Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical

¹¹¹In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, ¹¹¹In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether ¹¹¹In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6 MBq/μg) can amplify treatment-related DNA damage for therapeutic gain.MethodsMDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with ¹¹¹In-DTPA-anti-γH2AX-Tat (3 MBq, 6 MBq/μg) or a control radioimmunoconjugate, ¹¹¹In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. ¹¹¹In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies.ResultsThe number of γH2AX foci was greater after exposure of cells to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6 ± 2.5 versus 10.4 ± 2.3 foci/cell; P < .001).¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4 Gy) versus IR alone (5.2% ± 0.9% versus 47.8% ± 2.8%; P < .001). Similarly, bleomycin (25–200 μg/mL) plus ¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either ¹¹¹In-DTPA-anti-γH2AX-Tat or IR alone (? 0.002 ± 0.004 versus 0.036 ± 0.011 and 0.031 ± 0.014 mm³/day, respectively, P < .001).Conclusion¹¹¹In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.     

Phase I trial of intraoperative detection of tumor margins in patients with HER2-positive carcinoma of the breast following administration of 111In-DTPA-trastuzumab Fab fragments

IntroductionOur aim was to conduct a Phase I clinical trial to determine the feasibility of intraoperative detection of tumor margins in HER2 positive breast carcinoma using a hand-held γ-probe following administration of ¹¹¹In-DTPA-trastuzumab Fab fragments. Accurate delineation of tumor margins is important for preventing local recurrence.MethodsSix patients with HER2-positive in situ or invasive ductal carcinoma were administered 74 MBq (0.5 mg) of ¹¹¹In-DTPA-trastuzumab Fab fragments and counts in the tumor, surgical cavity wall and en face margins were measured intraoperatively at 72 h post-injection using the Navigator or C-Trak γ-probes. Margins were evaluated histologically. Quantitative whole body planar imaging was performed to estimate radiation absorbed doses using OLINDA/EXM software. SPECT imaging of the thorax was performed to evaluate tumor uptake. The pharmacokinetics of elimination from the blood and plasma were determined over 72 h.ResultsThere were no acute adverse reactions from ¹¹¹In-DTPA-trastuzumab Fab fragments and no changes in hematological or biochemical indices were found over a 3 month period. ¹¹¹In-DTPA-trastuzumab Fab fragments exhibited a biphasic elimination from the blood and plasma with t_1/2α = 11.9 h and 7.5 h, respectively, and t_1/2β = 26.6 and 20.7 h, respectively. The radiopharmaceutical accumulated in the liver, spleen and kidneys. SPECT imaging did not reveal tumor in any patient. The mean effective dose was 0.146 mSv/MBq (10.8 mSv for 74 MBq). Counts in excised tumors were low but were higher than in margins. Margins in two patients harboured tumor but this was not correlated with counts obtained using the γ-probes. Surgical cavity counts were high and likely due to detection of γ-photons outside the surgical field.ConclusionWe conclude that it was not feasible, at least at the administered amount of radioactivity used in this study, to reliably detect the margins of disease in patients with in situ or invasive ductal carcinoma intraoperatively using a hand-held γ-probe and ¹¹¹In-DTPA-trastuzumab Fab fragments due to low uptake in the tumor and involved margins.     

The human polynucleotide kinase/phosphatase (hPNKP) inhibitor A12B4C3 radiosensitizes human myeloid leukemia cells to Auger electron-emitting anti-CD123 111In-NLS-7G3 radioimmunoconjugates

IntroductionLeukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131^- phenotype uniquely displayed by LSCs.MethodsThe surviving fraction (SF) of CD123⁺/CD131^- AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33–266 nmols/L; 0.74 MBq/μg) or to γ-radiation (0.25-5 Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) or with γ-radiation (0.25–2 Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.ResultsBinding of ¹¹¹In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1 μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131^- epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25 Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25–0.5 Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6 Gy to AML-5 cells.ConclusionsWe conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.     

