| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Basic Science Investigations |
1 Research Service, Harry S. Truman Memorial VA Hospital, Columbia, Missouri
2 Department of Internal Medicine, University of Missouri-Columbia School of Medicine, Columbia, Missouri
3 Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, Missouri
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
Key Words: gastrin-releasing peptide • bombesin • 111In • prostate cancer
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
Significant progress has been made over the past few years in developing effective strategies to produce radiolabeled BBN analogs that specifically target GRP-receptor-expressing cancer cells in vitro and in vivo (9–24). Our laboratory has focused on developing radiometallated BBN analogs in which radiometal chelates are linked to the truncated BBN[7–14]NH2 sequence that serves as the highly specific GRP-receptor-binding motif (2,12–18). Use of this truncated BBN[7–14]NH2 sequence was selected since the BBN derivatives containing this sequence have been shown to bind in an agonist manner to the GRP receptors (7,14,17). As with other G-protein-coupled, 7-transmembrane segment (7-TMS) receptors, specific binding of an agonist to cell-surface GRP receptors typically leads to internalization of the agonist-receptor complex (25–27), providing an effective method to produce residualization of the radioactive metal atom that will be specifically delivered to the cancer cell by the BBN[7–14]NH2 moiety targeting vector (28). Studies with BBN[7–14]NH2 conjugates radiolabeled with 105Rh, 99mTc, and 111In have demonstrated receptor-mediated trapping of these radiotracers by GRP-receptor-expressing cancer cells (14,22,24). These results demonstrate the feasibility of producing BBN[7–14]NH2 analogs labeled with a variety of radiometals that hold potential for effective in vivo targeting of GRP-receptor cancer cells.
A fundamental concern in the design of radiometallated peptide constructs is to ensure in vivo stability of the radiometal incorporated by the ligand framework. In vivo stability can be controlled by virtue of the high thermodynamic stability and the kinetic inertness of a specific metal chelation framework (29–33). Because the dodecanetetraacetic acid (DOTA) macrocyclic ligand system is well known to form kinetically inert and thermodynamically stable chelates with indium, yttrium, and lanthanides, it has received wide acceptance for in vivo applications with several trivalent radiometals (29–33). Our research group is currently evaluating the potential of DOTA-BBN analogs labeled with trivalent radioactive metals, which are linked either directly to the N-terminal amine group of BBN[7–14]NH2 moiety or through hydrocarbon spacer groups, to specifically target GRP-receptor-expressing cancer cells (Fig. 1) (18). The length and composition of the spacer group, or tether, as well as the physicochemical properties of the radiolabeled moiety will influence the GRP-receptor-binding affinity, residualization of radioactivity in cancer cells, and pharmacokinetics of the BBN conjugate. The purpose of this study was to determine the effects of varying the length of the hydrocarbon spacer on the in vitro binding affinity of 111In+3-DOTA conjugates of BBN[7–14]NH2 with GRP receptors expressed on PC-3 androgen-independent human prostate cancer cells and their in vivo pharmacokinetics in CF-1 mice. The ultimate goal was identification of one or more lead candidates with optimum in vivo pharmacokinetics and high GRP-receptor-mediated uptake in tumor cells.
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
-amino alkyl carboxylic acids, from Advanced ChemTech. 125I-Tyr4-BBN was obtained from NEN Life Sciences Products, Inc., 111InCl3 was obtained from Mallinckrodt Medical, Inc., as a 0.05N HCl solution. Electrospray mass spectral analyses were performed by Synpep Corp. PC-3 cells were obtained from American Type Culture Collection and were maintained and grown in the University of Missouri Cell and Immunobiology Core Facility. CF-1 mice were purchased from Charles River Laboratories and were maintained in an in-house animal facility.
High-Performance Liquid Chromatography (HPLC)
HPLC analyses were performed on a Waters 600E system equipped with a Varian 2550 variable absorption detector, a Packard Radiometric 150TR flow scintillation analyzer, a sodium iodide crystal radiometric detector, an Eppendorf TC-50 column temperature controller, and Hewlett-Packard HP3395 integrators. HPLC solvents consisted of H2O containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B). A Phenomenex Jupiter C-18 (5 µm, 300 Å, 4.6 x 250 mm) column was used with a flow rate of 1.5 mL/min. This HPLC gradient system began with an initial solvent composition of 80% A and 20% B followed by a linear gradient to 70% A and 30% B in 30 min, after which the column was reequilibrated.
