Elaine M. Jagoda

[18F]FBEM-ZHER2:342-Affibody molecule—a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography

PurposeThe expression of human epidermal growth factor receptor-2 (HER2) receptors in cancers is correlated with a poor prognosis. If assessed in vivo, it could be used for selection of appropriate therapy for individual patients and for monitoring of the tumor response to targeted therapies. We have radiolabeled a HER2-binding Affibody molecule with fluorine-18 for in vivo monitoring of the HER2 expression by positron emission tomography (PET).Materials and methodsThe HER2-binding ZHER2:342-Cys Affibody molecule was conjugated with N-2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). The in vitro binding of the resulting radioconjugate was characterized by receptor saturation and competition assays. For in vivo studies, the radioconjugate was injected into the tail vein of mice bearing subcutaneous HER2-positive or HER2-negative tumors. Some of the mice were pre-treated with non-labeled ZHER2:342−Cys. The animals were sacrificed at different times post-injection, and the radioactivity in selected tissues was measured. PET images were obtained using an animal PET scanner.ResultsIn vitro experiments indicated specific, high-affinity binding to HER2. PET imaging revealed a high accumulation of the radioactivity in the tumor as early as 20 min after injection, with a plateau being reached after 60 min. These results were confirmed by biodistribution studies demonstrating that, as early as 1 h post-injection, the tumor to blood concentration ratio was 7.5 and increased to 27 at 4 h. Pre-saturation of the receptors with unlabeled ZHER2:342-Cys lowered the accumulation of radioactivity in HER2-positive tumors to the levels observed in HER2-negative ones.ConclusionOur results suggest that the [18F]FBEM-ZHER2:342 radioconjugate can be used to assess HER2 expression in vivo.

High resolution PET, SPECT and projection imaging in small animals

Positron emission tomography, single photon emission computed tomography and planar projection imaging of radioactive tracers have long been in use for detecting and diagnosing disease in human subjects. More recently, advanced versions of these same technologies have begun to be used across the breadth of modern biomedical research to study non-invasively small laboratory animals in a myriad of experimental settings. In this report, we describe some of the new instruments and techniques that make these measurements possible and illustrate, with a few examples, the potential power of these methods in modern biomedical research.

PET evaluation of [18F]FCWAY, an analog of the 5-HT1A receptor antagonist, WAY-100635

We synthesized [(18)F]FCWAY, an analog of [carbonyl-(11)C]WAY-100635 ¿[(11)C]N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl))-N-(2-(pyridi nyl))cyclohexanecarboxamide¿, by replacing the cyclohexanecarbonyl group acid with a trans-4-fluorocyclohexanecarbonyl group (FC). Control and preblocking studies were performed in anesthetized monkeys. Plasma radioactive metabolite analysis showed the presence of [(18)F]FC and [(18)F]fluoride. Tissue time-radioactivity curves were corrected for metabolite contamination based on separate positron-emission tomography studies of these two labeled metabolites. Analysis using a two-tissue compartment model gave distribution volume (V) estimates (mL/mL) ranging from 33 in frontal cortex to 4 in cerebellum. Preblocking data showed uniform V of 2-3 mL/mL. These studies demonstrate that [(18)F]FCWAY has very similar kinetic characteristics to [(11)C]WAY-100635.

Effects of Early-Life Stress on Serotonin1A Receptors in Juvenile Rhesus Monkeys Measured by Positron Emission Tomography

BACKGROUND Traumatic experiences in early childhood are associated with increased risk for developing mood and anxiety disorders later in life. Low serotonin(1A) receptor (5-HT(1A)R) density during development has been proposed as a trait-like characteristic leading to increased vulnerability of stress-related neuropsychiatric disorders. METHODS To assess the relationship between early-life stress and alterations in the serotonin system during development, we used positron emission tomography to measure in vivo 5-HT(1A)R density and apparent dissociation constant (K(D)(app)) in the brain of juvenile Rhesus monkeys exposed to the early-life stress of peer-rearing. RESULTS In general, 5-HT(1A)R density and K(D)(app) were decreased in peer-reared compared with control mother-reared animals. However, increase in receptor density was found in the dorsomedial prefrontal cortex of peer-reared females. CONCLUSIONS These findings suggest that exposure to an adverse early-life environment during infancy is associated with long-term alterations in the serotonin system and support previous studies suggesting that reduced 5-HT(1A)R density during development might be a factor increasing vulnerability to stress-related neuropsychiatric disorders. Furthermore, alterations in the serotonin system seemed to be gender- and region-specific, providing a biological basis for the higher prevalence of affective disorders in women.

