCP, a combination of lysyl chlorin p6 and PDT (17-24). They have enhanced molar extinction and elec chlorin p6, was synthesized and tested for effectiveness in photodynamic therapy using s.c. implanted 9L glioma tumors in rats. Tumors were tronic absorption peaks at substantially longer wavelengths irradiated with 664-nm light 4 h after LCP injection. Mean intratumoral than HpD and dihematoporphyrin ether ester, allowing for temperature elevations were less than 4"( ' using a power density of 50 both increased tissue penetrance and enhanced tumor photo- mW/cm2 for 33.3 min (100 .l/cnr). Subsequent experiments examining destruction (17-19). Furthermore, chlorins do not appear to histolÃ³gica!changes and tumor regrowth used a power density of 50 mW/ cause severe cutaneous photosensitization after 24 h (18-20). cm2and total energy densities of 25, 50, and 100 .I/cm'. Microscopically, However, some chlorin compounds are relatively difficult to an energy density-dependent coagulation necrosis of tumor cells occurred synthesize, such as monoaspartyl chlorin e6. in treated tumors. Long term inhibition of tumor growth was achieved only at an energy density of 100 .I/cm'. Side effects of treatment were Based on the potential advantages of chlorin photosensitiz ers, we decided to examine the effectiveness of a new chlorin, seen only in the irradiated area and consisted of coagulation necrosis of normal tissues in rats treated at 50 and 100 .I/cm", including severe skin LCP, for PDT using the s.c. 9L rat glioma model. LCP was necrosis. Exposure of rats to fluorescent room light did not cause any chosen because it is more readily synthesized than other com macroscopically detectable skin damage. Our data indicate that photo- monly used chlorin compounds, such as monoaspartyl chlorin dynamic destruction of s.c. 9L glioma tumors using LCP as a photosen e6. Tumor temperature changes during PDT, tumor necrosis sitizer results in significant tumor growth inhibition and that further and histolÃ³gica! changes 1 day after PDT, skin damage from study of LCP is warranted. irradiation and normal room light, and tumor regrowth follow ing treatment were examined. Our data indicate that LCP is a INTRODUCTION potentially effective photosensitizer for use in PDT and that PDT2 is a relatively new therapeutic strategy used in the further investigation of this photosensitizer is warranted. treatment of neoplasia. It has achieved selective tumoricidal effects without many of the serious side effects of more conven MATERIALS AND METHODS tional therapy in a variety of tumors, coming closer to the Photosensitizer. Lysyl chlorin p6 was prepared by treatment of pur cancer treatment paradigm of the selective destruction of tumor purin 18 methyl ester (obtained from chlorophyll-a) with lysine in a tissue without disruption of normal tissue function (1-4). PDT mixture of dichloromethane, p>ridine, and water. It was purified using depends on the uptake and retention of photosensitizers in a Waters Associate's CiÂ«SepPak cartridge. Reversed phase analytical tumors followed by activation of the photosensitizer by light at high-performance liquid chromatography indicated the product to con specific wavelengths in the presence of oxygen (2-5). The sist of 60% lysyl chlorin p6 and 40% chlorin p6 based