M. N. Tantawy

Novel Selective Allosteric Activator of the M1 Muscarinic Acetylcholine Receptor Regulates Amyloid Processing and Produces Antipsychotic-Like Activity in Rats

Recent studies suggest that subtype-selective activators of M1/M4 muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimers disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M1 receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1′-2-methylbenzyl)-1,4′-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M1 through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M1 by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M2 and M4. TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Aβ production in vitro. Together, these data suggest that selective activation of M1 may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimers disease.

Molecular Imaging of Therapeutic Response to Epidermal Growth Factor Receptor Blockade in Colorectal Cancer

Purpose: To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts. Experimental Design: Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging. Results: NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry. Conclusions: Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.

Aurora kinase A promotes inflammation and tumorigenesis in mice and human gastric neoplasia.

BACKGROUND & AIMS Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. METHODS We used quantitative real-time reverse transcription polymerase chain reaction, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1(-/-) mice, growth of tumor xenografts, and human tissues. RESULTS We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1(-/-) mice (r = 0.62; P = .0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1(-/-) mice. AURKA was found to regulate NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue. CONCLUSIONS In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

Fluorinated Cyclooxygenase-2 Inhibitors as Agents in PET Imaging of Inflammation and Cancer

COX-2 is a major contributor to the inflammatory response and cancer progression so it is an important target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an attractive target for molecular imaging. We report a series of novel fluorinated imaging agents, derived from indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorporation of 18F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient signal for in vivo positron emission tomography (PET) imaging. Selective uptake of 18F-7 was observed in inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by pretreatment with the COX-2 inhibitor, celecoxib. Uptake of 18F-7 was not observed when inflammation was induced in COX-2–null mice. In nude mice bearing both a COX-2–expressing human tumor xenograft (1483) and a COX-2–negative xenograft (HCT116), 18F-7 selectively accumulated in the COX-2–expressing tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of the COX-2 status of premalignant and malignant tumors. Cancer Prev Res; 4(10); 1536–45. ©2011 AACR.

Quantitative Preclinical Imaging of TSPO Expression in Glioma Using N,N-Diethyl-2-(2-(4-(2-18F-Fluoroethoxy)Phenyl)-5,7-Dimethylpyrazolo[1,5-a]Pyrimidin-3-yl)Acetamide

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-18F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (18F-DPA-714), as a translational probe for quantification of TSPO levels in glioma. Methods: Glioma-bearing rats were imaged with 18F-DPA-714 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-DPA-714 (130–200 MBq/0.2 mL). Blood was collected to derive the arterial input function (AIF), with high-performance liquid chromatography radiometabolite analysis performed on selected samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of 3H-PK 11195 with DPA-714 in vitro and displacement of 18F-DPA-714 with an excess of DPA-714 in vivo. Immediately after imaging, tumor and healthy brain tissues were harvested for validation by Western blotting and immunohistochemistry. Results: 18F-DPA-714 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced 18F-DPA-714 binding by greater than 60% on average. Tumor uptake of 18F-DPA-714 was similar to another high-affinity TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline, and agreed with ex vivo assay of TSPO levels in tumor and healthy brain. Conclusion: These studies illustrate the feasibility of using 18F-DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, 18F-DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of 18F-DPA-714 PET to serve as a novel predictive cancer imaging modality.

Quantitative, Preclinical PET of Translocator Protein Expression in Glioma Using 18F-N-Fluoroacetyl-N-(2,5-Dimethoxybenzyl)-2-Phenoxyaniline

Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. Methods: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70–100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. Results: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. Conclusion: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.

Multifunctional profiling of non-small cell lung cancer using 18F-FDG PET/CT and volume perfusion CT.

