microPET Studies Research Articles

DIFFERENT TIME COURSE OF ASTROCYTOSIS AND AMYLOID DEPOSITION IN ALZHEIMER'S APPSWE TRANSGENICE MICE: A MULTI-TRACER MICROPET STUDY

Emerging evidence suggests a link between neuroinflammation and beta-amyloid (A β) deposition in Alzheimer's disease (AD). Our recent in vivo microPET study showed higher 11 C-AZD2184 binding in 18-24 months APPswe mice, while elevated 11 C-deuterium-L-deprenyl binding in 6 months APPswe mice, suggesting different time courses of pathological events (Rodriguez-Vieitez, AAIC 2013). Here we investigate further the temporal and regionalrelation of astrocytosis, microgliosis and A β deposition in APPswe mice in vitro by autoradiography alsong with immunostaining. Binding characteristics of 3 H-AZD2184, 3 H-PIB, 3 H-L-deprenyl and 3 H-PK11195 were evaluated in APPswe mice brain. Distribution of Aβ, astrocytosis and microgliosis in APPswe mice (6, 8-15, 18-24 months, n=3-6/group) and wild-type mice (8-12, 18-24 months, n=3-6/group) were analyzed by in vitro autoradiography using the aforementioned ligands and immunostaining performed with antibodies for Aβ 42, GFAP, Iba-1 in sagittal brain slides. 3 H-AZD2184, 3 H-PIB, 3 H-L-deprenyl and 3 H-PK11195 autoradiography revealed presence of high affinity binding sites (nM range) in APPswe mice cortex. Both the 3 H-AZD2184 and 3 H-PIB binding wereelevated in the cortex and hippocampus of 18-24 months APPswe mice compared to wild-type mice, corroborated with increased A β 42 plaque deposition observed in aged APPswe mice. The 3 H-L-deprenyl binding was high already in youngAPPswemice and demonstrated no changes with age, while the 3 H-PK11195 binding was increased in the hippocampus of aged APPswe mice. A greater number of hypertrophic GFAP+ astrocytes were surrounding and distant to A β plaques, whereas Iba1+ activated microglia were in close proximity to A β plaques in aged APPswe mice. Our findings demonstrate that astrocytosis precedes amyloid deposition, and that microgliosis is more closely associated with A β plaque deposition in APPswe mice brain, supporting our in vivo microPET finding of early astrocytosis in APPswe mice.

Synthesis of 18F-FP-Lladtthhrpwt as a new probe for the detection of lung cancer by microPET

Objective To prepare 18F-2-fluoropropionyl (FP)-Lladtthhrpwt (18 F-FP-ZS-6) by a multifunctional 18F radiolabeling module and investigate its in vivo distribution in human lung cancer cell (NCI-H1299)-bearing nude mice by microPET.Methods Using a phage display peptide library to screen and identify Lladtthhrpwt (ZS-6) which has an affinity for human lung cancer.PET-MF-2V-IT-I synthesizer was used for the synthesis and purification of 18F-FP-ZS-6.MicroPET studies were carried out in NCI-H1299-bearing nude mice after injection of 18F-FP-ZS-6.Results 4-nitrophenyl 2-[18F] fluoropropionate (18F-NFP) was prepared by one-pot radiochemical procedure.The radiochemical yield of 18F-FP-ZS-6 was (5±2)％ (n=3,decay-corrected) from 18F and the radiochemical purity was more than 95％.MicroPET studies showed that the uptake of 18F-FP-ZS-6 was high in the tumor and stomach of NCI-H1299-bearing nude mice model.The tumor uptake of 18F-FP-ZS-6 was 0.329,0.350,0.405,0.433,0.420,0.415,0.402,0.403,0.390 ％ID/g at 5,15,25,35,45,55,65,75,85 min post-injection,respectively.The tumor uptake peak was at 35 min post-injection and then 18F-FP-ZS-6 was cleared slowly.Conclusions 18F-FP-ZS-6 could be successfully prepared.It might be a potential tracer for imaging human lung cancer. Key words: Lung neoplasms; Peptides; Fluorine radioisotopes; Chemical synthesis; Radionuclide imaging; Mice, nude

