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Simple Summary: The worldwide prevalence of liver cancer, an extremely lethal disease, continues to increase. Although there are many risk factors associated with liver cancer including a fatty liver, metabolic syndrome (diabetes), and lifestyle choices (poor diet and alcohol abuse), it is still challenging to predict which patients are most likely to develop the disease and identify the most effective interventions. In this study, we carried out a non-invasive, multi-modal study of liver cancer in a mouse model to identify imaging characteristics (imaging biomarkers) that could be used to predict the development of malignant disease. We found that multiple clinical imaging techniques could predict liver cancer development in male mice, while only a handful of imaging techniques had any predictive power in female mice. The results of this study provide evidence that imaging biomarkers can predict liver cancer in mouse models and that mouse gender can significantly influence liver tumor development. The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender. [ABSTRACT FROM AUTHOR]

Purpose: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. Conclusions: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake. [ABSTRACT FROM AUTHOR]

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma. [ABSTRACT FROM AUTHOR]

Purpose: Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. Procedures: Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. Results: Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. Conclusions: These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620. [ABSTRACT FROM AUTHOR]

High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus H2O2, but not H2O2 alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [18F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [18F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb−/− (Nox2−/−) gene deletion strongly abrogated [18F]4FN retention in vivo. Thus, [18F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT. Inflammation is imaged in mice with a PET reporter of high-energy reactive oxygen and nitrogen species. [ABSTRACT FROM AUTHOR]

In oncology, biomarker research aimed to provide insights on cancer biology via positron emission tomography (PET) and single photon emission tomography (SPECT) imaging has seen an incredible growth in the past two decades. Despite the increased number of publications on PET/SPECT radiopharmaceuticals, the field lacked standardization of in vitro and in vivo parameters necessary for the characterization of any radiotracer. Through the efforts of the World Molecular Imaging Society Education Committee, this white paper lays down validation studies that are essential to chemically and biologically characterize new radiopharmaceuticals derived from small molecules, peptides or proteins. Finally, a brief overview of the steps toward translation is also presented. Herein, we discuss the following: Chemistry and radiochemistry metrics to establish the identity of the imaging agent. In vitro and in vivo studies to examine the radiotracer's mechanism of action, which includes target specificity, pharmacokinetics and in vivo metabolism. [ABSTRACT FROM AUTHOR]

A drug delivery system (DDS) for combined therapy, based on a short oxidized multiwalled carbon nanotube, is reported. It was prepared by exploiting a synthetic approach which allowed loading of two drugs, doxorubicin and metformin, the targeting agent biotin and a radiolabeling tag, to enable labeling with Ga-68 or Cu-64 in order to perform an extensive biodistribution study by PET/CT. The DDS biodistribution profile changes with different administration methods. Once administered at therapeutic doses, the DDS showed a marginal beneficial effect on 4T1 tumor bearing mice, a syngeneic and orthotopic model of triple negative breast cancer, with survival extended by 1 week and 2 days in 20% of the mice. This is encouraging given the aggressiveness of the 4T1 tumor. Furthermore, our DDS was well tolerated, ruling out concerns regarding the toxicity of carbon nanotubes. [ABSTRACT FROM AUTHOR]

The progress of the chemistry of carbon nanotubes (CNT) and graphene derivatives [mainly graphene oxide (GO)] has produced a number of technologically advanced drug delivery systems (DDS) that have been used in the field of nanomedicine, mostly in studies related to oncology. However, such a demanding field of research requires continuous improvements in terms of efficiency, selectivity and versatility. The loading of two, or more, bioactive components on the same nanoparticle offers new possibilities for treating cancer, efficiently addressing issues related both to biodistribution and pharmacokinetics. Nanostructured carbon materials (NCM), with their high surface area, their efficient cellular membrane crossing and their chemical versatility are ideal candidates for easy hetero-decoration and exploitation as advanced DDS. This review describes the achievements obtained in this area focusing on those studies in which two or more active components were loaded onto the DDS. [ABSTRACT FROM AUTHOR]

Background: The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods: Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results: FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active mutant EGFR expressing NSCLC. Conclusion: [18F]FED20 binds EGFR but is devoid of ABC transporter activity, thus, has potential for EGFR imaging. [ABSTRACT FROM AUTHOR]

Molecular imaging is an attractive platform for noninvasive detection and assessment of cancer. In recent years, the targeted imaging of the C-X-C chemokine receptor 4 (CXCR4), a chemokine receptor that has been associated with tumor metastasis, has become an area of intensive research. This review article focuses on positron emission tomography (PET) and aims to provide useful and critical insights into the application of PET to characterize CXCR4 expression, including the chemical, radiosynthetic, and biological requirements for PET radiotracers. This discussion is informed by a summary of the different approaches taken so far and a comparison of their clinical translation. Finally, our expert opinions as to potential future advances in the field are expressed. [ABSTRACT FROM AUTHOR]

