Patent Publication Number: US-7709613-B2

Title: Rhenium-188 and rhenium-186 for treatment of tumors expressing a Na+/I− symporter

Description:
PRIORITY CLAIM 
     This application claims the benefit of U.S. Provisional Application No. 60/427,150, filed Nov. 18, 2002, the content of which is hereby incorporated by reference in its entirety into the subject application. 
    
    
     STATEMENT OF GOVERNMENT SUPPORT 
     The invention disclosed herein was made with U.S. Government support under grant numbers R01CA70897, R01CA75503, R01CA77552, and 5-P30-CA13330-26 from the National Institutes of Health, U.S. Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the use of Rhenium radioisotopes to detect and treat tumors that expresses a Na + /I −  symporter. 
     BACKGROUND OF THE INVENTION 
     Throughout this application various publications are referred to in parenthesis or brackets. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains. 
     Breast cancer remains the major cause of cancer death in women in the developed world. Novel therapeutic modalities are needed for patients with tumors resistant to conventional therapies such as chemotherapy, hormonal treatment and external radiation. Recently Tazebay et al. [31, 41] found that more than 80% of human mammary cancers, but not normal healthy non-lactating breast tissue, express a sodium/iodide symporter (NIS) which was named the mammary gland NIS (mgNIS). Kilbane et al. [11] found NIS expression to be a feature of both fibroadenomata and breast carcinoma tissues. 
     NIS mediates iodide accumulation in the thyroid gland [2], and the capability to concentrate and organify iodide allows the use of radioactive iodine isotope  131 I for the treatment of differentiated thyroid cancers and hyperthyroidism [10, 24]. However, as pointed out by Daniels and Harber [5], organification of iodide is unlikely to occur in breast cancer cells as the thyroid is the only organ known to organify iodide, a process that involves the conversion of inorganic iodide to an organic form by conjugation to tyrosine residues on the protein thyroglobulin, a precursor to iodinated forms of thyroid hormone [30]. The organification process causes radioiodine to be retained within the thyroid gland for several days [16]. This relatively long retention time matches the physical half-life of  131 I (8 days) and allows a significant radiation dose to be delivered to the tumor. 
     NIS gene expression has been detected in several tissues in humans, including the thyroid gland, mammary gland, salivary glands, and gastric mucosa [43]. 
     Several studies have reported transfection of NIS into different non-thyroid or undifferentiated thyroid tumors for the purpose of subsequent therapy with  131 I [1, 3, 15, 17, 21, 27]. In all of these studies, although  131 I uptake in NIS-expressing tumors was substantial (up to 27% injected dose in [27]), the residence times of  131 I in the tumors were relatively short and no tumor shrinkage was observed [1, 3, 27]. In a recent report by Spitzweg et al. [28], impressive therapeutic results were seen in NIS-transfected prostate tumor xenografts in mice when treated with a very high single 3 mCi dose of  131 I. However, as no biodistribution and no dose-escalation studies were reported, it is unclear why such high dose was administered. The lack of therapeutic gains observed by other investigators [1, 3, 27] can be attributable to the long physical half-life (8 days) and decay properties of  131 I, as the beta-particles emitted by  131 I are low energy (E average =0.134 MeV) and have an optimal tissue range of only 2.6-5.0 mm [22]. 
     Several different approaches to circumvent the problem of insufficient iodide radiation dose to NIS-expressing tumors have been suggested. Daniels and Haber [5] and Nakamoto [21] suggested that pharmacologic modulation of fast cellular radioiodide efflux from breast cancer cells might be possible by administering lithium salts which increase radioiodide half-life in thyroid tumors. Boland et al. [1] proposed to improve the efficiency of NIS gene transfer and thus the iodide uptake capacity of the target tissue by the use of modified vectors and/or higher viral doses. The same authors also proposed to increase the biological half-life of radioiodide in the tumor tissues by coupling transfer of the NIS gene with the delivery of a gene involved in the iodide organification process, such as thyroperoxidase. Such an approach is not easy to implement because of the inherent complexity of gene therapy procedures especially in the case of 2-gene transfer and the difficulty in transfecting only the target tissue (tumor) in vivo. 
     A short-lived isotope of technetium  99m Tc is used in ˜90% of all diagnostic nuclear medicine procedures [e.g., 42]. It has long been recognized by nuclear medicine practitioners that due to their common ionic characteristics, iodide and  99m Tc-pertechnetate ( 99m TcO 4   − ) behave similarly following intravenous administration [23]. Like iodide,  99m TcO 4   −  localizes in the thyroid, salivary glands, gastric mucosa, and choroid plexus of the brain. It is trapped but not organified in the thyroid gland and is used in nuclear medicine as an alternative to Na 123 I for assessing thyroid condition. 
     Rhenium is a chemical analogue of technetium and exhibits practically identical chemical and biodistribution properties [6]. 188-Rhenium ( 188 Re), a powerful beta-emitting radionuclide (E average =0.764 MeV) with a 16.7 hour half-life has been recently used in a number of therapeutic applications in humans including cancer radioimmunotherapy, palliation of skeletal bone pain, and endovascular brachytherapy to prevent restenosis after angioplasty [8, 12, 25], as well as in the pre-clinical development of novel therapeutics [4, 19]. 186-Rhenium ( 186 Re), which has a half-life of 3.7 days and E average =0.362 MeV, is also being used in clinical applications [33-36]. Because of its chemical similarity to pertechnetate, the perrhenate anion ( 188 ReO 4   − ) is concentrated in thyroid and stomach [14]. Co-injected  99m TcO 4   − ,  125 I − , and  188 ReO 4   −  have similar uptake and biodistribution in NIS-expressing (thyroid, stomach, salivary gland) and non-expressing tissues in normal, healthy mice, with the exception of the thyroid gland where only  125 I −  is retained by organification [32]. In contrast, a study employing rat thyroid NIS expressed in  Xenopus laevis  oocytes found that ReO 4   −  is a potent blocking agent for NIS, second to only perchlorate (ClO 4   − ), and that ReO 4   −  is transported via NIS only to a very small extent and only when the concentration of ReO 4   −  is high [44]. Accordingly, the ability of NIS to mediate the transport of ReO 4   −  in general, and its potential for doing so in tumor cells in particular, have not been resolved. 