Radiosyntheses and in vivo evaluation of carbon-11 PET tracers for PDE10A in the brain of rodent and nonhuman primate

The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioligands are reported here. Radiolabeling of [11C]1–4 was achieved by alkylation of their corresponding desmethyl precursors with [11C]CH3I. Preliminary biodistribution evaluation in Sprague-Dawley rats demonstrated that [11C]1 and [11C]2 had high striatal accumulation (at peak time) for [11C]1 and [11C]2 were 6.0-fold and 4.5-fold at 60min, respectively. Following MP-10 pretreatment, striatal uptake in rats of [11C]1 and [11C]2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET studies of [11C]1 and [11C]2 in nonhuman primates (NHP) also showed good tracer retention in the striatum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [11C]1 reached 1.8 at 30min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from NHP plasma samples suggested that [11C]1 had a very favorable metabolic stability. Our preclinical investigations suggest that [11C]1 is a promising candidate for quantification of PDE10A in vivo using PET.

A Rat Model of Photothrombotic Capsular Infarct with a Marked Motor Deficit: A Behavioral, Histologic, and microPET Study

We present a new method for inducing a circumscribed subcortical capsular infarct (SCI), which imposes a persistent motor impairment in rats. Photothrombotic destruction of the internal capsule (IC) was conducted in Sprague Dawley rats (male; n=38). The motor performance of all animals was assessed using forelimb placing, forelimb use asymmetry, and the single pellet reaching test. On the basis of the degree of motor recovery, rats were subdivided into either the poor recovery group (PRG) or the moderate recovery group (MRG). Imaging assessment of the impact of SCI on brain metabolism was performed using 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) microPET (positron emission tomography). Photothrombotic lesioning using low light energy selectively disrupted circumscribed capsular fibers. The MRG showed recovery of motor performance after 1 week, but the PRG showed a persistent motor impairment for >3 weeks. Damage to the posterior limb of the IC (PLIC) is more effective for producing a severe motor deficit. Analysis of PET data revealed decreased regional glucose metabolism in the ipsilesional motor and bilateral sensory cortex and increased metabolism in the contralesional motor cortex and bilateral hippocampus during the early recovery period after SCI. Behavioral, histologic, and functional imaging findings support the usefulness of this novel SCI rat model for investigating motor recovery.

Synthesis, [18F] radiolabeling, and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be 18F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC50=6.3nM). [18F]12 was synthesized under conventional conditions in high specific activity with 40–50% decay-corrected yield. MicroPET studies using [18F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [18F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [18F]12 in the tumor is specific to PARP-1 expression.

Radiosynthesis and in vivo evaluation of a novel σ1 selective PET ligand.

The σ1 receptor is an important target for CNS disorders. We previously identified a σ1 ligand TZ3108 having highly potent (Ki-σ1 = 0.48 nM) and selective affinity for σ1 versus σ2 receptors. TZ3108 was 18F-labeled with F-18 for in vivo evaluation. Biodistribution and blocking studies of [18F]TZ3108 in male Sprague-Dawley rats demonstrated high brain uptake, which was σ1-specific with no in vivo defluorination. MicroPET studies in cynomolgus macaques showed high brain penetration of [18F]TZ3108; the regional brain distribution was consistent with that of the σ1 receptor. Pseudo-equilibrium in the brain was reached ~ 45 min post-injection. Metabolite analysis of [18F]TZ3108 in NHP blood and rodent blood and brain revealed that ~ 70% parent remained in the plasma of NHPs 60 min post-injection and the major radiometabolite did not cross the blood-brain barrier in rats. In summary, the potent, selective and metabolically stable σ1 specific radioligand [18F]TZ3108 represents a potentially useful PET radioligand for quantifying the σ1 receptor in the brain.

Abstract B141: Molecular imaging of DNA damage and repair in cancer.

