18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models.

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer 18F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (18F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using 18F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using 18F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10-40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment 18F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate 18F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced 18F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment 18F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.

18F-5-fluorouracil dynamic positron emission tomography/computed tomography shows decreased tracer activity after bevacizumab in colorectal metastases.

OBJECTIVE: The aim of this study was to evaluate the potential of fluorine-18 (F)-5-fluorouracil (F-5-FU) positron emission tomography/computed tomography (PET/CT) to show differences in 5-FU activity in metastatic colorectal cancer before and after treatment with bevacizumab. METHODS: This was a pilot study of five patients with newly diagnosed and untreated metastatic colorectal adenocarcinoma. The presence of cancer was confirmed by histopathological analysis before enrollment. Patients underwent F-5-FU PET/CT scanning before treatment and at approximately 24 h postbevacizumab. PET/CT scanning consisted of a dynamic acquisition of images taken 0-20 min after injection of radiotracer. The degree of F-5-FU activity at the metastatic sites was assessed using visual interpretation and semiquantitative standardized uptake value analyses. RESULTS: The sizes of the metastatic lesions ranged from the smallest lesion measuring 3.04 × 1.50 cm to the largest measuring 4.19 × 2.76 cm. By drawing regions of interest, time-activity curves were generated at each tumor site and area under the curve (AUC) analyses were carried out. At baseline, during the first 5 min after F-5-FU injection the mean AUCtumor/AUCaorta ratio was 1.24 ± 0.30 (range, 0.424-2.14). Less than 24 h after the administration of bevacizumab, the AUCtumor/AUCaorta ratio decreased to 1.06 ± 0.32 (range, 0.23-2.13, P=0.04), which represented an average decline of 20.2% (range, 0.4-45%). Radiotracer uptake on the 5, 10, 15, and 20-min images did not show any significant change between baseline and posttreatment. Follow-up CT imaging showed stable tumor size in one patient and a decrease in metastasis size in the remaining four patients. CONCLUSION: In this pilot study of five patients with metastatic colorectal carcinoma, F-5-FU PET/CT scanning showed a significant perfusion-related decrease in tracer activity 24 h postbevacizumab.

PET of malignant melanoma using 18F-labeled metallopeptides.

UNLABELLED: Melanocortin type 1 receptor (MC1R), also known as alpha-melanocyte-stimulating hormone (alpha-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An (18)F-labeled linear alpha-MSH peptide ((18)F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2) [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized alpha-MSH peptide, ReO[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) (ReCCMSH(Arg(11))), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg(11)) could be a good platform for the further development of an (18)F-labeled probe for PET of MC1R-positive malignant melanoma. METHODS: In this study, the metallopeptide Ac-d-Lys-ReCCMSH(Arg(11)) was synthesized using conventional solid-phase peptide synthesis chemistry and a rhenium cyclization reaction. The resulting peptides were then labeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The (18)F-labeled metallopeptides were further tested for their in vitro receptor binding affinities, in vivo biodistribution, and PET imaging properties. RESULTS: Both isomers of Ac-d-Lys-ReCCMSH(Arg(11)), named as RMSH-1 and RMSH-2, were purified and identified by high-performance liquid chromatography. The binding affinities of RMSH-1 and RMSH-2 and their respective (19)F-SFB-conjugated peptides ((19)F-FB-RMSH-1 and (19)F-FB-RMSH-2) were all determined to be within nanomolar range. Both (18)F-labeled metallopeptides showed good tumor uptake in the B16F10 murine model, with high MC1R expression, but much lower uptake in the A375M human melanoma xenografted in mice, indicating low MC1R expression. (18)F-FB-RMSH-1, when compared with (18)F-FB-RMSH-2, displayed more favorable in vivo performance in terms of slightly higher tumor uptakes and much lower accumulations in the kidney and liver at 2 h after injection. Small-animal PET of (18)F-FB-RMSH-1 and -2 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptakes and poorer tumor-to-normal organ contrasts were observed for the A375M model than for the B16F10 model. (18)F-FB-RMSH-1 and -2 showed higher tumor uptake and better tumor retention than did (18)F-FB-NAPamide. CONCLUSION: Specific in vivo targeting of (18)F-FB-RMSH-1 to malignant melanoma was successfully achieved in preclinical models with high MC1R expression. Thus, the radiofluorinated metallopeptide (18)F-FB-RMSH-1 is a promising molecular probe for PET of MC1R-positive tumors.