This study reports the radiosynthesis of a new fluorine-18 glycosylated 'click' cyanoquinoline [18F]5 for positron emission tomography imaging of epidermal growth factor receptor (EGFR). The tracer was obtained in 47.7 ± 7.5% ( n = 3) decay-corrected radiochemical yield from 2-[18F]fluoro-2-deoxy-β- d-glucopyranosyl azide, and the overall nondecay-corrected radiochemical yield from aqueous fluoride was 8.6 ± 2.3% ( n = 3). An in vitro preliminary cellular uptake study showed selectivity of the tracer for EGFR-positive A431 cell lines versus EGFR-negative MCF-7 cell lines. [18F]5 tracer uptake in A431 cells was significantly reduced by addition of the cold isotope analogue compound 5. [ABSTRACT FROM AUTHOR]

Huisgen cycloaddition is attractive to label peptide because of its rapidity and bioorthogonality. However, for larger tracers, the physico-chemical differences between the precursor and the tracer are usually insufficient to allow their separation by HPLC, reducing the specific activity. This is of importance for peptidic tracers because the combination of their high-affinity receptor with low specific activity results in the precursor saturating the receptors, causing non-specific tracer binding. Here, we report a fast, one-pot, general strategy to circumvent this issue, yielding a tracer of improved specific activity. It consists in adding a lipophilic azide after the labeling step to scavenge unreacted precursor into a more lipophilic species that does not co-elute with the tracer. We applied this strategy to a new fluorinated cyclopentapeptidic CXCR4 antagonist for the PET imaging of cancer, CCIC15, for which we managed to reduce the apparent peptide concentration by a factor of 34 in 10 min. This tracer was radiolabeled by click chemistry with 2-[18F]fluoroethylazide, yielding the tracer in 18 ± 6% ( n = 5) end-of-synthesis radiochemical yields (EOS-RCY) in 1.5 h from [18F]fluoride with a specific activity of 19.4 GBq µmol−1. Preliminary biological evaluation of the probe confirmed potency and specificity for CXCR4; further biological evaluation is underway. [ABSTRACT FROM AUTHOR]

Acidosis is a useful biomarker for tumor diagnoses and for evaluating early response to anti-cancer treatments. Despite these useful applications, there are few methods for non-invasively measuring tumor extracellular pH, and none are routinely used in clinics. Responsive MRI contrast agents have been developed, and they undergo a change in MRI signal with pH. However, these signal changes are concentration-dependent, and it is difficult to accurately measure the concentration of an MRI contrast agent in vivo. PET/MRI provides a unique opportunity to overcome this concentration dependence issue by using the PET component to report on the concentration of the pH-responsive MRI agent. Herein, we synthesized PET/MRI co-agents based on the design of a pH-dependent MRI agent, and we have correlated pH with the r1 relaxivity of the MRI co-agent. We have also developed a procedure that uses PET radioactivity measurements and MRI R1 relaxation rate measurements to determine the r1 relaxivity of the MRI co-agent, which can then be used to estimate pH. This simultaneous PET/MRI procedure accurately measured pH in solution, with a precision that depended on the concentration of the MRI co-agent. We used our procedure to measure extracellular pH in a subcutaneous flank model of MIA PaCa-2 pancreatic cancer. Although the PET co-agents were stable in serum, post-imaging studies showed evidence that the PET co-agents were degraded in vivo. These results showed that tumor acidosis can be evaluated with simultaneous PET/MRI, although improvements are needed to more precisely measure MRI R1 relaxation rates, and ensure the in vivo stability of the agents. [ABSTRACT FROM AUTHOR]

The use of a germanium-functionalised non-cross-linked polystyrene (NCPS) support to aid the preparation of a 2-anilino-4- arylpyrimidine is described. This compound is a key intermediate en route to various IKK-2 and JNK inhibitors. [ABSTRACT FROM AUTHOR]

A range of 2-substituted 2,3-dihydro-1H-quinolin-4-ones have been synthesized from anilines by a two-step process involving Sonogashira coupling with a propargyl alcohol then acid-catalyzed cyclization of the resulting 2-(3′-hydroxypropynyl)anilines. The cyclization reaction appears to proceed via re�gioselective rearrangement of the propargyl alcohol to an α,β-unsaturated ketone (Rupe rearrangement) and then 6-endo-trig ring closure (Donnelly-Farrell cyclization). The isolation of the α,β-�unsaturated ketone intermediate in one example supports this pathway. [ABSTRACT FROM AUTHOR]

A range of 2-substituted 2,3-dihydro-1H-quinolin-4-ones have been synthesized from anilines by a two-step process involving Sonogashira coupling with a propargyl alcohol then acidcatalyzed cyclization of the resulting 2-(3'-hydroxypropynyl) anilines. The cyclization reaction appears to proceed via regioselective rearrangement of the propargyl alcohol to an α,β-unsaturated ketone (Rupe rearrangement) and then 6-endo-trig ring closure (Donnelly-Farrell cyclization). The isolation of the α,β-unsaturated ketone intermediate in one example supports this pathway. [ABSTRACT FROM AUTHOR]