Abstract Objectives: To directly assess apurinic/apyrimidinic (AP) sites induced by DNA-targeted chemotherapeutic agents for in vivo studies of DNA damage and repair. Background: Current cancer treatments rely heavily on chemotherapeutic agents to induce cytotoxic DNA damages and programmed cell death in ancer cells. However, the efficacy of DNA-targeted agents such as temozolomide are often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway results in formation of abasic or AP sites and inhibition of AP sites leads to significant reduction of drug resistance and enhancement of drug sensitivity. Thus, AP-site formation has been identified as an important biomarker in DNA-targeted chemotherapies. Methods: Design, synthesis, and evaluation of small molecular probes used in combination with positron emission tomography (PET) that allows for quantification of AP sites in vivo. Results: To date, we have design and synthesized a series of AP-binding agents for PET imaging studies. Following radiolabelling, microPET studies have been conducted to evaluate their pharmacokinetic profiles in melanoma and glioma xenograft tumor mouse models that are pre-treated with temozolomide to induce AP-site formation. Subsequent quantitative analysis showed that the radioactivity concentration were higher in tumor regions pre-treated with temozolomide relative to tumor regions without any treatment, indicating that AP-site formation was elevated following temozolomide treatment. In vivo blocking studies based on microPET also showed that the agents bound to AP sites with high specificity. Conclusion: Our studies have proved the concept that PET imaging agents can be developed to monitor BER pathway and demonstrate the feasibility for future clinical applications in cancer research. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B141. Citation Format: Yanming Wang, Chunying Wu, Allison Condie, Junqing Zhu, Stanton Gerson. Molecular imaging of DNA damage and repair in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B141.

Impact of partial volume effect correction on cerebral β-amyloid imaging in APP-Swe mice using [18F]-florbetaben PET

We previously investigated the progression of β-amyloid deposition in brain of mice over-expressing amyloid-precursor protein (APP-Swe), a model of Alzheimer's disease (AD), in a longitudinal PET study with the novel β-amyloid tracer [18F]-florbetaben. There were certain discrepancies between PET and autoradiographic findings, which seemed to arise from partial volume effects (PVE). Since this phenomenon can lead to bias, most especially in the quantitation of brain microPET studies of mice, we aimed in the present study to investigate the magnitude of PVE on [18F]-florbetaben quantitation in murine brain, and to establish and validate a useful correction method (PVEC).Phantom studies with solutions of known radioactivity concentration were performed to measure the full-width-at-half-maximum (FWHM) resolution of the Siemens Inveon DPET and to validate a volume-of-interest (VOI)-based PVEC algorithm. Several VOI-brain-masks were applied to perform in vivo PVEC on [18F]-florbetaben data from C57BL/6(N=6) mice, while uncorrected and PVE-corrected data were cross-validated with gamma counting and autoradiography. Next, PVEC was performed on longitudinal PET data set consisting of 43 PET scans in APP-Swe (13–20months) and age-matched wild-type (WT) mice using the previously defined masks. VOI-based cortex-to-cerebellum ratios (SUVR) were compared for uncorrected and PVE-corrected results. Brains from a subset of transgenic mice were ultimately examined by autoradiography ex vivo and histochemistry in vitro as gold standard assessments, and compared to VOI-based PET results.The phantom study indicated a FWHM of 1.72mm. Applying a VOI-brain-mask including extracerebral regions gave robust PVEC, with increased precision of the SUVR results. Cortical SUVR increased with age in APP-Swe mice compared to baseline measurements (16months: +5.5%, p<0.005; 20months: +15.5%, p<0.05) with uncorrected data, and to a substantially greater extent with PVEC (16months: +12.2% p<0.005; 20months: +36.4% p<0.05). WT animals showed no binding changes, irrespective of PVEC. Relative to autoradiographic results, the error [%] for uncorrected cortical SUVR was 18.9% for native PET data, and declined to 4.8% upon PVEC, in high correlation with histochemistry results. We calculate that PVEC increases by 10% statistical power for detecting altered [18F]-florbetaben uptake in aging APP-Swe mice in planned studies of disease modifying treatments on amyloidogenesis.