     SUMMARY OF THE INVENTION 
     The present application demonstrates that tumors expressing a Na + /I −  symporter uptake powerful beta-emitting Rhenium radioisotopes, a finding that is significant for the diagnosis and treatment of cancer. 
     The subject invention is directed to a method of treating a tumor that expresses a Na + /I −  symporter in a subject which comprises administering to the subject an amount of a Rhenium radioisotope effective to treat the tumor, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium, in particular  188 ReO 4   −  or  186 ReO 4   − . 
     The invention also provides a method for diagnosing the presence of a tumor that expresses a Na + /I −  symporter in a subject which comprises administering a Rhenium radioisotope to the subject&#39;s tumor and detecting the uptake of the Rhenium radioisotope by the tumor, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium, in particular  188 ReO 4   −  or  186 ReO 4   − . 
     The invention provides a method of making a composition for treating a tumor that expresses a Na + /I −  symporter in a subject, which comprises admixing a Rhenium radioisotope and a carrier, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium, in particular  188 ReO 4   −  or  186 ReO 4   − . The invention also provides a composition for treating a tumor that expresses a Na + /I −  symporter in a subject, which comprises a Rhenium radioisotope and a carrier, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium, in particular  188 ReO 4   −  or  186 ReO 4   − . 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Kinetic data on comparative  125 I −  and  188 ReO 4   −  accumulation in hNIS-transfected and non-transfected MDCK cells. 
         FIG. 2 . Comparative  125 I −  and  188 ReO 4   −  accumulation in hNIS-transfected and non-transfected MDCK cells as a function of radionuclide concentration. 
         FIGS. 3A-3E . Hematoxylin and eosin stained NIS-expressing tissues from control mice and mice treated with  188 ReO 4   −  or  131 I − : A) control thyroid, B) thyroid treated with 1.5 mCi  188 ReO 4   − , C) thyroid treated with 50 μCi  131 I − , D) control stomach, E) stomach treated with 1.5 mCi  188 ReO 4   − . 
         FIG. 4 . Treatment of nude mice bearing NAFA cells-xenografted tumors with  188 ReO 4   − . 
         FIGS. 5A-5B . Histological analysis for apoptosis of NAFA-cells xenografts in nude mice: A) control (untreated) tumor on day 35; B) tumor from a mouse treated with 2×1.5 mCi  188 Re-perrhenate, day 35 post-treatment. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The subject invention is directed to a method of treating a tumor that expresses a Na + /I −  symporter in a subject which comprises administering to the subject an amount of a Rhenium radioisotope effective to treat the tumor, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium. In different embodiments, the subject is treated using  188 ReO 4   −  or  186 ReO 4   − . 
     As used herein, the term “treat” a tumor means to eradicate the tumor, to reduce the size of the tumor, to stabilize the tumor so that it does not increase in size, or to reduce the further growth of the tumor. 
     In a preferred embodiment, the tumor is a breast tumor. In different embodiments, the tumor is a thyroid tumor or a gastrointestinal tract tumor. In one embodiment, the subject is not a lactating subject. 
     The Rhenium radioisotopes deliver a superior radiation dose to the tumor in comparison to radioactive iodine. The radiation dose that is delivered to the tumor using the Rhenium radioisotope is higher than the radiation dose that could be delivered to the tumor using an iodine radioisotope, where the concentration of the Rhenium radioisotope administered to the subject is the same as the concentration of the iodine radioisotope administered to the subject. Preferably, the radiation dose that is delivered to the tumor using the Rhenium radioisotope is at least four-fold higher than the radiation dose that could be delivered to the tumor using an iodine radioisotope, where the concentration of the Rhenium radioisotope administered to the subject is the same as the concentration of the iodine radioisotope administered to the subject. In one embodiment, the radiation dose that is delivered to the tumor using the Rhenium radioisotope is 4.5 times higher than the radiation dose that could be delivered to the tumor using an iodine radioisotope, where the concentration of the Rhenium radioisotope administered to the subject is the same as the concentration of the iodine radioisotope administered to the subject. 
     The Na + /I −  symporter can be transfected into tumor cells. In one embodiment, the tumor into which NIS is transfected is a prostate tumor. Methods for transfection of Na + /I −  symporter into tumors are well known in the art [1, 3, 15, 17, 21, 27, 28]. In one embodiment, the Na + /I −  symporter is transfected into tumor cells by direct injection into the tumor. Nucleic acid encoding the Na + /I −  symporter can be used, or Na + /I −  symporter protein can be used. 
     The Rhenium radioisotope can be administered in a single dose or in a fractionated manner. In different embodiments, the cumulative dose is between 0.5-500 mCi. 