Fluoro-, bromo-, and iodopaclitaxel derivatives: synthesis and biological evaluation

Paclitaxel (Taxol) is a clinically important chemotherapeutic agent. We describe the synthesis of fluoro-, bromo-, and iodopaclitaxel and their [(18)F]fluoro-, [(76)Br]bromo-, and [(124)I]iodo- analogues. [(18)F]Fluoropaclitaxel shows high uptake and rapid clearance from tissues in rats. Preadministration of paclitaxel in normal rats significantly increases (p < 0.005) retention of [(18)F]fluoropaclitaxel and [(76)Br]bromopaclitaxel in blood (33.0%), heart (32.0%), lung (37.6%) kidney (142.4%); and blood (33.4%), lung (42.3%), kidney (62.4%), respectively. [(18)F]Fluoropaclitaxel uptake in the brain of mdr1a/1b(-/-) mice is increased 1400% (p < 1.3e-07) relative to wild-type controls. Preadministration of paclitaxel or XR9576, a modulator, had little effect on the biodistribution in these mdr1a/1b(-/-) mice. As a result, [(18)F]fluoropaclitaxel will be a useful radiopharmaceutical for the study of multidrug resistant tumors.

Fluoro analogs of WAY-100635 with varying pharmacokinetics properties

Radiolabeled derivatives of WAY-100635 have been shown to be important for imaging in vivo because of their antagonist properties and their specificity for the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor. Our goal is to prepare a series of radiofluorinated derivatives of WAY-100635 that, in the rat, range in pharmacokinetic properties from nearly irreversible to reversible in their behavior. It appears that derivatives containing a cyclohexanecarboxylic acid (e.g., FCWAY) with its high affinity and high target to nontarget contrast, has properties suited to measure receptor concentration. Derivatives based on phenylcarboxamide (e.g., FBWAY and MeFBAWAY) have properties more suited to the measurement of changes in endogenous serotonin. The compound containing the pyrimidine moiety in place of the pyridine moeity in FBWAY (FBWAY 1,3N) appears to have intermediate properties.

Biologically stable [18F]-labeled benzylfluoride derivatives

Use of the [(18)F]-fluoromethyl phenyl group is an attractive alternative to direct fluorination of phenyl groups because the fluorination of the methyl group takes place under milder reaction conditions. However, we have found that 4-FMeBWAY showed femur uptake equal to that of fluoride up to 30 min in rat whereas 4-FMeQNB had a significantly lower percent injected dose per gram in femur up to 120 min. For these and other benzylfluoride derivatives, there was no clear in vivo structure-defluorination relationship. Because benzylchlorides (BzCls) are known alkylating agents, benzylfluorides may be alkylating agents as well, which may be the mechanism of defluorination. On this basis, the effects of substitution on chemical stability were evaluated by the 4-(4-nitro-benzyl)-pyridine (NBP) test, which is used to estimate alkylating activity with NBP. The effect of substitution on the alkylating activity was evaluated for nine BzCl derivatives: BzCl; 3- or 4-methoxy (electron donation) substituted BzCl; 2-, 3-, or 4-nitro (electron withdrawing) substituted BzCl; and 2-, 3-, or 4-chloro (electron withdrawing) substituted BzCl. Taken together, the alkylating reactivity of 3-chloro-BzCl was the weakest. This result was then applied to [(18)F]-benzylfluoride derivatives and in vivo and in vitro stability were evaluated. Consequently, 3-chloro-[(18)F]-benzylfluoride showed a 70-80% decrease of defluorination in both experiments in comparison with [(18)F]-benzylfluoride, as expected. Moreover, a good linear relationship between in vivo femur uptake and in vitro hepatocyte metabolism was observed with seven (18)F-labeled radiopharmaceuticals, which were benzylfluorides, alkylfluorides, and arylfluorides. Apparently, the [(18)F]-fluoride ion is released by metabolism in the liver in vivo. In conclusion, 3-chloro substituted BzCls are the most stable, which suggests that 3-chloro benzylfluorides will be the most chemically stable compound. This result should be important in future design of radioligands labeled with a benzylfluoride moiety.

