Evaluation of 99mTc-labeled photosan-3, a hematoporphyrin derivative, as a potential radiopharmaceutical for tumor scintigraphy

Nuclear Medicine & Biology, Vol. 27, pp. 419 – 426, 2000 Copyright © 2000 Elsevier Science Inc. All rights reserved.

ISSN 0969-8051/00/$–see front matter PII S00969-8051(00)00092-5

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. Chauhan3 and R. K. Sharma1 1

DEPARTMENT OF RADIOPHARMACEUTICALS, INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCES, LUCKNOW MARG,

DELHI, INDIA; 2DEPARTMENT OF RADIATION BIOLOGY, INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCES, LUCKNOW MARG, DELHI, INDIA; AND 31B/5A ASHOK VIHAR, 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 tissue distribution in a murine tumor model. The 99mTc-Photosan-3, which was prepared by using 99mTc-pertechnetate in place of reduced 99mTc, demonstrated better labeling efficiency (>90%) and reproducibility. The procedure also minimized radiation exposure to the radiochemist because handling time was considerably reduced. Due to the commercial availability of Photosan-3, 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 was preferentially taken up by the neoplastic tissue similar to the parent compound. In addition to its applications in tumor imaging, 99mTc-Photosan-3 could also be used for estimating tumor uptake of Photosan-3 as may be required for individualization of clinical protocols of PDT. NUCL MED BIOL 27;4:419 – 426, 2000. © 2000 Elsevier Science Inc. All rights reserved. KEY WORDS.

99m

Tc-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, especially melanomas (5, 15, 21). Among these, the hematoporphyrin derivative (Hpd) attracted the most attention of clinical researchers because of its 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 light-accessible 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 a Hpd fluorescence imaging technique has been restricted due to the invasiveness of the endoscopic 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 half-life gamma emitters were suggested to be better alternatives for tumor detection (1, 3, 8, 14, 16). Initially, efforts were made to label Hpd with 57Co and 64Cu, but lack of significant Address correspondence to R. K. Sharma, M.Pharm, Ph.D., Head, 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 18 February 2000.

tumor uptake did not permit imaging (1, 3). The porphyrin analogues coordinated with 57Co (16) and 109Pd (7), although accumulated in murine tumor models, 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-tobatch variation in the percent fraction of reduced/hydrolyzed 99mTc was observed. The same group further demonstrated that 111Inlabeled Hpd localized in murine breast tumors (19, 20). In comparison to 99mTc-labeled Hpd, the 111In-labeled product concentrated substantially in liver and spleen and appreciably more in malignant neoplasm. Lavallee and Fawwaz (13) also reported a convenient method of labeling 111In with the Hpd, but it did not gain widespread clinical use because 111In, being a cyclotron product, was not easily available at most of 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

Tc-pertechnetate

99m

Tc-pertechnetate was eluted from 99Mo-99mTc generator by solvent extraction method. 99Mo was supplied by BRIT (Mumbai, India).

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TABLE 1. Migration values (Rf) of 99mTc-pertechnetate, reduced/hydrolyzed (R/H) 99mTc, and 99mTc-Photosan-3 as determined by ascending instant thin layer chromatography with (SG) using different solvent systems

Radiochemical Purity

Rf value Solvent system 0.9% Sodium chloride Acetone:ethyl acetate:water: ammonia (7:3:3:0.3, v/v)

Free 99m Tc R/H 1.0 1.0

99m

Tc99m Tc Photosan-3 0.0 0.0

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.

0.0 1.0

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 physiologic 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) 99m Tc and free 99mTc-pertechnetate.

Radiolabeling One milligram of Photosan-3威 (gift of Dr. R. Baumgartner, Seehof Laboratorium GmbH, Munich, Germany) was thoroughly dissolved in 1 mL water for injection and 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 Chemical Co., St. Louis, MO USA) in 50 ␮L of 0.1 N HCl was added and pH was adjusted to 7.0. The contents were passed through 0.22 ␮M filter (Millipore Corporation, Bedford, MA USA) into an evacuated sterile sealed vial. One to two milliliters of sterile 99mTc-pertechnetate (75– 400 MBq) was added

FIG. 1. Effect of pH on labeling efficiency of 99mTc-Photosan-3.