     The invention also provides a method for diagnosing the presence of a tumor that expresses a Na + /I −  symporter in a subject which comprises administering a Rhenium radioisotope to the subject&#39;s tumor and detecting the uptake of the Rhenium radioisotope by the tumor, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium. In different embodiments, the Rhenium radioisotope is administered as  188 ReO 4 , or  186 ReO 4   − . Procedures for nuclear medicine imaging are well known in the art [23, 37]. The Rhenium radioisotope can be administered in vivo. The Rhenium radioisotope can also be administered to a tissue biopsy obtained from the subject. The tissue biopsy can be analyzed for the presence of a Na + /I −  symporter using immunohistochemistry [31, 41]. In a preferred embodiment, the tumor is a breast tumor and the subject is a non-lactating subject. 
     Gastric lavage can be used to decrease the binding of the Rhenium radioisotope to a Na + /I −  symporter in the gastric mucosa. Methods for gastric lavage are well known in the art [38]. Exogenous thyroid hormone can be administered to the subject to decrease the binding of the Rhenium radioisotope to a Na + /I −  symporter in the thyroid, using procedures that are well known in the art [39]. 
     The invention provides a method of making a composition for treating a tumor that expresses a Na + /I −  symporter in a subject, which comprises admixing a Rhenium radioisotope and a carrier, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium. In different embodiments, the Rhenium radioisotope is  188 ReO 4   −  or  186 ReO 4   − . The invention also provides a composition made according to this method. 
     The invention also provides a composition for treating a tumor that expresses a Na + /I −  symporter in a subject, which comprises a Rhenium radioisotope and a carrier, wherein the Rhenium radioisotope is 188-Rhenium or 186-Rhenium. In different embodiments, the Rhenium radioisotope is  188 ReO 4   −  or  186 ReO 4   − . 
     As used herein, the term “carrier” encompasses any of the standard pharmaceutical carriers, such as sterile isotonic saline, a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsions. 
     In one embodiment of any of the methods described herein, the subject is a mammal. In different embodiments, the mammal is a mouse, a rat, a cat, a dog, a horse, a sheep, a cow, a steer, a bull, livestock, a primate, a monkey, or preferably a human. 
     This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. 
     EXPERIMENTAL DETAILS 
     Methods and Materials 
     Radioisotopes.  125 I in the form of sodium iodide was purchased from NEN, Boston, Mass.  188 Re in the form of Na perrhenate Na 188 ReO 4  was eluted from a  188 W/ 188 Re generator (Oak Ridge National Laboratory (ORNL), Oak Ridge, Tenn.).  186 Re can be obtained through neutron irradiation of  185 Re in nuclear reactors, with high production levels achievable even in reactors with a moderate neuron flux. 
     Comparative  125 I and  188 Re uptake into the NIS-expressing cells. Both concentration dependence and kinetic experiments for  125 I and  188 ReO 4   −  in hNIS-transfected (hNIS in pcDNA 3.1 plasmid) and non-transfected MDCK (Madin-Darby canine kidney) cells were performed using the methods of Weiss et al. [45] and Dai et al. [46]. To initiate  125 I −  or  188 ReO 4   −  uptake, carrier-free Na 125 I or Na 188 ReO 4  was mixed with unlabeled NaI or NaReO 4 , respectively, to give a final concentration of 20 μM. The concentration of the NIS inhibitor Na perchlorate was 40 μM.  125 I − / 188 ReO 4   −  uptake was expressed as picomoles per μg DNA; the DNA was determined for each well as in Kissane and Robbins [47]. A 4-min time point was used to estimate initial rates for kinetic experiments. Saturation kinetics were analyzed by Lineweaver-Burk plot [48]. 
     Animal model. To investigate the binding of  125 I and  188 Re to NIS-expressing mammary tumors, a xenografted mammary adenocarcinoma model in nude mice was used. Since erbB-2 (Neu) is one of the better known human breast cancer oncogenes, a mammary tumor cell line (NAFA) induced by oncogenic Neu was used. The NAFA cell line derived from the MMTV-NeuT mouse [18] was grown in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin. Tazebay et al. previously demonstrated [31, 41] specific active iodide transport in mammary adenocarcinoma in MMTV-NeuT mice with  99m TcO 4   −  scintigraphy, confirmed by immunoblot analysis for mgNIS expression in Neu tumor. Female nude mice (Charles River Laboratories) 4-6 weeks old were injected SC with 10 6  NAFA cells into the left groin. Ten days after injection the tumors reached 0.5-0.7 cm in diameter. 
     All animal experiments were performed according to the protocol approved by the Animal Institute Committee, Albert Einstein College of Medicine. 
     Biodistribution. Each mouse was injected IV with a mixture of 18 μCi Na 188 ReO 4  and 1.7 μCi Na 125 I in 0.1 mL normal saline. In order to block mg-NIS in expressing organs and tumor, control animals were injected with the above mixture of isotopes combined with 2 mg NaClO 4 . At 20 minutes, 2 hours and 24 hours after administering radioactivity, the animals were sacrificed, and their organs and tumors were removed, weighed and counted in a gamma counter using the energy window for  188 Re which excludes  125 I emissions. One week later, after practically all  188 Re had already decayed, the organs were counted again in the  125 I energy window. The % ID/g for each organ was determined, and RIs (% ID/g in the tumor divided by the % ID/g in normal tissue) were calculated. 
     Dosimetry. The self-doses to major organs and tumors from  131 I and  188 Re in humans were calculated using MIRDOSE 3 software [29] (ORNL) applying S-values for a 70-kg reference adult. “S” is an absorbed dose per unit cumulated activity. S-values are tabulated in the Medical Internal Radiation Dose pamphlet #11 published by the Society of Nuclear Medicine. The following formula [26] was used for conversion of time-activity data in mice into time-activity data in humans:
 
