Hypoxia-activated prodrug and antiangiogenic therapies cooperatively treat pancreatic cancer but elicit immunosuppressive G-MDSC infiltration.

We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.

Production, isolation, and shipment of clinically relevant quantities of astatine-211: A simple and efficient approach to increasing supply.

The alpha emitter astatine-211 (211At) is a promising candidate for cancer treatment based on Targeted Alpha (α) Therapy (TAT). A small number of facilities, distributed across the United States, are capable of accelerating α-particle beams to produce 211At. However, challenges remain regarding strategic methods for shipping 211At in a form adaptable to advanced radiochemistry reactions and other uses of the radioisotope. PURPOSE: Our method allows shipment of 211At in various quantities in a form convenient for further radiochemistry. PROCEDURES: For this study, a 3-octanone impregnated Amberchrom CG300M resin bed in a column cartridge was used to separate 211At from the bismuth matrix on site at the production accelerator (Texas A&M) in preparation for shipping. Aliquots of 6 M HNO3 containing up to ≈2.22 GBq of 211At from the dissolved target were successfully loaded and retained on columns. Exempt packages (<370 MBq) were shipped to a destination radiochemistry facility, University of Texas MD Anderson Cancer Center, in the form of a convenient air-dried column. Type A packages have been shipped overnight to University of Alabama at Birmingham. MAIN FINDINGS: Air-dried column hold times of various lengths did not inhibit simple and efficient recovery of 211At. Solution eluted from the column was sufficiently high in specific activity to successfully radiolabel a model compound, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1), with 211At. The method to prepare and ship 211At described in this manuscript has also been used to ship larger quantities of 211At a greater distance to University of Alabama at Birmingham. PRINCIPAL CONCLUSIONS: The successful proof of this method paves the way for the distribution of 211At from Texas A&M University to research institutions and clinical oncology centers in Texas and elsewhere. Use of this simple method at other facilities has the potential increase the overall availability of 211At for preclinical and clinical studies.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

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.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization.

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.

PET/MR Imaging of a Lung Metastasis Model of Clear Cell Renal Cell Carcinoma with (2S,4R)-4-[18F]Fluoroglutamine.

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.

Imaging of innate immunity activation in vivo with a redox-tuned PET reporter.

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.

Radiometal-Based PET/MRI Contrast Agents for Sensing Tumor Extracellular pH.

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.

Development and Validation of a PET/SPECT Radiopharmaceutical in Oncology.

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.

RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging.

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.

An enolase inhibitor for the targeted treatment of ENO1-deleted cancers.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Evaluation of the Biodistribution of Serinolamide-Derivatized C60 Fullerene.

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.

Mechanism-Specific Pharmacodynamics of a Novel Complex-I Inhibitor Quantified by Imaging Reversal of Consumptive Hypoxia with [18F]FAZA PET In Vivo.

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.

Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis, radiolabeling, and preliminary biological studies of fluoroethyl analogues of crizotinib, alectinib, and ceritinib.

Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, is a therapeutic target in various cancers, including non-small cell lung cancer. Although several ALK inhibitors, including crizotinib, ceritinib, and alectinib, are approved for cancer treatment, their long-term benefit is often limited by the cancer's acquisition of resistance owing to secondary point mutations in ALK. Importantly, some ALK inhibitors cannot cross the blood-brain barrier (BBB) and thus have little or no efficacy against brain metastases. The introduction of a lipophilic moiety, such as a fluoroethyl group may improve the drug's BBB penetration. Herein, we report the synthesis of fluoroethyl analogues of crizotinib 1, alectinib 4, and ceritinib 9, and their radiolabeling with 18F for pharmacokinetic studies. The fluoroethyl derivatives and their radioactive analogues were obtained in good yields with high purity and good molar activity. A cytotoxicity screen in ALK-expressing H2228 lung cancer cells showed that the analogues had up to nanomolar potency and the addition of the fluorinated moiety had minimal impact overall on the potency of the original drugs. Positron emission tomography in healthy mice showed that the analogues had enhanced BBB penetration, suggesting that they have therapeutic potential against central nervous system metastases.

