Background

A noninvasive method of monitoring the loss of islet cells can provide an earlier and improved diagnosis for therapeutics development of preclinical phases of diabetes. The use of [(18)F]fallypride, a dopamine D2/D3 receptor radiotracer, has been developed as a surrogate marker to evaluate loss of pancreatic islet cells in a rodent model of type 1 diabetes.

Materials and methods

Healthy Sprague-Dawley rats were administered [(18)F]fallypride and imaged for 2 h in a positron emission tomography (PET)/computed tomography (CT) scan. Diabetes was then induced in the same rats by administration of streptozotocin, and a PET/CT scan was performed 4 days after establishing diabetes. Pancreata of a separate set of rats were evaluated by insulin immunostaining for loss of islet cells by streptozotocin.

Results

Blood glucose levels of 125 mg/dL and 550 mg/dL were established for those rats without and with diabetes, respectively. [(18)F]Fallypride uptake in the pancreas of both groups of rats was rapid, but the rats with diabetes showed a significantly lower uptake (less than 50%). The specific binding ratio was decreased by 77% in the diabetic rats.

Conclusions

[(18)F]Fallypride can be a useful surrogate marker for monitoring changes in pancreatic islet cells, thus providing a noninvasive method to evaluate efficacy of therapeutics.

Objectives

The spinal cord is known to be innervated with dopaminergic cells with catecholaminergic projections arising from the medulla and pons and dopaminergic transmission in the spinal cord is vital for sensory and motor function. Our goal was to evaluate and compare the imaging capability of dopamine D2/D3 receptors in the rat spinal cord using PET ligands (18)F-fallypride and (11)C-fallypride.

Methods

Male Sprague-Dawley rats were used in all in vitro and in vivo studies. Spinal cord and brain sections were used for in vitro autoradiography and ex vivo autoradiography. For in vivo studies animals received a (18)F-fallypride scan or a (11)C-fallypride PET scan. The spinal cord and the brain were then harvested, flash-frozen and imaged ex vivo. For in vivo analysis Logan plots with cerebellum as a reference was used to evaluate binding potentials (BP). Tissue ratios were used for ex vivo analysis. Drug effects were evaluated using clozapine, haloperidol and dopamine were evaluated on spinal cord sections in vitro.

Results

In vitro studies showed (18)F-fallypride binding to superficial dorsal horn (SDH), dorsal horn (DH), ventral horn (VH) and the pars centralis (PC). In the cervical section, the greatest amount of binding appeared to be in the SDH. Ex vivo studies showed approximately 6% of (18)F-fallypride in SDH compared to that observed in the striatum. In vivo analysis of both (18)F-fallypride and (11)C-fallypride in the spinal cord were comparable to that in the extrastriatal regions. Haloperidol and clozapine displaced more than 75% of the (18)F-fallypride in spinal cord sections.

Conclusions

Our studies showed (18)F-fallypride and (11)C-fallypride binding in the spinal cord in vitro and in vivo. The binding pattern correlates well with the known distribution of dopamine D2/D3 receptors in the spinal cord.

Background

Brown adipose tissue [BAT] metabolism in vivo is vital for the development of novel strategies in combating obesity and diabetes. Currently, BAT is activated at low temperatures and measured using 2-deoxy-2-18F-fluoro-D-glucose [18F-FDG] positron-emission tomography [PET]. We report the use of β3-adrenergic receptor-mediated activation of BAT at ambient temperatures using (R, R)-5-[2-[2,3-(3-chlorphenyl)-2-hydroxyethyl-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate, disodium salt [CL316,243] (a selective β3-adrenoceptor agonist) and measured by 18F-FDG PET/computed tomography [CT].

Methods

Control and CL316,243-treated (2 mg/kg) male Sprague-Dawley rats were administered with 18F-FDG for PET/CT studies and were compared to animals at cold temperatures. Receptor-blocking experiments were carried out using propranolol (5 mg/kg). Dose effects of CL316,243 were studied by injecting 0.1 to 1 mg/kg 30 min prior to 18F-FDG administration. Imaging results were confirmed by autoradiography, and histology was done to confirm BAT activation.