%  ID  human organ=%  ID /g animal organ ·(body mass aninal /body mass human )·organ mass human .
 
     It should be noted that dosimetry calculations in humans were performed with the sole purpose of comparing the therapeutic efficacy and potential damage to the stomach and thyroid from  131 I and  188 Re beta-particles, thus only “self” doses were calculated. Besides, absorbed fractions for beta-particles are equal to zero in a volume larger than 1 cm 3 [ 23] and thus do not contribute significantly to the “cross-fire” effect in humans. However, in more comprehensive dosimetry calculations organ cross-irradiation from highly penetrative  131 I gamma-rays should be taken into consideration. 
     Statistics. Biodistribution results obtained from the same animal were compared using paired t-test; results obtained from different animals were compared using unpaired t-test. P values of &lt;0.05 were considered statistically significant. 
     Results 
     In vitro studies. The comparative uptake of  125 I −  and  188 Re was determined using NIS-expressing Madin-Darby canine kidney (MDCK) cells. Kinetic data on comparative  125 I −  and  188 ReO 4   − , accumulation in hNIS-transfected and non-transfected MDCK cells are presented in  FIG. 1 . It is obvious from these data that  188 ReO 4   −  is transported by NIS with 14-fold concentration gradient in comparison to non-transfected cells. The K m  (26.1 μM) determined for  125 I −  influx in MDCK cells ( FIG. 2 ) was quite close to the K m  value of 30 μM determined for  125 I −  influx in FRTL-5 cells. The K m  determined for  188 ReO 4   −  (12.6 μM) reveals an even higher affinity of NIS for  188 ReO 4   −  than for  125 I − . The higher affinity of  188 ReO 4   −  for NIS than those of such actively transported ions as ClO 3 — (K m =277) and NO 3   −  (K m =739) [44] may be due to the significantly smaller ionic radii of ClO 3   −  and NO 3   −  (1.66 Å and 1.45 Å, respectively) in comparison to those of  188 ReO 4   −  (1.88 Å) and  125 I (2.2 Å), which make ClO 3   −  and NO 3   −  fit too “loosely” into the binding region of the symporter. 
     In vivo studies. The uptake of  188 ReO 4   −  compared to  125 I −  was determined in mice bearing mgNIS-expressing mammary tumors. The biodistribution results are given in Table 1. The tumors exhibited increased uptake of  125 I −  and  188 ReO 4   −  in comparison with the tissues that do not express NIS (liver, muscle, spleen, kidney). The uptake in the tumors plateaued over the initial 2 hours and then decreased over a 24 hour period. There was no statistically significant difference between  125 I −  and  188 ReO 4   −  % ID/g tumor at 20 minutes (P=0.05), but at later time points  188 ReO 4   −  uptake was higher than that of  125 I −  (P=0.039 and 0.033 for 2 and 24 hour time points, respectively). At all 3 time points, co-administration of ClO 4   − , a powerful blocking agent for NIS, significantly decreased uptake of both isotopes by the stomach, salivary glands and thyroid and resulted in their rapid clearance, paralleling the blood pool. In tumors, ClO 4   −  co-administration caused brief but significant elevation of both isotopes uptake at 20 minutes. This can be explained by a combination of the higher concentration of  125 I −  and  188 ReO 4   −  in the blood due to their decreased uptake by stomach and thyroid (“sink” effect) and, possibly, by different diffusion rates of ClO 4   − ,  125 I −  and  188 ReO 4   −  in tumor capillaries. By 2 hours, tumors were showing the same trend as other NIS-expressing tissue, i.e., decreased uptake of  125 I −  and  188 ReO 4   −  in the presence of ClO 4   − . Without ClO 4   − , the thyroid continued to increase  125 I −  uptake through 24 hours, presumably due to organification, while  188 ReO 4   −  was almost completely washed out of the thyroid by 24 hours. 
     The tumor to normal tissue ratios (RIs) shown in Table 2 exceeded unity at 20 minutes for all non-NIS expressing organs for both  125 I −  and  188 ReO 4   −  and steadily increased at later time points. RIs for salivary glands were also higher than unity reflecting less NIS activity in these organs than in tumor. Lower than 1 tumor to blood ratios for 20 minutes and 2 hours may be explained by poor vascularization of the tumors which were encapsulated in fatty tissue with very few blood vessels as well as by relatively slow blood clearance of  125 I −  and  188 ReO 4   −  during the first 2 hours after injection. 