Evaluation of P-glycoprotein (abcb1a/b) modulation of [18F]fallypride in MicroPET imaging studies

[18F]Fallypride ([18F]FP) is an important and routinely used D2/D3 antagonist for quantitative imaging of dopaminergic neurotransmission in vivo. Recently it was shown that the brain uptake of the structurally related [11C]raclopride is modulated by P-glycoprotein (P-gp), an important efflux transporter at the blood–brain barrier. The purpose of this study was to determine whether the brain uptake of [18F]FP is influenced by P-gp. For examination of this possible modulation microPET studies were performed in a rat and a mouse model. Hence, [18F]FP was applied to Sprague Dawley rats, half of them being treated with the P-gp inhibitor cyclosporine A (CsA). In a second experimental series the tracer was applied to three different groups of FVB/N mice: wild type, P-gp double knockout (abcb1a/1b (−/−)) and CsA-treated mice. In CsA-treated Sprague Dawley rats [18F]FP showed an elevated standard uptake value in the striatum compared to the control animals. In FVB/N mice a similar effect was observed, showing an increasing uptake from wild type to CsA-treated and double knockout mice. Since genetically or pharmacologically induced reduction of P-gp activity increased the uptake of [18F]FP markedly, we conclude that [18F]FP is indeed a substrate of P-gp and that the efflux pump modulates its brain uptake. This effect – if true for humans – may have particular impact on clinical studies using [18F]FP for assessment of D2/3 receptor occupancy by antipsychotic drugs.This article is part of the Special Issue Section entitled ‘Neuroimaging in Neuropharmacology’.

Effects of curcumin on glucose metabolism in the brains of rats subjected to chronic unpredictable stress: a 18 F-FDG micro-PET study

BackgroundChronic unpredictable stress (CUS) can cause behavioral and physiological abnormalities that are important to the prediction of symptoms of depression that may be associated with cerebral glucose metabolic abnormalities. Curcumin showed potential antidepressant effects, but whether or not it can reverse cerebral functional abnormalities and so ameliorate depression remains unknown.MethodsTo investigate the effects of curcumin on brain activity in CUS rats, rats were subjected to 3 weeks of CUS and then treated with curcumin orally at a dose of 40 mg/kg/day for one month. 18 F fluorodeoxyglucose (18 F-FDG)-micro positron emission tomography (micro-PET) neuroimaging was used to detect changes in cerebral metabolism. Body weight, sucrose preference, and open field tests were used to record depressive behaviors during CUS and after curcumin treatment.ResultsThree weeks of CUS significantly decreased body weight, sucrose preference, sucrose consumption, total distance travelling, and the number of rearing events. It also induced metabolic alterations in several parts of the brain, showing increased glucose metabolism in the right hemisphere. After curcumin treatment for one month, sucrose preference, sucrose consumption, total distance travelling, and the number of rearing events returned to normal levels. Curcumin treatment also induced strong deactivation of the left primary auditory cortex and activation of amygdalohippocampal cortex.ConclusionCurcumin was found to ameliorate the abnormalities in the behavior and brain glucose metabolism caused by CUS, which may account for its antidepressive effects.

2-[18F]Fludarabine, a Novel Positron Emission Tomography (PET) Tracer for Imaging Lymphoma: a Micro-PET Study in Murine Models

Fludarabine has proven to be of considerable efficacy in the treatment of low-grade lymphomas. We have developed the labeling of this drug with fluorine-18 and evaluated 2-[(18)F]fludarabine as a novel positron emission tomography (PET) probe for in vivo imaging. Preclinical studies were conducted with 2-[(18)F]fludarabine, in parallel with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG), in Swiss CD-1 and CB17 severely combined immunodeficient (SCID) mice, both as tumor-free control groups, and SCID mice bearing RL lymphomas. In Swiss mice, micro-PET studies with 2-[(18)F]fludarabine showed a distribution restricted to the organs of excretion and the spleen, the latter being less evident in SCID animals. In lymphoma-bearing SCID mice, 2-[(18)F]fludarabine demonstrated a rapid tumor uptake over the first 20min which subsequently plateaued and provided an improved contrast than that of [(18)F]FDG. This radiotracer merits further evaluation to establish its clinical usefulness to image low-grade lymphoma in humans in future clinical investigations.