Results

CL316,243-activated interscapular BAT [IBAT], cervical, periaortic, and intercostal BATs were clearly visualized by PET. 18F-FDG uptake of IBAT was increased 12-fold by CL316,243 vs. 1.1-fold by cold exposure when compared to controls. 18F-FDG uptake of the CL-activated IBAT was reduced by 96.0% using intraperitoneal administration of propranolol. Average 18F-FDG uptake of IBAT increased 3.6-, 3.5-, and 7.6-fold by doses of 0.1, 0.5, and 1 mg/kg CL, respectively. Ex vivo 18F-FDG autoradiography and histology of transverse sections of IBAT confirmed intense uptake in the CL-activated group and activated IBAT visualized by PET.

Conclusion

Our study indicated that BAT metabolic activity could be evaluated by 18F-FDG PET using CL316,243 at ambient temperature in the rodent model. This provides a feasible and reliable method to study BAT metabolism.

Objective

Pharmacologic approaches to study brown adipocyte activation in vivo with a potential of being translational to humans are desired. The aim of this study was to examine pre- and postsynaptic targeting of adrenergic system for enhancing brown adipose tissue (BAT) metabolism quantifiable by [(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET)/computed tomography (CT) in mice.

Methods

A β₃-adrenoreceptor selective agonist (CL 316243), an adenylyl cyclase enzyme activator (forskolin) and a potent blocker of presynaptic norepinephrine transporter (atomoxetine), were injected through the tail vein of Swiss Webster mice 30 minutes before intravenous (iv) administration of [(18)F]FDG. The mice were placed on the PET/CT bed for 30 min PET acquisition followed by 10 min CT acquisition for attenuation correction and anatomical delineation of PET images.

Results

Activated interscapular (IBAT), cervical, periaortic and intercostal BAT were observed in 3-dimentional analysis of [(18)F]FDG PET images. CL 316243 increased the total [(18)F]FDG standard uptake value (SUV) of IBAT 5-fold greater compared to that in placebo-treated mice. It also increased the [(18)F]FDG SUV of white adipose tissue (2.4-fold), and muscle (2.7-fold), as compared to the control. There was no significant difference in heart, brain, spleen and liver uptakes between groups. Forskolin increased [(18)F]FDG SUV of IBAT 1.9-fold greater than that in placebo-treated mice. It also increased the [(18)F]FDG SUV of white adipose tissue (2.2-fold) and heart (5.4-fold) compared to control. There was no significant difference in muscle, brain, spleen, and liver uptakes between groups. Atomoxetine increased [(18)F]FDG SUV of IBAT 1.7-fold greater than that in placebo-treated mice. There were no significant differences in all other organs compared to placebo-treated mice except liver (1.6 fold increase). A positive correlation between SUV levels of IBAT and CT Hounsfield unit (HU) (R(2)=0.55, p<0.001) and between CT HU levels of IBAT and liver (R(2)=0.69, p<0.006) was observed.

Conclusions

The three pharmacologic approaches reported here enhanced BAT metabolism by targeting different sites in adrenergic system as measured by [(18)F]FDG PET/CT.