Semiautomated radiosynthesis and biological evaluation of [18F]FEAU: a novel PET imaging agent for HSV1-tk/sr39tk reporter gene expression.

2'-deoxy-2'-[(18)F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil ([(18)F]FEAU) is a promising radiolabeled nucleoside designed to monitor Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene expression with positron emission tomography (PET). However, the challenging radiosynthesis creates problems for being able to provide [(18)F]FEAU routinely. We have developed a routine method using a commercial GE TRACERlab FX-FN radiosynthesis module with customized equipment to provide [(18)F]FEAU. All radiochemical yields are decay corrected to end-of-bombardment and reported as means +/- SD. Radiofluorination (33 +/- 8%; n = 4), bromination (85 +/- 8%; n = 4), coupling reaction (83 +/- 6%; n = 4), base hydrolysis steps, and subsequent high-performance liquid chromatography purification afforded purified [(18)F]FEAU beta-anomer in 5 +/- 1% overall yield (n = 3 runs) after approximately 5.5 h and a beta/alpha-anomer ratio of 7.4. Radiochemical/chemical purities and specific activity exceeded 99% and 1.3 Ci/micromol (48 GBq/micromol), respectively. In cell culture, [(18)F]FEAU showed significantly (P < 0.05) higher accumulation in C6 cells expressing HSV1-tk/sr39tk as compared to wild-type C6 cells. Furthermore, [(18)F]FEAU showed slightly higher accumulation than 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butylguanine ([(18)F]FHBG) in cells expressing HSV1-tk (P < 0.05), whereas [(18)F]FHBG showed significantly higher (P < 0.05) accumulation than [(18)F]FEAU in HSV1-sr39tk-expressing cells. micro-PET imaging of mice carrying tumor xenografts of C6 cells stably expressing HSV1-tk or HSV1-sr39tk are consistent with the cell uptake results. The [(18)F]FEAU mouse images also showed very low gastrointestinal signal with predominant renal clearance as compared to [(18)F]FHBG. The routine radiosynthesis of [(18)F]FEAU was successfully semiautomated using a commercial module along with customized equipment to provide the beta-anomer in modest yields. Although further studies are needed, early results also suggest [(18)F]FEAU is a promising PET radiotracer for monitoring HSV1-tk reporter gene expression.

64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression.

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.

Reproducibility of 3'-deoxy-3'-(18)F-fluorothymidine microPET studies in tumor xenografts in mice.

UNLABELLED: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been used to image tumor proliferation in preclinical and clinical studies. Serial microPET studies may be useful for monitoring therapy response or for drug screening; however, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FLT microPET studies. METHODS: C6 rat glioma xenografts were implanted into nude mice (n = 9) and grown to mean diameters of 5-17 mm for approximately 2 wk. A 10-min acquisition was performed on a microPET scanner approximately 1 h after (18)F-FLT (1.9-7.4 MBq [50-200 muCi]) was injected via the tail vein. A second microPET scan was performed approximately 6 h later on the same day after reinjection of (18)F-FLT to assess for reproducibility. Most of the mice were studied twice within the same week (for a total of 17 studies). Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility after subtraction of the estimated residual tumor activity from the first (18)F-FLT injection. RESULTS: The coefficient of variation (mean +/- SD) for %ID/g values between (18)F-FLT microPET scans performed 6 h apart on the same day was 14% +/- 10%. The difference in %ID/g values between scans was -0.06% +/- 1.3%. Serum thymidine levels were mildly correlated with %ID/g values (R(2) = 0.40). Tumor size, mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, controlling body temperature, the time of imaging after injection, and the ROI size. CONCLUSION: (18)F-FLT microPET mouse tumor xenograft studies are reproducible with moderately low variability. Serial studies may be performed to assess for significant changes in therapy response or for preclinical drug development.

Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments.

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.

Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model.