Low-frequency stimulation inhibits epileptogenesis by modulating the early network of the limbic system as evaluated in amygdala kindling model

Low-frequency stimulation (LFS) is emerging as a new option for the treatment of epilepsy. The present study was designed to determine whether there is a crucial period for the treatment of epileptogenesis with LFS. LFS was delivered at different time-points to evaluate its anti-epileptogenic effect on amygdala-kindling rats. (18)F-fluorodeoxyglucose small-animal positron-emission tomography (microPET) and multi-channel EEG recording (MER) were used to investigate the dynamics of brain networks during epileptogenesis and LFS treatment. Interestingly, LFS delivered in the first 7days significantly retarded the progression of behavioral seizure stages and shortened the afterdischarge duration (ADD), LFS delivered throughout the whole process resulted in similar effects. However, if LFS was delivered at the beginning of seizure stage 2 or 3 (5±0.3days during kindling acquisition), it had no anti-epileptogenic effect and even prolonged the ADD and enhanced synchronization of the EEGs. MicroPET study revealed a notable hypometabolism in the amygdala, piriform cortex, entorhinal cortex and other regions in the limbic system during the period from seizure stage 0 to stage 2 or 3. The glucose metabolism in those regions was specifically increased by LFS. MER further verified that an early network of afterdischarge spread was formed in those brain regions during kindling acquisition. Thus, we provided direct evidence that modulation of the early network in the limbic system is crucial for the anti-epileptogenic effect of LFS in amygdaloid-kindling rats.

The effects of exposure to 915 MHz radiofrequency identification on cerebral glucose metabolism in rat: A [F-18] FDG micro-PET study

Purpose: We investigated the effect of whole-body exposure to 915-MHz radiofrequency identification (RFID) on rat cortical glucose metabolism by using 18F-deoxyglucose positron emission tomography (FDG-PET).Materials and methods: Male Sprague-Dawley rats were divided into three groups: Cage-control, sham-exposed and RFID-exposed groups. Rats were exposed to the 915-MHz RFID for 8 h daily, 5 days per week, for 2 or 16 weeks. The whole-body average specific absorption rate (SAR) was 4 W/kg for the field of the 915 MHz RFID signal. FDG-PET images were obtained the day after RFID exposure, using micro-PET with a FDG tracer. With a Xeleris functional imaging workstation, absolute values in regions of interest (ROI) in the frontal, temporal and parietal cortexes and cerebellum were measured. Cortical ROI values were normalized to the cerebellar value and compared.Results: The data showed that the relative cerebral glucose metabolic rate was unchanged in the frontal, temporal and parietal cortexes of the 915 MHz RFID-exposed rats, compared with rats in cage-control and sham-exposed groups.Conclusion: Our results suggest that 915 MHz RFID radiation exposure did not cause a significant long lasting effect on glucose metabolism in the rat brain.

Abstract 318: Preclinical evaluation of monoclonal antibody H6-11 for prostate cancer imaging.

Abstract Altered post-translational modification (PTM) of glycosylations to various nuclear and cytosolic proteins is a universal feature of cancer cells and these modified glycan plan significant role in immune surveillance, tumor invasion, and increase malignancy. In this study, we reported a novel monoclonal antibody reacting to the tumor glycoproteins with molecular weight around 40-60 kDa. Deglycosylation examination suggested the antigenic isotope of the glycan is O-linked β-N-acetylglucosamine (O-GlcNAc) group. We examined the reactivity of H6-11 to cancer cells including PC-3, MCF-7 cells and implanted PC-3 and EMT-6 tumor tissues using immunofluorescence staining and autoradiography technique. The Zr-89 labeled H6-11 was successfully synthesized and the microPET study was performed in PC-3 xenograft prostate model in nude mice. microPET study showed that significant amount of radioactivity accumulation was observed in the excretory organs during the early period post-injection of radioactivity. At 24 hrs post-injection of radioactivity, significant amount of radioactivity was observed in the xenograft prostate tumor, while the radioactivity intensity was decreased in the liver and kidney. Radioactivity uptake in the tumor was still evident at 120 hrs post-injection of radioactivity. Our work suggested that the H6-11 antibody is capable of detecting prostate tumors both in vitro and in vivo. Citation Format: Hongjun Jin, Mai Xu, Prashanth K. Padakanti, Zhude Tu. Preclinical evaluation of monoclonal antibody H6-11 for prostate cancer imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2013-318