Immuno-PET of the Hepatocyte Growth Factor Receptor Met Using the 1-Armed Antibody Onartuzumab

The overexpression and overactivation of hepatocyte growth factor receptor (Met) in various cancers has been linked to increased proliferation, progression to metastatic disease, and drug resistance. Developing a PET agent to assess Met expression would aid in the diagnosis and monitoring of responses to Met-targeted therapies. In these studies, onartuzumab, the experimental therapeutic 1-armed monoclonal antibody, was radiolabeled with 76Br or 89Zr and evaluated as an imaging agent in Met-expressing cell lines and mouse xenografts. Methods: 89Zr-desferrioxamine (df)-onartuzumab was synthesized using a df-conjugate; 76Br-onartuzumab was labeled directly. Met-binding studies were performed using the human tumor–derived cell lines MKN-45, SNU-16, and U87-MG, which have relatively high, moderate, and low levels of Met, respectively. Biodistribution and small-animal PET studies were performed in MKN-45 and U87-MG xenografts. Results: 76Br-onartuzumab and 89Zr-df-onartuzumab exhibited specific, high-affinity Met binding (in the nanomolar range) that was concordant with established Met expression levels. In MKN-45 (gastric carcinoma) xenografts, both tracers cleared slowly from nontarget tissues, with the highest uptake in tumor, blood, kidneys, and lungs. 76Br-onartuzumab MKN-45 tumor uptake remained relatively constant from 18 h (5 percentage injected dose per gram of tissue [%ID/g]) to 48 h (3 %ID/g) and exhibited tumor-to-muscle ratios ranging from 4:1 to 6:1. In contrast, 89Zr-df-onartuzumab MKN-45 tumor uptake continued to accumulate from 18 h (10 %ID/g) to 120 h (23 %ID/g), attaining tumor-to-muscle ratios ranging from 20:1 to 27:1. MKN-45 tumors were easily visualized in imaging studies with both tracers at 18 h, but after 48 h 89Zr-df-onartuzumab image quality improved, with at least 2-fold-greater tumor uptake than nontarget tissues. MKN-45 tumor uptake for both tracers correlated significantly with tumor mass and Met expression and was not affected by the presence of plasma shed Met. Conclusion: 89Zr-df-onartuzumab and 76Br-onartuzumab specifically targeted Met in vitro and in vivo; 89Zr-df-onartuzumab achieved higher tumor uptake and tumor-to-muscle ratios than 76Br-onartuzumab at later times, suggesting that 89Zr-df-onartuzumab would be better suited to image Met for diagnostic and prognostic purposes.

In vivo muscarinic binding of 3-(alkylthio)-3-thiadiazolyl tetrahydropyridines†

Based on encouraging in vitro data indicating M2 subtype selectivity, we synthesized, radiolabeled with 18F, and evaluated 3‐(3‐(2‐fluoroethylthio)‐1,2,5‐thiadiazol‐4‐yl)‐1,2,5,6‐tetrahydro‐1‐methylpyridine [FE‐TZTP], and 3‐(3‐(3‐fluoropropylthio)‐1,2,5‐thiadiazol‐4‐yl)‐1,2,5,6‐tetrahydro‐1‐methylpyridine [FP‐TZTP] for muscarinic subtype selectivity in vivo. [18F]FE‐TZTP displays high uptake in vivo but is inhibited only weakly by coinjecting unlabeled P‐TZTP. Contrarily, [18F]FP‐TZTP shows significant inhibition of uptake by coinjecting unlabeled P‐TZTP or the muscarinic agonist L‐687,306 (3‐(3‐cyclopropyl‐1,2,4‐oxadiazol‐5‐yl)‐1‐azabicyclo[2.2.1]heptane). Using in vivo autoradiography, [18F]FP‐TZTP displays regional distribution consistent with M2 subtype distribution. In addition, [18F]FP‐TZTP shows specific uptake in the heart at 5 min. Analysis of metabolites in the awake rat brain revealed that the parent compound represents >95% of the extractable activity at 30 min. In vivo studies in rhesus monkeys revealed rapid brain uptake of [18F]FP‐TZTP, with clearance sustained over 2 h. Administration of P‐TZTP or FP‐TZTP (80 nmol/kg) at 60 min after injection of [18F]FP‐TZTP results in a significant displacement of brain activity in all regions. Metabolite analysis in monkey plasma shows that parent compound represents 20% of the extractable radioactivity at 40 min postinjection. One metabolite, which increases with time, has similar lipophilicity to the parent. However, based on metabolism in rat we believe metabolites are not in the brain to any significant extent in monkeys during the time of imaging studies. Regional uptake, autoradiographic distribution, and clearance rates in the brain are consistent with the hypothesis that [18F]FP‐TZTP is M2 selective in vivo. Synapse 31:29–40, 1999. Published 1999 Wiley‐Liss, Inc.

Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.