J Clin Endocrin Metab. First published ahead of print August 14, 2007 as doi:10.1210/jc.2007-0402
APPLICATION OF 188Re AS AN ALTERNATIVE RADIONUCLIDE FOR TREATMENT OF PROSTATE CANCER FOLLOWING TUMORSPECIFIC SODIUM IODIDE SYMPORTER GENE EXPRESSION Michael J. Willhauck1, Bibi-Rana Sharif Samani1, Franz Josef Gildehaus2, Ingo Wolf3, Reingard Senekowitsch-Schmidtke3, Hans-Jürgen Stark4, Burkhard Göke1, John C. Morris5, Christine Spitzweg1 Department of Internal Medicine II1 and Nuclear Medicine2, Ludwig-Maximilians-University, Munich, Germany, Department of Nuclear Medicine, Technical University Munich, Germany3, Department of Carcinogenesis of the Skin, German Cancer Research Center, Heidelberg, Germany4, Division of Endocrinology, Mayo Clinic College of Medicine, Rochester, MN, USA5 "This is an un-copyedited author manuscript copyrighted by The Endocrine Society. This may not be duplicated or reproduced, other than for personal use or within the rule of “Fair Use of Copyrighted Materials” (section 107, Title 17, U.S. Code) without permission of the copyright owner, The Endocrine Society. From the time of acceptance following peer review, the full text of this manuscript is made freely available by The Endocrine Society at http://www.endojournals.org/. The final copy edited article can be found at http://www.endojournals.org/. The Endocrine Society disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties.”
Short title: NIS-mediated 188Re therapy in prostate cancer Key words: sodium iodide symporter, prostate cancer, PSA promoter, gene therapy, 131I therapy, 188Re therapy Word count: 3596; Figures: 5 Correspondence and reprint requests: Christine Spitzweg, MD, Klinikum Grosshadern, Medizinische Klinik II, Marchioninistrasse 15, 81377 Muenchen, Germany. Phone:49-89-7095-0; Fax: 49-89-7095-8887; E-mail:
[email protected] Disclosure Statement: The authors have nothing to disclose Grant support: This study was supported by grants to C. Spitzweg (Sp 581/3-2, Sp 581/4-1, 4-2 (Forschergruppe FOR-411 ‘Radionuklidtherapie’)) from the Deutsche Forschungsgemeinschaft, Bonn, Germany, by the FöFoLe-Program of the University Munich to M. J. Willhauck (FöFoLe-442), and by the Mayo Foundation Prostate Cancer SPORE grant (CA91956) to J. C. Morris.
Copyright (C) 2007 by The Endocrine Society
2 ABSTRACT Context: We reported recently the induction of iodide accumulation in prostate cancer cells (LNCaP) by prostate-specific antigen (PSA) promoter-directed sodium iodide symporter (NIS) expression that allowed a significant therapeutic effect of 131I. These data demonstrated the potential of the NIS gene as novel therapeutic gene, although in some extrathyroidal tumors therapeutic efficacy may be limited by rapid iodide efflux due to lack of iodide organification. Objective: In the current study, we therefore studied the potential of 188Re, as an alternative radionuclide, also transported by NIS, with a shorter half-life and higher energy beta particles than 131I. Results: NIS-transfected LNCaP cells (NP-1) concentrated 8% of the total applied activity of 188Re as compared to 16% of 125I, which was sufficient for a therapeutic effect in an in vitro clonogenic assay. Gamma camera imaging of NP-1 cell xenografts in nude mice revealed accumulation of 8-16% ID/g of 188Re (t1/2 biol. 12.9 h) which resulted in a 4.7-fold increased tumor absorbed dose (450 mGy/MBq) for 188Re as compared to 131I. After application of 55.5 MBq of 131I or 188Re, smaller tumors showed a similar average volume reduction of 86%, while in larger tumors volume reduction was significantly increased from 73% after 131I treatment to 85% after application of 188Re. Conclusion: While in smaller prostate cancer xenografts both radionuclides seemed to be equally effective after PSA-promoter-mediated NIS gene delivery, a superior therapeutic effect has been demonstrated for 188Re in larger tumors.