on equal predominant tumoricidal mechanism in PDT is thought to absorbance at 403 nm. The long wavelength molar extinction coeffi involve a type II photochemical reaction with production of cients of lysyl chlorin p6 and chlorin p6 in methanol were 39,600 at reactive singlet oxygen, although type I free radical mechanisms 660 nm and 42,750 at 664 nm, respectively. The structures of lysyl also occur (2-4, 6, 7). In vivo, the major site of action of PDT chlorin p6 and chlorin p6 molecules are shown in Fig. 1. LCP in powder is claimed to be blood vessels, the destruction of which results form was used for all experiments. The powder was dissolved in PBS in ischemie necrosis of the treated tumor (2, 8-11). and 0.1 N NaOH (pH 10.0 to 11.0), brought to pH 7.35 with 0.1 N HC1, then sterilized with a 0.45-/im filter. Solutions were used the same Porphyrin photosensitizers, particularly HpD, and the sus day as prepared and always protected from light. Absorption spectros- pected active components of HpD, dihematoporphyrin ether copy and high-performance liquid chromatography showed no break ester and commercially prepared Photofrin II, are the best down of LCP when stored in the dark at 4Â°C,either when in solution known and studied photosensitizers, but they have several draw for 24 h or when stored in powder form for up to 4 months. backs. The most significant are cutaneous photosensitivity last Animal and Tumor Model. While under halothane anesthesia, male ing for 6 to 8 weeks following treatment (12-14), they are a Fischer 344 rats weighing 160 to 220 g were given injections in the left complex mixture of porphyrins which makes accurate study flank of 0.6 to 2 x 10" 9L glioma cells suspended in 0.3 ml of RPMI difficult (IS), and the wavelength of light absorption is not media containing 10% fetal calf serum. Experiments were initiated optimal for tissue penetration (2, 16). when tumors were between 14 and 22 mm maximum length or width Thus, examination of new photosensitizers for use in PDT (between 18 and 27 days after tumor injection). Rats were divided into has increased in an effort to minimize these shortcomings. three groups of 25 to 35 animals each for tumor temperature measure ments, for evaluation of tumor necrosis 1 day after irradiation, and for Received 5/6/91; accepted 12/13/91. tumor regrowth experiments. Within each experiment, rats were further The costs of publication of this article were defrayed in part by the payment divided into groups of 5 or 6 animals to form individual treatment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. groups. 1Part of this research was supported by the NIH (K. M. S.: CA 52997) and Determination of Irradiation Wavelength. Absorption spectra were by a National Institute of Environmental Health and Safety postdoctoral training obtained using a diode array HP 8450A spectrophotometer (Hewlett grant (M. W. L.: 5 T32 ESO7055). 2The abbreviations used are: PDT, photodynamic therapy; HpD, hematopor- Packard, Palo Alto, CA) from samples of photosensitizer dissolved in phyrin derivative; LCP, a combination of lysyl chlorin p6 and chlorin p6; PBS, PBS at a concentration of 0.04 mg/ml and at a pH of 7.4, either with phosphate-buffered saline. 10% fetal calf serum or with 1 x 10*9L glioma cells/ml. The absorption 1235

