Choline Analogues Research Articles

Alterations in target cell membrane phospholipids alter T cell but not NK cell killing

The ability of tumor cells to grow progressively in vivo despite the host immune response remains a major conundrum in tumor immunology. Various mechanisms have been proposed to explain how tumors evade immune destruction. The work presented herein shows that simple alterations in plasma membrane phospholipid composition can alter susceptibility to immune lysis. The phospholipid composition of target cells was specifically altered by growth in medium containing choline analogs. Manipulation of membrane phospholipids was observed to alter cell susceptibility to murine CTL but not NK cell lysis. The effects of such changes in phospholipid composition on CTL-mediated lysis appeared to occur during the recognition phase of lysis. This mechanism could be a means by which tumor cells, as well as other pathogenic organisms, escape immune detection and destruction.

Choline analogues in malaria chemotherapy.

Emerging resistance against well-established anti-malaria drugs warrants the introduction of new therapeutic agents with original mechanisms of action. Inhibition of membrane-based phospholipid biosynthesis, which is crucial for the parasite, has thus been proposed as a novel and promising therapeutic strategy. This review compiles literature concerning the design and study of choline analogues and related cation derivatives as potential anti-malarials. It covers advances achieved over the last two decades and describes: the concept validation, the design and selection of a clinical candidate (Albitiazolium), back-up derivatives while also providing insight into the development of prodrug approaches.

18F-fluorocholine for prostate cancer imaging: a systematic review of the literature

Positron emission tomography (PET or combined PET-computed tomography (PET/CT)) allows the non-invasive interrogation of metabolic processes using radiolabeled probes. Altered choline metabolism has been noted as a characteristic of prostate cancer (PCa), and radiolabeled choline and choline analogs have been investigated as PET/CT imaging agents for prostate cancer; [(18)F]fluoromethyl-dimethyl-2-hydroxyethyl-ammonium ((18)F-FCH) shows particular promise as a PCa imaging agent given its favorable physical and pharmacokinetic properties. We conducted a systematic review of results to date with (18)F-FCH. As the tracer was first described by DeGrado in 2001, we limited our search from January 2001 to August 2011. In all, 37 studies including 1244 patients met the inclusion criteria. Studies included those detailing the radiosynthesis of (18)F-FCH, preclinical and early clinical dosimetry, and biodistribution (n=7); evaluation of local disease (n=6), nodal disease (n=5), bone metastases and castrate-resistant disease (n=7), biochemical recurrence (n=11), radiotherapy planning (n=7) and sources of false-positive studies (n=2); and some studies reported on multiple indications. Potential sources of variations in the studies affecting reported performance included case series size, variation in extent of disease at imaging (including Gleason grade, and PSA), selection of gold standards for comparison and variations in scan technique. On the basis of the review, we suggest potential scenarios where this metabolic imaging might be considered for further evaluation in clinical trials for guiding PCa management.

Cancer de la prostate : utilité de la TEP-TDM à la 18F-fluorocholine

In oncology, positron emission computed tomography (PET/CT) has become an essential tool for initial staging, response evaluation and follow-up of cancer patients. Most of the frequent tumors (lung, breast, esophagus, and lymphomas) are highly avid for 18F-fluorodeoxyglucose ( 18FDG), but prostate cancer has not demonstrated significant uptake of FDG. The development of new tracers labeled with 18F such as choline analogs allowed already to obtain interesting results particularly in patients with biological relapse and inconclusive conventional imaging work-up. The impact of 18F-flurocholine PET/CT on patient management needs to be validated in large studies, but many centers use already this examination in order to guide further management, including radiotherapy planning.

Characterization of the Effects of Silver Nanoparticles on Liver Cell Using HR-MAS NMR Spectroscopy

AgNPs (silver nanoparticles) has been widely used for the commercial products, which have antimicrobial agent, medical devices, food industry and cosmetics. Despite, AgNPs have been reported as toxic to the mammalian cell, lung, liver, brain and other organs and many researchers have investigated the toxicity of AgNPs. In this study, we investigated toxicity of the AgNPs to the liver cell using metabolomics based on HRMAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) technics, which could apply to the intact tissues or cells, to avoid the sample destruction. Target profiling and multivariative statistical analysis were performed to analyze the 1D 1 H spectrum. The results show that the concentrations of many metabolites were affected by the AgNPs in the liver cell. The concentrations of glutathione (GSH), lactate, taurine, and glycine were decreased and most of amino acids, choline analogues, and pyruvate were increased by the AgNPs. Moreover, the levels of the metabolites were recovered upto similar level of metabolites in the normal cell by the pre-treatment of NAC, external antioxidant. The results suggest that the depletion of the GSH by the AgNPs might induce the conversion of lactate and taurine to the pyruvate.

Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.

PET imaging of hepatocellular carcinoma with 18F-fluoroethylcholine and 11C-choline

Choline-based radiotracers have been studied for PET imaging of hepatocellular carcinoma (HCC). Using an (18)F-labeled choline analog, instead of the (11)C-labeled native choline, would facilitate its widespread use in the clinic. In this study, PET with (18)F-fluoroethylcholine (FEC) and (11)C-choline (CHOL) were compared using an animal model of HCC. The effects of fasting on the performance of choline-based tracers were also investigated. A woodchuck model of HCC was used to compare the two tracers, which were administered and imaged in sequence during the same imaging session. Dynamic PET images were generated spanning 50 min starting from tracer injection. Time-activity curves and tracer contrast were calculated in liver regions with tracer accumulation, and the contrast at a late time-point with the two tracers, and between fasted and nonfasted states, were compared. Foci of HCC with increased uptake ranged in size from 1.0 to 1.6 cm, with mean tumor-to-background contrast of 1.3 with FEC and 1.5 with CHOL at 50 min after injection. The tracers show similar patterns of uptake immediately following administration, and both activities plateaued at 10 min after injection. No significant differences in uptake dynamics or final contrast were observed between the fasted and nonfasted states. PET imaging of HCC is possible with both CHOL and FEC. Fasting was not found to affect accumulation of either tracer. These results encourage further investigation into the clinical utility of FEC for HCC imaging.

Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na+ from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na+-free conditions was inhibited by a Na+/H+ exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na+-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H+ gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.

Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1-84-1 and JAY 2-22-33 in neuronal culture and Caenorhabditis elegans

BackgroundOur previous work indicated that novel analogs of choline have cytoprotective effects in vitro that might be useful in neurodegenerative conditions such as Alzheimer's disease (AD). Furthermore, two lead compounds (JWB1-84-1 and JAY2-22-33) from a library of more than 50 improved cognitive performances in a transgenic mouse model of AD. The purpose of these experiments was to more specifically investigate the neuroprotective capabilities of these lead compounds both in vitro and in vivo.ResultsWe used N2a cells which express a Swedish mutation in the amyloid precursor protein and presenilin 1 genes to investigate the effect of JWB1-84-1 and JAY2-22-33 on β-amyloid (Aβ) levels and found that both compounds significantly reduced Aβ levels. JWB1-84-1 and JAY2-22-33 also protected rat primary cortical neurons from Aβ toxicity. Subsequently, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism to identify potential molecular targets of these compounds. In the C. elegans model of Aβ toxicity, human Aβ is expressed intracellularly in the body wall muscle. The expression and subsequent aggregation of Aβ in the muscle leads to progressive paralysis.ConclusionWe found that JAY2-22-33 (but not JWB1-84-1) significantly reduced Aβ toxicity by delaying paralysis and this protective effect required both the insulin signaling pathway and nicotinic acetylcholine receptors (nAChRs).

A hyperpolarized choline molecular probe for monitoring acetylcholine synthesis

Choline as a reporter molecule has been investigated by in vivo magnetic resonance for almost three decades. Accumulation of choline metabolites (mainly the phosphorylated forms) had been observed in malignancy in preclinical models, ex-vivo, in vivo and in patients. The combined choline metabolite signal appears in (1) H-MRS of the brain and its relative intensity had been used as a diagnostic factor in various conditions. The advent of spin hyperpolarization methods for in vivo use has raised interest in the ability to follow the physiological metabolism of choline into acetylcholine in the brain. Here we present a stable-isotope labeled choline analog, [1,1,2,2-D(4) ,2-(13) C]choline chloride, that is suitable for this purpose. In this analog, the (13) C position showed 24% polarization in the liquid state, following DNP hyperpolarization. This nucleus also showed a long T(1) (35 s) at 11.8 T and 25 °C, which is a prerequisite for hyperpolarized studies. The chemical shift of this (13) C position differentiates choline and acetylcholine from each other and from the other water-soluble choline metabolites, namely phosphocholine and betaine. Enzymatic studies using an acetyltransferase enzyme showed the synthesis of the deuterated-acetylcholine form at thermal equilibrium conditions and in a hyperpolarized state. Analysis using a comprehensive model showed that the T(1) of the formed hyperpolarized [1,1,2,2-D(4) ,2-(13) C]acetylcholine was 34 s at 14.1 T and 37 °C. We conclude that [1,1,2,2-D(4) ,2-(13) C]choline chloride is a promising new molecular probe for hyperpolarized metabolic studies and discuss the factors related to its possible use in vivo.

