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Evaluation of 99mTc-labeled photosan-3, a hematoporphyrin derivative, as a potential radiopharmaceutical for tumor scintigraphy
Nuclear Medicine & Biology, Vol. 27, pp. 587–592, 2000 Copyright © 2000 Elsevier Science Inc. All rights reserved.
ISSN 0969-8051/00/$–see front matter PII S0969-8051(00)00123-2
Evaluation of 99mTc-Labeled Photosan-3, a Hematoporphyrin Derivative, as a Potential Radiopharmaceutical for Tumor Scintigraphy A. K. Babbar,1 A. K. Singh,1 H. C. Goel,2 U. P. S. Chauhan1 and R. K. Sharma1 DEPARTMENTS OF 1RADIOPHARMACEUTICALS AND 2RADIATION BIOLOGY, INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCES, LUCKNOW MARG, DELHI, INDIA
ABSTRACT. A quick and reproducible method for radiolabeling of Photosan-3威, a photosensitizer used worldwide for photodynamic therapy (PDT) of cancer, with radioisotope of technetium (99mTc) was developed. The radiotracer was evaluated for radiochemical purity, stability, and finally tissue distribution in a murine tumor model. The 99mTc-Photosan-3 prepared by using 99mTc-pertechnetate in place of reduced 99m Tc demonstrated better labeling efficiency (>90%) and reproducibility. The procedure also minimized the radiation exposure to the radiochemist as handling time was considerably reduced. Due to the commercial availability of Photosan-3威, the risk of batch-to-batch variation in the in situ synthesis of hematoporphyrin derivative, which is a complex mixture of at least five compounds, was also significantly reduced. The biodistribution studies and tumor scintigraphy confirmed that 99mTc-labeled Photosan-3威 was preferentially taken up by the neoplastic tissue in a manner similar to the parent compound. In addition to applications in tumor imaging, 99mTc-Photosan-3 could also be used for estimating tumor uptake of Photosan-3威 as may be required for individualization of clinical protocols of PDT. NUCL MED BIOL 27;6:587–592, 2000. © 2000 Elsevier Science Inc. All rights reserved. KEY WORDS. 99mTc-Photosan-3, Hematoporphyrin derivative, Ehrlich ascites tumor, Tumor scintigraphy
INTRODUCTION Porphyrin derivatives, the complex tetrapyrrole compounds capable of forming stable coordinated complexes with metal ions, have been documented for their widespread applications in photodynamic therapy (PDT) of localized tumors and especially melanomas (5, 15, 21). Among these, the hematoporphyrin derivative (Hpd) attracted maximum attention of clinical researchers because of the natural occurrence and better tumor localization than any other porphyrin compound (9, 15). Hpd, which is a complex mixture of hematoporphyrin diacetate, hematoporphyrin monoacetate, vinylporphyrins, protoporphyrins, dentoporphyrins, and several additional analogues, has been used successfully in the detection and phototherapy of lightaccessible tumors (10). It has already been approved by health boards in Canada, Japan, the Netherlands, and the United States for PDT and is now commercially available as Photosan-3威 and Photofrin威 (5). The use of Hpd fluorescence imaging technique has been restricted due to the invasive endoscopy procedure (22). Moreover, quenching of the fluorescence by body fluids, blood, and normal tissues restrained the data acquisition and recording of the fluorescence, radiographically or photographically. Therefore, radioactive porphyrins labeled with short halflife gamma emitters were suggested as a better alternative for tumor detection (1, 3, 8, 14, 16). Initially, efforts were made to label Hpd with 57Co and 64Cu, but lack of significant tumor
uptake did not permit imaging (1, 3). The porphyrin analogues coordinated with 57Co (16) and 109Pd (7), and though they accumulated in murine tumor models, they permitted less selective visualization of tumors in humans (11). Wong and his group (17, 18) produced 99mTc-labeled Hpd by in situ synthesis and demonstrated its potential for detection of murine tumors. However, the method involved careful treatment of 99mTc, otherwise batch-to-batch variation in the percent fraction of reduced/hydrolyzed 99mTc was observed. The same group further demonstrated that 111In-labeled Hpd localized in murine breast tumors (19, 20). In comparison to 99mTc-labeled Hpd, the 111 In-labeled product concentrated substantially in liver and spleen and appreciably more in malignant neoplasm. Lavallee and Fawwaz (13) also reported a convenient method to label 111 In with the Hpd, but it could not gain widespread clinical use because 111In, being a cyclotron product, was not easily available at most of the nuclear medicine centers. Considering the cost effectiveness, low radiation burden, and indigenous availability of short-lived gamma-emitting 99mTc, it was considered worthwhile to develop 99mTc-labeled Hpd for possible applications in the tumor scintigraphy (2). Here we describe a one-step method to radiolabel Photosan-3威 with 99mTc and report its utility for tumor visualization in a murine model.
MATERIALS AND METHODS 99m
Address correspondence to: Dr. R. K. Sharma, Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences, Lucknow Marg, Delhi 110-054, India; e-mail: [email protected]. Received 18 December 1999. Accepted 21 March 2000.
Tc-Pertechnetate
99m
Tc-pertechnetate was eluted from 99Mo-99mTc generator by solvent extraction method. The molybedenum-99 was supplied by BRIT, Mumbai, India.
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Radiolabeling One mg of Photosan-3威 (Seehof Laboratorium, GmbH, Munich, Germany gift from Dr. R. Baumgartner, Munich, Germany) was thoroughly dissolved in 1 mL of water for injection, and the pH of the solution was raised to 8.0 using 0.1 N sodium hydroxide solution. All the solutions used were nitrogen-purged. To this solution, 50 g of stannous chloride dihydrate (Sigma, St. Louis, MO, USA) in 50 L of 0.1 N HCl was added, and the pH was adjusted to 7.0. The contents were passed through a 0.22 M filter (Millipore Corporation, Bedford, MA, USA) into an evacuated sterile sealed vial. Sterile 99mTc-pertechnetate (1–2 mL; 75– 400 MBq) was added dropwise to the vial with continuous mixing over a period of 30 s. The reaction mixture was incubated at room temperature for 30 min with continuous nitrogen gas purging.
