Melanoma Therapy via Peptide-Targeted A-Radiation

Purpose:The therapeutic efficacy of a unique melanoma-targeting peptide conjugated with an in vivo generated a-particle-emitting radionuclide was evaluated in the B16/F1mouse melanoma animal model. a-Radiation is densely ionizing, resulting in high concentrations of destructive radicals and irreparable DNA double-strand breaks. This high linear energy transfer overcomes radiation-resistant tumor cells and oxygen effects resulting in potentially high therapeutic indices in tumors such asmelanoma. Experimental Design:The melanoma targeting peptide, 1,4,7,10-tetraazacyclodecane-1,4,7,10tetraacetic acid (DOTA)-Re(Arg)CCMSH, was radiolabeled with Pb, the parent of Bi, which decays via a and h decay. Biodistribution and therapy studies were done in the B16/F1 melanoma-bearing C57mouse flank tumor model. Results: Pb[DOTA]-Re(Arg)CCMSH exhibited rapid tumor uptake and extended retention coupled with rapid whole body disappearance. Radiation dose delivered to the tumor was estimated to be 61cGy/ACi Pb administered. Treatment of melanoma-bearing mice with 50, 100, and 200 ACiof Pb[DOTA]-Re(Arg)CCMSHextended their mean survival to 22, 28, and 49.8 days, respectively, comparedwith the14.6-daymean survivalof theplacebo control group. Fortyfive percent of the mice receiving 200 ACi doses survived the study disease-free. Conclusions: Treatment of B16/F1murine melanoma ^ bearing mice with Pb[DOTA]Re(Arg)CCMSH significantly decreased tumor growth rates resulting in extendedmean survival times, and in many cases, complete remission of disease. Pb-DOTA-Re(Arg)CCMSH seems to be a very promising radiopharmaceutical for targeted radionuclide therapy of melanoma. The incidence and mortality rates associated with melanoma have increased by 3% to 7% in recent years (1, 2). Early melanoma tumor diagnosis and prompt surgical removal are a patient’s best hope for a cure. Unfortunately, metastatic melanoma is resistant to current chemotherapy and immunotherapy regimens. Survival times for patients with lymph node metastases average 12 to 15 months, whereas patients with liver and bone metastases average 3 to 4 months (1). Despite extensive research, there have been no major improvements in advanced melanoma treatment outcomes during the past 30 years. Targeted radionuclide therapy, namely a-particle therapy, is a potentially important alternative to conventional therapeutic regimens. In comparison with external beam radiation therapy and chemotherapy, targeted radionuclide therapy offers the potential of tumor-selective radiotherapeutic treatment of distal metastases whereas sparing normal tissues and organs. a-Particles are doubly-charged helium ions with energies of 5 to 9 MeV characterized by high linear energy transfer over short path lengths (30-90 Am; refs. 3, 4). Only a few a-particle traversals per cell are necessary to cause irreversible damage resulting in cell death (5). Cytotoxicity of a-particle radiation is independent of dose rate (4) and unaffected by tissue oxygen levels (6), which allow tumors with hypoxic regions to be effectively irradiated. The short range and high ionization density associated with a-particles contribute to highly specific destruction within tumors, whereas minimizing collateral damage of healthy tissues. Promising preclinical a-particle radioimmunotherapy results with leukemia (7, 8) lung (9), ovarian (10), and prostate cancers (11) have been shown. In addition, early clinical trials have highlighted the potential of Bi and At labeled immunoconjugates for a-radiotherapy (12, 13). Over the past several years, our laboratory has developed a novel class of metal-cyclized melanotropin peptide analogues for melanoma imaging and therapy that target the melanocortin-1 receptor, which is overexpressed on melanoma tumor cells (14–16). Incorporation of a rhenium metal atom into the structure of the cyclic melanotropin peptide resulted in resistance to chemical and proteolytic degradation in vivo , whereas retaining high bioactivities. In this study, we report the melanoma therapeutic effects of targeted a-particle Cancer Therapy: Preclinical Authors’ Affiliations: Departments of Biochemistry, Internal Medicine, Radiology, and Veterinary Pathobiology, University of Missouri-Columbia, Harry S. TrumanVeterans Administration Hospital, Columbia, Missouri; AlphaMed, Inc., Acton, Massachusetts; and Pacific Northwest National Laboratory, Richland, Washington Received 3/18/05; revised 5/4/05; accepted 5/11/05. Grant support: NIH grant R42-CA85106 (T.P. Quinn), T32-RR07004 (C.T. Winkelmann) and U.S. Department of Energy grant ER61661 (T.P. Quinn). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Thomas P. Quinn, 117 Schweitzer Hall, Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211. Phone: 573882-6099; Fax: 573-884-4812; E-mail: [email protected]. F2005 American Association for Cancer Research. www.aacrjournals.org Clin Cancer Res 2005;11(15) August1, 2005 5616 Cancer Research. on October 15, 2017. © 2005 American Association for clincancerres.aacrjournals.org Downloaded from radiotherapy using the 1,4,7,10-tetraazacyclodecane-1,4,7,10tetraacetic acid (DOTA)–conjugated melanotropin analogue, DOTA-Re(Arg)CCMSH (17), radiolabeled with Pb. High tumor to normal tissue uptake ratios of Pb[DOTA]Re(Arg)CCMSH coupled with rapid whole body disappearance of activity, resulted in large and selective radiation doses to the tumors. Treatment of melanoma-bearing mice with Pb[DOTA]-Re(Arg)CCMSH yielded a dose-dependent reduction in tumor growth and total eradication of many tumors at higher activity levels. Despite being classified as a radiation-resistant neoplasm, melanoma was efficaciously treated by peptide-targeted a-radiation in tumor-bearing