Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level.

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [(18)F]-labeled STI-571 was prepared with high specific activity (75 GBq/µmol) by nucleophilic displacement and an average radiochemical yield of 12%. [(131)I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [(18)F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [(18)F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor.

INTRODUCTION: The cannabinoid receptor type 2 (CB(2)) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB(2) receptor ligands. Some of the compounds demonstrated to be good CB(2)-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their (18)F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB(2) receptor expression. Here, we report the radiosynthesis of two (18)F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB(2)-specific radioligand. METHODS: 4-[(18)F]fluorobenzyl amine [(18)F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH(4) and then coupled with acid chlorides 11 and 12 to afford [(18)F]-13 and [(18)F]-14. In vitro CB(2) receptor binding assay was performed using U87 cells transduced with CB(2) and CB(1) receptor. Ex vivo autoradiography was performed with [(18)F]-14 on spleen and on CB(2)- and CB(1)-expressing and wild-type U87 subcutaneous tumors grown in mice. RESULTS: The radiochemical yields of [(18)F]-13 and [(18)F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/µmol. The dissociation constant Kd for [(18)F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [(18)F]-14 in the CB(2)-expressing tumor. CONCLUSION: Two new [(18)F]-labeled CB(2) ligands have been synthesized. Compound [(18)F]-14 appears to be a potential PET imaging agent for the assessment of CB(2) receptor expression; however, poor solubility restrain its use in vivo.

Fluorinated cannabinoid CB2 receptor ligands: synthesis and in vitro binding characteristics of 2-oxoquinoline derivatives.

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-1618, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [(3)H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (K(i)) values of compounds 13-16, 18, 19, 21-24, 27, and 28 for CB2 were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar K(i) value, could be radiolabeled with (18)F and explored for PET imaging of CB2 expression.

An anticancer C-Kit kinase inhibitor is reengineered to make it more active and less cardiotoxic.

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

A human-derived reporter gene for noninvasive imaging in humans: mitochondrial thymidine kinase type 2.

UNLABELLED: A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular-genetic processes for potential clinical use. METHODS: The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (DeltahTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of DeltahTK2 and DeltahTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of (124)I-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-iodouracil (FIAU), (18)F-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil (FEAU), or (18)F-9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed. RESULTS: DeltahTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing DeltahTK2 and DeltahTK2 GFP were at least 10-fold higher than in wild-type cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for DeltahTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that DeltahTK2 transduced cells can be eliminated by treatment with d-arabinofuranosyl-cytosine. CONCLUSION: We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular-genetic processes by nuclear imaging techniques in humans. Using (124)I-FIAU, (18)F-FIAU, or (18)F-FEAU, it should be possible to image DeltahTK2 reporter gene expression with PET in preclinical and clinical studies.

A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging.

Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Delta45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Delta45HSV1-tk/GFP/luciferase (Delta45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (~130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Delta45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Delta45-TGL cells compared to nontransduced control cells. The Ki of (14)C-FIAU was 0.49+/-0.02, 0.51+/-0.03, and 0.003+/-0.001 ml/min/g in U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/ nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Delta45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [(131)I]FIAU (7.4 MBq/animal) or [(124)I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity in the wild-type xenografts. Tissue sampling yielded values of 0.47%+/-0.08%, 0.86%+/-0.06%, and 0.03%+/-0.01%dose/g [(131)I]FIAU in U87-NES-TGL, U87-Delta45-TGL, and U87 xenografts, respectively. The TGL triple-fusion reporter gene preserves the functional activity of its subunits and is very effective for multimodality imaging. It provides for the seamless transition from fluorescence microscopy and FACS to whole-body bioluminescence imaging, to nuclear (PET, SPET, gamma camera) imaging, and back to in situ fluorescence image analysis.

Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging.

To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a "cryptic" testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [(131)I]FIAU and a gamma-camera.