Single-step radiosynthesis and in vivo evaluation of a novel fluorine-18 labeled hippurate for use as a PET renal agent

ObjectiveThe objective of this study was to investigate a new fluorine-18 labeled hippurate, m-cyano-p-[¹⁸ F]fluorohippurate ([¹⁸ F]CNPFH), as a potential radiopharmaceutical for evaluating renal function by PET.Methods[¹⁸ F]CNPFH was synthesized by a direct one-step nucleophilic aromatic substitution using an ¹⁸ F-for-[N(CH₃)₃]⁺-reaction. In vivo stability was determined by HPLC analysis of urine collected from a healthy rat at 30 min p.i. of [¹⁸ F]CNPFH. The plasma protein binding (PPB) and erythrocyte uptake of [¹⁸ F]CNPFH were determined using blood collected from healthy rats at 5 min p.i. Biodistribution studies were conducted in healthy rats at 10 min and 1 h p.i. of [¹⁸ F]CNPFH. Dynamic PET/CT imaging data were acquired in normal rats. For comparison, the same rats underwent an identical imaging study using the previously reported p-[¹⁸ F]fluorohippurate ([¹⁸ F]PFH) renal agent.Results[¹⁸ F]CNPFH demonstrated high in vivo stability with no metabolic degradation. The in vivo PPB and erythrocyte uptake of [¹⁸ F]CNPFH were found to be comparable to those of [¹⁸ F]PFH. Biodistribution and dynamic PET/CT imaging studies revealed a rapid clearance of [¹⁸ F]CNPFH primarily through the renal–urinary pathway. However, unlike [¹⁸ F]PFH, a minor (about 12%) fraction was eliminated via the hepatobiliary route. The PET-derived [¹⁸ F]CNPFH renograms revealed an average time-to-peak (T_max) of 3.2 ± 0.4 min which was similar to [¹⁸ F]PFH, but the average time-to-half-maximal activity (11.4 ± 2.8 min) was found to be higher than that of [¹⁸ F]PFH (7.1 ± 1.3 min).ConclusionsOur in vivo results indicate that [¹⁸ F]CNPFH has renogram characteristics similar to those of [¹⁸ F]PFH, however, the unexpected hepatobiliary elimination is adding undesirable background signal in the PET images.     

Visualizing inflammation activity in rheumatoid arthritis with Tc-99 m Anti-CD4-mAb fragment scintigraphy

PurposeT-cell-located CD4 antigen represents one of the therapeutic targets in rheumatoid arthritis (RA). However, up to now there is no established imaging tool to visualize this target in vivo. The aim of our study was to assess the safety and tolerability of a technetium-99 m labelled murine anti-human CD4 IgG1-Fab fragment ([^99mTc]-anti-CD4-Fab, [^99mTc]-EP1645) in patients with active synovitis due to RA, and to evaluate its potential as a marker of disease activity.MethodsIn the present phase I proof of principle study five patients with RA were examined. Planar scans of the whole body, hands, and feet were taken 30 min up to 24 h after application of 550 ± 150 MBq [^99mTc]-anti-CD4-Fab, followed by visual analyses, comparison with clinical data in 68 joints per patient and semiquantitative analysis of hand and wrist joints.ResultsNeither infusion related adverse events nor adverse events during follow up were observed. No increase in human anti-murine antibody titres was seen. All patients had positive scans in almost 70% of clinically affected joints. Positive scans were also found in 8% of joints without evidence of swelling or tenderness.ConclusionScintigraphy with [^99mTc]-anti-CD4-Fab is a promising technique for evaluation of inflammatory activity in patients with RA, pre-therapeutical evaluation of CD4 status and therapy control. Tracer uptake in clinically inconspicuous joints strongly indicates diagnostic potential of [^99mTc]-anti-CD4-Fab. Whether this technique is eligible as a prognostic factor in RA needs to be analysed in further studies as well as the pathophysiological background of clinically affected joints lacking tracer uptake.     

Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

Preliminary evaluation of 1′-[18F]fluoroethyl-β-D-lactose ([18F]FEL) for detection of pancreatic cancer in nude mouse orthotopic xenografts

IntroductionEarly detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [¹⁸F]FDG, has inadequate resolution for detection of small (2–3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an ¹⁸F-labelled lactose derivative, [¹⁸F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[¹⁸F]fluoroethyl lactose ([¹⁸F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice.MethodsXenografts were developed in nude mice by injecting L3.6pl/GL⁺ pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2–3 mm, the animals were injected with [¹⁸F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [¹⁸F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression.ResultsTumour growth was rapid, as observed by BLI and MRI. Blood clearance of [¹⁸F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [¹⁸F]FEL in the peritumoural pancreatic tissue was 1.29 ± 0.295 %ID/g, and that in the normal pancreas of control animals was 0.090 ± 0.101 %ID/g. [¹⁸F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [¹⁸F]FEL binding and HIP/PAP expression.Conclusion[¹⁸F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.     

Human whole-body biodistribution and dosimetry of a new PET tracer, [11C]ketoprofen methyl ester,for imagings of neuroinflammation

IntroductionNeuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease and other brain disorders, and nonsteroidal anti-inflammatory drugs (NSAIDs) are considered therapeutic candidates. As a biomarker of neuroinflammatory processes, ¹¹C-labeled ketoprofen methyl ester ([¹¹C]KTP-Me) was designed to allow cerebral penetration of ketoprofen (KTP), an active form of a selective cyclooxygenase-1 inhibitor that acts as an NSAID. Rat neuroinflammation models indicate that [¹¹C]KTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. [¹¹C]KTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study.Methods[¹¹C]KTP-Me was synthesized by rapid C-[¹¹C]methylation of [¹¹C]CH₃I and the corresponding arylacetate precursor, purified with high-performance liquid chromatography, and prepared as an injectable solution including PEG400, providing radiochemical purity of > 99% and specific activity of > 25 GBq/μmol at injection. Six young healthy male humans were injected with [¹¹C]KTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40 min post-injection, together with sequential blood sampling and labeled metabolite analysis.ResultsNo adverse effects were observed during PET scanning after [¹¹C]KTP-Me injection. [¹¹C]KTP-Me was rapidly metabolized to ¹¹C-labeled ketoprofen ([¹¹C]KTP) within 2–3 min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2 min. The cortical activity was gradually washed out. Whole-body images indicated that the urinary bladder was the major excretory pathway. The organ with the highest radiation dose was the urinary bladder (average dose of 41μGy/MBq, respectively). The mean effective dose was 4.7 μSv/MBq, which was comparable to other ¹¹C-labeled radiopharmaceuticals.Conclusion[¹¹C]KTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. [¹¹C]KTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.     

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

⁶⁴Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of ⁶⁴Cu-DOTA-AE105.MethodsFive mice received iv tail injection of ⁶⁴Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another ⁶⁴Cu-DOTA peptide-based tracer, ⁶⁴Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using ⁶⁴Cu-DOTA-TATE.ResultsHuman estimates of ⁶⁴Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04 mSv/MBq. The mean effective whole-body dose of ⁶⁴Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for ⁶⁴Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision (predicted value: 0.0252 mSv/Mbq, Observed value: 0.0315 mSv/MBq) thus validating our approach for human dosimetry estimation.ConclusionFavorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of ⁶⁴Cu-DOTA-AE105.     

A heterodimeric [RGD-Glu-[64Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors

IntroductionIn the present study, we describe a ⁶⁴Cu-radiolabeled heterodimeric peptide conjugate for dual α_vβ₃/GRPr (α_vβ₃ integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for α_vβ₃ integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting).MethodsRGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization–mass spectrometry (ESI-MS). Radiolabeling of the conjugate with ⁶⁴Cu produced [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥ 95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model.ResultsA competitive displacement receptor binding assay in human prostate PC-3 cells using ¹²⁵I-[Tyr⁴]BBN as the radioligand showed high binding affinity of [RGD-Glu-[^natCu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09 ± 0.34 nM). A similar assay in human, glioblastoma U87-MG cells using ¹²⁵I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvβ₃ integrin (518 ± 37.5 nM). In vivo studies of [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86 ± 1.01 %ID/g, 1 h post-intravenous injection (p.i.)) and prolonged retention (4.26 ± 1.23 %ID/g, 24 h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4 h p.i.ConclusionsThe favorable pharmacokinetics and enhanced tumor uptake of ⁶⁴Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy.     