mice, highlighting the clinical potential of targeted a-radiotherapy for disseminated melanoma. Materials andMethods Synthesis of Pb-DOTA-Re(Arg)CCMSH. DOTA-Re(Arg)CCMSH, was synthesized and purified by a method previously described (17). DOTA-Re(Arg)CCMSH was radiolabeled with Pb obtained from a Ra-Pb/Bi radioisotope generator (ref. 18; AlphaMed, Inc., Acton, MA). Preferential elution of Pb was done by rinsing the generator column with 0.5 N HCl to remove the majority of Bi and its daughters followed by a 2 N HCl rinse to elute the Pb along with any remaining Bi. Shortly following the time of elution, Pb was in dynamic equilibrium with Bi and its daughters. DOTARe(Arg)CCMSH was radiolabeled by combining 100 to 350 AL of the generator eluant, 85 AL of 5 mol/L NaOH, 250 AL of 0.5 mol/L NH4OAc (pH 5.4), and 25 AL of 1 mg/mL DOTA-Re(Arg )CCMSH into a reaction vial and incubated at 75jC for 40 minutes. Pb-DOTARe(Arg)CCMSH was purified to a single species using HPLC with a C-18 reverse phase analytic column. Prior to the HPLC collection, 25 mg of L-ascorbic acid was added into the collection vial to minimize the radiolysis of Pb-DOTA-Re(Arg)CCMSH. The radioactivity of Pb-DOTA-Re(Arg)CCMSH preparation was determined immediately after HPLC purification in a Capintec CRC-15R dose calibrator calibrated with a standardized Pb sample obtained from Pacific Northwest National Laboratory (Richland, WA). Purified peptide solutions were purged with nitrogen gas for 20 minutes to remove the acetonitrile and adjusted to pH 5 with 0.9% NaCl. Animal studies. Animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. Pharmacokinetic and therapy studies were done in C57 black mice bearing B16/F1 murine melanoma tumors. Melanocortin-1 receptors on B16/F1 murine melanoma and human melanoma have indistinguishable affinities for the radionuclide targeting peptide (16), suggesting that the results obtained in the B16 melanoma model will be translatable to human melanoma models and potential clinical use. Mice were inoculated s.c. with 1 10 B16/F1 murine melanoma cells in the right flank for biodistribution studies. When the weight of tumors reached f0.2 g, 0.16 to 4.40 ACi of Pb-DOTA-Re(Arg)CCMSH was injected into each mouse through the tail vein. Groups of four mice per each time point were used for the biodistribution studies. The mice were sacrificed at 5 and 30 minutes, and 1, 2, 4, 24, and 48 hours postinjection, and tumors and organs of interest were harvested, weighed and counted in a Wallac 1480 automated gamma counter using the following energy windows, Pb/190 to 260 keV and Bi/ 420 to 810 keV. Blood values were taken as 6.5% of the whole body weight. The results were expressed as a percentage of the injected dose per gram (%ID/g) and as the percentage of injected dose (%ID). The tumor uptake specificity of Pb-DOTA-Re(Arg)CCMSH was determined by blocking tumor uptake at 2 hours postinjection with the coinjection of 10 Ag of unlabeled NDP (19), a linear a-MSH peptide analogue with picomolar affinity for the a-MSH receptor present on murine melanoma cells. Dosimetry studies. The biodistribution of Pb-DOTA-Re(Arg)CCMSH over time was determined to calculate radiation absorbed doses from Pb-DOTA-Re(Arg)CCMSH in tumor and normal organs and tissues using methods described previously (20–22). Time-activity curves were generated for 13 organs and tissues (blood, brain, heart, lung, liver, spleen, stomach, kidney, large intestine, small intestine, muscle, pancreas, and tumor). Cumulative activities of Pb and Bi were determined for each organ by integrating the area under the time-activity curves. The cumulative activities were then used with a dosimetric model (21, 22) developed specifically for the laboratory mouse, accounting for the aand h-radiation deposited locally. The dosimetric model is used to evaluate dose from activity within tissues as well as the cross-organ h dose contributions. Radionuclide therapy of Pb-DOTA-Re(Arg)CCMSH. The therapeutic efficacy of Pb-DOTA-Re(Arg)CCMSH was examined in B16/ F1 murine melanoma–bearing C57 mice. C57 mice were inoculated s.c. with 1 10 B16/F1 murine melanoma cells in the right flank. Palpable dark melanoma tumors were observed 3 days following tumor cell inoculation. Three treatment groups of 8 to 10 mice were administrated single doses of 50, 100, and 200 ACi of Pb-DOTA-Re(Arg)CCMSH through the tail vein on the 4th day after tumor cell implantation. An untreated tumor control group received 100 AL of normal saline. After the administration of the therapeutic infusion, tumor size, body weight, and animal body condition were determined daily. Tumor volume was calculated by measuring the length, width, and depth of the tumors with a caliper and using the following formula: tumor volume = (length width depth) k / 6. Mice were removed from the therapy study and sacrificed if body weight loss was >20% of initial body weight, tumor size exceeded 1.0 cm, or the appearance of skin ulcerations at the tumor site. The total study period was 120 days, which was 10 times the average survival period of untreated tumor-bearing animals. Toxicity of PbDOTA-Re(Arg)CCMSH to the kidneys of 100 and 200 ACi treatment groups was evaluated by pathologic examination after completion of the therapy study. The kidney and tumor site skin biopsies from therapy animals were examined by a veterinary pathologist at the University of Missouri School of Veterinary Medicine Research Animal Diagnostic Laboratory. Kaplan-Meier survival curves were obtained by using SPSS software (SPSS Inc., Chicago, IL). Statistical analysis was done using Student’s t test for unpaired data. A 95% confidence level was chosen to determine the significance between untreated and treated groups, with P < 0.05 being significantly different.