Lin Zhu

Metabolic Imaging of Glutamine in Cancer

Glucose and glutamine are the most abundant nutrients for producing energy and building blocks in normal and tumor cells. Increased glycolysis in tumors, the Warburg Effect, is the basis for 18F-FDG PET imaging. Cancer cells can also be genetically reprogrammed to use glutamine. 5-11C-(2S)-glutamine and 18F-(2S,4R)4-fluoroglutamine may be useful complementary tools to measure changes in tumor metabolism. In glioma patients, the tracer 18F-(2S,4R)4-fluoroglutamine showed tumor-to-background contrast different from that of 18F-FDG and differences in uptake in glioma patients with clinical progression of disease versus stable disease (tumor-to-brain ratio > 3.7 in clinically active glioma tumors, minimal or no specific uptake in clinically stable tumors). These preliminary results suggest that 18F-(2S,4R)4-fluoroglutamine PET may be a new tool for probing in vivo metabolism of glutamine in cancer patients and for guiding glutamine-targeted therapeutics. Further studies of uptake mechanism, and comparison of kinetics for 18F-(2S,4R)4-fluoroglutamine versus the 11C-labeled native glutamine, will be important and enlightening.

Study the effect of a pseudo-carrier on pharmacokinetics of 9-fluoropropyl-(+)-dihydrotetrabenazine in rat plasma by ultra-performance liquid chromatography–tandem mass spectrometry

To evaluate the effect of a pseudo-carrier (9-hydroxypropyl-(+)-dihydrotetrabenazine, AV-149) on pharmacokinetics of 9-fluoropropyl-(+)-dihydrotetrabenazine (AV-133), an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the determination of AV-133 and AV-149 in rat plasma. AV-133 and AV-149 were extracted from plasma following protein precipitation. The chromatographic analysis was performed on an ACQUITY UPLC BEH™ C₁₈ column (50 mm x 2.1 mm x 1.7 μm) by a gradient elution. The mass spectrometer was operated in positive mode using electrospray ionization. The analytes were measured using the multiple reaction-monitoring mode (MRM). An external calibration was used, and the calibration curves were linear in the range of 1.00-800 ng/mL for AV-133 and AV-149. The accuracy ranged from 90.8% to 113.2% and the precision ranged from 2.7% to 9.9% for each analyte. The effect of a pseudo-carrier on pharmacokinetics of AV-133 was studied using the presented method.

Determination of the penetration of 9-fluoropropyl-(+)-dihydrotetrabenazine across the blood–brain barrier in rats by microdialysis combined with liquid chromatography–tandem mass spectrometry

To evaluate the penetration of the blood-brain barrier by 9-fluoropropyl-(+)-dihydrotetrabenazine (AV-133), microdialysis probes were implanted simultaneously into rat blood and brain, and a liquid chromatography-tandem mass spectrometric method was developed and validated to monitor the AV-133 concentration in the microdialysates. The chromatographic separation was performed on an XTerra C(18) column (150 mm × 2.1 mm i.d., 5 μm particles) with gradient elution. The mass spectrometer was operated in positive mode using electrospray ionization. The analytes were measured using the multiple-reaction-monitoring mode. The calibration curves were linear over the range of 5.00-1000 ng/mL AV-133, with a coefficient of determination >0.995. The accuracies ranged from 99.5% to 105.0% and the precisions were <10% for AV-133. This method was used to determine the concentrations of AV-133 and its pharmacokinetics in the brains and blood of rats. The blood and brain concentration-time profiles for AV-133 were obtained, and the blood-brain barrier penetration was evaluated.

Study the pharmacokinetics of AV‐45 in rat plasma and metabolism in liver microsomes by ultra‐performance liquid chromatography with mass spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed and validated to determine AV-45 in rat plasma. After the addition of the internal standard benzophenone, plasma samples were pretreated by protein precipitation. Chromatographic separation was achieved on an Acquity UPLC BEH C₁₈ column (50u2009×u20092.1u2009mm, 1.7u2009µm) by gradient elution at a flow rate of 0.4u2009mL/min. Detection of analytes and internal standard (IS) was done by tandem mass spectrometry, operating in positive-ion and multiple reaction monitoring mode. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect and stability study. The calibration curve showed good linearity over the concentration range 2.00-1000 ng/mL for AV-45. Intra- and inter-day precisions were less than 7.6%, and accuracy ranged from 100.6 to 107.8%. There was no matrix effect. The validated method was successfully applied to a pharmacokinetic study of AV-45 in rats. Additionally, the metabolism of AV-45 in rat liver microsomes was also studied by ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC/TOF-MS). With the help of chromatographic behavior and accurate mass measurements, the metabolites were characterized.

Progressive loss of striatal dopamine terminals in MPTP-induced acute parkinsonism in cynomolgus monkeys using vesicular monoamine transporter type 2 PET imaging ([(18)F]AV-133).