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 physiologic 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 withdrawn blood samples at different time intervals, which were subjected to ITLC.

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Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy

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FIG. 2. Effect of amount of stannous chloride dihydrate on the labeling efficiency of 99mTc-Photosan-3.

Blood Clearance and Plasma Protein Binding 99m

Blood clearance of Tc-Photosan-3 was studied in rabbits. Forty MBq of the radiolabel were 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 a fraction of total activity in the sample.

Biodistribution In vivo distribution of 99mTc-Photosan-3 was studied in 2–3-monthold Balb/c mice. Fifty microliters of 99mTc-Photosan-3 (40 KBq) was administered through the tail vein to each mice weighing 25–30 g. The animals were sacrificed at different time intervals and different 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 mice by injecting 2.5 ⫻ 107 cells intraperitoneally. The cells were harvested the seventh day after administration. These ascites cells were subcutaneously inoculated in soft tissue to produce solid tumors. In our study, 0.2 mL ascites fluid containing 1.5 ⫻ 107 cells was subcutaneously injected in the right thigh of mice (strain A). After 6 –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 the 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,

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FIG. 3. In vitro and in vivo stability of 99mTc-Photosan-3.

which successfully resolved labeled product from R/H and free 99m Tc (Table 1). The labeled preparations comprised 90% of the 99m Tc-Photosan-3, the rest being R/H 99mTc (6%) and free 99mTc (4%). To achieve optimum labeling efficiency, the pH of the reaction mixture was varied from 6.5 to 8.0, while the rest of the factors were kept constant. Similarly, the amount of stannous chloride dihydrate was varied from 12.5– 400 ␮g, while the pH, amount of Photosan-3, and volume of the reaction were kept 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 99mTcpertechnetate fractions were very high, whereas at pH 8.0, although the percentage of free 99mTc 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 ␮g to 400 ␮g, while keeping other conditions constant, are shown in Figure 2. At low concentrations of stannous chloride dihydrate, the fraction of free 99m Tc was greater, whereas at high concentrations the R/H 99mTc increased to 40%. Fifty micrograms of SnCl2 䡠 2H2O and a pH of 7.0 yielded the highest labeling efficiency of 99mTc-Photosan-3. 99m Tc-Photosan-3 complex was fairly stable in vitro up to 2 h but the stability deteriorated gradually thereafter, and at 24 h 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 h, 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 h postadministration, 18% of the injected radioactivity was present in blood, which reduced to 3.5% by 24 h. In vivo protein binding was 84% at 30 min, reaching to 88% 2 h postadministration of the complex (Figure 5). The protein binding reduced to 71% at 24 h. In vitro protein binding was maximum at 30 min (75.6%) and it reduced to 60% at 24 h of incubation. The biodistribution data of 99mTc-Photosan-3 in 2–3-month-old Balb/c mice is summarized in Table 2. Based on the percent injected dose per whole organ, the highest uptake of 99mTc-Photosan-3 was found in liver, blood, kidneys, intestines, and muscle, averaging 23.8, 9.01, 4.34, 4.65, and 3.8%, respectively, 1 h postadministration. Very little uptake of the radiotracer was seen in the lungs. The radioactivity in all the organs except liver was reduced considerably at 24 h. Scintigraphy in mice bearing EAT after administration of 99mTcPhotosan-3 showed a very early uptake at 2 h; however, maximum tumor to muscle ratio of 7.9 was achieved 4 h postadministration (Fig. 6). The ratio slowly decreased subsequently, showing a slight rise at 24 h. DISCUSSION Hematoporphyrins and other photosensitizers have been used for fluorescence detection of tumors using endoscopy (4, 22). However,

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Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy

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FIG. 4. Blood clearance of 99mTcPhotosan-3 in rabbit (average data of 2 rabbits).

radioactive porphyrins were predicted to be a better alternative for tumor detection than was 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 with 99mTc and demonstrated its detection and localization in tumors. Radiolabeling of Photosan-3 was carried out by adding 99mTc-pertechnetate instead of reduced 99mTc, as was reported by Wong’s group (17, 18). The earlier method used for labeling Hpd with 99mTc involved the addition of stannous reduced 99m Tc, which produced a complex with a major fraction of reduced 99m Tc. 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 procedure that can be completed within 30 min and 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 an 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 stannous concentration and pH are quite narrow. We have critically studied the stability of the radiopharmaceutical, its blood kinetics, its biodistribution, its tumor specificity, 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, which is 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, whereas 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 approx-

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FIG. 5. In vitro and in vivo plasma protein binding of 99mTc-Photosan-3 expressed as a fraction of total radioactivity in the sample.

imately 2–3 h. There is no evidence of in vivo dissociation of the complex. The biodistribution data suggest that 99mTc-Photosan-3 is excreted through kidneys and reticulo-endothelial system. The efficacy of 99mTc-Photosan-3 as a tumor-imaging agent was investigated in a murine tumor model. Radioactivity was found mainly in the abdominal region and corresponded with the tissue biodistribution data. The tumor scintigraphy data clearly demonstrated that

TABLE 2. Tissue distribution in normal mice at 1, 3, and 24 h after intravenous administration of 99mTc-Photosan-3 Percent injected dose/whole organ Organ Blood Heart Lungs Liver Kidneys Intestines Stomach Muscle

1h

3h

24 h

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 5 animals.

99m

Tc-Photosan-3 localizes in tumor with good tumor to background ratio. The early uptake by the tumor at 2 h postadministration and maximum tumor to muscle ratio of 7.9 at 4 h postadministration proves that it is a successful tumor marker. Small vascular tumors (approximately 0.9 cm3) concentrated more of radiolabeled Photosan-3 than did the larger ones (data not shown). In large tumors (approximately 2 cm3) with potential necrosis and limited blood supply, early (2 and 4 h) scans showed only faint localization, which disappeared in the delayed images (24 h). Imaging studies in viable tumor with 99mTc-Photosan-3 indicated that a late delayed scan is preferred. A delay of more than 10 h may be needed to minimize background radioactivity and to allow continuous build-up 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 h postadministration was in agreement 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 hyper-intense area of the liver. Other than liver and implanted tumor, no other major internal organs and bone marrow were notably visible in the images.

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Tc-Photosan-3, a Promising Agent for Tumor Scintigraphy

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FIG. 6. Whole-body scintigrams of Ehrlich ascites tumor-bearing mice obtained at 4, 8, 16, and 24 h postintravenous administration of 4 MBq of 99mTc-Photosan-3.

Preliminary results from biodistribution studies and imaging demonstrate that radiolabeled Photosan-3, like its 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 by the tumor are not clear, but the rate of its uptake could be a function of its hydrophilicity. The specificity of 99mTcPhotosan-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 the commercial availability of standardized Hpd in the form of Photosan-3, more reliability is ensured, resulting in better reproducibility of the radiopharmaceutical preparation. Before accepting it

for clinical use, several other parameters require detailed investigations. Intratumoral distribution, dependence of accumulation on the tumor volume, optimum time scheduling for the best image, specificity, and selectivity of the tumor uptake over infections or inflammatory lesions remain the important factors for its clinical acceptance. 99m Tc-labeled Photosan-3 appears promising as a tumor-imaging agent because of its highly favorable tumor to blood and other organ ratio, which allows 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 of relevance in PDT as well, because it could be employed for prediction and quantitative measurement of the photosensitizer in tumor tissue for individualization of the clinical treatment protocols.

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The authors are grateful to the director of the INMAS for providing necessary facilities for the study and to Mr. T. Singh and Mr. Surender Singh for technical help.

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