     The results of dosimetry calculations for  131 ′ and  188 ReO 4   −  administered to a reference 70-kg adult bearing a 2 g tumor are presented in Table 3. The dose delivered to the tumor by  188 ReO 4   −  is 4.5 times higher than by  131 I − . The doses delivered by  131 I −  and  188 ReO 4   −  to NIS-expressing tumors produce an estimated peak dose intensity of 0.1 Gy/h (10 rad/h) and are within the domain of low-dose rate radiation (LDR). In contrast, high-dose radiation (HDR), such as external-beam radiation therapy (RT), typically delivers 60 Gy/h (6000 rad/h) [20]. LDR has been shown to be effective in specific applications, such as radioimmunotherapy, through mechanisms such as cell cycle effects, induction of apoptosis, and tumor bed effects (death of adjacent, non-irradiated cells) [13]. 
     Using S-values for the whole body from  131 I −  and  188 ReO 4   − [ 29], the whole body dose from  188 ReO 4 — was estimated to be ˜2 times higher than from  131 I −  (0.71 rad/mCi) [23] which is generally very well tolerated. The dose to the stomach is higher for  188 ReO 4   −  but is still well within the tolerance level of this organ [7]. Also, methods of decreasing  188 ReO 4   −  binding to NIS in gastric mucosa, such as gastric lavage, can be employed if needed. Regarding the dose to the thyroid, it should be pointed out that while 0.93 rad/mCi from  188 ReO 4   −  is the maximum dose as there is no  188 ReO 4   −  present in the thyroid after 24 hours, 1.26 rad/mCi from  131 I −  within 24 hours is only a fraction of the actual dose to the thyroid, due to iodine organification, which is 1300 rad/mCi [23]. In order to protect the thyroid, exogenous thyroid hormone should be administered prior to  131 I treatment. 
     To establish the safe dose of  188 ReO 4   −  to treat the NIS-expressing tumors in mice, healthy mice were treated with 1.5 mCi  188 ReO 4   −  and observed for 80 days. No weight loss or other gastrointestinal toxicity was seen in treated animals. After 80 days the animals were sacrificed, their thyroids and stomachs removed and analyzed histologically.  FIGS. 3A-3C  present the hematoxylin and eosin stained thyroid tissue from a control mouse, a mouse treated with 1.5 mCi  188 ReO 4   − , and a mouse treated with 50 μCi  131 I − .  FIGS. 3D-3E  show the stomachs from the control mouse and the mouse treated with 1.5 mCi  188 ReO 4   − , respectively. The absence of damage by  188 ReO 4   −  to normal NIS-containing structures in the mouse can be explained by the ≦1 mm thickness of these tissues in comparison with the &gt;10 mm optimal tissue range of high-energy betas emitted by  188 ReO 4   − . These results are confirmed by the well-known tolerance of gastric mucosa in patients treated with high activities of  131 I −  and indirectly by recently published therapy results in patients treated with  90 Y (which is a high energy beta-emitter like  188 Re) labeled gastrin receptor ligands [40]. 
     A xenografted mammary adenocarcinoma model in nude mice was used to demonstrate the efficiency of treatment with  188 ReO 4   − . When the tumors reached 0.5-0.7 cm in diameter, the animals were treated with 1.5 mCi  188 ReO 4   −  followed by a 2 nd  dose of 1.5 mCi  188 ReO 4   −  one week later. Tumor growth stabilization was observed in treated mice in comparison with the aggressive growth of the untreated tumors ( FIG. 4 ). 
     At the completion of the experimental treatment with  188 Re-perrhenate, the animals were sacrificed, their tumors removed, fixed in buffered formaline, cut and analyzed for apoptosis using an Apoptag Plus® kit. Briefly, 3′-OH termini of DNA fragments, which are the hallmark of apoptosis, were labeled with digoxigenin-modified nucleotides by terminal deoxynucleotidyl transferase (TdT). This enzyme selectively detects apoptotic over necrotic cells. The labeled DNA was detected using an anti-digoxigenin antibody, and the detection was performed using a chromagen. As a result the apoptotic cells were stained brown. The results are shown in  FIGS. 5A-5B , where apoptotic cells appear lightly stained in the black-and-white image. The treated tumors ( FIG. 5B ) showed significantly higher number of apoptotic cells in comparison with control tumors ( FIG. 5A ) as well as the areas of necrosis. This observation is consistent with the fact that particulate ionizing radiation is known to cause cell death through both apoptotic [13] and necrotic pathways [49, 50]. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Biodistribution of  125 I −  and  188 ReO 4   −  in NIS-expressing 
               