Radiochemical Purity The radiochemical purity of 99mTc-Photosan-3 was assessed by ascending instant thin layer chromatography (ITLC) using silica gel-coated fiber glass sheets (Gelman Sciences Inc., Ann Arbor, MI, USA) and dual solvent systems, namely physiological saline (0.9% NaCl) and a solvent mixture of acetone, ethyl acetate, water, and ammonium hydroxide (7:3:3:0.3, v/v) as mobile phase. The radioactive contaminants were identified as reduced/hydrolyzed (R/H) technetium-99m and free 99mTc-pertechnetate.
In Vitro and in Vivo Stability The radiolabel was tested for its in vitro and in vivo stability by ascending ITLC. For in vitro stability in physiological saline and serum, 100 L of the radiolabel was mixed in triplicate with 2 mL each of 0.9% saline and human serum, respectively. ITLC was carried out to assess the labeling efficiency after incubating at 37°C for different time intervals. In vivo stability was assessed by administering 100 L of 99mTc-Photosan-3 to New Zealand Albino rabbits through the ear vein and blood samples withdrawn at different time intervals were subjected to ITLC.
Blood Clearance and Plasma Protein Binding Blood clearance of 99mTc-Photosan-3 was studied in rabbits. Radiolabel (40 MBq) was administered to each rabbit through the ear vein and blood samples were collected at different time intervals. The radioactivity in blood was calculated as percentage of the injected dose. From the blood samples, plasma was separated by centrifugation and the proteins were precipitated by adding equal volumes of 12.5% trichloroacetic acid (TCA) and plasma. The radioactivity in the precipitate and supernatant was measured in a well-type gamma spectrometer. For in vitro protein binding, 0.1 mL of 99mTc-Photosan-3 was mixed with 2 mL of plasma. Samples were processed as above and evaluated for protein binding at different time intervals. The plasma protein binding was expressed as fraction of total activity in the sample.
Biodistribution In vivo distribution of 99mTc-Photosan-3 was studied in 2- to 3-month-old Balb/c mice. 99mTc-Photosan-3 (50 L, 40 KBq) was administered to each mice weighing 25–30 g through the tail vein. The animals were sacrificed at different time intervals, and different
TABLE 1. Migration Values (Rf) of 99mTc-pertechnetate, R/H 99mTc and 99mTc-Photosan-3 as Determined by Ascending ITLC (SG) Using Different Solvent Systems Rf value Solvent system 0.9% sodium chloride Acetone: ethyl acetate: water: ammonia (7:3:3:0.3, v/v)
Free Tc
99m
1.0 1.0
R/H Tc
99m
0.0 0.0
99m
Tc-Photosan-3 0.0 1.0
organs were removed, washed with normal saline, and dried in the paper folds. The radioactivity in each organ was counted using well-type gamma spectrometer and expressed as percent injected dose per organ.
Tumor Implantation and Scintigraphy Ehrlich ascites tumor (EAT) cells were grown in the ascites fluid of mouse by injecting 2.5 ⫻ 107 cells intraperitoneally. The cells were harvested on the seventh day after administration. These ascites cells were subcutaneously inoculated in soft tissue to produce solid tumors. In our study, 0.2 mL of ascites fluid containing 1.5 ⫻ 107 cells were subcutaneously injected in the right thigh of mice (Strain A). After 6 to 7 days, tumors were in the volume range of 0.9 ⫾ 0.1 cm3. The scintigraphy in normal as well as EAT-bearing mice was carried out after intravenous administration of 0.1 mL of 99mTcPhotosan-3 (4 MBq) through caudal vein. The animals were anesthetized by intramuscular injection of ketamine hydrochloride (20 mg/kg body weight) 10 min before imaging. The animal was fixed on a board in posterior anterior position, and imaging was performed at different time intervals using a planar gamma camera. RESULTS Photosan-3, a hematoporphyrin derivative, was labeled with 99mTc at pH 7.0 using 50 g of stannous chloride dihydrate as a reductant. The radiochemical purity of the product was evaluated by ITLC, which successfully resolved labeled product from R/H and free 99m Tc (Table 1). The labeled preparations comprised of 90% of the 99m Tc-Photosan-3, with the rest being R/H 99mTc (6%) and free 99m Tc (4%). To achieve optimum labeling efficiency, the pH of the reaction mixture was varied from 6.5 to 8.0, while keeping rest of the factors constant. Similarly, the amount of stannous chloride dihydrate was varied from 12.5– 400 g, while keeping the pH, amount of photosan-3, and volume of the reaction constant. Results of the effect of pH and amount of stannous chloride dihydrate on labeling efficiency are shown in Figures 1 and 2, respectively. At pH 6.5, both the R/H 99mTc and 99mTc-pertechnetate fractions were very high, However, at pH 8.0, although the percentage of free 99m Tc was low, the R/H 99mTc fraction was found to be 35%. Similarly the effects of varying the concentration of stannous chloride dihydrate from 12.5 to 400 g, keeping other conditions constant, is shown in Figure 2. At low concentrations of stannous chloride dihydrate, the fraction of free 99mTc was greater, while at high concentrations the R/H 99mTc increased up to 40%. Fifty g of SnCl2 䡠 2H2O and pH of 7.0 yielded the highest labeling efficiency of 99mTc-Photosan-3.
99m
Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy
FIG. 1. Effect of pH on labeling efficiency of san-3.
99m
Tc-Photo-
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FIG. 3. In vitro and in vivo stability of
99m
Tc-Photosan-3.
Tc-Photosan-3 complex was fairly stable in vitro up to 2 hours, but the stability deteriorated gradually thereafter, and at 24 hours the percent of labeled product was only 75.9% as determined by ITLC (Fig. 3). In serum, the radiolabel was sufficiently stable even
up to 24 hours, possibly due to a protective effect of some serum component. The blood clearance data in rabbits after intravenously administering 40 MBq of 99mTc-Photosan-3 exhibited slow and biphasic clearance (Fig. 4). At 1 hour postadministration, 18% of the injected radioactivity was present in blood, which reduced to 3.5% by 24 hours.
