Synthesis and Characterization of 9-(4-[18F]Fluoro-3-(hydroxymethyl)butyl)-2-(phenylthio)-6-oxopurine as a Novel PET Agent for Mutant Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene Imaging.

PURPOSE: [18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a reporter gene for cell and gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of reporter gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging reporter gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. PROCEDURES: [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (-) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1 mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. RESULTS: The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10 % radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (-) cells. In the presence of GCV (1 mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75 min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 - 57.75 min, P > 0.207). CONCLUSIONS: Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk reporter gene imaging agents for additional research in the future.

A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant.

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

Specific Imaging of Bacterial Infection Using 6″-18F-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria.

6″-18F-fluoromaltotriose is a PET tracer that can potentially be used to image and localize most bacterial infections, much like 18F-FDG has been used to image and localize most cancers. However, unlike 18F-FDG, 6″-18F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in gram-positive and gram-negative strains of bacteria. Methods: 6″-18F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains in cultures and in living mice. Results: 6″-18F-fluoromaltotriose was taken up in both gram-positive and gram-negative bacterial strains. 6″-18F-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6″-18F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6″-18F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients with infectious diseases of bacterial origin.

A Systematic Comparison of 18F-C-SNAT to Established Radiotracer Imaging Agents for the Detection of Tumor Response to Treatment.

PURPOSE: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of (18)F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3-triggered nanoaggregation. EXPERIMENTAL DESIGN: Here, we compared the preclinical utility of (18)F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo. RESULTS: In drug-treated lymphoma cells, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R(2) > 0.8; P < 0.001), with no correlation measured for (18)F-ML-10 (R(2) = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of (99m)Tc-Annexin V and (18)F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although (99m)Tc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either (18)F-FDG or (18)F-ML-10. CONCLUSIONS: We have demonstrated here that (18)F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with (18)F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility.

Investigation of 6-[¹8F]-fluoromaltose as a novel PET tracer for imaging bacterial infection.

UNLABELLED: Despite advances in the field of nuclear medicine, the imaging of bacterial infections has remained a challenge. The existing reagents suffer from poor sensitivity and specificity. In this study we investigate the potential of a novel PET (positron emission tomography) tracer that overcomes these limitations. METHODS: 6-[¹8F]-fluoromaltose was synthesized. Its behavior in vitro was evaluated in bacterial and mammalian cultures. Detailed pharmacokinetic and biodistribution profiles for the tracer were obtained from a murine model. RESULTS: 6-[¹8F]-fluoromaltose is taken up by multiple strains of pathogenic bacteria. It is not taken up by mammalian cancer cell lines. 6-[¹8F]-fluoromaltose is retained in infected muscles in a murine model of bacterial myositis. It does not accumulate in inflamed tissue. CONCLUSION: We have shown that 6-[¹8F]-fluoromaltose can be used to image bacterial infection in vivo with high specificity. We believe that this class of agents will have a significant impact on the clinical management of patients.

Expression of the novel human gene, UBE2Q1, in breast tumors.

The novel human gene, designated ubiquitin-conjugating enzyme E2Q family member 1 (UBE2Q1) maps to chromosome 1q21.3. The gene has an open reading frame corresponding to 422 amino acids and contains a RWD domain and an E2 ubiquitin conjugating enzyme domain. Here, we investigated the expression levels of both mRNA and protein of UBE2Q1 gene in cancerous versus normal parts of breast specimens from 26 patients. Real-time PCR data showed that the relative expression level of UBE2Q1 mRNA was significantly greater in cancers than in non-cancerous tissues of breast specimens (Mean ± SEM, 0.064 ± 0.015 for cancers and 0.026 ± 0.01 for noncancerous tissues, P < 0.05 Mann-Whitney test). A rabbit polyclonal antibody was generated against an amino acid sequence predicted from the DNA sequence of UBE2Q1 gene. This antibody was used to perform Western blotting on 21 cases in our cohort of breast specimens. Thus, 13 (61.904%) of the cases showed an increase in the UBE2Q1 immunoreactivity in their cancerous tissues as compared with the corresponding normal tissues. This result along with the real-time PCR data shows that the novel human gene, UBE2Q1, is expressed in human breast and may have implications for pathogenesis of breast cancer.

A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor.