The synthesis of α-substituted prolines has been accomplished by microwave-assisted Huisgen 1,3-dipolar cycloaddition between azides and orthogonally protected α-propynyl proline in the presence of Cu(I). [ABSTRACT FROM AUTHOR]

Carbon nanoparticles have consistently been of great interest in medicine. However, there are currently no clinical materials based on carbon nanoparticles, due to inconsistent biodistribution and excretion data. In this work, we have synthesized a novel C60 derivative with a metal chelating agent (1,4,7-Triazacyclononane-1,4,7-triacetic acid; NOTA) covalently bound to the C60 cage and radiolabeled with copper-64 (t1/2 = 12.7 h). Biodistribution of the material was assessed in vivo using positron emission tomography (PET). Bingel-Hirsch chemistry was employed to functionalize the fullerene cage with highly water-soluble serinolamide groups allowing this new C60 conjugate to clear quickly from mice almost exclusively through the kidneys. Comparing the present results to the larger context of reports of biocompatible fullerene derivatives, this work offers an important evaluation of the in vivo biodistribution, using experimental evidence to establish functionalization guidelines for future C60-based biomedical platforms. [ABSTRACT FROM AUTHOR]

Tumors lack a well-regulated vascular supply of O2 and often fail to balance O2 supply and demand. Net O2 tension within many tumors may not only depend on O2 delivery but also depend strongly on O2 demand. Thus, tumor O2 consumption rates may influence tumor hypoxia up to true anoxia. Recent reports have shown that many human tumors in vivo depend primarily on oxidative phosphorylation (OxPhos), not glycolysis, for energy generation, providing a driver for consumptive hypoxia and an exploitable vulnerability. In this regard, IACS-010759 is a novel high affinity inhibitor of OxPhos targeting mitochondrial complex-I that has recently completed a Phase-I clinical trial in leukemia. However, in solid tumors, the effective translation of OxPhos inhibitors requires methods to monitor pharmacodynamics in vivo. Herein, 18F-fluoroazomycin arabinoside ([18F]FAZA), a 2-nitroimidazole-based hypoxia PET imaging agent, was combined with a rigorous test-retest imaging method for non-invasive quantification of the reversal of consumptive hypoxia in vivo as a mechanism-specific pharmacodynamic (PD) biomarker of target engagement for IACS-010759. Neither cell death nor loss of perfusion could account for the IACS-010759-induced decrease in [18F]FAZA retention. Notably, in an OxPhos-reliant melanoma tumor, a titration curve using [18F]FAZA PET retention in vivo yielded an IC50 for IACS-010759 (1.4 mg/kg) equivalent to analysis ex vivo. Pilot [18F]FAZA PET scans of a patient with grade IV glioblastoma yielded highly reproducible, high-contrast images of hypoxia in vivo as validated by CA-IX and GLUT-1 IHC ex vivo. Thus, [18F]FAZA PET imaging provided direct evidence for the presence of consumptive hypoxia in vivo, the capacity for targeted reversal of consumptive hypoxia through the inhibition of OxPhos, and a highly-coupled mechanism-specific PD biomarker ready for translation. [ABSTRACT FROM AUTHOR]

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312. [ABSTRACT FROM AUTHOR]

Colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women worldwide. We have recently reported that curcuminoid complexes labelled with gallium-68 have demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compared to normal human lymphocytes. In the present study, we report a new gallium-68-labelled curcumin derivative (68Ga-DOTA-C21) and its initial validation as marker for early detection of colorectal cancer. The precursor and non-radioactive complexes were synthesized and deeply characterized by analytical methods then the curcuminoid was radiolabelled with gallium-68. The in vitro stability, cell uptake, internalization and efflux properties of the probe were studied in HT29 cells, and the in vivo targeting ability and biodistribution were investigated in mice bearing HT29 subcutaneous tumour model. 68Ga-DOTA-C21 exhibits decent stability (57 ± 3% after 120 min of incubation) in physiological media and a curcumin-mediated cellular accumulation in colorectal cancer cell line (121 ± 4 KBq of radiotracer per mg of protein within 60 min of incubation). In HT29 tumour-bearing mice, the tumour uptake of 68Ga-DOTA-C21 is 3.57 ± 0.3% of the injected dose per gram of tissue after 90 min post injection with a tumour to muscle ratio of 2.2 ± 0.2. High amount of activity (12.73 ± 1.9% ID/g) is recorded in blood and significant uptake of the radiotracer occurs in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). Further studies are needed to understand the mechanism of accumulation and clearance; however, 68Ga-DOTA-C21 provides a productive base-structure to develop further radiotracers for imaging of colorectal cancer. [ABSTRACT FROM AUTHOR]