2-[18F]Fluoroethanol and 3-[18F]fluoropropanol: facile preparation,biodistribution in mice,and their application as nucleophiles in the synthesis of [18F]fluoroalkyl aryl ester and ether PET tracers

Introduction2-[¹⁸F]Fluoroethoxy and 3-[¹⁸F]fluoropropoxy groups are common moieties in the structures of radiotracers used with positron emission tomography. The objectives of this study were (1) to develop an efficient one-step method for the preparation of 2-[¹⁸F]fluoroethanol (2-[¹⁸F]FEtOH) and 3-[¹⁸F]fluoropropanol (3-[¹⁸F]FPrOH); (2) to demonstrate the feasibility of using 2-[¹⁸F]FEtOH as a nucleophile for the synthesis of 2-[¹⁸F]fluoroethyl aryl esters and ethers; and (3) to determine the biodistribution profiles of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH in mice.Methods2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were prepared by reacting n-Bu₄N[¹⁸F]F with ethylene carbonate and 1,3-dioxan-2-one, respectively, in diethylene glycol at 165 °C and purified by distillation. 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were prepared by coupling 2-[¹⁸F]FEtOH with 4-fluorobenzoyl chloride and 1-fluoro-4-nitrobenzene, respectively. Biodistribution and PET/CT imaging studies of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were performed in normal female Balb/C mice.ResultsThe preparation of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH took 60 min, and their decay-corrected yields were 88.6 ± 2.0% (n = 9) and 65.6 ± 10.2% (n = 5), respectively. The decay-corrected yields for the preparation of 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were 36.1 ± 5.4% (n = 3) and 27.7 ± 10.7% (n = 3), respectively. Imaging/biodistribution studies in mice using 2-[¹⁸F]FEtOH showed high initial radioactivity accumulation in all major organs followed by very slow clearance. On the contrary, by using 3-[¹⁸F]FPrOH, radioactivity accumulated in all major organs was cleared rapidly, but massive in vivo defluorination (31.3 ± 9.57%ID/g in bone at 1 h post-injection) was observed.ConclusionsUsing 2-[¹⁸F]FEtOH/3-[¹⁸F]FPrOH as a nucleophile is a competitive new strategy for the synthesis of 2-[¹⁸F]fluoroethyl/3-[¹⁸F]fluoropropyl aryl esters and ethers. Our biodistribution data emphasize the importance of in vivo stability of PET tracers containing a 2-[¹⁸F]fluoroethyl or 3-[¹⁸F]fluoropropyl group due to high background and high bone uptake resulting from 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH, respectively. This is especially important for their aryl ester derivatives which are prone to in vivo hydrolysis.     

Evaluation of metabolism,plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

Introduction(−)-[¹⁸ F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α₄β₂ receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.MethodsPlasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min.ResultsA fraction of 15% ± 2% of (−)-[¹⁸ F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[¹⁸ F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[¹⁸ F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts.Discussion(−)-[¹⁸ F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.     

The natBr(p,x)73,75Se nuclear processes: a convenient route for the production of radioselenium tracers relevant to amino acid labelling

A possible route for the production of no-carrier-added (n.c.a.) ⁷³Se (T_1/2=7.1 h) and ⁷⁵Se (120 d) is introduced. d,l-2-Amino-4-([⁷³Se]methyl-seleno) butanoic acid (d,l-[⁷³Se]selenomethionine) with an overall radiochemical yield of >40% could be prepared via a 3-step polymer-supported synthesis after successful separation of ⁷³Se from KBr targets. Excitation functions for the ^natBr(p,x) ^72,73,75Se processes were measured from threshold up to 100 MeV utilizing pellets of pressed KBr. Targets were irradiated at the NAC cyclotron with proton beams having primary energies of 40.4, 66.8 and 100.9 MeV. The calculated ⁷³Se yield (EOB) for 1 h irradiation in 1 μA of beam at the optimum proton energy range of 62→42 MeV is 81.4 MBq (2.2 mCi), and the calculated ⁷⁵Se yield (EOB) for the overall range 62 MeV→threshold for the same irradiation conditions is 0.97 MBq (0.026 mCi).     