Symmetrical choline-derived dications display strong anti-kinetoplastid activity

to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

Promising role of [18F] fluorocholine PET/CT vs [18F] fluorodeoxyglucose PET/CT in primary brain tumors—Early experience

Primary brain tumors (PBT), in particular gliomas, are among the most difficult neoplasms to treat, necessitating good quality imaging to guide clinicians at many junctures. Current imaging modalities, including [18F] fluorodeoxyglucose (FDG) PET/CT, MRI and MR spectroscopy (MRS), have various limitations, particularly with regard to differentiating tumor from radiation induced necrosis (RIN) and from normal cerebral metabolic uptake. [18F] fluorocholine (FCH) is an analog of choline with potentially optimal imaging characteristics, as pharmacokinetic studies with FCH conducted in patients showed minimal FCH uptake by normal brain parenchyma, whereas high-grade tumors are known to have increased choline uptake. We present two cases of our early experience with FCH PET/CT for patients with PBT and discuss the potential use and comparative limitations of this imaging modality.

Radiosynthesis and pre-clinical evaluation of [18F]fluoro-[1,2-2H4]choline

Choline radiotracers are widely used for clinical PET diagnosis in oncology. [(11)C]Choline finds particular utility in the imaging of brain and prostate tumor metabolic status, where 2-[(18)F]fluoro-2-deoxy-D-glucose ('FDG') shows high background uptake. More recently we have extended the clinical utility of [(11)C]choline to breast cancer where radiotracer uptake correlates with tumor aggressiveness (grade). In the present study, a new choline analog, [(18)F]fluoro-[1,2-(2)H(4)]choline, was synthesized and evaluated as a potential PET imaging probe. [(18)F]Fluorocholine, [(18)F]fluoro-[1-(2)H(2)]choline and [(18)F]fluoro-[1,2-(2)H(4)]choline were synthesized by alkylation of the relevant precursor with [(18)F]fluorobromomethane or [(18)F]fluoromethyl tosylate. Radiosynthesis of [(18)F]fluoromethyl tosylate required extensive modification of the existing method. [(18)F]Fluorocholine and [(18)F]fluoro-[1,2-(2)H(4)]choline were then subjected to in vitro oxidative stability analysis in a chemical oxidation model using potassium permanganate and an enzymatic model using choline oxidase. The two radiotracers, together with the corresponding di-deuterated compound, [(18)F]fluoro-[1-(2)H(2)]choline, were then evaluated in vivo in a time-course biodistribution study in HCT-116 tumor-bearing mice. Alkylation with [(18)F]fluoromethyl tosylate proved to be the most reliable radiosynthetic route. Stability models indicate that [(18)F]fluoro-[1,2-(2)H(4)]choline possesses increased chemical and enzymatic (choline oxidase) oxidative stability relative to [(18)F]fluorocholine. The distribution of the three radiotracers, [(18)F]fluorocholine, [(18)F]fluoro-[1-(2)H(2)]choline and [(18)F]fluoro-[1,2-(2)H(4)]choline, showed a similar uptake profile in most organs. Crucially, tumor uptake of [(18)F]fluoro-[1,2-(2)H(4)]choline was significantly increased at late time points compared to [(18)F]fluorocholine and [(18)F]fluoro-[1-(2)H(2)]choline. Stability analysis and biodistribution suggest that [(18)F]fluoro-[1,2-(2)H(4)]choline warrants further in vivo investigation as a PET probe of choline metabolism.

Reverse-benzamidine antimalarial agents: Design, synthesis, and biological evaluation

In the frame of the development of bis-cationic choline analogs, the RSA of bis- N-alkylamidines were studied and a new series of reverse-benzamidine derivatives was designed. Contrary to the lipophilicity, the basicity of alkylamidine compounds directly influences their antimalarial potencies.

PET with 18F-labelled choline-based tracers for tumour imaging: a review of the literature

To give an up-to-date overview of the potential clinical utility of (18)F-labelled choline derivatives for tumour imaging with positron emission tomography. A PubMed search for (18)F-labelled choline analogues was performed. Review articles and reference lists were used to supplement the search findings. (18)F-labelled choline analogues have been investigated as oncological PET probes for many types of cancer on the basis of enhanced cell proliferation. To date, studies have focused on the evaluation of prostate cancer. Available studies have provided preliminary results for detecting local and metastatic disease. Experience with (18)F-fluorocholine PET in other tumour types, including brain and liver tumours, is still limited. In the brain, excellent discrimination between tumour and normal tissue can be achieved due to the low physiological uptake of (18)F-fluorocholine. In the liver, in which there is a moderate to high degree of physiological uptake in normal tissue, malignancy discrimination may be more challenging. PET/CT with (18)F-fluorocholine can be used to detect (recurrent) local prostate cancer, but seems to have limited value for T (tumour) and N (nodal) staging. In patients presenting with recurrent biochemical prostate cancer, it is a suitable single-step examination with the ability to exclude distant metastases when local salvage treatment is intended. In the brain, high-grade gliomas, metastases and benign lesions can be distinguished on the basis of (18)F-fluorocholine uptake. Moreover, PET imaging is able to differentiate between radiation-induced injury and tumour recurrence. In the liver, (18)F-fluorocholine PET/CT seems promising for the detection of hepatocellular carcinoma.