FIG. 2. Effect of amount of stannous chloride dihydrate on the labeling efficiency of 99mTc-Photosan-3.
FIG. 4. Blood clearance of erage data of 2 rabbits).
99m
99m
Tc-Photosan-3 in rabbit (av-
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FIG. 5. In vitro and in vivo plasma protein binding of 99m Tc-Photosan-3 expressed as a fraction of total radioactivity in the sample. In vivo protein binding was 84% at 30 min, reaching to 88% at 2 hours postadministration of the complex as shown in Figure 5. The protein binding reduced to 71% at 24 hours. In vitro protein binding was maximum at 30 min (75.6%), and it reduced to 60% at 24 hours of incubation. The biodistribution data of 99mTc-Photosan-3 in 2- to 3-monthold Balb/c mice is summarized in Table 2. Based on the percent injected dose per whole organ, the highest uptake of 99mTcPhotosan-3 was found in liver, blood, kidneys, intestines, and muscle averaging 23.8, 9.01, 4.34, 4.65, and 3.8%, respectively, at 1 hour postadministration. Very little uptake of the radiotracer is seen in the lungs. The radioactivity in all the organs except liver was reduced considerably at 24 hours. After administration of 99mTc-Photosan-3, scintigraphy in mice bearing Ehrlich ascites tumor showed a very early uptake at 2 hours. TABLE 2. Tissue Distribution in Normal Mice at 1, 3, and 24 hour After Intravenous Administration of 99mTc-Photosan-3 Percent Injected Dose/Whole Organ Organ Blood Heart Lungs Liver Kidneys Intestines Stomach Muscle
1 hour
3 hour
24 hour
9.01 ⫾ 0.77 0.10 ⫾ 0.02 0.37 ⫾ 0.04 23.79 ⫾ 1.21 4.34 ⫾ 0.08 4.65 ⫾ 0.20 0.23 ⫾ 0.01 3.80 ⫾ 0.67
3.81 ⫾ 0.36 0.07 ⫾ 0.01 0.22 ⫾ 0.01 17.71 ⫾ 0.76 3.54 ⫾ 0.50 7.60 ⫾ 0.82 0.27 ⫾ 0.05 3.63 ⫾ 0.38
0.65 ⫾ 0.10 0.03 ⫾ 0.003 0.08 ⫾ 0.01 12.66 ⫾ 0.49 1.00 ⫾ 0.07 1.06 ⫾ 0.24 0.20 ⫾ 0.03 0.71 ⫾ 0.03
Data are expressed as percent injected dose per whole organ ⫾ SE of five animals.
FIG. 6. Whole body scintigrams of Ehrlich ascites tumorbearing mice obtained at 4, 8, 16, and 24 hours post-iv administration of 4 MBq of 99mTc-Photosan-3. However, maximum tumor-to-muscle ratio of 7.9 was achieved at 4 hours postadministration (Fig. 6). The ratio slowly decreased, subsequently showing a slight rise at 24 hours. DISCUSSION Hematoporphyrins and other photosensitizers have been tried for fluorescence detection of tumors using endoscopy procedure (4, 22). However, radioactive porphyrins were predicted as a better alternative for tumor detection than Hpd fluorescence endoscopy (18). Biodistribution data of 99mTc-labeled Hpd (2, 18) demonstrated highly favorable tumor-to-blood and organ ratios, allowing tumor delineation away from the abdominal organs. We have successfully radiolabeled Photosan-3威 with 99mTc and demonstrated its detection and localization in tumors. Radiolabeling of Photosan-3威 was carried out by adding 99mTc-pertechnetate instead of reduced 99mTc as reported by Wong’s group (17, 18). The earlier method used for labeling Hpd with 99mTc involved the addition of stannous reduced 99m Tc and the complex thus produced had a major fraction of reduced 99mTc. Our method has the advantage of increased yield of 99m Tc-Photosan-3 and simple handling of the radioisotope, resulting in lower radiation exposure. The modified labeling procedure deserves special attention because it is a simple radiolabeling
99m
Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy
procedure within 30 min, which can be transformed into a one-vial kit for instant labeling (data not shown). The method gave reproducible labeling efficiency. Radiolabeling at pH 7.0 ensured that microcolloid formation was prevented. Lower labeling efficiency at acidic pH could be due to precipitation of labeled and unlabeled Photosan-3 from the solution as reported by Wong et al. (17). The radiochemical purity was studied by ITLC, and the radioactive contaminants were identified as R/H and free pertechnetate. Free 99mTc-pertechnetate moved with the solvent front, whereas reduced and labeled 99mTc remained at the point of application when 0.9% NaCl was used as the mobile phase. Labeled 99mTc moved along with free 99mTc, leaving only reduced 99mTc at the base in the case of the second solvent mixture. The use of two solvent systems was found to be a very accurate method to clearly distinguish and quantitate the relative amounts of free 99mTc, R/H 99mTc, and 99mTc-Photosan-3. Studies on the optimization of labeling parameter reveal that the optimal range of Sn (ous) concentration and pH are quite narrow. We have critically studied the stability, blood kinetics, biodistribution, and tumor specificity of the radiopharmaceutical and its accumulation as a function of time, which are the key factors for clinical acceptance of the radiopharmaceutical. The in vivo stability of the complex is evident from the lack of affinity of 99mTcPhotosan-3 for stomach, the target organ for free 99mTc. Kessel et al. (12) suggested that tumor localization of the dyes is strongly affected by their binding to specific plasma components. Hydrophobic dyes tend to associate with lipoproteins, leading to direct dye accumulation in neoplastic cells, while hydrophilic dyes bind to albumin, leading to dye accumulation in the stromal elements of the neoplastic tissue. Therefore, analysis of the protein binding properties is important to assess the clinical usefulness of this radiopharmaceutical. High protein binding of the labeled dye to the plasma protein was observed both in vitro and in vivo in our study. Irrespective of the nature of the porphyrin derivatives, the mechanism of their preferential uptake by neoplastic tissues is possibly due to their high specificity for lipoprotein receptors, which are overwhelmingly expressed in neoplasm (6, 20). Fawwaz et al. (7) reported the use of metalloporphyrins for enhancing the sensitivity of tumor detection. Biodistribution studies performed using 99mTc-Photosan-3 showed that the volume of distribution of the labeled dye is initially confined to plasma. The rate of disappearance is inversely related to the amount injected. The half-time disappearance of the radiopharmaceutical is approximately 2 to 3 hours. There is no evidence of in vivo dissociation of the complex. The biodistribution