Synthesis and biological evaluation of [11C]SB366791: A new PET-radioligand for in vivo imaging of the TRPV1 receptor

IntroductionThe transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor, a non-selective cation channel, is known for its key role in pain nociception and neurogenic inflammation. TRPV1 expression has been demonstrated in diverse tissues and an essential role for TRPV1 in various disorders has been suggested. A TRPV1-specific PET-radioligand can serve as a useful tool for further in vivo research in animals and directly in humans. In this study, we report the synthesis and biological evaluation of a carbon-11 labelled analogue of N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) which was reported as a specific high-affinity antagonist for TRPV1.MethodsThe new tracer was evaluated with respect to log D and biodistribution in control, pretreated and TRPV1^?/? mice. The percentage of radiometabolites of [¹¹C]SB366791 was determined in mouse plasma and brain.Results[¹¹C] SB366791 was obtained in good yield (69% ± 11%; isolated amounts 3034–5032 MBq) and high specific activity (390 ± 215 GBq/μmol). The tracer was efficiently cleared from blood and all major organs via hepatobiliary and renal pathways. Initial brain uptake was high (1.6% ID) and wash-out from brain was rapid. The retention of [¹¹C] SB366791 in the trigeminal nerve of control mice was prominent. The in vitro binding affinity of SB366791 was determined to be 280 ± 56 nM and 780 ± 140 nM for human and rat TRPV1, respectively.Conclusions[¹¹C] SB366791 has favourable biodistribution characteristics in mice. However the obtained low binding affinity for TRPV1 may not be sufficient to use the current compound as PET tracer.     

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy

IntroductionLow dose-rate radioimmunotherapy (RIT) using ¹²⁵I-labelled monoclonal antibodies (¹²⁵I-mAbs) is associated with unexpected high cytotoxicity per Gy.MethodsWe investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage was measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53^−/− and p53^+/+ HCT116 cells exposed to increasing activities of internalizing anti-HER1 ¹²⁵I-mAbs or non-internalizing anti-CEA ¹²⁵I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined.ResultsCell hypersensitivity to low absorbed doses of anti-CEA ¹²⁵I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4 °C during exposure but were repaired under standard RIT conditions (37 °C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei.ConclusionHypersensitivity to non-internalizing ¹²⁵I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing ¹²⁵I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of ¹²⁵I-mAbs.     

Enhanced radiosyntheses of [11C]raclopride and [11C]DASB using ethanolic loop chemistry

IntroductionTo improve the synthesis and quality control of carbon-11 labeled radiopharmaceuticals, we report the fully automated loop syntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanol as the only organic solvent for synthesis module cleaning, carbon-11 methylation, HPLC purification, and reformulation.MethodsEthanolic loop chemistry is fully automated using a GE TRACERLab FX_C-Pro synthesis module, and is readily adaptable to any other carbon-11 synthesis apparatus. Precursors (1 mg) were dissolved in ethanol (100 μL) and loaded into the HPLC loop. [¹¹C]MeOTf was passed through the HPLC loop and then the labeled products were purified by semi-preparative HPLC and reformulated into ethanolic saline.ResultsBoth [¹¹C]raclopride (3.7% RCY; > 95% RCP; SA = 20831 Ci/mmol; n = 64) and [¹¹C]DASB, both with (3.0% RCY; > 95% RCP; SA = 15152 Ci/mmol; n = 9) and without (3.0% RCY; > 95% RCP; SA = 10931 Ci/mmol; n = 3) sodium ascorbate, have been successfully prepared using the described methodology. Doses are suitable for human use and the described methods are now employed for routine clinical production of both radiopharmaceuticals at the University of Michigan.ConclusionsEthanolic loop chemistry is a powerful technique for preparing [¹¹C]raclopride and [¹¹C]DASB, and we are in the process of adapting it for other carbon-11 radiopharmaceuticals prepared in our laboratories ([¹¹C]PMP, [¹¹C]PBR28 etc.).     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.     

