Search

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM. Experimental Design: [18F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/μmol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma. Results: In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood–brain barrier and was rapidly cleared. In patients with GBM, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [18F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation. Conclusions: We developed and translated [18F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy–induced normalization of aberrant cancer metabolism.

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form of alternating electric fields therapy on the aberrant glycolysis of hGBM. [18F]FDG is the most common radiotracer in cancer metabolic imaging, but its utility in hGBM is impaired due to high glucose uptake in normal brain tissue. With TTFields, radiochemistry, Western blot, and immunofluorescence microscopy, we identified pyruvate kinase M2 (PKM2) as a biomarker of hGBM response to therapeutic TTFields. We used [18F]DASA-23, a novel radiotracer that measures PKM2 expression and which has been shown to be safe in humans, to detect a shift away from hGBM aberrant glycolysis in response to TTFields. Compared to unexposed hGBM, [18F]DASA-23 uptake was reduced in hGBM exposed to TTFields (53%, P< 0.05) or temozolomide chemotherapy (33%, P > 0.05) for 3 d. A 6-d TTFields exposure resulted in a 31% reduction (P = 0.043) in 60-min uptake of [18F]DASA-23. [18F]DASA-23 was retained after a 10 but not 30-min wash-out period. Compared to [18F]FDG, [18F]DASA-23 demonstrated a 4- to 9-fold greater uptake, implying an improved tumor-to-background ratio. Furthermore, compared to no-TTFields exposure, a 6-d TTFields exposure caused a 35% reduction in [18F]DASA-23 30-min uptake compared to only an 8% reduction in [18F]FDG 30-min uptake. Quantitative Western blot analysis and qualitative immunofluorescence for PKM2 confirmed the TTFields-induced reduction in PKM2 expression. This is the first study to demonstrate that TTFields impairs hGBM aberrant glycolytic metabolism through reduced PKM2 expression, which can be non-invasively detected by the [18F]DASA-23 radiotracer.

The aim of this study was development of an improved PET radiotracer for measuring xC− activity with increased tumor uptake and reduced uptake in inflammatory cells compared with (S)-4-(3-18F-fluoropropyl)-l-glutamate (18F-FSPG). Methods: A racemic glutamate derivative, 18F-hGTS13, was evaluated in cell culture and animal tumor models. 18F-hGTS13 was separated into C5 epimers, and the corresponding 18F-hGTS13-isomer1 and 18F-hGTS13-isomer2 were evaluated in H460 tumor–bearing rats. Preliminary studies investigated the cellular uptake of 18F-hGTS13-isomer2 in multiple immune cell populations and states. Results:18F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor-associated radioactivity was significantly higher for 18F-hGTS13 (7.5 ± 0.9 percentage injected dose [%ID]/g, n = 3) than for 18F-FSPG (4.6 ± 0.7 %ID/g, n = 3, P = 0.01). 18F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3 ± 1.1 %ID/g, n = 3) and significantly reduced uptake in multiple immune cell populations relative to 18F-FSPG. 18F-hGTS13-isomer2 exhibited increased liver uptake relative to 18F-FSPG (4.6 ± 0.8 vs. 0.7 ± 0.01 %ID/g), limiting its application in hepatocellular carcinoma. Conclusion:18F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of xC− activity that may provide information on tumor oxidation states. 18F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax because of the low background signal in healthy tissue.

Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

The aim of this study was to develop a positron emission tomography (PET) tracer to visualize and monitor therapeutic response to bacterial infections. In our continued efforts to find maltose based PET tracers that can image bacterial infections, we have designed and prepared 6''-[18 F]fluoromaltotriose as a second generation PET imaging tracer targeting the maltodextrin transporter of bacteria. We have developed methods to synthesize 6''-deoxy-6''-[18 F]fluoro-α-D-glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4)-O-D-glucopyranose (6''-[18 F]-fluoromaltotriose) as a bacterial infection PET imaging agent. 6''-[18 F]fluoromaltotriose was prepared from precursor, 2'',3'',4''-tri-O-acetyl-6''-O-nosyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose (per-O-acetyl-6''-O-nosyl-maltotriose 4). This method utilizes the reaction between precursor 4 and anhydrous [18 F]KF/Kryptofix 2.2.2 in dimethylformamide (DMF) at 85°C for 10 minutes to yield per-O-acetyl-6''-deoxy-6-'' [18 F]-fluoromaltotriose (7). Successive acidic and basic hydrolysis of the acetyl protecting groups in 7 produced 6''-[18 F]fluoromaltotriose (8). Also, cold 6''- [19 F]fluoromaltotriose was prepared from per-O-acetyl-6''-hydroxymaltotriose via a diethylaminosulfur trifluoride reaction followed by a basic hydrolysis. A successful synthesis of 6''-[18 F]-fluoromaltotriose has been accomplished in 8 ± 1.2% radiochemical yield (decay corrected). Total synthesis time was 120 minutes. Serum stability of 6''-[18 F]fluoromaltotriose at 37°C indicated that 6''-[18 F]-fluoromaltotriose remained intact up to 2 hours. In conclusion, we have successfully synthesized 6''-[18 F]-fluoromaltotriose via direct fluorination of an appropriate precursor of a protected maltotriose.