Solid-Phase Peptide Synthesis
DOTA-X-BBN[7–14]NH2 conjugates (Fig. 1) were synthesized on a Perkin Elmer-Applied Biosystems model 432 automated peptide synthesizer using traditional Fmoc chemistry, with 2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation of carboxyl groups on the reactant, and with the N-terminal amino group on the growing peptide anchored through the C-terminus to the resin. Rink amide MBHA resin (25 µmol), Fmoc-protected amino acids with appropriate side-chain protections (75 µmol), Fmoc-protected -amino alkyl carboxylic acids (75 µmol), and DOTA-tris(t-butyl ester) (75 µmol) were used for the synthesis. The final products were cleaved by a standard procedure using a cocktail containing thioanisole, water, ethane dithiol, and trifluoroacetic acid in a ratio of 2:1:1:36 and were precipitated into methyl-t-butyl ether. Typical yields of the crude peptides were 80%–85%. Crude peptides were purified by HPLC, and the solvents were removed on a SpeedVac concentrator (Thermo Savant). The purified peptides were characterized by electrospray mass spectrometry (Table 1).
|
|
|
-counting system. The percentage of 125I-Tyr4-BBN bound to cells was plotted versus increasing concentrations of In-DOTA-X-BBN[7–14]NH2 conjugates to determine the respective IC50 values. In vitro studies to determine the degree of internalization of 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate were performed by a method similar to that described by Reile et al. (9). These studies were performed by incubating 3 x 104 PC-3 cells (suspended in RPMI medium 1640, at pH 7.4, and containing 4.8 mg/mL HEPES, 0.1 µg/mL Bacitracin, and 2 mg/mL BSA) for 40 min at 37°C and in 5% CO2 in the presence of 20,000 cpm 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate. After 40 min of incubation, the binding was stopped through aspiration of the reaction medium and washing of the cells with culture medium. The radioactivity-bound cells were then incubated in culture medium at 37°C. After 1, 2, 3, and 4 h of incubation in culture medium, the percentage of radioactivity trapped in the cells was determined after removal of activity bound to the surface of the cells by washing with a pH 2.5 (0.2 mol/L acetic acid and 0.5 mol/L NaCl) buffer.
In Vivo Biodistribution Studies
The biodistribution and uptake of 111In-DOTA-X-BBN[7–14]NH2 conjugates in healthy CF-1 mice was studied. Each mouse (average weight, 25 g) was injected with an aliquot (50–100 µL) of the HPLC-purified radiolabeled peptide solution (55–75 kBq) through the tail vein. Tissues and organs were excised from the animals sacrificed at 1 h after injection. The activity was counted in a NaI counter, and the percentage injected dose (%ID) per organ and per gram was calculated. The %ID in the blood was estimated assuming a blood volume equal to 6.5% of the total body weight. Receptor-blocking studies were also performed when excess (100 µg) BBN was administered to animals along with the 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate.
Pharmacokinetic Studies of the 111In-DOTA-8-Aoc-BBN[7–14]NH2 Analog in PC-3 Tumor-Bearing SCID Mice
Four- to 5-wk-old female ICR SCID (severely compromised immunodeficient) outbred mice were obtained from Taconic. Five mice were housed per sterile Microisolator cage (Alternative Design) in a temperature- and humidity-controlled room with a daily schedule of 12 h of light followed by 12 h of darkness. The animals were fed autoclaved rodent chow (Ralston Purina Co.) and water ad libitum. They were housed for 1 wk before inoculation of tumor cells, and they were anesthetized for injections with isoflurane (Baxter Healthcare Corp.) at a rate of 2.5% with 0.4 L oxygen through a nonrebreathing anesthesia vaporizer.
PC-3 cells were injected bilaterally and subcutaneously in the flank, with 
5 x 106 cells in a suspension of 100 µL normal sterile saline injected per site. The PC-3 cells were allowed to grow in vivo for 2–3 wk after inoculation, and tumors ranging from 0.2 to 1.0 g developed. The pharmacokinetic studies of the 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate were determined in SCID mice bearing PC-3 tumors. Each mouse (average weight, 25 g) was injected with an aliquot (50–100 µL) of the HPLC-purified 111In-labeled peptide solution (55–75 kBq) through the tail vein. The mice were euthanized, and tissues and organs were excised at 15 min; 30 min; and 1, 4, 24, 48, and 72 h after injection. Subsequently, the tissues and organs were weighed and counted in a NaI well counter, and the %ID and %ID/g of each organ or tissue were calculated. The %ID in whole blood was estimated assuming a whole-blood volume of 6.5% of the total body weight. Receptor-blocking studies were also performed when excess (100 µg) BBN was administered to the animals along with the 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate. All studies were conducted in accordance with protocols approved by the institutional animal care and use committee.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
In vitro competitive cell-binding assays performed on PC-3 cells for the In-DOTA-X-BBN[7–14]NH2 analogs against 125I-Tyr4-BBN demonstrated a high specificity and affinity (i.e., IC50 < 2.5 nmol/L; Table 2 and Fig. 4) for the analogs in which the spacer contains 3–8 carbon atoms.