The 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP)-induced parkinsonism model, particularly in non-human primates, remains the gold-standard for studying the pathogenesis and assessing novel therapies for Parkinson’s disease. However, whether the loss of dopaminergic neurons in this model is progressive remains controversial, mostly due to the lack of objective in vivo assessment of changes in the integrity of these neurons. In the present study, parkinsonism was induced in cynomolgus monkeys by intravenous administration of MPTP (0.2 mg/kg) for 15 days; stable parkinsonism developed over 90 days, when the symptoms were stable. Noninvasive positron emission tomographic neuroimaging of vesicular monoamine transporter 2 with 9-[18F] fluoropropyl-(+)-dihydrotetrabenazine ([18F]AV-133) was used before, and 15 and 90 days after the beginning of acute MPTP treatment. The imaging showed evident progressive loss of striatal uptake of [18F]AV-133. The dopaminergic denervation severity had a significant linear correlation with the clinical rating scores and the bradykinesia subscores. These findings demonstrated that [18F]AV-133 PET imaging is a useful tool to noninvasively evaluate the evolution of monoaminergic terminal loss in a monkey model of MPTP-induced parkinsonism.

Mapping the Target Localization and Biodistribution of Non-Radiolabeled VMAT2 Ligands in rat Brain

Imaging targeting vesicular monoamine transporter (VMAT2) alterations is a sensitive tool for early diagnosis of Parkinson’s disease. Our group has reported several novel 2-amino-DTBZ derivatives as potential VMAT2 imaging agents. The objective of this paper is to develop a non-radiolabeled methodology to screen the candidate compounds for accelerating the drug discovery process. 9-[18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]AV-133) is a PET imaging agent targeting VMAT2 binding sites in the brain. Nonradioactive AV-133 was injected (iv) into rats, at the end of the allotted time, the animals were killed and six regions of brain and plasma from each animal were processed for quantitative measurement of AV-133 by LC-MS/MS. These data were converted to the percentage injected dose per gram tissue weight (%ID/g tissue) and the brain target tissue to background ratios to allow direct comparison with data obtained by gamma counting of the injected radioactive [18F]AV-133. The %ID/g and the brain target tissue to background ratios calculated using the LC-MS/MS method were highly correlated to the values obtained by standard radioactivity measurements of [18F]AV-133. The pattern of AV-133 in rat brain was consistent with the known distribution of VMAT2. The concordance indicated that high-sensitivity LC-MS/MS is an indispensable tool in evaluating the quantity of administered chemical in tissue as part of the development of new molecular imaging probes. Furthermore, several novel 2-amino-DTBZ derivatives were detected using this methodology, and their biodistribution data in rat brain were obtained. The information about target engagements of candidates was provided.

Dose–response relationships of FMISO between trace dose and various macro-doses in rat by ultra-performance liquid chromatography with mass spectrometry and radioactivity analysis

Screening the pharmacokinetics of candidates using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) may be efficacious and safe for the research and development of new PET imaging agents. However, the PET imaging agent is administered as trace dose and the sensitivity of LC-MS/MS is often insufficient. If the dose was increased to be quantifiable, it should be necessary to prove whether the pharmacokinetics between trace and macro-doses is consistent or not. In this paper, fluoromisonidazole (FMISO), a tumor PET imaging agent, was chosen to evaluate the dose-response pharmacokinetics by administering various single intravenous doses (0.1, 0.4, 1.6 and 6.4 mg/kg) in male Sprague-Dawley rats. The plasma concentration of FMISO was determined by an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method, and the blood radioactivity of [(18)F]FMISO was detected by a gamma counter. By calculating and comparing the pharmacokinetic parameters, the total area under the plasma concentration-time curve from time zero to infinity (AUC(0-∞)) and peak plasma concentration (C(max)) values increased with the selected FMISO doses, and showing linear dose-dependent. On the other hand, some parameters related to time, such as the elimination half-lives (t(1/2)) and elimination rate constant (K(e)) were dose-independent, and there is no significant deference between trace dose and various macro-doses. The data should be useful to evaluate the novel 2-nitroimidazole derivatives as potential PET tumor imaging agents.