data suggest the excretion of 99m Tc-Photosan-3 through kidneys and reticulo-endothelial system. The efficacy of 99mTc-Photosan-3 as a tumor-imaging agent was investigated in a murine tumor model. The radioactivity was mainly found in the abdominal region and corresponds with the tissue biodistribution data. The tumor scintigraphy data clearly demonstrate that 99mTc-Photosan-3 localizes in tumor with good tumorto-background ratio. The early uptake by the tumor at 2 hours postadministration and maximum tumor-to-muscle ratio of 7.9 at 4 hours postadministration proves it to be a successful tumor marker. Small vascular tumor (⬃0.9 cm3) concentrated more radiolabeled Photosan-3 than the larger ones (data not shown). In large tumors (⬃2 cm3) with potential necrosis and limited blood supply, early (2 and 4 hours) scans showed only faint localization, which disappeared in the delayed images (24 hours). Imaging studies in viable tumor with 99mTc-Photosan-3 indicate that a late delayed scan is preferred. A delay of more than 10 hours may be needed to minimize background radioactivity and to allow continuous buildup
591
of the radiopharmaceutical in the tumor. Blood pool radioactivity was minimal as evident by nonvisualization of the heart and validated by the mice biodistribution data. High localization in liver and kidneys observed, even at 24 hours postadministration, was in conformity with similar observations reported for certain metalloporphyrins by Fawwaz et al. (7, 8). Therefore, the radiopharmaceutical appears to be metabolized in liver and partially eliminated by the kidneys and gastrointestinal tract. Radioactivity from spleen and stomach is partly obscured in the image by the hyperintense area of the liver. Besides liver and implanted tumor, no other major internal organs and bone marrow were notably visible in the images. Preliminary results from biodistribution studies and imaging demonstrate that the radiolabeled Photosan-3威, like the parent compound, is preferentially taken up by the neoplastic tissue. A close look at the time-dependent distribution pattern of activity inside the tumor is indicative of the role played by tumor morphology, vascularity, and necrotic areas. The precise reasons for retention of Photosan-3威 by the tumor are not clear, but the rate of its uptake could be a function of its hydrophilicity. The specificity of 99m Tc-Photosan-3 as a tumor-imaging agent has not been completely investigated. One of the serious limitations of the porphyrin derivatives with regard to their concentration in the tumor is their high variability due to pH-dependent aggregation. However, due to commercial availability of standardized Hpd in the form of Photosan-3威, more reliability is ensured, resulting in better reproducibility of the radiopharmaceutical preparation. Before its clinical use, several other parameters require detailed investigations. Intratumoral distribution, dependence of accumulation on the tumor volume, the optimum time scheduling for the best image, and specificity and selectivity of the tumor uptake over infections or inflammatory lesions remain the important factors for its clinical acceptance. 99mTc-labeled Photosan-3 appears promising as a tumor-imaging agent due to highly favorable tumor-to-blood and other organ ratio, allowing tumor delineation away from the abdominal organs. Scintigraphy using 99mTc-Photosan-3 has immense potential in oncology as predictive assay. Data of its accumulation in tumors could be relevant in the photodynamic therapy as well, since it could be employed for prediction and quantitative measurement of the photosensitizer in tumor tissue for individualization of the clinical treatment protocols. The authors are grateful to Director of INMAS for providing necessary facilities for the study and Mr. T. Singh and Mr. Surender Singh for technical help.
References 1. Anghileri L. J., Heidbreder M. and Mathes R. (1976) 57Co haematoporphyrin accumulation by experimental tumors. Nucl. Med. 15, 183–184. 2. Babbar A. K., Singh T., Chauhan U. P. S. and Jain V. (1995) Radiolabeling of haemetoporphyrin derivatives with 99mTc and study of its kinetics in mice. Ind. J. Nucl. Med. 10, p-43 (Abstract). 3. Base R., Brodie S. S. and Rubenfield S. (1958) Attempts at tumor localization using 64Cu labeled copper porphyrins. Cancer 11, 259 –263. 4. Benson R., Farrow G. M., Kinsley J. H., Cortese D. A., Zincke H. and Utz D. C. (1982) Detection and localization in situ carcinoma of the bladder with Hpd. Mayo Clin. Proc. 57, 548 –555. 5. Boyle R. W. and Dolphin D. (1996) Structure and biodistribution relationship of photodynamic sensitizers. Photochem. Photobiol. 64, 469 – 435. 6. Daugherty T. J., Kaufman J. E., Goldfare A., Weishaupt K. R., Boyle D. and Mittleman A. (1978) Photoradiation therapy for the treatment of malignant tumors. Cancer Res. 38, 2628 –2635. 7. Fawwaz R., Hemphill W. and Winchell H. (1971) Potential use of
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Pd-porphyrin complexes for selective lymphatic ablation. J. Nucl. Med. 12, 231–236. Fawwaz R., Bohdiewicz P., Lavallee D., Wang T., Oluwole S., New House J. and Alderson P. (1990) Use of metalloporphyrins in diagnostic imaging. Nucl. Med. Biol. 17, 65–72. Foster N., Woo D. V., Kaltovich F., Emrich J. and Djungquist C. (1985) Delineation of a transplanted malignant melanoma with Indium-111 labeled porphyrin. J. Nucl. Med. 26, 756 –760. Gregorie H. B., Horger E. O., Ward J. L., Green J. F., Richards T., Robertson H. C. Jr. and Stevenson T. R. (1968) Haematoporphyrin derivative fluorescence in malignant neoplasm. Ann. Surg. 167, 820 – 828. Hambright P., Fawwaz R. A., Valk P., McRae J. and Bearden A. J. (1975) The distribution of various water-soluble radioactive metalloporphyrins in tumor bearing mice. Bioinorg. Chem. 5, 87–92. Kessel D., Thompson P., Saatio K. and Nantwi K. D. (1987) Tumor localization and photosensitization by sulfonated derivatives of tetraphenylporphine. Photochem. Photobiol. 45, 787–790. Lavallee D. K. and Fawwaz R. (1986) The synthesis and characterization of 111In haematoporphyrin derivative. Nucl. Med. Biol. 13, 639 – 641. Thaller R. A. and Lyster D. M. (1983) Potential use of radiolabeled porphyrins for tumor scanning. In: Porphyrin Photosensitization (Edited by Kessel D. and Doughherty T. J.). Adv. Exp. Med. Biol. 160, 265–278. Tronconi M., Colombo A., DeCesare M., Marchesini R., Woodburn