The percentage of tumor necrosis was calculated as (25) CH3

*p Hl . p"I CH2CH3 CH3 CHjCHj P., Pâ€ž x 100

where Pâ€ž,isthe number of points over necrotic tumor, section from first block; /'â€ž,isthe number of points over necrotic tumor, section from second block; P,Â¡is the number of points over viable tumor, CHjOjC' CH3O2C' section from first block; and /',. is the number of points over viable tumor, section from second block. H2N-CH PDT-mediated Inhibition of Tumor Regrowth. Tumors were moni CO2H tored every 2 days until tumor volume had increased 6-fold over Lysyl Chlorin p6 Chlorin p6 measurements made immediately pretreatment. Tumor size was calcu Fig. 1. Chemical structures of lysyl chlorin p6 (left) and chlorin p6 (right). lated as (20)

Table 1 Grading system for skin photosensitization reaction Score Observation PDT-mediated Skin Response. Skin changes in rats used for tumor Reaction regrowth experiments were evaluated within the irradiated areas over 0 Normal 1 Edema lying tumors, the ears, and the feet using a quantitative scoring system 2 Erythema (described in Table 1) to document the degree of skin damage induced 3 Small area of necrosis <3 mm in diameter by each treatment and by normal fluorescent room light. Evaluation of 4 Moderate area of necrosis 3-10 mm in diameter the skin was made daily. The score given to each animal represents the 5 Patchy areas of necrosis >10 mm in diameter 6 Complete necrosis of skin >15 mm in diameter most severe reaction (the highest score) seen in that animal during the experiment. Statistical Analysis. Individual group means from the percentage of tumor necrosis and tumor regrowth following PDT experiments were peak of LCP was at 664 nm in both samples, and this wavelength was compared to the group receiving no injection or light, using the Wil used for irradiation of rats. coxon 2-sample test, normal approximation, with continuity correction In Vivo Light Source and Delivery System. An argon-pumped dye of 0.5. laser system ( 2040 and 375B; Spectra Physics, Mountain View, CA) tuned to emit light at 664 nm was used. A quartz Tiber fitted with a microlens (LaserTherapeutics, Inc., Buellton, CA) was interfaced to the RESULTS dye laser to deliver a uniform field of light. Laser output was measured The absorption spectrum of LCP in PBS with 10% fetal calf with a power meter (66XLA; Photodyne, Newbury Park, CA). Treat ment areas included a minimum of 1 mm of normal tissue adjacent to serum is shown in Fig. 2. Major absorption peaks are at 412 and 664 nm. These peaks are red-shifted by 6 nm compared to the tumor. Photosensitizer Injections and Preparation for Tumor Irradiation. Rats LCP in PBS without protein (data not shown). were anesthetized with pentobarbital (45 mg/kg i.p.) and then given Fig. 3 shows the temperature changes in tumors when rats injections of LCP in the left femoral vein (2.5 mg/kg). Between 4 and were given injections of PBS or 2.5 mg/kg LCP and irradiated 4.25 h later rats were again anesthetized with pentobarbital (55 mg/kg with 664-nm light. Five or six tumors were examined in each Â¡.p.),placedon a water circulation heating pad maintained at 37'C, and group, with the average temperature change plotted during and irradiated. immediately following irradiation. The average starting tem perature was 34.5Â°C(range, 33.0-36.3Â°C). A power density of Tumor Temperature Measurements. Temperature profiles as a func tion of fluence and time were obtained. A 26-gauge hypodermic needle 100 mW/cm2 resulted in an average temperature rise of 6.6Â°C microprobe (Sensortek, Clifton, NJ) was placed in the tumors perpen in LCP-injected rats, compared to 3.1 Â°Cin PBS-injected rats. dicularly to the plane of delivered light 3 mm below the skin surface. At a power density of 50 mW/cm:, the mean temperature rise Temperature changes were measured within tumors during and imme diately after treatment at power densities of 50 and 100 mW/cm2. Total irradiation times were 33 min 20 s, resulting in energy fluences of 100 and 200 J/cm2, respectively. Controls were rats given injections of an equal volume of PBS and similarly irradiated. PDT Tumor Treatments. Rats were irradiated at a power density of 50 mW/cm2 with total laser energy densities of 25, 50, and 100 J/cm2. Irradiation times were 8 min 20 s, 16 min 40 s, and 33 min 20 s, respectively, per animal. Controls were rats which were either not given injections or irradiated, given injections of photosensitizer but not irradiated, or given injections of an equal volume of PBS and irradiated at 100 J/cm2. HistolÃ³gica! Examination 1 Day Postirradiation. Animals were eu thanized 24 h after completion of irradiation and immediately necrop- sied. Tissues were evaluated after immersion fixation in 10% neutral buffered formalin, routine processing, sectioning, and then staining with hematoxylin and eosin. Morphometric determination of tumor necrosis was done by examining sections from 2 blocks of tumor tissue WAVELENGTH (nm) cut perpendicular to the skin surface and taken at 90-degree angles to Fig. 2. Absorption spectrum of LCP in PBS with 10% fetal calf serum. Major one another, using a 42-point grid (Graticules, Ltd., Kent, England). absorption peaks are at 412 and 664 nm. 1236

tosensitizer prior to irradiation. The amount of skin damage in the irradiated field was related to the energy dose, with minimal PDT started 4 to 4.25 hours after skin damage at 25 J/cm2 and a large area of necrosis of skin photosensitizer injection. overlying the tumor at 100 J/cm2.