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. Significance: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells.

OBJECTIVES We developed 1-((2-fluoro-6-(fluoro-[18F])phenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) as a novel radiopharmaceutical to measure pyruvate kinase M2 levels by positron emission tomography (PET). PKM2 catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in the healthy brain, making it an important biomarker of cancer glycolytic re-programming. Here, we report the first evaluation of [18F]DASA-23 in human healthy volunteers and subjects with low-grade (LGG) and high-grade glioma (HGG). METHODS [18F]DASA-23 was synthesized under GMP conditions. Brain [18F]DASA-23 PET/MRI scans (3T) were performed in human healthy volunteers (n=5) and subjects with LGG (n=3) and HGG (n=2). The PET imaging duration was 60 min and standardized uptake value (SUV) calculations were performed on the 30–60 min summed images. The maximum SUV in the tumor (TumorSUVmax) and contralateral white matter (WMSUVmax) were calculated. RESULTS [18F]DASA-23 specific activity was 2961±873 mCi/µmol (n=10) with radiochemical purity >95%, injected mass of 1.8±0.7 mcg, and dose of 0.3±0.02 mcg per kg body weight. In healthy volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared through the bladder and also showed uptake in the gallbladder, liver, and intestines over time. [18F]DASA-23 was found to be intact in plasma up to 10 min post-injection and 75% intact at 30 min post-injection. In subjects with glioma, TumorSUVmax was significantly greater in HGG (2.2±0.4, n=2) compared to LGG (0.8±0.3m n=3), p=0.02. In this early human series, the normalized ratio of TumorSUVmax/WMSUVmax was not significantly different between subjects with HGG (2.0±0.6) and LGG (1.0±0.4), p=0.1. CONCLUSION [18F]DASA-23 is a promising new imaging agent for the non-invasive delineation of LGG and HGG based on aberrantly expressed PKM2. An ongoing study is evaluating the utility of this agent in additional patients with intracranial malignancies (NCT03539731).

A hallmark of cancer is metabolic reprogramming, which is exploited by cancer cells to ensure rapid growth and survival. Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key step in tumor metabolism and growth. Recently, we reported the radiosynthesis of the first positron emission tomography tracer for visualizing PKM2 in vivo—i.e., [11C]DASA-23. Due to the highly promising imaging results obtained with [11C]DASA-23 in rodent model glioblastoma, we set out to generate an F-18-labeled version of this tracer, with the end goal of clinical translation in mind. Herein, we report the radiosynthesis of 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) and our initial investigation of its binding properties in cancer cells. We synthesized [18F]DASA-23 via fluorination of 1-((2-fluoro-6-nitrophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine (10) with K[18F]F/K2.2.2 in N,N-dimethylformamide at 110 °C for 20 min. Subsequently, we evaluated uptake of [18F]DASA-23 in HeLa cervical adenocarcinoma cells and in vitro stability in human and mouse serum. We successfully prepared [18F]DASA-23 in 2.61 ± 1.54 % radiochemical yield (n = 10, non-decay corrected at end of synthesis) with a specific activity of 2.59 ± 0.44 Ci/μmol. Preliminary cell uptake experiments revealed high uptake in HeLa cells, which was effectively blocked by pretreating cells with the structurally distinct PKM2 activator, TEPP-46. [18F]DASA-23 remained intact in human and mouse serum up to 120 min. Herein, we have identified a F-18-labeled PKM2 specific radiotracer which shows potential for in vivo imaging. The promising cell uptake results reported herein warrant the further evaluation of [18F]DASA-23 for its ability to detect and monitor cancer noninvasively.

Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form of alternating electric fields therapy on the aberrant glycolysis of hGBM. [18F]FDG is the most common radiotracer in cancer metabolic imaging, but its utility in hGBM is impaired due to high glucose uptake in normal brain tissue. With TTFields, radiochemistry, Western blot, and immunofluorescence microscopy, we identified pyruvate kinase M2 (PKM2) as a biomarker of hGBM response to therapeutic TTFields. We used [18F]DASA-23, a novel radiotracer that measures PKM2 expression and which has been shown to be safe in humans, to detect a shift away from hGBM aberrant glycolysis in response to TTFields. Compared to unexposed hGBM, [18F]DASA-23 uptake was reduced in hGBM exposed to TTFields (53%, P< 0.05) or temozolomide chemotherapy (33%, P > 0.05) for 3 d. A 6-d TTFields exposure resulted in a 31% reduction (P = 0.043) in 60-min uptake of [18F]DASA-23. [18F]DASA-23 was retained after a 10 but not 30-min wash-out period. Compared to [18F]FDG, [18F]DASA-23 demonstrated a 4- to 9-fold greater uptake, implying an improved tumor-to-background ratio. Furthermore, compared to no-TTFields exposure, a 6-d TTFields exposure caused a 35% reduction in [18F]DASA-23 30-min uptake compared to only an 8% reduction in [18F]FDG 30-min uptake. Quantitative Western blot analysis and qualitative immunofluorescence for PKM2 confirmed the TTFields-induced reduction in PKM2 expression. This is the first study to demonstrate that TTFields impairs hGBM aberrant glycolytic metabolism through reduced PKM2 expression, which can be non-invasively detected by the [18F]DASA-23 radiotracer.

RCS-4 [(4-methoxyphenyl)-1-yl-(1-pentyl-1H-indol-3-yl)methanone] represents the first of several N-alkyl-3-(methoxybenzoyl)indoles identified by forensic scientists as synthetic cannabinoid (SC) designer drugs. Despite the detection of RCS-4 and several analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4, and RCS-4-C4) in products intended for human consumption, relatively little is known about this class of cannabinoids. The synthesis of all regioisomers of RCS-4 and their C4 homologues is described. This study also systematically explored the structure–activity relationships of this class of SCs at human CB1 and CB2 receptors using an in vitro fluorometric imaging plate reader membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 54–574 nM) and CB2 (EC50 = 4.5–46 nM) receptors, with the C4 homologues showing a preference for CB2 receptors over CB1 receptors (31–42 times). Since most of the analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4 and RCS-4-C4) are not subject to regulation in much of the world, despite their activities towards CB1 and CB2 receptors, there is a possibility that these analogues will emerge on the black market.

Synthetic cannabinoid (SC) designer drugs featuring bioisosteric fluorine substitution are identified by forensic chemists and toxicologists with increasing frequency. Although terminal fluorination of N-pentyl indole SCs is sometimes known to improve cannabinoid type 1 (CB1) receptor binding affinity, little is known of the effects of fluorination on functional activity of SCs. This study explores the in vitro functional activities of SC designer drugs JWH-018, UR-144, PB-22, and APICA, and their respective terminally fluorinated analogues AM-2201, XLR-11, 5F-PB-22, and STS-135 at human CB1 and CB2 receptors using a FLIPR membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 2.8–1959 nM) and CB2 (EC50 = 6.5–206 nM) receptors, with the fluorinated analogues generally showing increased CB1 receptor potency (∼2–5 times). Additionally, the cannabimimetic activities and relative potencies of JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135 in vivo were evaluated in rats using biotelemetry. All SCs dose-dependently induced hypothermia and reduced heart rate at doses of 0.3–10 mg/kg. There was no consistent trend for increased potency of fluorinated SCs over the corresponding des-fluoro SCs in vivo. Based on magnitude and duration of hypothermia, the SCs were ranked for potency (PB-22 > 5F-PB-22 = JWH-018 > AM-2201 > APICA = STS-135 = XLR-11 > UR-144).