Radiosynthesis and biological evaluation of alpha-[F-18]fluoromethyl phenylalanine for brain tumor imaging

Radiolabeled amino acids have proven utility for imaging brain tumors in humans, particularly those that target system L amino acid transport. We have prepared the novel phenylalanine analogue, (FMePhe, 9), as part of an effort to develop new system L tracers that can be prepared in high radiochemical yield through nucleophilic [(18)F]fluorination. The tumor imaging properties of both enantiomers of this new tracer were evaluated through cell uptake, biodistribution and microPET studies in the mouse DBT model of high grade glioma. The non-radioactive form of 9 and the cyclic sulfamidate labeling precursor were prepared from commercially available racemic α-benzylserine. Racemic [(18)F]9 was prepared from the labeling precursor in two steps using standard[(18)F]fluoride nucleophilic reaction conditions followed by acidic deprotection. The individual enantiomers [(18)F]9a and [(18)F]9b were isolated using preparative chiral HPLC. In vitro uptake inhibition assays were performed with each enantiomer using DBT cells. Biodistribution and microPET/CT studies were performed with each enantiomer in male BALB/c mice at approximately 2 weeks after implantation of DBT tumor cells. Radiolabeling of the cyclic sulfamidate precursor 5 provides racemic [(18)F]9 in high radiochemical yield (60%-70%, n=4) and high radiochemical purity (>96%, n=4). In vitro uptake assays demonstrate that both [(18)F]9a and [(18)F]9b undergo tumor cell uptake through system L transport. The biodistribution studies using the single enantiomers [(18)F]9a and [(18)F]9b demonstrated good tumor uptake with lower uptake in most normal tissues, and [(18)F]9a had higher tumor uptake than [(18)F]9b. MicroPET imaging demonstrated good tumor visualization within 10 min of injection, rapid uptake of radioactivity, and tumor to brain ratios of approximately 6:1 at 60 min postinjection. The novel PET tracer, [(18)F]FMePhe, is readily synthesized in good yield from a cyclic sulfamidate precursor. Biodistribution and microPET studies in the DBT model demonstrate good tumor to tissue ratios and tumor visualization, with enantiomer [(18)F]9a having higher tumor uptake. However, the brain availability of both enantiomers was lower than expected for system L substrates, suggesting the [(18)F]fluorine group in the β-position affects uptake of these compounds by system L transporters.

18 F-click labeling and preclinical evaluation of a new 18 F-folate for PET imaging

BackgroundThe folate receptor (FR) is a well-established target for tumor imaging and therapy. To date, only a few 18 F-folate conjugates via 18 F-prosthetic group labeling for positron emission tomography (PET) imaging have been developed. To some extent, they all lack the optimal balance between efficient radiochemistry and favorable in vivo characteristics.MethodsA new clickable olate precursor was synthesized by regioselective coupling of folic acid to 11-azido-3,6,9-trioxaundecan-1-amine at the γ-position of the glutamic acid residue. The non-radioactive reference compound was synthesized via copper-catalyzed azide-alkyne cycloaddition of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne and γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide. The radiosynthesis was accomplished in two steps: at first a 18 F-fluorination of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate, followed by a 18 F-click reaction with the γ-azido folate. The in vitro, ex vivo, and in vivo behaviors of the new 18 F-folate were investigated using FR-positive human KB cells in displacement assays and microPET studies using KB tumor-bearing mice.ResultsThe new 18 F-folate with oligoethylene spacers showed reduced lipophilicity in respect to the previously developed 18 F-click folate with alkyl spacers and excellent affinity (Ki = 1.6 nM) to the FR. Combining the highly efficient 18 F-click chemistry and a polar oligoethylene-based 18 F-prosthetic group facilitated these results. The overall radiochemical yield of the isolated and formulated product averages 8.7%. In vivo PET imaging in KB tumor-bearing mice showed a tumor uptake of 3.4% ID/g tissue, which could be reduced by FR blockade with native folic acid. Although the new 18 F-oligoethyleneglycole (OEG)-folate showed reduced hepatobiliary excretion over time, a distinct unspecific abdominal background was still observed.ConclusionsA new 18 F-folate was developed, being available in very high radiochemical yields via a fast and convenient two-step radiosynthesis. The new 18 F-OEG-folate showed good in vivo behavior and lines up with several recently evaluated 18 F-labeled folates.