Identification of dopamine D3 receptors (D3R) in vivo is important to understand several brain functions related to addiction. The goal of this work was to identify D3R binding of the dopamine D2 receptor (D2R)/D3R imaging agent, (18)F-fallypride. Brain slices from male Sprague-Dawley rats (n = 6) and New Zealand White rabbits (n = 6) were incubated with (18)F-fallypride and D3R selective agonist (R)-7-OH-DPAT (98-fold D3R selective). Rat slices were also treated with BP 897 (68-fold D3R selective partial agonist) and NGB 2904 (56-fold D3R selective antagonist). In vivo rat studies (n = 6) were done on Inveon PET using 18-37 MBq (18)F-fallypride and drug-induced displacement by (R)-7-OH-DPAT, BP 897 and NGB 2904. PET/CT imaging of wild type (WT, n = 2) and D2R knock-out (KO, n = 2) mice were carried out with (18)F-fallypride. (R)-7-OH-DPAT displaced binding of (18)F-fallypride, both in vitro and in vivo. In vitro, at 10 nM (R)-7-OH-DPAT, (18)F-fallypride binding in the rat ventral striatum (VST) and dorsal striatum (DST) and rabbit nucleus accumbens were reduced by ∼10-15%. At 10 μM (R)-7-OH-DPAT all regions in rat and rabbit were reduced by ≥85%. In vivo reductions for DST and VST before and after (R)-7-OH-DPAT were: low-dose (0.015 mg kg(-1)) DST -22%, VST -29%; high-dose (1.88 mg kg(-1)) DST -58%, VST -77%, suggesting D3R/D2R displacement. BP 897 and NGB 2904 competed with (18)F-fallypride in vitro, but unlike BP 897, NGB 2904 did not displace (18)F-fallypride in vivo. The D2R KO mice lacked (18)F-fallypride binding in the DST. In summary, our findings suggest that up to 20% of (18)F-fallypride may be bound to D3R sites in vivo.

Serotonin 5-HT3 receptors are involved in various brain functions including as an emesis target during cancer chemotherapy. We report here the development of (S)-2,3-dimethoxy-5-(3'-[(18)F]fluoropropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide ([(18)F]fesetron) as a potential PET imaging agent for serotonin 5-HT3 receptors. By radiolabeling((S)-2,3-dimethoxy-5-(3'-tosyloxypropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide) with fluorine-18, (S)-[(18)F]fesetron was obtained in 5 to 10% decay-corrected yields and with specific activities >74GBq/μmol at the end of radiosynthesis. PET imaging in rats showed low uptake of [(18)F]fesetron in the brain with retention of binding in the striatal and cerebellar regions. Using colliculi as a reference region, ratios were 3.4 for striata and 2.5 for cerebellum. Ex vivo brain PET analysis displayed binding of [(18)F]fesetron in the hippocampus, striatum and cerebellar regions. Cerebellar regions corresponded to area postrema and nucleus tract solitaris known to contain 5-HT3 receptors. Dorsal hippocampus showed the highest uptake with ratio of >17 with respect to colliculi, while area postrema and striata had ratios of >10. Thus, [(18)F]fesetron exhibited a unique binding profile to rat brain regions known to contain significant amounts of serotonin 5-HT3 receptors. However, the very low brain uptake limits its usefulness as a PET radiotracer in this animal model.

We report the use of β3 adrenergic receptor mediated activation of rat brain frontal cortex using mirabegron (a selective β3 adrenoceptor agonist), measured by (18)F-FDG PET/CT. Another β3 agonist, CL 316,243, did not have this effect due to impermeability through the blood brain barrier (BBB), while atomoxetine, a norepinephrine transporter blocker, did increase (18)F-FDG uptake in the frontal cortex. Mirabegron exhibited a dose-dependent increase in frontal cortex (18)F-FDG uptake. These findings suggest a possible use of selective β3 adrenoceptor agonists in reversing regional glucose hypometabolism in the brain.

Elevated levels of histone deacetylases (HDACs) have been indicated in the development of some cancers. HDAC has been imaged using (18)F-FAHA and may serve as a marker to study epigenetics. We report evaluation of (18)F-FAHA as a probe in the early diagnosis of lung cancer using (18)F-FAHA PET/CT studies of A/J mice treated with NNK. (18)F-FAHA radiosynthesis was carried out in specific activity of ~2 Ci/μmol. A/J mice were divided into 2 groups: 1. Controls; 2. NNK treatment group with NNK (100 mg/kg, ip, weekly for 4 wks). Mice were injected 100-200 μCi i.v. (18)F-FAHA and then scanned in Inveon PET/CT under anesthesia using 2.0% isoflurane. Midbrain, cerebellum and brainstem uptake of (18)F-FAHA was displaced by the known HDAC inhibitor, suberanilohydroxamic acid (SAHA) with less than 10% activity remaining. CT revealed presence of lung nodules in 8 to 10-month old NNK mice while control mice were free of tumors. Little uptake of (18)F-FAHA was observed in the control mice lungs while significant (18)F-FAHA uptake occurred in the lungs of NNK-treated mice with tumor/nontumor >2.0. Ex vivo scans of the excised NNK and control mice lungs confirmed presence of extensive amounts of lung nodules seen by CT and confirmed by (18)F-FAHA in the NNK mice with tumor/nontumor >6.0. Our preliminary imaging studies with A/J mice lung cancer model suggest (18)F-FAHA PET may allow the study of epigenetic mechanisms involved in NNK-induced tumorigenesis in the lungs.