               
                 xenografted mammary adenocarcinoma in nude mice. 
               
            
           
           
               
               
            
               
                   
                 ID/g, % (SD) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Time 
                 Organ 
                   125 I −   
                   125 I −  + ClO 4   − * 
                   188 ReO 4   −   
                   188 ReO 4   −  + ClO 4   −   
               
               
                   
               
               
                 20 min 
                 blood 
                  5.40 (1.68) 
                 10.15 (1.06) 
                  9.34 (3.08) 
                 19.79 (0.54) 
               
               
                   
                 spleen 
                  1.93 (1.60) 
                  0.60 (0.14) 
                  2.77 (1.77) 
                  2.10 (0.94) 
               
               
                   
                 liver 
                  1.83 (0.49) 
                  4.35 (0.64) 
                  3.05 (1.47) 
                  7.06 (1.82) 
               
               
                   
                 kidneys 
                  2.38 (0.53) 
                  6.50 (0.28) 
                  3.16 (1.31) 
                  8.58 (2.18) 
               
               
                   
                 muscle 
                  0.23 (0.06) 
                  0.60 (0.00) 
                  0.47 (0.13) 
                  1.02 (0.06) 
               
               
                   
                 stomach 
                 29.03 (16.31) 
                  3.45 (0.77) 
                 37.03 (16.09) 
                  5.05 (0.84) 
               
               
                   
                 salivary 
                  1.40 (0.85) 
                  0.65 (0.07) 
                  2.64 (0.25) 
                  1.51 (0.03) 
               
               
                   
                 glands 
                  5.10 (2.64) 
                  0.90 (0.14) 
                  7.47 (3.21) 
                  1.93 (0.10) 
               
               
                   
                 thyroid 
                  3.08 (0.68) 
                  5.85 (0.77) 
                  3.86 (0.97) 
                  9.23 (2.88) 
               
               
                   
                 tumor 
               
               
                  2 hr 
                 blood 
                  4.10 (0.65) 
                  7.85 (0.91) 
                  6.53 (0.61) 
                 11.10 (1.41) 
               
               
                   
                 spleen 
                  1.07 (0.99) 
                  2.35 (0.21) 
                  1.17 (0.70) 
                  2.09 (0.30) 
               
               
                   
                 liver 
                  1.37 (0.05) 
                  2.25 (0.36) 
                  2.44 (0.28) 
                  4.39 (0.16) 
               
               
                   
                 kidneys 
                  1.87 (0.21) 
                  3.75 (0.36) 
                  2.50 (0.29) 
                  4.45 (0.36) 
               
               
                   
                 muscle 
                  0.43 (0.31) 
                  0.30 (0.00) 
                  0.67 (0.35) 
                  0.41 (0.03) 
               
               
                   
                 stomach 
                 29.00 (8.13) 
                  3.60 (0.43) 
                 45.20 (10.32) 
                  3.47 (0.43) 
               
               
                   
                 salivary 
                  2.30 (1.19) 
                  0.50 (0.14) 
                  6.47 (2.10) 
                  0.86 (0.18) 
               