Several lines of experimental evidence support the involvement of the α7 nAChR in schizophrenia and Alzheimer's disease. Modulators of the α7 nAChR have been extensively reviewed for the treatment of the cognitive deficits associated with these pathologies. SEN12333 represents a novel α7 nAChR agonist chemotype with potential for reduced side effects but requiring further SAR exploration. The present work investigates the amide bond of SEN12333, specifically its connectivity and replacement with the tetrazole functionality, a known cis amide isostere. The results reveal the original amide bond connectivity of SEN12333 to be favorable for binding affinity and agonist activity at α7 nAChRs. The use of a tetrazole isostere completely abolishes affinity and functional activity and suggests that SEN12333 binds in a linear conformation. Results reported herein also suggest the pyridine nitrogen within the terminal aromatic ring of SEN12333 is not essential for binding affinity or functional activity. Further SAR investigations involving manipulation of other moieties contained within SEN12333 are warranted.

INTRODUCTION: Pyruvate kinase M2 (PKM2) catalyzes the final and rate-limiting step in glycolysis, a key step in tumor growth and metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in the healthy brain, making it an important marker of cancer glycolytic re-programming. Our laboratory has developed 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23), a novel radiotracer to noninvasively measure the expression of PKM2 and are currently conducting first-in-human studies with positron emission tomography (PET) imaging. METHODS: In this study we evaluated the ability of [18F]DASA-23 to detect GBM metabolic changes in response to seven classes of anti-GBM chemotherapy (n=11 drugs total). Human U87 GBM cells were subjected to the IC(50) of alkylating agents (temozolomide [TMZ], carmustine, lomustine), a topoisomerase I inhibitor (irinotecan), inhibitors of vascular endothelial and epidermal growth factor receptors (cediranib and erlotinib, respectively) an anti-metabolite (5-fluorouracil), an anti-microtubule agent (vincristine), and metabolic therapies (AG-120 and dichloroacetate). After three or six days of exposure (n≥5/condition), the cellular uptake of [18F]DASA-23 was evaluated and PKM2 protein expression determined. PKM2 protein expression was compared via correlation analysis between two methods, [18F]DASA-23 uptake and Western blot. Two-way ANOVA was used with corrections for multiple comparisons. Results are reported as mean±SD. RESULTS: There was a significant interaction between the interventions (n=13 including vehicle, media-only, and 11 drugs) and treatment duration (3 or 6 days) on PKM2 expression as measured by [18F]DASA-23 uptake (p=0.0001). The most significant change in [18F]DASA-23 uptake at XX minutes was evident in response to treatment with alkylating agents (p=0.0003), irinotecan (p=0.0055), erlotinib (p=0.04), and 5-fluorouracil (p=0.02). Western blot analysis revealed a moderate correlation between PKM2 protein expression [18F]DASA-23 uptake (R(2)=0.44, p=0.15, Pearson correlation). CONCLUSIONS: These studies highlight the potential for evaluation of PKM2 expression with [18F]DASA-23 as a means to non-invasively monitor response to multiple classes of anti-GBM agents.

Pyruvate kinase M2 (PKM2) is a key marker of cancer metabolic reprogramming since it catalyzes the final step in glycolysis. [18F]DASA-23 is a novel radiotracer that measures aberrantly-expressed PKM2 in glioblastoma (GBM). Tumor treating fields (TTFields), the application of alternating electric fields (100–300 kHz, 1–4 V/cm) to tumors, is emerging as the fourth therapeutic modality in GBM after surgery, temozolomide (TMZ) chemotherapy, and radiotherapy. There is an important need to assess early on whether a patient’s GBM is responding to a given therapy. We evaluated the ability of [18F]DASA-23 to detect changes in GBM metabolism in response to standard-of-care (TMZ) and emerging (TTFields) therapies. Human U87 GBM cells were subjected to 200 kHz TTFields, the IC(50) of TMZ, or vehicle for three or six days (n≥3/condition), followed by evaluation of [18F]DASA-23 uptake. Immunofluorescence for PKM2 was performed to confirm the [18F]DASA-23 uptake results. Finally, Western blot analysis was performed to determine the effect of TMZ and TTFields exposure on the expression of PKM2 and a downstream enzyme, lactate dehydrogenase (LDH). 2-way ANOVA with multiple comparisons was performed. Data are reported as mean±SD. There was a significant interaction between the treatment (TTFields, TMZ, or vehicle) and treatment duration (3 or 6 days) on PKM2 expression as measured by [18F]DASA-23 uptake (p=0.005) at 30 minutes post-addition of radioactivity. Immunofluorescence independently confirmed reduced PKM2 expression due to TTFields. Western blot analysis revealed reduced PKM2 and LDH (normalized to loading control) without/with TMZ (0.43 ± 0.13 vs. 0.30 ± 0.05 [p=0.14] and 0.61 ± 0.16 vs. 0.43 ± 0.04 [p=0.12], respectively) and without/with TTFields (0.70 ± 0.23 vs. 0.15 ± 0.09 [p=0.01] and 1.14 ± 0.36 vs. 0.54 ± 0.14 [p=0.04], respectively). These data highlight the potential for non-invasive assessment of GBM’s glycolytic response to standard and emerging therapies using [18F]DASA-23. A clinical study is underway to evaluate the ability of [18F]DASA-23 to predict responders vs. non-responders to anti-GBM therapy.