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J. A., Phillips D. R. and Zunino F. (1995) Biodistribution of haemetoporphyrin analogues in a lung carcinoma model. Cancer Lett. 88, 41– 48. Winkelman J., Rubenfeld S. and McAfee J. (1964) The metabolism and excretion of 57Co tetraphenyl porphine sulfonate in cancer patients. J. Nucl. Med. 5, 462– 470. Wong D. W. (1983) A simple chemical method of labeling haematoporphyrin derivative with 99mTc. J. Labeled Compd. Radiopharm. 20, 351–361. Wong D. W., Mandal A., Reese I. C., Brown J. and Siegler R. (1983) In vivo assessment of 99mTc-labeled haematoporphyrin derivative on tumor-bearing animals. Nucl. Med. Biol. 10, 211–218. Wong D. W. (1984) A simple and efficient method of labeling haematoporphyrin derivative with indium-111. Int. J. Appl. Radiat. Isot. 35, 691– 692. Wong D. W., Mandal A., Brown J., Reese I. C., Siegler R. and Hyman S. (1989) In vivo assessment of 111In labeled haematoporphyrin derivatives in breast tumor bearing animals. Nucl. Med. Biol. 16, 269 –281. Woodburn K. W., Stylli S., Hill J. S., Kaye A. H., Reiss J. A. and Phillips D. R. (1992) Evaluation of tumor and tissue distribution of porphyrins for use in photodynamic therapy. Br. J. Cancer 65, 321–328. Zaneli G. D. and Kaelin A. D. (1981) Synthetic porphyrins as tumor localizing agents. Br. J. Rad. 54, 403– 407.
ISSN 0969-8051/00/$–see front matter PII S0969-8051(00)00123-2
Evaluation of 99mTc-Labeled Photosan-3, a Hematoporphyrin Derivative, as a Potential Radiopharmaceutical for Tumor Scintigraphy A. K. Babbar,1 A. K. Singh,1 H. C. Goel,2 U. P. S. Chauhan1 and R. K. Sharma1 DEPARTMENTS OF 1RADIOPHARMACEUTICALS AND 2RADIATION BIOLOGY, INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCES, LUCKNOW MARG, DELHI, INDIA
ABSTRACT. A quick and reproducible method for radiolabeling of Photosan-3威, a photosensitizer used worldwide for photodynamic therapy (PDT) of cancer, with radioisotope of technetium (99mTc) was developed. The radiotracer was evaluated for radiochemical purity, stability, and finally tissue distribution in a murine tumor model. The 99mTc-Photosan-3 prepared by using 99mTc-pertechnetate in place of reduced 99m Tc demonstrated better labeling efficiency (>90%) and reproducibility. The procedure also minimized the radiation exposure to the radiochemist as handling time was considerably reduced. Due to the commercial availability of Photosan-3威, the risk of batch-to-batch variation in the in situ synthesis of hematoporphyrin derivative, which is a complex mixture of at least five compounds, was also significantly reduced. The biodistribution studies and tumor scintigraphy confirmed that 99mTc-labeled Photosan-3威 was preferentially taken up by the neoplastic tissue in a manner similar to the parent compound. In addition to applications in tumor imaging, 99mTc-Photosan-3 could also be used for estimating tumor uptake of Photosan-3威 as may be required for individualization of clinical protocols of PDT. NUCL MED BIOL 27;6:587–592, 2000. © 2000 Elsevier Science Inc. All rights reserved. KEY WORDS. 99mTc-Photosan-3, Hematoporphyrin derivative, Ehrlich ascites tumor, Tumor scintigraphy
INTRODUCTION Porphyrin derivatives, the complex tetrapyrrole compounds capable of forming stable coordinated complexes with metal ions, have been documented for their widespread applications in photodynamic therapy (PDT) of localized tumors and especially melanomas (5, 15, 21). Among these, the hematoporphyrin derivative (Hpd) attracted maximum attention of clinical researchers because of the natural occurrence and better tumor localization than any other porphyrin compound (9, 15). Hpd, which is a complex mixture of hematoporphyrin diacetate, hematoporphyrin monoacetate, vinylporphyrins, protoporphyrins, dentoporphyrins, and several additional analogues, has been used successfully in the detection and phototherapy of lightaccessible tumors (10). It has already been approved by health boards in Canada, Japan, the Netherlands, and the United States for PDT and is now commercially available as Photosan-3威 and Photofrin威 (5). The use of Hpd fluorescence imaging technique has been restricted due to the invasive endoscopy procedure (22). Moreover, quenching of the fluorescence by body fluids, blood, and normal tissues restrained the data acquisition and recording of the fluorescence, radiographically or photographically. Therefore, radioactive porphyrins labeled with short halflife gamma emitters were suggested as a better alternative for tumor detection (1, 3, 8, 14, 16). Initially, efforts were made to label Hpd with 57Co and 64Cu, but lack of significant tumor
uptake did not permit imaging (1, 3). The porphyrin analogues coordinated with 57Co (16) and 109Pd (7), and though they accumulated in murine tumor models, they permitted less selective visualization of tumors in humans (11). Wong and his group (17, 18) produced 99mTc-labeled Hpd by in situ synthesis and demonstrated its potential for detection of murine tumors. However, the method involved careful treatment of 99mTc, otherwise batch-to-batch variation in the percent fraction of reduced/hydrolyzed 99mTc was observed. The same group further demonstrated that 111In-labeled Hpd localized in murine breast tumors (19, 20). In comparison to 99mTc-labeled Hpd, the 111 In-labeled product concentrated substantially in liver and spleen and appreciably more in malignant neoplasm. Lavallee and Fawwaz (13) also reported a convenient method to label 111 In with the Hpd, but it could not gain widespread clinical use because 111In, being a cyclotron product, was not easily available at most of the nuclear medicine centers. Considering the cost effectiveness, low radiation burden, and indigenous availability of short-lived gamma-emitting 99mTc, it was considered worthwhile to develop 99mTc-labeled Hpd for possible applications in the tumor scintigraphy (2). Here we describe a one-step method to radiolabel Photosan-3威 with 99mTc and report its utility for tumor visualization in a murine model.