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
The biologic activity of the DOTA-X-BBN[7–14]NH2 analogs was determined through in vitro competitive cell-binding assays on PC-3 cells against 125I-Tyr4-BBN. Results from these cell-binding studies demonstrated that the In-DOTA-X-BBN[7–14]NH2 analogs containing ß-Ala, 5-Ava, and 8-Aoc spacers bind to the GRP receptors on PC-3 cells with high specificity and affinity (i.e., IC50 < 2.5 nmol/L; Table 2 and Fig. 4). The In-DOTA-X-BBN[7–14]NH2 analogs containing no spacer and 11-Aun spacer exhibited high GRP-receptor specificity, but the affinity was more than 100-fold less than that of the 8-carbon-spacer analog (Table 2 and Fig. 4). These results were consistent with those obtained in our previous studies with 105Rh-S4-X-BBN[7–14]NH2 and 99mTc-N3S-X-BBN[7–14]NH2 conjugates—studies showing that increasing the hydrophobicity of the linker group to excessive levels will reduce the receptor-binding affinity (14,16,17). These cell-binding studies indicated that analogs of this design with ß-Ala, 5-Ava, and 8-Aoc spacers have a dissociation constant suitable for further in vivo targeting studies of cells expressing GRP receptors.
For optimizing the diagnostic and therapeutic efficacy of a radioactive drug, it is important that the radioactivity be residualized in the tumor cell after drug localization at the tumor site (21,22,25,28,34). Internalization and efflux studies in PC-3 cells were performed with the 111In-DOTA-8-Aoc-BBN[7–14]NH2 analog, the analog that exhibited the lowest IC50 values with PC-3 cell–expressed GRP receptors (Table 2). GRP receptors are 7-TMS-G-protein-coupled receptors that can internalize agonist-receptor complexes (26,27,35). Since the BBN[7–14]NH2 peptide sequence confers agonistic GRP-receptor-binding capability, the GRP-receptor-mediated endocytosis of the 111In-DOTA-X-BBN[7–14]NH2 conjugates used in this study was not unexpected. Previous studies with 111In-cycloheximide-B-diethylenetriaminepentaacetic acid-8-Aoc-BBN[7–14]NH2, 105Rh-S4-X-BBN[7–14]NH2, and 99mTc-N3S-X-BBN[7–14]NH2 conjugates also demonstrated GRP-receptor-mediated trapping of radioactivity (9,14,16,17). In all these derivatives, the BBN[7–14]NH2 binding motif was present in the conjugate constructs. The specific intracellular trapping mechanism of the 111In activity in these PC-3 cells is not understood. It is likely that after internalization of 111In-DOTA-8-Aoc-BBN[7–14]NH2, lysosomal proteases degrade the 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate to peptide fragments (36,37). The identity of 111In-containing fragments retained by the cell is not known, and further work is needed to identify the structure of the 111In-fragments to help elucidate the trapping mechanisms (36,38).