MicroPET imaging studies using (18)F-nifene, a new positron emission tomography (PET) radiotracer for nicotinic acetylcholinergic receptors (nAChR) α4β2 receptors in rats, have been carried out. Rats were imaged for 90 min after intravenous injection of (18)F-nifene (0.8 to 1 mCi), and binding potential (BP(ND)) was measured. (18)F-Nifene binding to thalamic and extrathalamic brain regions was consistent with the α4β2 nAChR distribution in the rat brain. Using the cerebellum as a reference, the values for the thalamus varied less than 5% (BP(ND) = 1.30, n = 3), confirming reproducibility of (18)F-nifene binding. (18)F-Nifene microPET imaging was also used to evaluate effects of nicotine in a group of Sprague-Dawley rats under isoflurane anesthesia. Nicotine challenge postadministration of (18)F-nifene demonstrated reversibility of (18)F-nifene binding in vivo. For α4β2 nAChR receptor occupancy (nAChR(OCC)), various doses of nicotine (0, 0.02, 0.1, 0.25, and 0.50 mg/kg nicotine free base) 15 min prior to (18)F-nifene were administered. Low-dose nicotine (0.02 mg) reached > 80% nAChR(OCC) while at higher doses (0.25 mg) > 90% nAChR(OCC) was measured. The small amount of (18)F-nifene binding with reference to the cerebellum affects an accurate evaluation of nAChR(OCC). Efforts are underway to identify alternate reference regions for (18)F-nifene microPET studies in rodents.

Imaging the high-affinity, functional state (HA) of dopamine D2 and D3 receptors has been pursued in PET imaging studies of various brain functions. We report further evaluation of 18 F-5-OH-FPPAT, and the newer 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT. Syntheses of 18 F-5-OH-FHXPAT and 18 F-7-OH-FHXPAT were improved by modifications of our previously reported procedures. Brain slices and brain homogenates from male Sprague-Dawley rats were used with the 3 radiotracers (74-111 kBq/cc). Competition with dopamine (1-100 nM) and Gpp(NH)p (10-50 µM) were carried out to demonstrate binding to dopamine D2 and D3 HA-states and binding kinetics of 18 F-5-OH-FPPAT measured. Ex vivo brain slice autoradiography was carried out on rats administered with 18 F-5-OH-FHXPAT to ascertain HA-state binding. PET/CT imaging in rats and wild type (WT) and D2 knock-out mice were carried out using 18 F-7-OH-FHXPAT (2-37 MBq). Striatum was clearly visualized by the three radiotracers in brain slices and dopamine displaced more than 80% of binding, with dissociation rate in homogenates of 2.2 × 10-2 min-1 for 18 F-5-OH-FPPAT. Treatment with Gpp(NH)p significantly reduced 50-80% striatal binding with faster dissociation rates (5.0 × 10-2 min-1 ), suggesting HA-state binding of 18 F-5-OH-FPPAT and 18 F-5-OH-FHXPAT. Striatal binding of 18 F-5-OH-FHXPAT in ex vivo brain slices were sensitive to Gpp(NH)p, suggesting HA-state binding in vivo. PET binding ratios of 18 F-7-OH-FHXPAT in rat brain were ventral striatum/cerebellum = 2.09 and dorsal striatum/cerebellum = 1.65; similar binding ratios were found in the D2 WT mice. These results suggest that in vivo PET measures of agonists in the brain at least in part reflect binding to the membrane-bound HA-state of the dopamine receptor.