PURPOSE: Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an (18)F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. METHODS: An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I(2) oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-(18)F-fluorobenzyl aldehyde ((18)F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, (18)F-FBO-MUT-DS, was performed at 37°C. (18)F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 µCi/µg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. RESULTS: Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of (18)F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K(D)) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of (18)F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 µg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05). CONCLUSION: F-FBO-MUT-DS displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of (18)F-based PET probes.

Synthesis of 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine: a novel agent for imaging T-cell activation with PET.

PURPOSE: 9-(ß-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2'-deoxy-2'-[(18)F]fluoro-9-ß-D-arabinofuranosylguanine ([(18)F]F-AraG) and investigated its uptake in T cells. PROCEDURE: We have synthesized [(18)F]F-AraG via a direct fluorination of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-trifyl-ß-D-ribofuranosyl)guanine with [(18)F]KF/K.2.2.2 in DMSO at 85°C for 45 min. [(18)F]F-AraG uptake in both a CCRF-CEM leukemia cell line (unactivated) and activated primary thymocytes was evaluated. RESULTS: We have successfully prepared [(18)F]F-AraG in 7-10% radiochemical yield (decay corrected) with a specific activity of 0.8-1.3 Ci/µmol. Preliminary cell uptake experiments showed that both a CCRF-CEM leukemia cell line and activated primary thymocytes take up the [(18)F]F-AraG. CONCLUSION: For the first time to the best of our knowledge, [(18)F]F-AraG has been successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach maybe also useful for the synthesis of other important positron emission tomography (PET) probes such as [(18)F]FEAU, [(18)F]FMAU, and [(18)F]FBAU which are currently synthesized by multiple steps and involve lengthy purification. The cell uptake studies support future studies to investigate the use of [(18)F]F-AraG as a PET imaging agent of T cells.

Pharmacokinetic assessment of the uptake of 16beta-18F-fluoro-5alpha-dihydrotestosterone (FDHT) in prostate tumors as measured by PET.

UNLABELLED: The aim of this study was to develop a clinically applicable noninvasive method to quantify changes in androgen receptor (AR) levels based on (18)F-16beta-fluoro-5alpha-dihydrotestosterone ((18)F-FDHT) PET in prostate cancer patients undergoing therapy. METHODS: Thirteen patients underwent dynamic (18)F-FDHT PET over a selected tumor. Concurrent venous blood samples were acquired for blood metabolite analysis. A second cohort of 25 patients injected with (18)F-FDHT underwent dynamic PET of the heart. These data were used to generate a population-based input function, essential for pharmacokinetic modeling. Linear compartmental pharmacokinetic models of increasing complexity were tested on the tumor tissue data. Four suitable models were applied and compared using the Bayesian information criterion (BIC). Model 1 consisted of an instantaneously equilibrating space, followed by a unidirectional trap. Models 2a and 2b contained a reversible space between the instantaneously equilibrating space and the trap, into which metabolites were excluded (2a) or allowed (2b). Model 3 built on model 2b with the addition of a second reversible space preceding the unidirectional trap and from which metabolites were excluded. RESULTS: The half-life of the (18)F-FDHT in blood was between 6 and 7 min. As a consequence, the uptake of (18)F-FDHT in prostate cancer lesions reached a plateau within 20 min as the blood-borne activity was consumed. Radiolabeled metabolites were shown not to bind to ARs in in vitro studies with CWR22 cells. Model 1 produced reasonable and robust fits for all datasets and was judged best by the BIC for 16 of 26 tumor scans. Models 2a, 2b, and 3 were judged best in 7, 2, and 1 cases, respectively. CONCLUSION: Our study explores the clinical potential of using (18)F-FDHT PET to estimate free AR concentration. This process involved the estimation of a net uptake parameter such as the k(trap) of model 1 that could serve as a surrogate measure of AR expression in metastatic prostate cancer. Our initial studies suggest that a simple body mass-normalized standardized uptake value correlates reasonably well to model-based k(trap) estimates, which we surmise may be proportional to AR expression. Validation studies to test this hypothesis are underway.

64Cu-labeled affibody molecules for imaging of HER2 expressing tumors.

INTRODUCTION: The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets. DISCUSSION: In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with (64)Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of (64)Cu-DOTA-Z(HER2:477) and (64)Cu-DOTA-(Z(HER2:477))(2) were 6.12 +/- 1.44% and 1.46 +/- 0.50% ID/g, respectively, in nude mice (n = 3 each) at 4 h postinjection. Moreover, (64)Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of (64)Cu-DOTA-Z(HER2:477) in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that (64)Cu-labeled Z(HER2:477) is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

A novel method for direct site-specific radiolabeling of peptides using [18F]FDG.