In vivo biodistribution studies of the 111In-DOTA-X-BBN[7–14]NH2 analogs were performed on healthy CF-1 mice. Table 3 summarizes the results of the biodistribution for the 111In-DOTA-X-BBN[7–14]NH2 analog series on CF-1 mice at 1 h after injection. Because the pancreas is the primary normal tissue in these animals that expresses a high density of bloodstream-accessible GRP receptors, the accumulation of 111In in the pancreas is a direct reflection of the efficacy of radiolabeled BBN analogs for in vivo targeting of cell-surface-expressed GRP receptors (10). In the present study, all 111In-DOTA-X-BBN[7–14]NH2 conjugates cleared efficiently from the blood, with <0.35 %ID/g remaining in the blood at 1 h after injection (Table 3). The primary route of radioactivity clearance from the body is renal/urinary, with more than 70 %ID observed in the urine at 1 h after injection for analogs with no spacer or with the ß-Ala, 5-Ava, and 8-Aoc spacers (Table 3). 111In-DOTA-11-Aun-BBN[7–14]NH2 is cleared to a significantly greater extent through the hepatobiliary route (averaging 26 %ID at 1 h after injection) than are the other derivatives (Table 3); this finding is assumed to be related to the higher lipophilicity of this analog. Retention of radioactivity in the kidneys after administration of all these 111In-DOTA-X-BBN[7–14]NH2 analogs, at <2.5 %ID/g at 1 h after injection (Table 3), was lower than that of most other small peptide-based radioactive drugs (32,33,39). Uptake of radioactivity in the pancreas increased as the lipophilicity of the conjugate increased; uptake ranged from 0.20 ± 0.04 %ID/g at 1 h after injection for the analog with no spacer (the least lipophilic analog in the series) to 26.97 ± 3.97 %ID/g at 1 h after injection for the analog using the 8-Aoc spacer (second most lipophilic in the series) (Table 3). Biodistribution studies in which an excess (100 µg) of nonradioactive BBN was coinjected with the 111In-DOTA-8-Aoc-BBN[7–14]NH2 in CF-1 mice demonstrated that uptake in the pancreas is specific and receptor mediated. These studies showed that uptake of this analog was reduced to 0.43 ± 0.10 %ID/g in the pancreas at 1 h after injection (Table 3)—nearly 98% lower than pancreatic uptake of 111In-DOTA-8-Aoc-BBN[7–14]NH2 when no blocking agent was coinjected (Table 3). The pharmacokinetics and pancreatic accumulation of this short series of 111In-DOTA-X-BBN[7–14]NH2 analogs indicates that constructs in which the spacer group (X) ranges from 5 to 8 carbon atoms appear to be the most promising for further development as GRP-receptor-targeting radiopharmaceuticals.
On the basis of these observations, we selected the analog containing 8-Aoc spacer for in vivo pharmacokinetic studies on PC-3 tumor-bearing SCID mice. Table 4 summarizes the results of the pharmacokinetic studies on PC-3 tumor-bearing SCID mice. The 111In conjugate cleared efficiently from the bloodstream within 1 h after injection. For example, 0.60 ± 0.39 %ID/g remained in the blood at 1 h after injection. Most of the activity was excreted through the renal/urinary pathway (i.e., 68.6 ± 7.0 %ID at 1 h after injection and 87.2 ± 4.3 %ID at 24 h after injection), with the remainder of the radioactivity being excreted through the hepatobiliary pathway. Receptor-mediated tumor targeting of the PC-3-xenografted SCID mice resulted in tumor uptake and retention values of 3.63 ± 1.11, 1.56 ± 0.45, 0.68 ± 0.24, and 0.34 ± 0.10 %ID/g at, respectively, 1, 24, 48, and 72 h residualization (i.e., 43%, 19%, and 9% of the radioactivity in the tumor at 1 h after injection was retained at, respectively, 24, 48, and 72 h after injection). At 1 h after injection, tumor-to-blood and tumor-to-muscle ratios of approximately 6:1 and 45:1 were achieved. Although 111In in the tumor was not optimal, the fact that the half-life of retention was approximately 24 h indicates that the 111In- DOTA-8-Aoc-BBN[7–14]NH2 conjugate was internalized agonistically. The observation that coadministration of 100 µg of BBN along with the 111In-DOTA-8-Aoc-BBN[7–14]NH2 conjugate blocked about 98% of the receptor-mediated radioactivity uptake in pancreas but only 64% in tumor may be related to the fact that these human tumors are supported by murine vasculature architecture (Table 4). Student t tests of blocked-versus-unblocked in vivo tumor uptake showed a t value of 4.67 and a P value of 0.002, indicating a statistical difference among datasets. Accumulation of activity in the kidneys was significant (i.e., 5.66 ± 1.33 %ID/g at 1 h); however, the level of activity decreased with time (i.e., to 1.18 ± 0.14 %ID/g at 24 h and 0.48 ± 0.97 %ID/g at 48 h). It may be possible to reduce the initial accumulation of 111In conjugate in the kidney or accelerate its washout by administration of excess lysine or other positively charged molecules (38). Accretion of 111In-DOTA-8-Aoc-BBN[7–14]NH2 in other tissues or organs was minimal.
The DOTA chelator framework can form complexes with a variety of trivalent and divalent radiometals to produce radiolabeled bioconjugates with high in vitro and in vivo stability (29,30). Radiometallation of the DOTA-X-BBN[7–14]NH2 analogs with particle-emitting radionuclides available in high specific activities (including 90Y+3, 64Cu+2, 177Lu+3, 149Pm+3, and 166Ho+3) should produce GRP-receptor-avid conjugates with potential for therapeutic applications (31,40).