BACKGROUND Efforts are underway to uncover novel, unorthodox therapies against glioblastoma (GBM). Tumor treating fields (TTFields) disrupt mitotic spindle formation and stymie proliferation in tumor cells. Combining TTFields with Withaferin A, synergistically inhibited proliferation in GBM. We wished to uncover the relevant mechanism. METHODS Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary tumors. Cells were engineered to stably express firefly luciferase (fLuc). Proliferation was assessed by bioluminescence imaging (using D-Luciferin as a substrate for fLuc) or cell counting. Dextran-FITC binding and scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes. RESULTS TTFields (200 kHz, 4 V/cm) significantly inhibited growth of cells (n=3/time point, p≤0.02; 2-way ANOVA, no TTFields vs. TTFields). Combination of Withaferin A with TTFields significantly inhibited growth of glioma cells synergistically beyond that of Withaferin A or TTFields alone (p< 0.01, at least n=3 experiments). Bioluminescence imaging suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, 2-way ANOVA, TTFields vs. no TTFields, n=3 experiments). SEM revealed significantly greater and larger number of holes on the membrane surface of TTFields-exposed U87-MG cancer cells (53.5±19.1 holes per field of view and mean size=240.6±91.7 nm2) compared to unexposed cells (23.9±11.0 holes per field of view and mean size=129.8±31.9 nm2, p< 0.005: TTFields exposed vs. non-exposed, n=3 samples, univariate Mann-Whitney test). CONCLUSION The findings suggest a novel combinatorial approach to treat GBM in a manner that is better than single treatment (drug or TTFields) alone and that is synergistic. Synergy is achieved through TTFields inducing increased permeability on membranes thus conferring greater accessibility to drug. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma.

In our continued exploration of trishomocubane derivatives with central nervous system (CNS) activity, N -arylalkyl-8-aminopentacyclo[5.4.0.0 2,6 .0 3,10 .0 5,9 ]undecanes ( 10 – 13 ) displaying affinity for the sigma (σ) receptor were also found, in several cases, to interact with the dopamine transporter (DAT). Compound 12 was identified as the first trishomocubane-derived high affinity DAT ligand ( K i = 1.2 nM), with greater than 8300-fold selectivity over the monoamine transporters NET and SERT, and only low to moderate affinity for σ 1 and σ 2 receptors.

Synthetic cannabinoid (SC) designer drugs based on indole and indazole scaffolds and featuring l-valinamide or l-tert-leucinamide side chains are encountered with increasing frequency by forensic researchers and law enforcement agencies and are associated with serious adverse health effects. However, many of these novel SCs are unprecedented in the scientific literature at the time of their discovery, and little is known of their pharmacology. Here, we report the synthesis and pharmacological characterization of AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, 5F-ADBICA, and several analogues. All synthesized SCs acted as high potency agonists of CB1 (EC50 = 0.24-21 nM) and CB2 (EC50 = 0.88-15 nM) receptors in a fluorometric assay of membrane potential, with 5F-ADB-PINACA showing the greatest potency at CB1 receptors. The cannabimimetic activities of AB-FUBINACA and AB-PINACA in vivo were evaluated in rats using biotelemetry. AB-FUBINACA and AB-PINACA dose-dependently induced hypothermia and bradycardia at doses of 0.3-3 mg/kg, and hypothermia was reversed by pretreatment with a CB1 (but not CB2) antagonist, indicating that these SCs are cannabimimetic in vivo, consistent with anecdotal reports of psychoactivity in humans.