Rescuing of the hydride transfer reaction in the Glu312Asp variant of choline oxidase by a substrate analogue

In the active site of choline oxidase, Glu312 participates in binding the trimethylammonium group of choline, thereby positioning the alcohol substrate properly for efficient hydride transfer to the enzyme-bound flavin. Previous studies have shown that substitution of Glu312 with aspartate results in a perturbed mechanism of hydride transfer, with a 260-fold decrease in the rate associated with the mutation. Here, the reaction of alcohol oxidation catalyzed by the Glu312Asp enzyme has been investigated with 3-hydroxypropyl-trimethylamine (3-HPTA), a choline analogue with an extra methylene, as substrate. The results of the kinetic investigation using steady state and rapid reaction approaches showed that the impaired ability of the Glu312Asp enzyme to catalyze a hydride transfer reaction can be effectively, but not completely, rescued in the presence of an extra methylene group on the substrate that compensates for the equivalent shortening of the side chain on residue 312. This observation is consistent with choline oxidase having evolved to optimally catalyze the oxidation of choline.

The bisnaphthalimides as new active lead compounds against Plasmodium falciparum

The bisquaternary bisnaphthalimides are a versatile class of compounds being active against the malaria parasite Plasmodium falciparum in the lower nanomolar range of concentration combined with no cytotoxicity. The series of compounds is designed as choline analogues and interfering agents of the phosphatidylcholine biosynthesis. The qualitative analysis of the structure–activity relationships (SAR) revealed the importance of a long methylene middle chain of at least 8 methylene groups between the two bisquaternary naphthalimides or a monoquaternary naphthalimide consisting of a long alkyl chain attached to the positively charged nitrogen atom. Since the SARs are different from reported biscationic antimalarial drugs the mode of action remains to be elucidated.

Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.

Efficient automated one-step synthesis of 2-[18F]fluoroethylcholine for clinical imaging: optimized reaction conditions and improved quality controls of different synthetic approaches

[(18)F]-labelled choline analogues, such as 2-[(18)F]fluoroethylcholine ((18)FECH), have suggested to be a new class of choline derivatives highly useful for the imaging of prostate and brain tumours. In fact, tumour cells with enhanced proliferation rate usually exhibit an improved choline uptake due to the increased membrane phospholipids biosynthesis. The aim of this study was the development of a high yielding synthesis of (18)FECH. The possibility of shortening the synthesis time by reacting all the reagents in a convenient and rapid one-step reaction was specially considered. (18)FECH was synthesized by reacting [(18)F]fluoride with 1,2-bis(tosyloxy)ethane and N,N-dimethylaminoethanol. The synthesis was carried out using both a one- and a two-step reaction in order to compare the two procedures. The effects on the radiochemical yield and purity by using different [(18)F]fluoride phase transfer catalysts, reagents amounts and purification methods were assessed. Quality controls on the final products were performed by means of radio-thin-layer chromatography, gas chromatography and high-performance liquid chromatography equipped with conductimetric, ultraviolet and radiometric detectors. In the optimized experimental conditions, (18)FECH was synthesized with a radiochemical yield of 43+/-3% and 48+/-1% (not corrected for decay) when the two-step or the one-step approach were used, respectively. The radiochemical purity was higher than 99% regardless of the different synthetic pathways or purification methods adopted. The main chemical impurity was due to N,N-dimethylmorpholinium. The identity of this impurity in (18)FECH preparations was not previously reported. An improved two-step and an innovative one-step reaction for synthesizing (18)FECH in a high yield were reported. The adaptation of a multistep synthesis to a single step process, opens further possibilities for simpler and more reliable automations.

Iron oxide‐based magnetic nanostructures bearing cytotoxic organosilicon molecules for drug delivery and therapy

AbstractThe results of our own investigation on synthesis, physico‐chemical and biological study of iron oxide based magnetic nanoparticles bearing cytotoxic organosilicon molecules of choline and colamine analogues, as potential agents for antitumor therapy, are summarized. These molecules contain hydrophilic head and long lipophilic tails, which are able to deepen inside the first surfactant shell (oleic acid), forming bilayer membrane like structures. Such compositions have a great privilege possessing magnetic properties, which in some cases could be essential moment in targeted drug delivery. The methodological approach has been developed and applied to the preparation of water soluble single or mixed coated biologically active nanoparticles of different types. Copyright © 2010 John Wiley & Sons, Ltd.