Comparison of trans-1-amino-3-[18 F]fluorocyclobutanecarboxylic acid (anti-[18 F]FACBC) accumulation in lymph node prostate cancer metastasis and lymphadenitis in rats

IntroductionTrans-1-amino-3-[¹⁸ F]fluorocyclobutanecarboxylic acid (anti-[¹⁸ F]FACBC) is a positron emission tomography (PET) tracer used to visualize prostate cancer (PCa). In this study, we investigated the differences in anti-[¹⁸ F]FACBC accumulation between metastatic and inflamed lymph node (LN) lesions.MethodsA PCa LN metastasis (PLM) model was developed by inoculating a rat PCa cell line, MAT-Ly-Lu-B2, into popliteal LNs of Copenhagen rats. Acute lymphadenitis (AL) was induced by injecting concanavalin A (Con A) into the hind footpad, and chronic lymphadenitis (CL) was induced by daily injection of Con A into the tissues surrounding the popliteal LNs for 2 weeks. Main lesions of all animal models were established in lumbar and/or inguinal LNs. Biodistribution and dynamic PET imaging data were acquired after tracer injection. T2-weighted magnetic resonance (MR) images were registered with PET images.ResultsIn the biodistribution study, the uptake ratios of PLM-to-lymphadenitis in lesional lumbar and inguinal LNs were 0.97 − 1.57 and 1.47 − 2.08 at 15 and 60 min post-anti-[¹⁸ F]FACBC injection respectively. In PET imaging, the lesional lumbar LNs of CL and PLM, but not of AL, were visualized on anti-[¹⁸ F]FACBC-PET/MR fusion images without disturbance from radioactivity from urine, and the rank order of anti-[¹⁸ F]FACBC accumulation at 50 − 60 post-injection in lesional lumbar LNs was PLM > CL > AL.ConclusionsAnti-[¹⁸ F]FACBC accumulation in LNs with PLM was higher than that in inflamed LNs.Advances in knowledgeThe study showed that although low but significant levels of anti-[¹⁸ F]FACBC uptake by chronic inflamed lesions might cause false-positives in anti-[¹⁸ F]FACBC-PET in some PCa patients, uptake of the tracer at acutely inflamed sites was minimal.Implications for patient careThe findings of this study suggest the potential of Anti-[¹⁸ F]FACBC for distinguishing between tumors and acute inflammation in clinical practice.     

Biodistribution and radiation dosimetry in humans of [11C]FLB 457, a positron emission tomography ligand for the extrastriatal dopamine D2 receptor

Purpose[¹¹C]FLB 457, a radioligand with very high affinity and selectivity for dopamine D_2/3 receptors, is used to measure receptor binding in extrastriatal regions showing low density of the receptors. The purpose of this study was to estimate the whole-body biodistribution of radioactivity and the radiation absorbed doses to organs after intravenous injection of [¹¹C]FLB 457 in healthy human subjects.MethodsWhole-body images were acquired for 2 h after an injection of [¹¹C]FLB 457 in six healthy humans. Radiation absorbed doses were estimated by the MIRD scheme implemented in OLINDA/EXM 1.1 software.ResultsOrgans with the longest residence time were the liver, lungs, and brain. The organs with the highest radiation doses were the kidneys, liver, and pancreas. The effective dose delivered by [¹¹C]FLB 457 is 5.9 μSv/MBq, similar to those of other ¹¹C-labeled tracers.ConclusionsThis effective dose would allow multiple scans in the same individual based on prevailing maximum recommended-dose guidelines in the USA and Europe.     

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

IntroductionTwo 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA_A/benzodiazepine receptor by comparing them to [¹¹C]flumazenil in rhesus monkey.MethodsRadiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [¹⁸F]AH114726 and [¹⁸F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results[¹⁸F]GEH120348 and [¹⁸F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [¹⁸F]AH114726 and [¹⁸F]GEH120348 were determined in a non-human primate and directly compared with [¹¹C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [¹¹C]flumazenil and the known concentrations of GABA_A/benzodiazepine receptors in the monkey brain. [¹⁸F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [¹¹C]flumazenil. In contrast, [¹⁸F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.ConclusionThe ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [¹¹C]flumazenil, support that [¹⁸F]AH114726 is a suitable option for imaging the GABA_A receptor.