DISCUSSION 2.5 100 200 25 50 100 Our data demonstrate that photodynamic therapy of s.c. 9L 0 100 200 glioma tumors using LCP as the photosensitizer results in 0 50 100 significant tumor destruction and growth inhibition. Skin photosensitization was not seen except in tissues which received direct irradiation. No systemic toxic effects were noted clinically 0 5 10 15 20 25 30 35 or pathologically at the dose of photosensitizer used (2.5 mg/ kg). Time (Minutes) Our investigation into the effectiveness of LCP was initiated Fig. 3. Temperature rise in the s.c. 9L glioma as a function of time during exposure to 664-nm light. Temperatures were measured 3 mm below the skin based on earlier reports which indicated that chlorin com surface and each point represents the average temperature rise from 5 or 6 tumors. pounds may have superior photosensitizer characteristics com Bars, SD. pared to porphyrins. Additionally, LCP is easier to synthesize than chlorins such as monoaspartyl chlorin e6. LCP is a mixture was only 3.8Â°Cin LCP-injected rats, while PBS-injected rats of lysyl chlorin p6 and chlorin p6, is hydrophilic, and has a had an average rise of 2.6Â°C.All subsequent experiments used major absorption peak at 664 nm when in the presence of a power density of 50 mW/cm2 to minimize hyperthermic protein. Chlorin p6 is the hydrolytic by-product of the opening effects during tumor treatments. of the anhydride ring in purpurin 18 and was present in the Microscopic changes seen in tumors 24 h after PDT consisted photosensitizer tested due to the synthetic methods we initially of congestion, thrombosis, and coagulation necrosis of tumor used. Because of our encouraging results, we have investigated cells. The percentage of tumor necrosis in each treatment group and developed methods for synthesis of the pure compound, lysyl chlorin p6, by avoiding aqueous solvents during its syn- is shown in Table 2. Less than 1% tumor necrosis was seen in tumors from the untreated group, and there was no significant difference in tumor necrosis between the untreated group, the Table 2 Percentage of necrosis in 9L tumors 24 h after PDT rats given injections of photosensitizer only, or the rats irradi Rats were killed 24 h after PDT. Tumors were evaluated microscopically after ated but not given injections of photosensitizer. The percentage immersion fixation in 10% neutral buffered formalin, routine processing, section ing, and then staining with hematoxylin and eosin. Means Â±SD expressed in of tumor necrosis in rats given injections of LCP and then percentage of necrosis are shown. W = 5/group. irradiated increased with higher energy densities. Treatment Treatment parameters % of necrosis with 100 J/cm2 resulted in a mean of 98.4% tumor necrosis, No injection, no light 0.14 Â±0.24 with viable tumor cells observed only at the deep border of the PBS, 100 J/cm2 0.28 Â±0.63 tumor in the 100-J/cm2 treatment group. All groups given 2.5 mg/kg LCP, no light 0.12 Â±0.16 2.5 mg/kg LCP, 25 J/cm2 26.66 Â±25.85Â° injections of LCP and irradiated had significantly greater tumor 2.5 mg/kg LCP, 50 J/cm2 79.85 Â±9.14Â° necrosis compared to the untreated group (P < 0.02). The 2.5 mg/kg LCP, 100 J/cm2 98.37 Â±0.84Â° extent of tumor necrosis in the group treated at 25 J/cm2 was ' Significantly different from no injection, no light group: P < 0.02. quite variable between individual animals, while in other groups results were more consistent. Additional microscopic changes Table 3 Tumor regrowth following PDT in adjacent fat, muscle, and overlying skin were congestion, Rats were given LCP i.v. followed 4 h later by irradiation with 664-nm light. edema, and necrosis. The severity of changes in normal tissues Means Â±SD expressed in days until a 6-fold increase in tumor volume was paralleled that seen in tumors, with more normal tissue damage detected are shown. N = 5 rats/group. seen at higher energy doses. Minimal normal tissue damage Time (days) to 6-fold increase was seen at 25 J/cm2. Treatment parameters in tumor volume No injection, no light Â±2.61 Results from tumor regrowth studies are shown in Table 3. PBS, 100 J/cm2 13.6 Â±4.34 Five rats were examined in each group. Tumor growth was 2.5 mg/kg LCP, no light 11.2 Â±1.01 2.5 mg/kg LCP, 25 J/cm2 similar in all groups except those given 2.5 mg/kg LCP and a 11.2Â± 1.79 2.5 mg/kg LCP, 50 J/cm2 14.4 Â±3.58 light dose of 100 J/cm2. This treatment significantly lengthened 2.5 mg/kg LCP, 100 J/cm212.4 47.6 Â±10.43" the mean of the days to a 6-fold tumor volume increase from ' Significantly different from no injection, no light group: /' < 0.02. 12.4 days in untreated controls to 47.6 days (P < 0.02). Al though there was a slight increase in mean tumor regrowth time of rats treated with LCP and irradiated with 50 J/cm2 compared Table 4 Skin scores from rats treated with PDT to controls, the differences were not statistically significant. Rats were given LCP i.v. followed 4 h later by irradiation with 664-nm light. Table 4 shows the skin responses observed in the irradiated Mean skin score Â±SD is shown. N = 5 rats/group. field. Skin responses were very uniform within groups, with the parametersNoTreatment scorefield0.00 in irradiated exception of the group treated at 25 J/cm2 in which scores injection, no light Â±0.00 PBS, 100 J/cm2 ranged from 0 to 3. There was no skin damage on the ears or 0.00 Â±0.00 2.5 mg/kg LCP, no light 0.00 Â±0.00 feet in any group, despite exposure to fluorescent room lights 2.5 mg/kg LCP, 25 J/cm2 1.20Â± 1.64 throughout the duration of the experiments. Within the irradi 2.5 mg/kg LCP, 50 J/cm2 5.00 Â±0.00 2.5 mg/kg LCP, 100 J/cm2Skin ated field, responses were only seen in animals receiving pho 6.00 Â±0.00 1237