Rapid Detection of the Residual Kryptofix 2.2.2 Levels in [18F]-Labeled Radiopharmaceuticals by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

A fast and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for determination of the residual levels of Kryptofix 2.2.2 (K222) in [18F]-labeled radiopharmaceuticals. The analytical time was only 3 min, and the injection volume was 5 μL. An electrospray ionization source was used in the positive mode (ESI+) for UPLC/MS/MS. The analytical measurements were performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 2–500 ng/mL for K222 showed good linearity. The intra- and inter-day precisions were not more than 5%. The accuracy satisfied the requirement of quality control analysis, the recoveries were found to be 80–120%. This method was successfully applied to detect the residue of K222 in [18F]-fluorodeoxyglucose [(18F)FDG], [18F]-fluoromisonizole[(18F)FMISO], 3′-deoxy-3′-[18F]-fluorothymidine [(18F)FLT], and two new [18F]-labeled radiopharmaceuticals 4-[-(2-[18F]fluoroethoxy) methyl]-1-[2-(2-methyl-5-nitro-1H- imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as 18F-BNU-1) and 4-[-(2-[18F] fluoroethoxy) methyl]-1-[2-(2-nitro-1H-imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as 18F-BNU-2) produced in our lab.

Brain uptake of a non-radioactive pseudo-carrier and its effect on the biodistribution of [18 F]AV-133 in mouse brain

INTRODUCTIONn9-[(18)F]Fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133) is a new PET imaging agent targeting vesicular monoamine transporter type II (VMAT2). To shorten the preparation of [(18)F]AV-133 and to make it more widely available, a simple and rapid purification method using solid-phase extraction (SPE) instead of high-pressure liquid chromatography (HPLC) was developed. The SPE method produced doses containing the non-radioactive pseudo-carrier 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). The objectives of this study were to evaluate the brain uptake of AV-149 by UPLC-MS/MS and its effect on the biodistribution of [(18)F]AV-133 in the brains of mice.nnnMETHODSnThe mice were injected with a bolus including [(18)F]AV-133 and different doses of AV-149. Brain tissue and blood samples were harvested. The effect of different amounts of AV-149 on [(18)F]AV-133 was evaluated by quantifying the brain distribution of radiolabelled tracer [(18)F]AV-133. The concentrations of AV-149 in the brain and plasma were analyzed using a UPLC-MS/MS method.nnnRESULTSnThe concentrations of AV-149 in the brain and plasma exhibited a good linear relationship with the doses. The receptor occupancy curve was fit, and the calculated ED50 value was 8.165mg/kg. The brain biodistribution and regional selectivity of [(18)F]AV-133 had no obvious differences at AV-149 doses lower than 0.1mg/kg. With increasing doses of AV-149, the brain biodistribution of [(18)F]AV-133 changed significantly.nnnCONCLUSIONnThe results are important to further support that the improved radiolabelling procedure of [(18)F]AV-133 using an SPE method may be suitable for routine clinical application.

[ 18 F]Diphenyl sulfide derivatives for imaging serotonin transporter (SERT) in the brain

OBJECTIVESnSerotonin transporters (SERT) play an important role in controlling serotonin concentration in the synaptic cleft and in managing postsynaptic signal transduction. Inhibitors of SERT binding are well known as selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, sertraline, paroxetine, and escitalopram, that are commonly prescribed antidepressants. Positron emission tomography (PET) and single photon emission tomography (SPECT) imaging agents targeting SERT may be useful for studying its function and providing a tool for monitoring drug treatment.nnnMETHODSnA series of novel 18F-labeled diphenyl sulfide derivatives were prepared and tested for their binding affinity. Among them, 2-((2-((dimethylamino)-methyl)-4-(2-(2-fluoroethoxy)ethoxy)phenyl)thio)aniline, 1, which showed excellent binding toward serotonin transporter (SERT) in the brain (Kiu202f=u202f0.09u202fnM), was selected for further evaluation. An active OTs intermediate, 7, was treated with [18F]F-/K222 to provide [18F]1 in one step and in high radiochemical yields. This new SERT targeting agent was evaluated in rats by biodistribution studies and animal PET imaging studies.nnnRESULTSnThe radiolabeling reaction led to the desired [18F]1. After HPLC purification no-carrier-added [18F]1 was obtained (radiochemical yield, 23-47% (nu202f=u202f10,); radiochemical purity >99%; molar activity, 15-28u202fGBq/μmol). Biodistribution studies with [18F]1 showed good brain uptake (1.04% dose/g at 2u202fmin post-injection), high uptake into the hypothalamus (1.55% dose/g at 30u202fmin), and a high target-to-non-target (hypothalamus to cerebellum) ratio of 6.1 at 120u202fmin post-injection. A PET imaging study in normal rats showed excellent uptake in the midbrain and thalamus regions known to be rich in SERT binding sites at 60u202fmin after iv injection. Chasing experiment with escitalopram (iv, 2u202fmg/kg) in a rat at 60u202fmin after iv injection caused a noticeable reduction in the regional radioactivity and the target-to-non-target ratio, suggesting binding by [18F]1 was highly specific and reversible for SERT binding sites in the brain.nnnCONCLUSIONSnA novel diphenyl sulfide derivative, [18F]1 for SERT imaging was successfully prepared and evaluated. Results suggest that this new chemical entity is targeting SERT binding sites in the brain, and it is a suitable candidate for future commercial development.