The aim of this study was to investigate correlations between glucose metabolism registered by 18F-FDG PET/CT and tumor perfusion quantified by volume perfusion CT and immunohistochemical markers Ki67 and microvessel density (MVD) in patients with non–small cell lung cancer (NSCLC). Methods: Between February 2010 and April 2011, 24 consecutive patients (21 women, 3 men; mean age ± SD, 67.6 ± 6.8 y; age range, 55.6–81.3 y) with histologically proven NSCLC (14 adenocarcinoma, 9 squamous cell lung carcinoma [SCC], and 1 mixed adenocarcinoma and SCC) underwent 18F-FDG PET/CT and additional volume perfusion CT. Maximum standardized uptake value (SUVmax), mean SUV, and the metabolic tumor volume were used for 18F-FDG uptake quantification. Blood flow (BF), blood volume (BV), flow extraction product (Ktrans), and standardized perfusion value (SPV) were determined as CT perfusion parameters. Both perfusion parameters and 18F-FDG uptake values were subsequently related to the histologic subtypes, proliferation marker Ki67, MVD according to CD34 staining, and total tumor volume. Results: Mean SUV, SUVmax, and the metabolic tumor volume (mL) were 5.8, 8.7, and 32.3, respectively, in adenocarcinoma and 8.5, 12.9, and 16.8, respectively, in SCC. Mean BF (mL/100 mL/min), mean BV (mL/100 mL), and Ktrans (mL/100 mL/min) were 35.4, 7.3, and 27.8, respectively, in adenocarcinoma and 35.5, 10.0, and 27.8, respectively, in SCC. Moderate correlations were found between the 18F-FDG PET/CT parameters and Ki67 as well as between CT perfusion parameters and MVD but not vice versa. For all tumors, the following correlations were found: between SUVmax and Ki67, r = 0.762 (P = 0.017); between SUVmax and MVD, r = −0.237 (P = 0.359); between mean BF and Ki67, r = −0.127 (P = 0.626); and between mean BF and MVD, r = 0.467 (P = 0.059). Interestingly, correlations between the BF–metabolic relationship and total tumor volume were higher in SCC (r = 0.762, P = 0.017) than in adenocarcinoma (r = −0.0791, P = 0.788). Conclusion: 18F-FDG uptake correlates with Ki67, whereas BF, BV, and Ktrans correlate with MVD. Therefore, 18F-FDG uptake and perfusion parameters provide complementary functional information. An improved tumor profiling will be beneficial for both prognosis and therapy response evaluation in these tumors.

Simplified [18F]FDG image-derived input function using the left ventricle, liver, and one venous blood sample.

A relatively simple, almost entirely noninvasive imaging-based method is presented for deriving arterial blood input functions for quantitative [18F]2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomographic (PET) studies in rodents. It requires only one venous blood sample at the end of the scan. MicroPET images and arterial blood time-activity curves (TACs) were downloaded from the Mouse Quantitation Program database at the University of California, Los Angeles. Three-dimensional regions of interest were drawn around the blood-pool region of the left ventricle and within the liver to derive their respective TACs. To construct the “hybrid” image-derived input function (IDIF), the initial part of the left ventricle TAC, containing the peak concentration of [18F]FDG in the arterial blood, was corrected for spillout (ie, partial-volume effect yielding a recovery coefficient < 1) and then joined to the liver TAC (normalized to the 60-minute arterial blood sample) immediately after it peaks. To validate our method, the [18F]FDG influx constant (Ki) was estimated using a two-tissue compartment model and compared to estimates of Ki obtained using measured arterial blood TACs. No significant difference in the Ki estimates was obtained with the arterial blood input function and our hybrid IDIF. We conclude that the normalized hybrid IDIF can be used in practice to obtain reliable Ki estimates.

[123I]-Celecoxib Analogues as SPECT Tracers of Cyclooxygenase-2 in Inflammation

We report the synthesis and evaluation of a series of iodinated celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission computerized tomography (SPECT) imaging agents for the detection of inflammation. The structure−activity relationship identified 5-(4-iodophenyl)-1-{4-(methylsulfonyl)phenyl}-3-(trifluoromethyl)-1H-pyrazole (8) as a promising compound with IC50 values of 0.05 μM against purified COX-2 and 0.03 μM against COX-2 in activated macrophages. The arylstannane of 8 undergoes facile radio-[123I]-iodination upon treatment with Na123I/NaI and chloramine T using an EtOAc/H2O two-phase system. The [123I]-8 was produced in a radiochemical yield of 85% and a radiochemical purity of 99%. In vivo SPECT imaging demonstrated that the radiotracer was taken up by inflamed rat paws with an average 1.7-fold enrichment over contralateral noninflamed paws. This study suggests that conversion of celecoxib into its isomeric iodo-[123I]-analogues is a useful approach for generating novel and efficacious agents for COX-2-targeted SPECT imaging of inflammation.

Engineering a Material Surface for Drug Delivery and Imaging using Layer-by-Layer Assembly of Functionalized Nanoparticles

[*] Prof. V. P. Shastri, T. Soike, A. K. Streff, C. Guan, R. Ortega, M. Tantawy, Dr. C. Pino Institute for Macromolecular Chemistry University of Freiburg Stefan-Meier Strasse 31, 79104 Freiburg (Germany) E-mail: prasad.shastri@bioss.uni-freiburg.de; prasad.shastri@makro.uni-freiburg.de Prof. V. P. Shastri, T. Soike, A. K. Streff, C. M. Guan Department of Biomedical Engineering Vanderbilt University Nashville, TN 37232 (USA)