Sixteen new phenyl alkyl ether derivatives (12, 14e28) of the 5,7-dimethylpyrazolo[1,5-a]pyrimidin-3ylacetamide (DPA) class were synthesized and evaluated in a competition binding assay against [ 3 H] PK11195 using 18 kDa translocator protein (TSPO) derived from rat kidney mitochondrial fractions. All analogues showed superior binding affinities for TSPO compared to DPA-713 (5) and DPA-714 (6). Picomolar affinities were observed for this class of TSPO ligands in this assay for the first time, with phenethyl ether 28 showing the greatest affinity (Ki ¼ 0.13 nM). Additionally, all analogues increased pregnenolone biosynthesis (134e331% above baseline) in a rat C6 glioma cell steroidogenesis assay. © 2015 Published by Elsevier Masson SAS.

Alpha7 nicotinic acetylcholine receptors (nAChRs) have implications in the regulation of cognitive processes such as memory and attention and have been identified as a promising therapeutic target for the treatment of the cognitive deficits associated with schizophrenia and Alzheimer's disease (AD). Structure affinity relationship studies of the previously described α7 agonist SEN12333 (8), have resulted in the identification of compound 45, a potent and selective agonist of the α7 nAChR with enhanced affinity and improved physicochemical properties over the parent compound (SEN12333, 8).

The preparation by living polymerization and the investigation of the properties of polymers with cyclic chain architectures have been recently undertaken. The cyclization involves a unimolecular end-to-end reaction of a heterodifunctional linear precursor The elementary steps of the cyclization mechanism are examined in the light of matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) data obtained on linear polystyrene precursors and cyclized products. The synthetic procedures developed to prepare macrocyclic polymers of various structures, as well as macrocyclic random and block copolymers are then reviewed and discussed. The main characteristics of the cyclic (co)polymers are compared to the corresponding linear chains and some of the original properties that are induced by the cyclic chain architecture are presented.

GluN2B subtype-selective NMDA antagonists represent promising therapeutic targets for the symptomatic treatment of multiple CNS pathologies. A series of N-benzyl substituted benzamidines were synthesised and the benzyl ring was further replaced with various polycyclic moieties. Compounds were evaluated for activity at GluN2B containing NMDA receptors where analogues 9, 12, 16 and 18 were the most potent of the series, replacement of the benzyl ring with polycycles resulted in a complete loss of activity.

We recently proposed a new method allowing the controlled synthesis of ring-shaped polymers with predictable molar masses and narrow molar mass distribution. The cyclization is based on a unimolecular “end-to-end” coupling reaction of an heterodifunctional linear polymer precursor. In this paper, this methodology is applied to the synthesis of semicyclic block copolymers comprising a cyclic hydrophilic block on which a linear hydrophobic polymeric tail is attached, i.e., a cyclic diblock copolymer of poly(hydroxyethyl vinyl ether) and linear polystyrene. The general strategy for the synthesis involves three main steps: (i) preparation of a linear α-acetal-γ-styrenyl-poly(chloroethyl vinyl ether-block-styrene) precursor, (ii) intramolecular coupling involving the activation and reaction of the two complementary functions under high dilution, yielding the cyclized poly(CEVE) block, and (iii) substitution by hydroxy functions of the chloro side groups of poly(CEVE) block in order to form the amphiphilic sem...

A series of ligands based on SEN12333, containing either contracted or elongated alkyl chains, were synthesized and evaluated in molecular docking studies against a homology model of the α7 nicotinic acetylcholine receptor (nAChR) subtype. The predicted binding of all ligands was highly similar, with the exception of the analog containing a 5 methylene unit spacer. However, in vitro competition binding assays revealed that the ligands possessed dissimilar binding affinities, with a K(i) range of more than an order of magnitude (K(i)=0.50 to >10 μM), and only SEN12333 itself exhibited functional activity at the α7 nAChR.