MATERIALS AND METHODS 99m
Address correspondence to: Dr. R. K. Sharma, Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences, Lucknow Marg, Delhi 110-054, India; e-mail: [email protected]. Received 18 December 1999. Accepted 21 March 2000.
Tc-Pertechnetate
99m
Tc-pertechnetate was eluted from 99Mo-99mTc generator by solvent extraction method. The molybedenum-99 was supplied by BRIT, Mumbai, India.
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Radiolabeling One mg of Photosan-3威 (Seehof Laboratorium, GmbH, Munich, Germany gift from Dr. R. Baumgartner, Munich, Germany) was thoroughly dissolved in 1 mL of water for injection, and the pH of the solution was raised to 8.0 using 0.1 N sodium hydroxide solution. All the solutions used were nitrogen-purged. To this solution, 50 g of stannous chloride dihydrate (Sigma, St. Louis, MO, USA) in 50 L of 0.1 N HCl was added, and the pH was adjusted to 7.0. The contents were passed through a 0.22 M filter (Millipore Corporation, Bedford, MA, USA) into an evacuated sterile sealed vial. Sterile 99mTc-pertechnetate (1–2 mL; 75– 400 MBq) was added dropwise to the vial with continuous mixing over a period of 30 s. The reaction mixture was incubated at room temperature for 30 min with continuous nitrogen gas purging.
Radiochemical Purity The radiochemical purity of 99mTc-Photosan-3 was assessed by ascending instant thin layer chromatography (ITLC) using silica gel-coated fiber glass sheets (Gelman Sciences Inc., Ann Arbor, MI, USA) and dual solvent systems, namely physiological saline (0.9% NaCl) and a solvent mixture of acetone, ethyl acetate, water, and ammonium hydroxide (7:3:3:0.3, v/v) as mobile phase. The radioactive contaminants were identified as reduced/hydrolyzed (R/H) technetium-99m and free 99mTc-pertechnetate.
In Vitro and in Vivo Stability The radiolabel was tested for its in vitro and in vivo stability by ascending ITLC. For in vitro stability in physiological saline and serum, 100 L of the radiolabel was mixed in triplicate with 2 mL each of 0.9% saline and human serum, respectively. ITLC was carried out to assess the labeling efficiency after incubating at 37°C for different time intervals. In vivo stability was assessed by administering 100 L of 99mTc-Photosan-3 to New Zealand Albino rabbits through the ear vein and blood samples withdrawn at different time intervals were subjected to ITLC.
Blood Clearance and Plasma Protein Binding Blood clearance of 99mTc-Photosan-3 was studied in rabbits. Radiolabel (40 MBq) was administered to each rabbit through the ear vein and blood samples were collected at different time intervals. The radioactivity in blood was calculated as percentage of the injected dose. From the blood samples, plasma was separated by centrifugation and the proteins were precipitated by adding equal volumes of 12.5% trichloroacetic acid (TCA) and plasma. The radioactivity in the precipitate and supernatant was measured in a well-type gamma spectrometer. For in vitro protein binding, 0.1 mL of 99mTc-Photosan-3 was mixed with 2 mL of plasma. Samples were processed as above and evaluated for protein binding at different time intervals. The plasma protein binding was expressed as fraction of total activity in the sample.
Biodistribution In vivo distribution of 99mTc-Photosan-3 was studied in 2- to 3-month-old Balb/c mice. 99mTc-Photosan-3 (50 L, 40 KBq) was administered to each mice weighing 25–30 g through the tail vein. The animals were sacrificed at different time intervals, and different
TABLE 1. Migration Values (Rf) of 99mTc-pertechnetate, R/H 99mTc and 99mTc-Photosan-3 as Determined by Ascending ITLC (SG) Using Different Solvent Systems Rf value Solvent system 0.9% sodium chloride Acetone: ethyl acetate: water: ammonia (7:3:3:0.3, v/v)
Free Tc
99m
1.0 1.0
R/H Tc
99m
0.0 0.0
99m
Tc-Photosan-3 0.0 1.0
organs were removed, washed with normal saline, and dried in the paper folds. The radioactivity in each organ was counted using well-type gamma spectrometer and expressed as percent injected dose per organ.