3 INTRODUCTION Because of the lack of curative therapy for metastatic disease, prostate cancer represents an important health issue as the second leading cause of cancer death in men (1, 2), which requires the exploration of innovative treatment strategies including gene therapy. Based upon the effective application of radioiodine that has been used for over 60 years in the management of follicular-cell derived thyroid cancer due to endogenous expression of the sodium iodide symporter (NIS), cloning of NIS has provided us with a promising suicide gene (3-5). As one of the oldest targets of molecular imaging and therapy, characterization of NIS as a novel therapeutic gene offers the possibility of NIS gene transfer into nonthyroidal tumors followed by radioiodine application. The dual function of NIS as a diagnostic and therapeutic gene thereby allows to easily monitor functional NIS expression by scintigraphic imaging before proceeding to the application of a therapeutic 131I dose – an essential prerequisite for the exact planning and controlling of gene therapy applications in the clinical setting. In order to ensure tumor specificity of radiation exposure, the application of tumorspecific promoters offers the ability to transcriptionally target NIS gene expression to tumor cells. In earlier studies we have reported the application of the PSA and probasin promoters to achieve prostate-specific iodide accumulation in the human prostatic adenocarcinoma cell line LNCaP in vitro and in vivo (6-10). Further, the amount of accumulated 131I was shown to be sufficiently high to selectively kill NIS-transfected LNCaP cells in vitro as well as in vivo in stably transfected LNCaP cell xenografts with an average tumor volume reduction of more than 95% after application of 3 mCi (111 MBq) 131I (6, 7). A similar therapeutic effect of 131I was observed after adenoviral in vivo NIS gene delivery using the replication-defective tissueunspecific Ad5-CMV-NIS (11). In preparation of a first phase I clinical study these data were confirmed in beagle dogs using Ad5-CMVNIS for local intraprostatic injection. Dosimetry calculations after application of 3 mCi 131I revealed an average absorbed dose to the prostate of 23 ± 42 cGy/mCi 131I indicating that a dose of 85 mCi 131I would be sufficient to obtain a target dose of 2000 cGy to the
prostate (12). These studies clearly showed for the first time that tissue-specific NIS gene expression into nonthyroidal tumors might represent an effective and potentially curative therapy for tumors without endogenous iodide accumulation. In the thyroid gland thyroid peroxidase (TPO)-catalyzed oxidation and incorporation of trapped iodide into tyrosyl residues along the thyroglobulin backbone, a process called iodide organification, increases the retention time of accumulated radioiodide (5), thereby increasing the therapeutic effect of 131I due to prolongation of its effective half-life. Since extrathyroidal tissues are generally not able to organify iodide after NIS gene transfer, therapeutic efficacy of 131I can be limited by rapid iodide efflux. The application of alternative radioisotopes also transported by NIS with a shorter physical half-life and decay properties superior to 131I may provide a powerful means to enhance therapeutic efficacy of NIS-targeted radionuclide therapy in the presence of a limited effective half-life of 131I. In addition to I-, NIS has been demonstrated to transport other structurally similar anions like ClO3-, SCN-, SeCN-, NO3-, Br-, TcO4- as well as ReO4- (13, 14). 188 Rhenium (188ReO4-) represents a betaemitting radionuclide with a short physical half-life (16.7 h), that has recently been used in a variety of therapeutic applications in humans, including cancer radioimmunotherapy, palliation of skeletal bone pain, and endovascular brachytherapy after angioplasty (15-23). Due to its higher energy and shorter physical half-life as compared to 131I, administration of 188Re offers the possibility of higher energy deposition in a shorter time period. In the current study we therefore examined accumulation and therapeutic efficacy of 188Re in direct comparison to radioiodine in our prostate cancer model following PSA promoter-driven NIS gene transfer in vitro and in vivo (6, 7). MATERIALS AND METHODS Stably transfected LNCaP cell lines Generation of the stably transfected LNCaP cell lines NP-1 (NIS/PSA-pEGFP-1) and P-1 (PSA-pEGFP-1) has been performed as described previously (6, 7). Cell culture
4 LNCaP cells were grown in RPMI 1640 medium with 10% fetal bovine serum supplemented with L-glutamine, pen/strep and 200μg/ml geneticin (Invitrogen) and maintained at 37° C and 5% CO2. Cell viability assay Cell viability was measured using the commercially available MTS-assay (Promega Corp., Mannheim, Germany) according to the manufacturer’s recommendations as described previously (9). Radionuclide uptake studies in vitro Uptake of 188Re and 125I was determined according to the method by Weiss et al. (24) as described previously (7). Radionuclide uptake studies were performed in HBSS supplemented with 10 μM NaReO4 or 10 μM NaI, 0.1 μCi (3.7 MBq) Na188Re or Na125I/ml, and 10 mM HEPES at pH 7.3. Results were normalized to cell survival measured by cell proliferation assay (see above) and expressed as cpm/A490 nm. Radionuclide efflux studies in vitro Radionuclide efflux was determined according to the method by Weiss et al. (24) as described previously (9). In vitro clonogenic assay LNCaP cells stably transfected with the expression vector (NP-1) or the control vector (P-1) were incubated for 7 h with 29.6 MBq (0.8 mCi) 188Re in HBSS supplemented with 10 μM NaReO4 and 10 mM HEPES at pH 7.3 and 37 °C. After incubation with 188Re, a clonogenic assay was performed as described previously (6, 25). Establishment of LNCaP cell xenotransplants Xenotransplants derived from NP-1 (right flank) and P-1 (left flank) were established in male CD-1 nu/nu mice (Charles River Labs. Sulzfeld, Germany) as described previously (6). The experimental protocol was approved by the regional governmental commission for animal protection (Regierung von Oberbayern). Radionuclide uptake studies in vivo Eight to ten weeks after s.c. injection of cells (tumor diameter ~10 mm), mice were switched to a low-iodine diet and received T4 supplementation (5 mg/l) in their drinking water for 2 weeks to maximize radioiodine
uptake in the tumor and reduce uptake by the thyroid gland. After i.p. injection of 111 MBq (3 mCi) 188Re or 18.5 MBq (0.5 mCi) 123I, radionuclide imaging was performed using a gamma camera (Forte, ADAC Laboratories) equipped with a medium energy (MEAP) collimator (188Re) and an ultra high resolution (VXHR) collimator (123I). Regions of uptake have been quantified and expressed as a fraction of the total amount of applied radionuclide. Radionuclide retention time in the tumor was determined by serial scanning (0.25, 0.5, 1, 3, 5, 10, 16, 24, 48 h p. i.). Dosimetric calculations were performed using OLINDA/EXM (26) (supplemental data). Radionuclide therapy studies in vivo Xenografts of NP-1 and P-1 cells were established in six groups of mice (each group n= 6). Two groups of mice were administered 55.5 MBq (1.5 mCi) 131I or 55.5 MBq (1.5 mCi) 188Re by a single i.p. injection after 8–10 weeks of tumor growth (tumor volume > 0.2 cm³), and two other groups of mice were administered 55.5 MBq of either 131I or 188Re after 4–6 weeks of tumor growth (tumor volume