A Novel Mitochondrial Inhibitor Blocks MAPK Pathway and Overcomes MAPK Inhibitor Resistance in Melanoma.

PURPOSE: The purpose of this study is to determine if inhibition of mitochondrial oxidative phosphorylation (OxPhos) is an effective strategy against MAPK pathway inhibitor (MAPKi)-resistant BRAF-mutant melanomas.Experimental Design: The antimelanoma activity of IACS-010759 (OPi), a novel OxPhos complex I inhibitor, was evaluated in vitro and in vivo. Mechanistic studies and predictors of response were evaluated using molecularly and metabolically stratified melanoma cell lines. 13C-labeling and targeted metabolomics were used to evaluate the effect of OPi on cellular energy utilization. OxPhos inhibition in vivo was evaluated noninvasively by [18F]-fluoroazomycin arabinoside (FAZA) PET imaging. RESULTS: OPi potently inhibited OxPhos and the in vivo growth of multiple MAPKi-resistant BRAF-mutant melanoma models with high OxPhos at well-tolerated doses. In vivo tumor regression with single-agent OPi treatment correlated with inhibition of both MAPK and mTOR complex I activity. Unexpectedly, antitumor activity was not improved by combined treatment with MAPKi in vitro or in vivo. Signaling and growth-inhibitory effects were mediated by LKB1-AMPK axis, and proportional to AMPK activation. OPi increased glucose incorporation into glycolysis, inhibited glucose and glutamine incorporation into the mitochondrial tricarboxylic acid cycle, and decreased cellular nucleotide and amino acid pools. Early changes in [18F]-FAZA PET uptake in vivo, and the degree of mTORC1 pathway inhibition in vitro, correlated with efficacy. CONCLUSIONS: Targeting OxPhos with OPi has significant antitumor activity in MAPKi-resistant, BRAF-mutant melanomas, and merits further clinical investigation as a potential new strategy to overcome intrinsic and acquired resistance to MAPKi in patients.

The 3S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.

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.

Multiwalled Carbon Nanotubes for Combination Therapy: a Biodistribution and Efficacy Pilot Study.

A drug delivery system (DDS) for combined therapy, based on a short oxidized multiwalled carbon nanotube, is reported. It was prepared 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.

Development of a Potential Gallium-68-Labelled Radiotracer Based on DOTA-Curcumin for Colon-Rectal Carcinoma: From Synthesis to In Vivo Studies.

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.

Caspase-3 Substrates for Noninvasive Pharmacodynamic Imaging of Apoptosis by PET/CT.

Quantitative imaging of apoptosis in vivo could enable real-time monitoring of acute cell death pathologies such as traumatic brain injury, as well as the efficacy and safety of cancer therapy. Here, we describe the development and validation of F-18-labeled caspase-3 substrates for PET/CT imaging of apoptosis. Preliminary studies identified the O-benzylthreonine-containing substrate 2MP-TbD-AFC as a highly caspase 3-selective and cell-permeable fluorescent reporter. This lead compound was converted into the radiotracer [18F]-TBD, which was obtained at 10% decay-corrected yields with molar activities up to 149 GBq/µmol on an automated radiosynthesis platform. [18F]-TBD accumulated in ovarian cancer cells in a caspase- and cisplatin-dependent fashion. PET imaging of a Jo2-induced hepatotoxicity model showed a significant increase in [18F]-TBD signal in the livers of Jo2-treated mice compared to controls, driven through a reduction in hepatobiliary clearance. A chemical control tracer that could not be cleaved by caspase 3 showed no change in liver accumulation after induction of hepatocyte apoptosis. Our data demonstrate that [18F]-TBD provides an immediate pharmacodynamic readout of liver apoptosis in mice by dynamic PET/CT and suggest that [18F]-TBD could be used to interrogate apoptosis in other disease states.