Tumor Implantation and Scintigraphy Ehrlich ascites tumor (EAT) cells were grown in the ascites fluid of mouse by injecting 2.5 ⫻ 107 cells intraperitoneally. The cells were harvested on the seventh day after administration. These ascites cells were subcutaneously inoculated in soft tissue to produce solid tumors. In our study, 0.2 mL of ascites fluid containing 1.5 ⫻ 107 cells were subcutaneously injected in the right thigh of mice (Strain A). After 6 to 7 days, tumors were in the volume range of 0.9 ⫾ 0.1 cm3. The scintigraphy in normal as well as EAT-bearing mice was carried out after intravenous administration of 0.1 mL of 99mTcPhotosan-3 (4 MBq) through caudal vein. The animals were anesthetized by intramuscular injection of ketamine hydrochloride (20 mg/kg body weight) 10 min before imaging. The animal was fixed on a board in posterior anterior position, and imaging was performed at different time intervals using a planar gamma camera. RESULTS Photosan-3, a hematoporphyrin derivative, was labeled with 99mTc at pH 7.0 using 50 g of stannous chloride dihydrate as a reductant. The radiochemical purity of the product was evaluated by ITLC, which successfully resolved labeled product from R/H and free 99m Tc (Table 1). The labeled preparations comprised of 90% of the 99m Tc-Photosan-3, with the rest being R/H 99mTc (6%) and free 99m Tc (4%). To achieve optimum labeling efficiency, the pH of the reaction mixture was varied from 6.5 to 8.0, while keeping rest of the factors constant. Similarly, the amount of stannous chloride dihydrate was varied from 12.5– 400 g, while keeping the pH, amount of photosan-3, and volume of the reaction constant. Results of the effect of pH and amount of stannous chloride dihydrate on labeling efficiency are shown in Figures 1 and 2, respectively. At pH 6.5, both the R/H 99mTc and 99mTc-pertechnetate fractions were very high, However, at pH 8.0, although the percentage of free 99m Tc was low, the R/H 99mTc fraction was found to be 35%. Similarly the effects of varying the concentration of stannous chloride dihydrate from 12.5 to 400 g, keeping other conditions constant, is shown in Figure 2. At low concentrations of stannous chloride dihydrate, the fraction of free 99mTc was greater, while at high concentrations the R/H 99mTc increased up to 40%. Fifty g of SnCl2 䡠 2H2O and pH of 7.0 yielded the highest labeling efficiency of 99mTc-Photosan-3.
99m
Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy
FIG. 1. Effect of pH on labeling efficiency of san-3.
99m
Tc-Photo-
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FIG. 3. In vitro and in vivo stability of
99m
Tc-Photosan-3.
Tc-Photosan-3 complex was fairly stable in vitro up to 2 hours, but the stability deteriorated gradually thereafter, and at 24 hours the percent of labeled product was only 75.9% as determined by ITLC (Fig. 3). In serum, the radiolabel was sufficiently stable even
up to 24 hours, possibly due to a protective effect of some serum component. The blood clearance data in rabbits after intravenously administering 40 MBq of 99mTc-Photosan-3 exhibited slow and biphasic clearance (Fig. 4). At 1 hour postadministration, 18% of the injected radioactivity was present in blood, which reduced to 3.5% by 24 hours.
FIG. 2. Effect of amount of stannous chloride dihydrate on the labeling efficiency of 99mTc-Photosan-3.
FIG. 4. Blood clearance of erage data of 2 rabbits).
99m
99m
Tc-Photosan-3 in rabbit (av-
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FIG. 5. In vitro and in vivo plasma protein binding of 99m Tc-Photosan-3 expressed as a fraction of total radioactivity in the sample. In vivo protein binding was 84% at 30 min, reaching to 88% at 2 hours postadministration of the complex as shown in Figure 5. The protein binding reduced to 71% at 24 hours. In vitro protein binding was maximum at 30 min (75.6%), and it reduced to 60% at 24 hours of incubation. The biodistribution data of 99mTc-Photosan-3 in 2- to 3-monthold Balb/c mice is summarized in Table 2. Based on the percent injected dose per whole organ, the highest uptake of 99mTcPhotosan-3 was found in liver, blood, kidneys, intestines, and muscle averaging 23.8, 9.01, 4.34, 4.65, and 3.8%, respectively, at 1 hour postadministration. Very little uptake of the radiotracer is seen in the lungs. The radioactivity in all the organs except liver was reduced considerably at 24 hours. After administration of 99mTc-Photosan-3, scintigraphy in mice bearing Ehrlich ascites tumor showed a very early uptake at 2 hours. TABLE 2. Tissue Distribution in Normal Mice at 1, 3, and 24 hour After Intravenous Administration of 99mTc-Photosan-3 Percent Injected Dose/Whole Organ Organ Blood Heart Lungs Liver Kidneys Intestines Stomach Muscle
1 hour
3 hour
24 hour
9.01 ⫾ 0.77 0.10 ⫾ 0.02 0.37 ⫾ 0.04 23.79 ⫾ 1.21 4.34 ⫾ 0.08 4.65 ⫾ 0.20 0.23 ⫾ 0.01 3.80 ⫾ 0.67
3.81 ⫾ 0.36 0.07 ⫾ 0.01 0.22 ⫾ 0.01 17.71 ⫾ 0.76 3.54 ⫾ 0.50 7.60 ⫾ 0.82 0.27 ⫾ 0.05 3.63 ⫾ 0.38
0.65 ⫾ 0.10 0.03 ⫾ 0.003 0.08 ⫾ 0.01 12.66 ⫾ 0.49 1.00 ⫾ 0.07 1.06 ⫾ 0.24 0.20 ⫾ 0.03 0.71 ⫾ 0.03
Data are expressed as percent injected dose per whole organ ⫾ SE of five animals.