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

Direct site-specific radiolabeling of an Affibody protein with 4-[18F]fluorobenzaldehyde via oxime chemistry.

PURPOSE: In this study, we introduce a methodology for preparing 18F-labeled Affibody protein, specifically 18F-Anti-HER2 dimeric Affibody (14 kDa), for in vivo imaging of HER2neu with positron emission tomography (PET). PROCEDURES: We have used 4-[18F]fluorobenzaldehyde as a synthon to prepare 18F-Anti-HER2 Affibody. Aminooxy-functionalized Affibody (Anti-HER2-ONH2) was incubated with 4-[18F]fluorobenzaldehyde in ammonium acetate buffer at pH 4 in the presence of methanol at 70 degrees C for 15 min. The resulting 18F-labeled Affibody molecule was evaluated as a PET probe in xenograft models expressing HER2. RESULTS: We have successfully prepared 18F-Anti-HER2 dimeric Affibody (14 kDa), N-(4-[18F]fluorobenzylidine)oxime-Anti-HER2 Affibody, [18F]FBO-Anti-HER2, in 26-30% radiochemical yields (decay corrected). High-contrast small-animal PET images with relatively moderate tumor uptake (1.79 +/- 0.40% ID/g) were observed for the 18F-Anti-HER2 Affibody. CONCLUSION: Site-specific 18F-labeled Affibody against HER2 has been synthesized via chemoselective oxime formation between an aminooxy-functionalized Affibody and 18F-fluorobenzaldehyde. The results have implications for radiolabeling of other affibodies and macromolecules and should also be important for advancing Affibody imaging with PET.

Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules.

UNLABELLED: Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. METHODS: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z(HER2:477) and (Z(HER2:477))(2), respectively] were radiofluorinated at a reasonable radiochemical yield (13%-18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor-bearing mouse model. RESULTS: The 4-(18)F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [(18)F-N-(4-fluorobenzylidene)oxime (FBO)-Z(HER2:477) and (18)F-FBO-(Z(HER2:477))(2)] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that (18)F-FBO-Z(HER2:477) showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas (18)F-FBO-(Z(HER2:477))(2) showed poor in vivo performance (low tumor uptake and tumor-to-normal tissue ratios). The specificity of (18)F-FBO-Z(HER2:477) for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z(HER2) and trastuzumab. Moreover, small-animal PET imaging studies revealed that (18)F-FBO-Z(HER2:477) produced higher-quality tumor imaging than (18)F-FBO-(Z(HER2:477))(2). (18)F-FBO-Z(HER2:477) could clearly identify HER2-positive tumors with good contrast. CONCLUSION: Overall, these data demonstrate that (18)F-FBO-Z(HER2:477) is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with (18)F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.

Synthesis and biological evaluation of a fluorine-18 derivative of dasatinib.

Tyrosine kinases often play pivotal roles in the pathogenesis of cancer and are good candidates for therapeutic intervention and targeted molecular imaging. The precursor synthesis, radiosynthesis, and biological characterization of a fluorine-18 analog of dasatinib, a multitargeted kinase inhibitor, are reported. Compound 5 potently inhibits Abl, Src, and Kit kinases and inhibits K562 and M07e/p210bcr-abl human leukemic cell growth. Using positron emission tomography, we visualized K562 tumor xenografts in mice with [18F]-5.

Fluorescent fructose derivatives for imaging breast cancer cells.

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

Synthesis and in vitro examination of [124I]-, [125I]- and [131I]-2-(4-iodophenylamino) pyrido[2,3-d]pyrimidin-7-one radiolabeled Abl kinase inhibitors.

