Rat Imaging and In Vivo Stability Studies using [11C]-Dimethyl-Diphenyl Ammonium,a Candidate Agent for PET-Myocardial Perfusion Imaging

BackgroundPET myocardial perfusion imaging (MPI) holds several advantages over SPECT for diagnosing coronary artery disease. The short half-lives of prevailing PET-MPI agents hamper wider clinical application of PET in nuclear cardiology; prompting the development of novel PET-MPI agents. We have previously reported on the potential of radiolabeled ammonium salts, and particularly on that of [¹¹C]dimethyl-diphenyl-ammonium ([¹¹C]DMDPA), for cardiac PET imaging. This study was designed to improve the radiosynthesis and increase the yield of [¹¹C]DMDPA, characterize more meticulously the kinetics of radioactivity distribution after its injection via micro-PET/CT studies, and further explore its potential for PET-MPI.MethodsThe radiosynthetic procedure of [¹¹C]DMDPA was improved with respect to the previously reported one. The kinetics of radioactivity distribution following injection of [¹¹C]DMDPA were investigated in juvenile and young adult male SD rats using microPET/CT, and compared to those of [¹³N]NH₃. Furthermore, the metabolic fate of [¹¹C]DMDPA in vivo was examined after its injection into rats.ResultsFollowing a radiosynthesis time of 25–27 min, 11.9 ± 1.1 GBq of [¹¹C]DMDPA was obtained, with a 43.7% ± 4.3% radiochemical yield (n = 7). Time activity curves calculated after administration of [¹¹C]DMDPA indicated rapid, high and sustained radioactivity uptake in hearts of both juvenile and young adult rats, having a two-fold higher cardiac radioactivity uptake compared to [¹³N]NH₃. Accordingly, at all time points after injection to both juvenile and young adult rats, image quality of the left ventricle was higher with [¹¹C]DMDPA compared to [¹³N]NH₃. In vivo stability studies of [¹¹C]DMDPA indicate that no radioactive metabolites could be detected in plasma, liver and urine samples of rats up to 20 min after injection, suggesting that [¹¹C]DMDPA is metabolically stable in vivo.ConclusionsThis study further illustrates that [¹¹C]DMDPA holds, at least in part, essential qualities required from a PET-MPI probe. Owing to the improved radiosynthetic procedure reported herein, [¹¹C]DMDPA can be produced in sufficient amounts for clinical use.     

Evaluation of radiolabeled ML04, a putative irreversible inhibitor of epidermal growth factor receptor, as a bioprobe for PET imaging of EGFR-overexpressing tumors

Overexpression of epidermal growth factor receptor (EGFR) has been implicated in tumor development and malignancy. Evaluating the degree of EGFR expression in tumors could aid in identifying patients for EGFR-targeted therapies and in monitoring treatment. Nevertheless, no currently available assay can reliably quantify receptor content in tumors. Radiolabeled inhibitors of EGFR-TK could be developed as bioprobes for positron emission tomography imaging. Such imaging agents would not only provide a noninvasive quantitative measurement of EGFR content in tumors but also serve as radionuclide carriers for targeted radiotherapy. The potency, reversibility, selectivity and specific binding characteristics of ML04, an alleged irreversible inhibitor of EGFR, were established in vitro. The distribution of the F-18-labeled compound and the extent of EGFR-specific tumor uptake were evaluated in tumor-bearing mice. ML04 demonstrated potent, irreversible and selective inhibition of EGFR, combined with specific binding to the receptor in intact cells. In vivo distribution of the radiolabeled compound revealed tumor/blood and tumor/muscle activity uptake ratios of about 7 and 5, respectively, 3 h following administration of a radiotracer. Nevertheless, only minor EGFR-specific uptake of the compound was detected in these studies, using either EGFR-negative tumors or blocking studies as controls. To improve the in vivo performance of ML04, administration via prolonged intravenous infusion is proposed. Detailed pharmacokinetic characterization of this bioprobe could assist in the development of a kinetic model that would afford accurate measurement of EGFR content in tumors.     

High-affinity epidermal growth factor receptor (EGFR) irreversible inhibitors with diminished chemical reactivities as positron emission tomography (PET)-imaging agent candidates of EGFR overexpressing tumors

Previous studies with the anilinoquinazoline epidermal growth factor receptor (EGFR) irreversible inhibitor [(11)C]-ML03 demonstrated a rapid metabolism of the tracer, which led to its low in vivo accumulation in EGFR overexpressing tumors. To enhance tumor uptake, the chemical structure of the compound was modified, and four new groups of EGFR inhibitors with a wide range of chemical reactivities were synthesized. Chemical reactivity assay of the compounds, performed with reduced glutathione (GSH), revealed that the group C (4-(dimethylamino)-but-2-enoic amide) derivative was the least chemically reactive against the nucleophilic attack of GSH. Nonetheless, it demonstrated a high inhibitory potency and bound irreversibly to the EGFR. Consequently, the blood stability of the group C compound (5a, ML04) labeled with (11)C was studied. In a time frame of 60 min, no radioactive metabolites were detected in blood. The stability of [(11)C]-5a, as indicated both from in vitro blood-stability assays and injection into nude rats, was significantly higher as compared to [(11)C]-ML03. Since group C presented a greater promise for tumor accumulation, it represents, to date, the most suitable candidate for radiolabeling with long-lived positron emission tomography (PET) radioisotopes.     