The results of studies with these 111In-DOTA-X-BBN[7–14]NH2 constructs can be used as a basis to design potential therapeutic analogs labeled with ß-particle-emitting radionuclides. In some cases, substitution of another metal for 111In+3 may not significantly modify the in vitro and in vivo properties of DOTA bioconjugates (29,33). However, the physicochemical properties of the appended radiometallated DOTA moieties may be metal specific and can measurably alter the receptor-binding and pharmacokinetic properties of the bioconjugate (2,33). Clearly, it will be important to formulate DOTA-X-BBN[7–14]NH2 analogs complexed with therapeutically useful radiometals and to individually evaluate their utility for specific in vivo targeting of GRP-receptor-expressing cancers.
|
CONCLUSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
For correspondence or reprints contact: Wynn A. Volkert, PhD, Radiopharmaceutical Sciences Institute, 143 Major Hall, DC200.0 HSC, University of Missouri-Columbia, Columbia, MO 65212.
E-mail: VolkertW{at}health.missouri.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. Cescato, T. Maina, B. Nock, A. Nikolopoulou, D. Charalambidis, V. Piccand, and J. C. Reubi Bombesin Receptor Antagonists May Be Preferable to Agonists for Tumor Targeting J. Nucl. Med., February 1, 2008; 49(2): 318 - 326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Garrison, T. L. Rold, G. L. Sieckman, S. D. Figueroa, W. A. Volkert, S. S. Jurisson, and T. J. Hoffman In Vivo Evaluation and Small-Animal PET/CT of a Prostate Cancer Mouse Model Using 64Cu Bombesin Analogs: Side-by-Side Comparison of the CB-TE2A and DOTA Chelation Systems J. Nucl. Med., August 1, 2007; 48(8): 1327 - 1337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. F. Prasanphanich, P. K. Nanda, T. L. Rold, L. Ma, M. R. Lewis, J. C. Garrison, T. J. Hoffman, G. L. Sieckman, S. D. Figueroa, and C. J. Smith [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues PNAS, July 24, 2007; 104(30): 12462 - 12467. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. de Visser, W. M. van Weerden, C. M.A. de Ridder, S. Reneman, M. Melis, E. P. Krenning, and M. de Jong Androgen-Dependent Expression of the Gastrin-Releasing Peptide Receptor in Human Prostate Tumor Xenografts J. Nucl. Med., January 1, 2007; 48(1): 88 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, W. Cai, F. Cao, E. Schreibmann, Y. Wu, J. C. Wu, L. Xing, and X. Chen 18F-Labeled Bombesin Analogs for Targeting GRP Receptor-Expressing Prostate Cancer J. Nucl. Med., March 1, 2006; 47(3): 492 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schuhmacher, H. Zhang, J. Doll, H. R. Macke, R. Matys, H. Hauser, M. Henze, U. Haberkorn, and M. Eisenhut GRP Receptor-Targeted PET of a Rat Pancreas Carcinoma Xenograft in Nude Mice with a 68Ga-Labeled Bombesin(6-14) Analog J. Nucl. Med., April 1, 2005; 46(4): 691 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zitzmann, W. Mier, A. Schad, R. Kinscherf, V. Askoxylakis, S. Kramer, A. Altmann, M. Eisenhut, and U. Haberkorn A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor Imaging and Therapy Clin. Cancer Res., January 1, 2005; 11(1): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Reubi, H. R. Macke, and E. P. Krenning Candidates for Peptide Receptor Radiotherapy Today and in the Future J. Nucl. Med., January 1, 2005; 46(1_suppl): 67S - 75S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Scheffel and M. G. Pomper PET Imaging of GRP Receptor Expression in Prostate Cancer J. Nucl. Med., August 1, 2004; 45(8): 1277 - 1278. [Full Text] [PDF]
|
|
|
|
|
|
X. Chen, R. Park, Y. Hou, M. Tohme, A. H. Shahinian, J. R. Bading, and P. S. Conti microPET and Autoradiographic Imaging of GRP Receptor Expression with 64Cu-DOTA-[Lys3]Bombesin in Human Prostate Adenocarcinoma Xenografts J. Nucl. Med., August 1, 2004; 45(8): 1390 - 1397. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY | THE JOURNAL OF NUCLEAR MEDICINE |
= 280 nm) (B).