The pyridopyrimidinones are a potent class of inhibitors of c-Abl kinase and Bcr-Abl kinase, the causative fusion protein in chronic myelogenous leukemia and Src family kinases. A novel method for routine, high-yield no-carrier-added synthesis of [(124)I]-, [(125)I]- and [(131)I]-6-(2,6-dichlorophenyl)-2-(4-iodophenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one has been developed. The 4'-trimethylstannyl- or 4'-tri-n-butylstannyl-pyridopyrimidinone precursors were prepared from the aryl bromide via a palladium-mediated coupling with hexaalkylditin (dioxane/microwave irradiation/10 min at 160 degrees C). The radioiodination of 4'-stannylpyridopyrimidinones was found to optimally occur via an iododestannylation with Na(124)I, Na(125)I or Na(131)I in the presence of an oxidant [30% H(2)O(2)/HOAc (1:3)/10 min] in 79-87% radiochemical yield with >99% radiochemical purity. The total radiosynthesis time was 30 min. The 4-iodophenylpyridopyrimidinone 2 inhibited recombinant Abl kinase activity with an IC(50) of 2.0 nM. Cell proliferation of K562 and A431 cells was inhibited with an IC(50) of 2.0 and 20 nM, respectively. Rapid cellular uptake and equilibrium were observed within 10-15 min using [(131)I]-4-iodophenylpyridopyrimidinone 6c in K562 and A431 cells and demonstrated a 2.8-fold uptake selectivity for the Bcr-Abl-expressing K562 cells at 60 min. These results suggest that pyridopyrimidinone radiotracers may be useful in imaging Abl-, Bcr-Abl- or Src-expressing malignancies.

Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography.

Molecular imaging of a suicide transgene's expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for re-growth of tumors unable to accumulate [18F]FHBG.

A tracer kinetic model for 18F-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET.

UNLABELLED: Reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and reporter gene mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) have been used for imaging reporter gene expression with PET. Current methods for quantitating the images using the percentage injected dose per gram of tissue do not distinguish between the effects of probe transport and subsequent phosphorylation. We therefore investigated tracer kinetic models for 18F-FHBG dynamic microPET data and noninvasive methods for determining blood time-activity curves in an adenoviral gene delivery model in mice. METHODS: 18F-FHBG (approximately 7.4 MBq [approximately 200 microCi]) was injected into 4 mice; 18F-FHBG concentrations in plasma and whole blood were measured from mouse heart left ventricle (LV) direct sampling. Replication-incompetent adenovirus (0-2 x 10(9) plaque-forming units) with the E1 region deleted (n = 8) or replaced by HSV1-sr39tk (n = 18) was tail-vein injected into mice. Mice were dynamically scanned using microPET (approximately 7.4 MBq [approximately 200 microCi] 18F-FHBG) over 1 h; regions of interest were drawn on images of the heart and liver. Serial whole blood 18F-FHBG concentrations were measured in 6 of the mice by LV sampling, and 1 least-squares ratio of the heart image to the LV time-activity curve was calculated for all 6 mice. For 2 control mice and 9 mice expressing HSV1-sr39tk, heart image (input function) and liver image time-activity curves (tissue curves) were fit to 2- and 3-compartment models using Levenberg-Marquardt nonlinear regression. The models were compared using an F statistic. HSV1-sr39TK enzyme activity was determined from liver samples and compared with model parameter estimates. For another 3 control mice and 6 HSV1-sr39TK-positive mice, the model-predicted relative percentage of metabolites was compared with high-performance liquid chromatography analysis. RESULTS: The ratio of 18F-FHBG in plasma to whole blood was 0.84 +/- 0.05 (mean +/- SE) by 30 s after injection. The least-squares ratio of the heart image time-activity curve to the LV time-activity curve was 0.83 +/- 0.02, consistent with the recovery coefficient for the partial-volume effect (0.81) based on independent measures of heart geometry. A 3-compartment model best described 18F-FHBG kinetics in mice expressing HSV1-sr39tk in the liver; a 2-compartment model best described the kinetics in control mice. The 3-compartment model parameter, k3, correlated well with the HSV1-sr39TK enzyme activity (r2 = 0.88). CONCLUSION: 18F-FHBG equilibrates rapidly between plasma and whole blood in mice. Heart image time-activity curves corrected for partial-volume effects well approximate LV time-activity curves and can be used as input functions for 2- and 3-compartment models. The model parameter k3 from the 3-compartment model can be used as a noninvasive estimate for HSV1-sr39TK reporter protein activity and can predict the relative percentage of metabolites.

Positron-emission tomography reporter gene expression imaging in rat myocardium.

BACKGROUND: This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET). METHODS AND RESULTS: In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1x10(6) to 1x10(9) pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P<0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1x10(7) pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17. CONCLUSIONS: HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.