Cancer molecular imaging: radionuclide-based biomarkers of the epidermal growth factor receptor (EGFR)

Overexpression of the epidermal growth factor receptor tyrosine kinase (EGFR-TK) has been documented in numerous human cancers of epithelial origin, and was found to correlate with resistance to treatment and poor prognosis. Recognizing the central role that this receptor plays in cancer development and progression, various approaches have been developed to target it in order to more specifically eradicate cancer cells. These methods include, among others, low-molecular weight inhibitors of the TK domain that are commonly designed to treat those tumors that overexpress the EGFR. Nevertheless, no currently available assay provides non-invasive, longitudinal and sensitive quantitation of receptor levels in tumors so as to better identify candidate patients for EGFR-targeted therapies. Hence, attempts have been made to develop radiolabeled molecular imaging agents as potential bioprobes to quantitatively monitor treatment efficiency. Such EGFR-targeted bioprobes could not only improve patient selection and treatment monitoring, but also allow a direct delivery of radionuclides for radiotherapy. In this review, the role that EGFR plays in cancer development and therapy is briefly presented, followed by a short review of prominent milestones in the development of EGFR-TK inhibitors. These inhibitors constitute the fundamental core structure for the development of radiolabeled probes to visualize the EGFR in vivo. The considerations that need to be taken into account for the development of such probes will be presented, along with a critical examination on the progress that has been made thus far in the field.     

Development of a Fluorinated Analogue of Erlotinib for PET Imaging of EGFR Mutation–Positive NSCLC

Molecular Imaging and Biology - Positron emission tomography (PET) using [11C]erlotinib identifies non-small cell lung carcinoma (NSCLC) tumors with activating mutations in the epidermal growth...     

The effect of the [18F]-PEG group on tracer qualification of [4-(phenylamino)-quinazoline-6-YL]-amide moiety--an EGFR putative irreversible inhibitor.

Previous reports have designated the labeled derivatives of [4-(phenylamino)-quinazoline-6-yl]-amide group as the most promising EGFR-PET imaging agent candidates. To further improve tracer qualifications and increase stability and solubility, additional derivatives of this group substituted at the 7-position with various lengths of fluoro-polyethyleneglycol (F-PEG) chains were synthesized. These novel derivatives inhibited EGFR autophosphorylation with IC(50) values of 5-40 nM. The compounds were successfully labeled with fluorine-18 at the PEG chain via a three-step radiosynthesis route. The labeled final products were obtained with a 13-32% decay corrected radiochemical yield, 99% radiochemical purity, and high specific activity.     

Novel carbon-11 labeled 4-dimethylamino-but-2-enoic acid [4-(phenylamino)-quinazoline-6-yl]-amides: potential PET bioprobes for molecular imaging of EGFR-positive tumors

We have previously reported of labeled reversible and irreversible EGFR inhibitors, such as 4-(3,4-dichloro-6-fluoroanilino)-6,7-dimethoxyquinazoline (ML01) and 6-acrylamido-4-(3,4-dichloro-6-fluoroanilino)quinazoline (ML03), to be suboptimal as imaging agents. On the basis of these studies, a new generation of novel, more chemically stable irreversible inhibitors was labeled with carbon-11 as potential positron emission tomography (PET) biomarkers for molecular imaging of epidermal growth factor receptor (EGFR)-positive tumors. In these new labeled, irreversible inhibitors the acryl-amide group at the 6-position of the quinazoline ring was replaced with a 4-dimethylamino-but-2-enoic amide. The nonlabeled compounds were evaluated in vitro to determine their EGFR autophosphorylation IC(50) values. The IC(50) values indicated that these new irreversible compounds possess similar potencies towards the EGFR, as the parent compound, ML03. These compounds were labeled with carbon-11 at the dimethylamine moiety, using the well known labeling reagent C-11 MeI. The labeling procedure was automated using a commercial module. The final products were obtained with 10% decay corrected radiochemical yield, 99% radiochemical purity, 96% chemical purity, and a high specific activity of 2.7 Ci/micromol EOB. The high potency of these new labeled bioprobes towards the EGFR establishes their potential to serve as PET agents for molecular imaging of EGFR-positive tumors.     

Imaging microglial/macrophage activation in spinal cords of experimental autoimmune encephalomyelitis rats by positron emission tomography using the mitochondrial 18 kDa translocator protein radioligand [1?F]DPA-714

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Activated microglia/macrophages play a key role in the immunopathogenesis of MS and its corresponding animal models, experimental autoimmune encephalomyelitis (EAE). Microglia activation begins at early stages of the disease and is associated with elevated expression of the 18 kDa mitochondrial translocator protein (TSPO). Thus, positron emission tomography (PET) imaging of microglial activation using TSPO-specific radioligands could be valuable for monitoring disease-associated neuroinflammatory processes. EAE was induced in rats using a fragment of myelin basic protein, yielding acute clinical disease that reflects extensive spinal cord inflammation. Enhanced TSPO expression in spinal cords of EAE rats versus those of controls was confirmed by Western blot and immunohistochemistry. Biodistribution studies in control and EAE rats were performed using the TSPO radioligand [1?F]DPA-714 [N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide]. At 1 h after injection, almost fivefold higher levels of [1?F]DPA-714 were measured in spinal cords of EAE rats versus controls. The specific binding of [1?F]DPA-714 to TSPO in spinal cords was confirmed in competition studies, using unlabeled (R,S)-PK11195 [(R,S)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide)] or DPA-714 in excess. MicroPET studies affirm that this differential radioactivity uptake in spinal cords of EAE versus control rats could be detected and quantified. Using [1?F]DPA-714, neuroinflammation in spinal cords of EAE-induced rats could be visualized by PET, offering a sensitive technique for monitoring neuroinflammatory lesions in the CNS and particularly in the spinal cord. In addition to current MRI protocols, this approach could provide molecular images of neuroinflammation for detection, monitoring, and research in MS.