               
                   
                 glands 
                 21.10 (13.74) 
                  0.60 (0.14) 
                 13.98 (6.92) 
                  0.75 (0.07) 
               
               
                   
                 thyroid 
                  2.80 (0.91) 
                  1.05 (0.21) 
                  3.50 (0.56) 
                  1.35 (0.07) 
               
               
                   
                 tumor 
               
               
                 24 hr 
                 blood 
                  0.15 (0.07) 
                  0.02 (0.01) 
                  0.28 (0.10) 
                 0.005 (0.001) 
               
               
                   
                 spleen 
                  0.10 (0.00) 
                 0.010 (0.001) 
                  0.04 (0.01) 
                  0.00 (0.00) 
               
               
                   
                 liver 
                  0.04 (0.01) 
                 0.010 (0.001) 
                 0.050 (0.003) 
                 0.003 (0.001) 
               
               
                   
                 kidneys 
                  0.09 (0.01) 
                 0.010 (0.001) 
                  0.13 (0.03) 
                 0.001 (0.001) 
               
               
                   
                 muscle 
                  0.04 (0.01) 
                 0.010 (0.001) 
                 0.032 (0.003) 
                 0.009 (0.001) 
               
               
                   
                 stomach 
                  1.30 (0.28) 
                 0.020 (0.01) 
                  2.40 (0.28) 
                 0.020 (0.001) 
               
               
                   
                 salivary 
                  0.10 (0.01) 
                  0.01 (0.00) 
                  0.14 (0.01) 
                 0.020 (0.001) 
               
               
                   
                 glands 
                 42.60 (2.68) 
                  0.13 (0.04) 
                  0.20 (0.01) 
                 0.010 (0.001) 
               
               
                   
                 thyroid 
                  0.25 (0.07) 
                 0.060 (0.002) 
                  0.40 (0.05) 
                  0.08 (0.002) 
               
               
                   
                 tumor 
               
               
                   
               
               
                 *2 mg NaClO 4  per animal was co-injected with  125 I −  and  188 ReO 4   − . ClO 4   −  is a blocking agent for NIS. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Tumor to normal organ ratio (RIs) of  125 I −  and  188 ReO 4   −  in 
               
               
                 NIS-expressing xenografted mammary adenocarcinoma in nude mice. 
               
            
           
           
               
               
               
            
               
                   
                 RI 
                   
               
            
           
           
               
               
               
               
            
               
                 Time 
                 Organ 
                   125 I −   
                   188 ReO 4   −   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                       20 min 
                 blood 
                 0.57 
                 0.41 
               
               
                   
                 spleen 
                 1.60 
                 1.39 
               
               
                   
                 liver 
                 1.68 
                 1.27 
               
               
                   
                 kidneys 
                 1.29 
                 1.22 
               
               
                   
                 muscle 
                 13.39 
                 8.21 
               
               
                   
                 stomach 
                 0.11 
                 0.10 
               
               
                   
                 salivary glands 
                 2.20 
                 1.46 
               
               
                   
                 thyroid 
                 0.60 
                 0.52 
               
               
                   
                 tumor 
                 1.00 
                 1.00 
               
               
                  2 hr 
                 blood 
                 0.68 
                 0.54 
               
               
                   
                 spleen 
                 2.62 
                 2.99 
               
               
                   
                 liver 
                 2.04 
                 1.43 
               
               
                   
                 kidneys 
                 1.50 
                 1.40 
               
               
                   
                 muscle 
                 12.17 
                 5.22 
               
               
                   
                 stomach 
                 0.11 
                 0.08 
               
               
                   
                 salivary glands 
                 1.21 
                 0.54 
               
               
                   
                 thyroid 
                 0.13 
                 0.25 
               
               
                   
                 tumor 
                 1.00 
                 1.00 
               
               
                 24 hr 
                 blood 
                 1.67 
                 1.45 
               
               
                   
                 spleen 
                 2.50 
                 10.00 
               
               
                   
                 liver 
                 6.25 
                 8.00 
               
               
                   
                 kidneys 
                 2.77 
                 3.08 
               
               
                   
                 muscle 
                 6.25 
                 12.50 
               
               
                   
                 stomach 
                 0.19 
                 0.17 
               
               
                   
                 salivary glands 
                 2.50 
                 2.85 
               
               
                   
                 thyroid 
                 0.01 
                 2.00 
               
               
                   
                 tumor 
                 1.00 
                 1.00 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Doses delivered during 24 hour period by  131 I −  or  188 ReO 4   −  to 
               
               
                 the selected organs and 2 g tumor in 70-kg reference adult. 
               