Radiolabelling and preliminary evaluation of 68Ga-tetrapyrrole derivatives as potential tracers for PET

Tetrapyrroles are multisided natural products which are of relevance in clinical medicine. Owing to their specific accumulation in tumour tissue, porphyrins, metalloporphyrins and chlorins have been used as in photodynamic therapy and optical imaging. Moreover, their specific uptake into inflammatory atheromatous plaques via LDL endocytosis has been reported. The present study is concerned with the synthesis of (68)Ga labelled porphyrin derivatives and an in vitro assessment of the utility of radiotracers in positron emission tomography. A set of five porphyrin derivatives were labelled using (68)Ga from a commercially obtained radionuclide generator. Dedicated post-processing of the generator eluate was conducted to allow for labelling in aqueous media and also under anhydrous conditions. Challenge studies and incubation in human serum confirmed the stability of the tracers. Plasma protein binding was investigated in order to confirm the presence of freely diffusible radioligand in plasma. A preliminary microPET study in a tumour-bearing rat resulted in a clear visualisation of the tumour.

Synthesis and Evaluation of 64Cu-Labeled Monomeric and Dimeric NGR Peptides for MicroPET Imaging of CD13 Receptor Expression

The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated (64)Cu-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid (DOTA) and labeled with (64)Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The resulting monomeric ((64)Cu-DOTA-NGR1) and dimeric ((64)Cu-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of (64)Cu-DOTA-NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of (64)Cu-DOTA-NGR1. For small animal microPET studies, (64)Cu-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to (64)Cu-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. (64)Cu-DOTA-NGR2 showed higher tumor uptake and better tumor retention than (64)Cu-DOTA-NGR1, presumably due to bivalency effect and increase in apparent molecular size. (64)Cu-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo.

Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known αvβ3 integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1±1.2%) in a short reaction time (10min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). αvβ3 integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on αvβ3-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.

In vivo evaluation of [18F]FEAnGA-Me: a PET tracer for imaging β-glucuronidase (β-GUS) activity in a tumor/inflammation rodent model

The PET tracer, 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate ([(18)F]FEAnGA), was recently developed for PET imaging of extracellular β-glucuronidase (β-GUS). However, [(18)F]FEAnGA exhibited rapid renal clearance, which resulted in a relatively low tracer uptake in the tumor. To improve the pharmacokinetics of [(18)F]FEAnGA, we developed its more lipophilic methyl ester analog, [(18)F]FEAnGA-Me. [(18)F]FEAnGA-Me was obtained by alkylation of the O-protected glucuronide methyl ester precursor with [(18)F]-fluoroethylamine ([(18)F]FEA), followed by removal of the acetate protecting groups with NaOMe/MeOH. The PET tracer was evaluated by in vitro and in vivo studies. [(18)F]FEAnGA-Me was obtained in 5%-10% overall radiochemical yield. It is 10-fold less hydrophilic than [(18)F]FEAnGA and it is stable in PBS and in the presence of β-GUS for 1h. However, in the presence of esterase or plasma [(18)F]FEAnGA-Me is converted to [(18)F]FEAnGA, and subsequently converted to [(18)F]FEA by β-GUS. MicroPET studies in Wistar rats bearing a C6 glioma and a sterile inflammation showed similar uptake in tumors after injection of either [(18)F]FEAnGA-Me or [(18)F]FEAnGA. Both tracers had a rapid two-phase clearance of total plasma radioactivity with a half-life of 1 and 8min. The [(18)F]FEAnGA fraction generated from [(18)F]FEAnGA-Me by in vivo hydrolysis had a circulation half-life of 1 and 11min in plasma. Similar distribution volume in the viable part of the tumor was found after injection of either [(18)F]FEAnGA-Me or [(18)F]FEAnGA. The imaging properties of [(18)F]FEAnGA-Me were not significantly better than those of [(18)F]FEAnGA. Therefore, other strategies should be applied in order to improve the kinetics of these tracers.