FIG. 6. Whole body scintigrams of Ehrlich ascites tumorbearing mice obtained at 4, 8, 16, and 24 hours post-iv administration of 4 MBq of 99mTc-Photosan-3. However, maximum tumor-to-muscle ratio of 7.9 was achieved at 4 hours postadministration (Fig. 6). The ratio slowly decreased, subsequently showing a slight rise at 24 hours. DISCUSSION Hematoporphyrins and other photosensitizers have been tried for fluorescence detection of tumors using endoscopy procedure (4, 22). However, radioactive porphyrins were predicted as a better alternative for tumor detection than Hpd fluorescence endoscopy (18). Biodistribution data of 99mTc-labeled Hpd (2, 18) demonstrated highly favorable tumor-to-blood and organ ratios, allowing tumor delineation away from the abdominal organs. We have successfully radiolabeled Photosan-3威 with 99mTc and demonstrated its detection and localization in tumors. Radiolabeling of Photosan-3威 was carried out by adding 99mTc-pertechnetate instead of reduced 99mTc as reported by Wong’s group (17, 18). The earlier method used for labeling Hpd with 99mTc involved the addition of stannous reduced 99m Tc and the complex thus produced had a major fraction of reduced 99mTc. Our method has the advantage of increased yield of 99m Tc-Photosan-3 and simple handling of the radioisotope, resulting in lower radiation exposure. The modified labeling procedure deserves special attention because it is a simple radiolabeling
99m
Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy
procedure within 30 min, which can be transformed into a one-vial kit for instant labeling (data not shown). The method gave reproducible labeling efficiency. Radiolabeling at pH 7.0 ensured that microcolloid formation was prevented. Lower labeling efficiency at acidic pH could be due to precipitation of labeled and unlabeled Photosan-3 from the solution as reported by Wong et al. (17). The radiochemical purity was studied by ITLC, and the radioactive contaminants were identified as R/H and free pertechnetate. Free 99mTc-pertechnetate moved with the solvent front, whereas reduced and labeled 99mTc remained at the point of application when 0.9% NaCl was used as the mobile phase. Labeled 99mTc moved along with free 99mTc, leaving only reduced 99mTc at the base in the case of the second solvent mixture. The use of two solvent systems was found to be a very accurate method to clearly distinguish and quantitate the relative amounts of free 99mTc, R/H 99mTc, and 99mTc-Photosan-3. Studies on the optimization of labeling parameter reveal that the optimal range of Sn (ous) concentration and pH are quite narrow. We have critically studied the stability, blood kinetics, biodistribution, and tumor specificity of the radiopharmaceutical and its accumulation as a function of time, which are the key factors for clinical acceptance of the radiopharmaceutical. The in vivo stability of the complex is evident from the lack of affinity of 99mTcPhotosan-3 for stomach, the target organ for free 99mTc. Kessel et al. (12) suggested that tumor localization of the dyes is strongly affected by their binding to specific plasma components. Hydrophobic dyes tend to associate with lipoproteins, leading to direct dye accumulation in neoplastic cells, while hydrophilic dyes bind to albumin, leading to dye accumulation in the stromal elements of the neoplastic tissue. Therefore, analysis of the protein binding properties is important to assess the clinical usefulness of this radiopharmaceutical. High protein binding of the labeled dye to the plasma protein was observed both in vitro and in vivo in our study. Irrespective of the nature of the porphyrin derivatives, the mechanism of their preferential uptake by neoplastic tissues is possibly due to their high specificity for lipoprotein receptors, which are overwhelmingly expressed in neoplasm (6, 20). Fawwaz et al. (7) reported the use of metalloporphyrins for enhancing the sensitivity of tumor detection. Biodistribution studies performed using 99mTc-Photosan-3 showed that the volume of distribution of the labeled dye is initially confined to plasma. The rate of disappearance is inversely related to the amount injected. The half-time disappearance of the radiopharmaceutical is approximately 2 to 3 hours. There is no evidence of in vivo dissociation of the complex. The biodistribution data suggest the excretion of 99m Tc-Photosan-3 through kidneys and reticulo-endothelial system. The efficacy of 99mTc-Photosan-3 as a tumor-imaging agent was investigated in a murine tumor model. The radioactivity was mainly found in the abdominal region and corresponds with the tissue biodistribution data. The tumor scintigraphy data clearly demonstrate that 99mTc-Photosan-3 localizes in tumor with good tumorto-background ratio. The early uptake by the tumor at 2 hours postadministration and maximum tumor-to-muscle ratio of 7.9 at 4 hours postadministration proves it to be a successful tumor marker. Small vascular tumor (⬃0.9 cm3) concentrated more radiolabeled Photosan-3 than the larger ones (data not shown). In large tumors (⬃2 cm3) with potential necrosis and limited blood supply, early (2 and 4 hours) scans showed only faint localization, which disappeared in the delayed images (24 hours). Imaging studies in viable tumor with 99mTc-Photosan-3 indicate that a late delayed scan is preferred. A delay of more than 10 hours may be needed to minimize background radioactivity and to allow continuous buildup
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of the radiopharmaceutical in the tumor. Blood pool radioactivity was minimal as evident by nonvisualization of the heart and validated by the mice biodistribution data. High localization in liver and kidneys observed, even at 24 hours postadministration, was in conformity with similar observations reported for certain metalloporphyrins by Fawwaz et al. (7, 8). Therefore, the radiopharmaceutical appears to be metabolized in liver and partially eliminated by the kidneys and gastrointestinal tract. Radioactivity from spleen and stomach is partly obscured in the image by the hyperintense area of the liver. Besides liver and implanted tumor, no other major internal organs and bone marrow were notably visible in the images. Preliminary results from biodistribution studies and imaging demonstrate that the radiolabeled Photosan-3威, like the parent compound, is preferentially taken up by the neoplastic tissue. A close look at the time-dependent distribution pattern of activity inside the tumor is indicative of the role played by tumor morphology, vascularity, and necrotic areas. The precise reasons for retention of Photosan-3威 by the tumor are not clear, but the rate of its uptake could be a function of its hydrophilicity. The specificity of 99m Tc-Photosan-3 as a tumor-imaging agent has not been completely investigated. One of the serious limitations of the porphyrin derivatives with regard to their concentration in the tumor is their high variability due to pH-dependent aggregation. However, due to commercial availability of standardized Hpd in the form of Photosan-3威, more reliability is ensured, resulting in better reproducibility of the radiopharmaceutical preparation. Before its clinical use, several other parameters require detailed investigations. Intratumoral distribution, dependence of accumulation on the tumor volume, the optimum time scheduling for the best image, and specificity and selectivity of the tumor uptake over infections or inflammatory lesions remain the important factors for its clinical acceptance. 99mTc-labeled Photosan-3 appears promising as a tumor-imaging agent due to highly favorable tumor-to-blood and other organ ratio, allowing tumor delineation away from the abdominal organs. Scintigraphy using 99mTc-Photosan-3 has immense potential in oncology as predictive assay. Data of its accumulation in tumors could be relevant in the photodynamic therapy as well, since it could be employed for prediction and quantitative measurement of the photosensitizer in tumor tissue for individualization of the clinical treatment protocols. The authors are grateful to Director of INMAS for providing necessary facilities for the study and Mr. T. Singh and Mr. Surender Singh for technical help.
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