            
           
           
               
               
               
            
               
                   
                 Doses, rad/mCi 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Organ 
                   131 I −   
                   188 ReO 4   −   
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 spleen 
                 0.016 
                 0.058 
               
               
                   
                 liver 
                 0.028 
                 0.155 
               
               
                   
                 kidneys 
                 0.04 
                 0.190 
               
               
                   
                 stomach 
                 0.42 
                 1.97 
               
               
                   
                 thyroid* 
                 1.26 
                 0.930 
               
               
                   
                 tumor 
                 0.04 
                 0.18 
               
               
                   
                   
               
               
                   
                 *Doses have been calculated without thyroid suppression by exogenous thyroid hormone. 
               
            
           
         
       
     
                     TABLE 4                  Comparison of the properties of  188 Re,  186 Re and  131 I.                                   188 Re     186 Re     131 I                                                     Energy of   155   keV   137   keV   364   keV       photons                             Abundance of   15%   9%   81%       photons                                         Average energy   0.764   MeV   0.362   MeV   0.192   MeV       of beta particles       Half-life of   16.7   hours   3.7   days   8   days       radionuclides       Optimal tissue   23-32   mm   7-12   mm   2.6-5   mm       range of beta       particles                             # atoms/g tumor   5.34 × 10 11     2.34 × 10 12     6.38 × 10 12         for 90% cure       probability at       optimal range       Radiation dose   4.5×   n.d.   1.0×       delivered to       tumor,       arbitrary units               n.d. = not determined.            
Discussion
 
     The present application demonstrates that the Na + /I −  symporter mediates the uptake of the powerful beta-emitting radiometal Rhenium in mammary tumors, using as an example  188 Re-perrhenate. This finding is significant for the diagnosis of breast cancer and treatment of cancer. Before the present study, it was not obvious that tumors expressing a Na + /I −  symporter would take up ReO 4   − , since it had been reported that ReO 4   −  is a potent blocking agent for NIS in a  Xenopus  oocyte expression system [44]. 
     The present application further demonstrates that Rhenium radioisotopes, as exemplified in the form of  188 ReO 4   − , can effectively inhibit the in vivo growth of tumors expressing a Na + /I −  symporter. The application also demonstrates that in the absence of organification of the radioisotopes, Rhenium radioisotope delivers a superior radiation dose to the tumor in comparison to radioactive iodine, a finding which would not have been previously obvious. Similar to radioiodine,  188 Re-perrhenate exhibited NIS-dependent uptake into the tumors, but, like  99m Tc-pertechnetate and unlike radioiodine, no long-term organification and accumulation of Rhenium occurs in the thyroid gland. 
       188 Re is conveniently obtained from  188 W/ 188 Re generators [9] which are available from Oak Ridge National Laboratory (ORNL). The long half-life (69 days) of the parent  188 W permits extensive distribution to nuclear medicine departments and on-site access to the radioisotope in a fashion similar to  99m Tc. The superior properties of Rhenium radioisotopes compared to those of  131 I are summarized in Table 4. The emission characteristics and physical properties of  188 Re, as well as those of  186 Re, are superior in comparison to those of  131 I. The higher energy beta particles of  188 Re and  186 Re (E average =0.764 MeV for  188 Re and E average =0.362 MeV for  186 Re compared to E average =0.134 MeV for  131 I) are effective over a greater range, sufficient to eradicate medium or large tumors by a “cross-fire” effect, while their lower energy and low abundance gamma photons (155 keV, 15% abundance for  188 Re, and 137 keV, 9% abundance for  186 Re) can be imaged yet are easier to shield than the 364 keV photons of  131 I. As a consequence of their emission characteristics, the optimal tissue range is 23.0-32.0 mm for  188 Re and 7.0-12.0 mm for  186 Re. In contrast, the lower energy beta-particles emitted by  131 I have an optimal tissue range of only 2.6-5.0 mm [22]. The number of atoms of radioisotope per gram of tumor needed to produce a cure probability of 90% at the optimal range is 6.38×10 12  for  131 I, 2.34×10 12  for  186 Re, and 5.34×10 11  for  188 Re [22]. These figures clearly show that  188 Re and  186 Re have the potential of delivering larger doses of radiation to tumors expressing NIS than does  131 I even if the uptake of  188 Re-perrhenate or  186 Re-perrhenate is significantly lower than that of  131 I. Dosimetry calculations performed by extrapolation of biodistribution data to humans, showed that  188 Re-perrhenate is able to deliver 4.5 times higher dose to the tumor than  131 I. The use of Rhenium radioisotopes in especially indicated for tumors larger than 5.0 mm in diameter which cannot be covered by the emission range of  131 I beta-particles. 
     In summary, the present study has shown that Rhenium radioisotopes, as exemplified in the form of  188 ReO 4   − , can be used effectively to inhibit the in vivo growth of tumors expressing a Na + /I −  symporter and that Rhenium radioisotope delivers a superior radiation dose to the tumor in comparison to radioactive iodine. The application further discloses the use of Rhenium radioisotopes to diagnose the presence of a breast tumor that expresses a Na + /I −  symporter. 
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