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. PHOTODYNAMIC THERAPY USING A NEW CHLORIN p6 MIXTURE thesis. We are currently initiating experiments using the pure and then irradiated with 664-nm light. Fluorescent room light compound, lysyl chlorin p6. did not appear to cause any cutaneous effects at the photosen On the basis of data from other investigators using related sitizer dose of 2.5 mg/kg. Normal tissue damage (coagulation chlorin compounds, we have used a treatment schedule with necrosis with subsequent scarring) was seen in tissues which irradiation of tumors 4 h after i.v. injection of the photosensi- were directly irradiated, including skin, fat, and muscle adjacent tizer (20). Longer time intervals after photosensitizer injection to the tumor, and in the immediately underlying colon and were not examined in this study since they have not given kidney (data not shown). Normal tissue damage was mild at a consistent tumor necrosis when using monoaspartyl chlorin e6 fluence of 50 mW/cm2 and more severe at 100 mW/cm2. Rats (NPe6) (18, 20). Thus, it is possible that another time interval repaired such damage, as indicated by the ability of several to may result in more effective tumor necrosis. Pharmacological survive in good health for over 50 days after irradiation in the experiments remain to be performed with LCP and may provide group receiving the highest energy dose. information regarding the optimal time of irradiation following Our study indicates that LCP is an effective photosensitizer LCP injection. for use in PDT. While PDT using LCP does not cause cuta Initial studies were done to determine temperature changes neous photosensitization in nonirradiated skin, directly irradi in tumors during PDT to minimize thermal effects during ated tissues including skin were damaged during treatment. subsequent experiments. Intratumoral temperatures at a power This probably relates to the 4-h interval between photosensitizer density of 50 mW/cm2 remained below 40Â°C(data not shown), injection and irradiation, which was probably not adequate to which alone would not cause significant tumor necrosis (26, allow serum and normal tissue clearance of the LCP. Further 27), and thus further experiments were done at this power investigation of LCP, including preparation of pure material density. and pharmacokinetic studies, is warranted to indicate treatment Microscopic changes in tumors and surrounding tissues ex schedules which provide for maximal tumor destruction and amined 24 h following PDT were similar to those described minimal normal tissue damage. using other photosensitizers, namely congestion, edema, and coagulation necrosis (18, 28). Necrosis was minimal in control ACKNOWLEDGMENTS animals, and neither drug alone nor light alone appeared to have any effect on the microscopic appearance or growth of the The authors would like to acknowledge Sharon Anglin and John tumors. The percentage of necrosis in PDT-treated tumors Picanso for their technical assistance. These studies were performed at increased as the energy density was increased, indicating more the Comparative Cancer Center, University of California-Davis. activation of the photosensitizer. Within most treatment groups there was a consistent amount of necrosis. However, within the REFERENCES 25-J/cm2 group there was wide variation in the percentage of necrosis between individual animals. It is possible this energy 1. Dougherty, T. J. Photodynamic therapy: status and potential. Oncology, .): 67-73, 1989. density is near the threshold required for inducing tumor necro 2. Corner, C. J., Rucker, N., Ferrano, A., and Wong, S. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. Effectiveness of a Lysyl Chlorin p6/Chlorin p6 Mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat

Michael W. Leach, Robert J. Higgins, James E. Boggan, et al.

Cancer Res 1992;52:1235-1239.

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