Epidemiological studies and clinical observations suggest that nonsteroidal anti-inflammatory drugs and certain selective cyclooxygenase (COX)-2 inhibitors may reduce the relative risk of clinically evident prostate cancer. This prompted us to investigate the chemopreventive potential of celecoxib, a selective COX-2 inhibitor, against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Similar to prostate cancer in humans, prostate malignancies in TRAMP mice progress from precursor intraepithelial lesions, to invasive carcinoma that metastasizes to lymph nodes, liver, lungs, and occasionally to bone. The basal enzyme activity and protein expression of COX-2 is significantly higher (>4-fold) in the dorsolateral prostate of TRAMP mice up to 24 weeks of age compared with their nontransgenic littermates. Eight-week-old TRAMP mice were randomly divided and fed either control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500-ppm celecoxib ad libitum for 24 weeks, a dosage that would compare with the normal recommended dose for the treatment of human disease. Studies from two independent experiments, each consisting of 10 mice on test, showed that the cumulative incidence of prostate cancer development at 32 weeks of age in animals fed with AIN 76A diet was 100% (20 of 20) as observed by tumor palpation, whereas 65% (13 of 20), 35% (7 of 20), and 20% (4 of 20) of the animals exhibited distant site metastases to lymph nodes, lungs, and liver. Celecoxib supplementation to TRAMP mice from 8-32 weeks of age exhibited significant reduction in tumor development (5 of 20) with no signs of metastasis. Celecoxib feeding resulted in a significant decrease in prostate (56%; P < 0.0003) and genitourinary weight (48%; P < 0.008). Sequential magnetic resonance imaging analysis of celecoxib-fed mice documented lower prostate volume compared with the AIN 76A-fed group. Histopathological examination of celecoxib-fed animals showed reduced proliferation, and down-modulation of COX-2 and prostaglandin E2 levels in the dorsolateral prostate and plasma, respectively. These results correlated with retention of antimetastasis markers, viz E-cadherin, and alpha- and beta-catenin, along with a significant decrease in vascular endothelial growth factor protein expression. Celecoxib supplementation also resulted in enhanced in vivo apoptosis in the prostate as monitored by several techniques including a recently perfected technique of 99mTc-labeled annexin V in live animals followed by phosphor imaging. One striking observation in an additional study was that celecoxib feeding to mice with established tumors (16 weeks of age) significantly improved their overall survival (P = 0.014), compared with AIN 76A-fed group. Our findings suggest that celecoxib may be useful in chemoprevention of prostate cancer.

A simplified method for preparation of 99mTc-annexin V and its biologic evaluation for in vivo imaging of apoptosis after photodynamic therapy.

UNLABELLED: Apoptosis is the likely mechanism behind the effects of chemotherapeutic and radiation treatments, rejection of organ transplants, and cell death due to hypoxic-ischemic injury. A simplified method capable of imaging apoptosis in living animals could have many applications leading to understanding of the involvement of apoptosis in biologic and therapeutic processes. To accomplish this goal we developed a method for the preparation of (99m)Tc-annexin V and evaluated its usefulness to detect apoptosis that occurs during tumor shrinkage after photodynamic therapy (PDT). PDT is a cancer treatment modality that leads to rapid induction of apoptosis both in vitro and in in vivo animal models. METHODS: A novel synthesis of (99m)Tc-annexin V was performed in a simple 1-pot procedure. Radiation-induced fibrosarcoma (RIF-1) tumors grown in C(3)H mice were treated with PDT, followed by injection of (99m)Tc-annexin V and measurement of its tumor uptake at 2, 4, and 7 h after PDT by autoradiography. RESULTS: The radiochemical purity of (99m)Tc-annexin V was >95%. At all time points, (99m)Tc-annexin V was clearly imaged in the PDT-treated tumors, whereas the untreated tumors showed no uptake of the radiolabeled compound. Histopathology and immunohistochemistry of PDT-treated tumors confirmed the evidence of apoptosis compared with untreated tumors. CONCLUSION: These data demonstrate the detection of apoptosis using (99m)Tc-annexin V in tumor tissue in living animals after PDT treatment. This novel technique could be used as a noninvasive means to detect and serially image tissues undergoing apoptosis after cancer treatment protocols in humans. Other potential applications of this technique could be the diagnosis of several human disorders, such as myocardial ischemia or infarct, rejection of organ transplantation, and neonatal cerebral ischemia.