A series of N-(benzofuran-2-ylmethyl)-N'-benzylpiperazines bearing alkyl or fluoroalkyl aryl ethers were synthesized and evaluated at various central nervous system receptors. Examination of in vitro sigma1 {[3H]+-pentazocine} and sigma2 ([3H]DTG) receptor binding profiles of piperazines 11-13 and 25-36 revealed several highly potent and sigma1 selective ligands, notably, N-(benzofuran-2-ylmethyl)-N'-(4'-methoxybenzyl)piperazine (13, Ki=2.7 nM, sigma2/sigma1=38) and N-(benzofuran-2-ylmethyl)-N'-(4'-(2''-fluoroethoxy)benzyl)piperazine (30, Ki=2.6 nM, sigma2/sigma1=187). Structural features for optimal sigma1 receptor affinity and selectivity over the sigma2 receptor were identified. On the basis of its favorable log D value, 13 was selected as a candidate for the development of a sigma1 receptor positron emission tomography radiotracer. [11C]13 showed high uptake in the brain and other sigma receptor-rich organs of a Papio hamadryas baboon. The in vivo evaluation of [11C]13 indicates that this radiotracer is a suitable candidate for imaging the sigma1 receptor in neurodegenerative processes.

The effect of chronic administration of phosphatidylserine derived from brain cortex on the neuroendocrine responses to physical stress has been examined in a placebo-controlled study in 9 healthy men. Phosphatidylserine 800 mg/d for 10 days significantly blunted the ACTH and cortisol responses to physical exercise (P = 0.003 and P = 0.03, respectively), without affecting the rise in plasma GH and PRL. Physical exercise significantly increased the plasma lactate concentration both after placebo and phosphatidylserine. The results suggest that chronic oral administration of phosphatidylserine may counteract stress-induced activation of the hypothalamo-pituitary-adrenal axis in man.

The existence of two distinct sigma (σ) receptor subtypes was established in the early 1990s. Sigma-1 and sigma-2 receptors (S1Rs and S2Rs, respectively) were shown to possess distinct molecular size, anatomical distribution, and ligand discrimination. S2R is overexpressed in numerous human cancers, and has therapeutic potential for the imaging and treatment of certain tumours. In contrast, S1R is more broadly involved in a wide variety of central nervous system (CNS) diseases including motor disorders, memory deficits, depression, schizophrenia, anxiety, pain, drug addiction, and many more. Since the human S1R was cloned in 1996, numerous high affinity ligands with excellent selectivity for S1R have been developed. This review focuses on recent developments in the generation of structurally diverse S1R-selective ligands and novel therapeutic candidates targeting S1Rs.

Background Several Hyaluronan based products are available for intra-articular administration. These products differ in their composition and contain either natural hyaluronan of differing molecular weight or chemically crosslinked hyaluronan. Numerous clinical studies have shown that a series of 3–5 weekly intra-articular injections of hyaluronan of molecular weight 500–730 kDa (Hyalgan) reduces pain and improves function in patients with osteoarthritis of the knee and that this effect can be long lasting (6–12 months). Moreover, there is some evidence from pilot clinical studies to suggest that this product may slow osteoarthritis. Objectives The primary objective of the present study was to examine the structure-modifying activity of intra-articular hyaluronan of molecular weight 500–730 kDa.(Hyalgan). Methods A multicentre, randomised, placebo-controlled, double-blind (masked observer) study in patients with primary knee osteoarthritis (ACR criteria) was conducted in 17 centres in the UK. The primary end point was the change in radiological joint space between baseline and one year in the medial tibio-femoral compartment, measured by digital image analysis. Weight-bearing antero-posterior radiographs were taken using a standardised technique. Patients were randomly assigned to receive 3 courses of 3 intra-articular injections of hyaluronan or 2 ml of placebo (saline solution) during the one year study. Uncontrolled use of analgesics and NSAIDs were allowed. Results 408 patients with knee osteoarthritis (II-III Kellgren-Lawrence) were enrolled in the trial and 319 patients completed the one year study period. Mean patient age was 64.2 yrs (sd = 9.3), 68.4% of patients were female. The two groups, placebo and control, were comparable at entry for disease state and demographics. The change in joint space width between baseline and one year in the two groups was compared by analysis of variance. The group of patients with greater joint space at entry, treated with hyaluronan, showed statistically reduced progression of joint space narrowing. In the hyaluronan group the mean loss was only -0.22 (sd = 1.1), while in the placebo group the mean loss was -0.60 (sd = 1.1). This was statistically significant, p Conclusion Repeat treatment cycles of intra-articular hyaluronan of molecular weight 500–730 kDa (Hyalgan) result in a statistically significant delay in radiological joint space narrowing within one year, in patients with osteoarthritis of the knee.