Bradley T. Christian

Evaluation of dopamine D-2 receptor occupancy by clozapine, risperidone, and haloperidol in vivo in the rodent and nonhuman primate brain using 18F-fallypride

We have used the high-affinity dopamine D-2 receptor radioligand, 18F-fallypride for evaluating receptor occupancy by the antipsychotic drugs, clozapine, risperidone, and haloperidol in rodents and nonhuman primates. In rodents, clozapine (0.1 mg/kg to 100 mg/kg) competed with 18F-fallypride at all the doses administered. At doses over 40 mg/kg, clozapine was able to displace all the administered 18F-fallypride. A pseudobiphasic profile of receptor occupancy by clozapine was observed. This behavior was compared with such other neuroleptics as risperidone and haloperidol that exhibited over 90% receptor occupancy at doses over 0.1 mg/kg and did not exhibit a biphasic nature. Dopamine D-2 receptor occupancy in the monkeys was studied using positron emission tomography (PET) after acute subcutaneous doses of the various drugs. At therapeutically relevant doses, clozapine, risperidone, and haloperidol were able to compete significantly with the binding of 18F-fallypride in all brain regions in rhesus monkeys, and our analyses indicate that these drugs (clozapine, risperidone, and haloperidol) do not discriminate between the striatal (caudate and putamen) and the extrastriatal (thalamus and cortical regions) dopamine receptors. The following extent of D-2 receptor occupancies were measured in the monkey brain using PET: clozapine ≈70% (dose of 9.7 mg/kg), risperidone ≈75% (0.05 mg/kg), and haloperidol ≈90% (0.05 mg/kg).

Quantitation of striatal and extrastriatal D‐2 dopamine receptors using PET imaging of [18F]fallypride in nonhuman primates

[18F]Fallypride is a highly selective, high‐affinity dopamine D‐2 receptor ligand. The high affinity, KD = 30 pM, makes it a suitable candidate for visualizing both striatal and extrastriatal binding in the brain. In this work, dynamic PET studies of two macaque monkeys were acquired along with arterial plasma samples. Compartmental analysis and Logan plots were used to analyze the striatum, thalamus, frontal, and temporal cortices and to validate a reference region of analysis which yields a distribution volume ratio (DVR). The cerebellum was used as the reference region. The results indicate that all methods of analysis are in close agreement over all the analyzed regions in the brain. The average DVRs for the two monkeys was found to be: caudate = 26, putamen = 29, thalamus = 3.8, frontal ctx = 1.7, and temporal ctx = 1.7 on a high‐resolution PET scanner. It was found that a scan time of 2 h is needed to accurately estimate the DVR for all regions of the brain. The striatal regions require the longest to linearize and are the most sensitive to variations in the average tissue‐to‐plasma efflux constant, k̄2. For the extrastriatal regions, the effect of the k̄2 term on DVR calculation is negligible. Repeatability measurements for all regions were found to be within 10% using the DVR parameter. Synapse 38:71–79, 2000.

Measuring the in Vivo Binding Parameters of [18F]-Fallypride in Monkeys Using a PET Multiple-Injection Protocol

The goal of this work was to quantify the in vivo transport and binding parameters of [F-18]fallypride and the D2/D3 receptor density (B′max) in both the striatal (putamen, caudate, ventral striatum) and extrastriatal regions (thalamus, amygdala, cerebellum, temporal and frontal cortices) of the rhesus monkey brain. Multiple-injection PET experimental protocols with injections of radiolabeled and unlabeled doses of fallypride were used to estimate the K1, k2, kon/VR, koff and B′max kinetic parameters. The experimental design was chosen using the D-optimal criterion to maximize the precision of the estimated binding parameters for the various brain regions. There was a significant range in B′max for the putamen (27pmol/mL), caudate (23pmol/mL), ventral striatum (14pmol/mL), thalamus (1.8pmol/mL) and amygdala (0.9pmol/mL). Significant receptor binding was also found in the cortical regions. Knowledge of these in vivo rate constants serves as a necessary step in using [F-18]fallypride PET to measure D2/D3 receptor density and drug occupancy in clinical research applications. We believe the precise parameter estimates derived from these complicated experimental protocols are necessary for proper application of drug occupancy and clinical research studies with [F-18]fallypride, which often rely on the validity of assumptions regarding the model parameters.

Measurement of d-amphetamine-induced effects on the binding of dopamine D-2/D-3 receptor radioligand, 18F-fallypride in extrastriatal brain regions in non-human primates using PET.

The ability to measure amphetamine-induced dopamine release in extrastriatal brain regions in the non-human primates was evaluated by using the dopamine D-2/D-3 receptor radioligand, (18)F-fallypride. These regions included the thalamus, amygdala, pituitary, temporal cortex and frontal cortex as well as putamen, caudate and ventral striatum. The positron emission tomography (PET) studies involved control studies, which extended to 3 h, and the amphetamine-challenge studies, which involved administration of d-amphetamine (approx. 0.5-1 mg/kg, i.v.). PET data analysis employed the distribution volume ratio method (DVR) in which the cerebellum was used as a reference region. Our results show a substantial decrease in the binding potential of (18)F-fallypride in extrastriatal regions: thalamus (-20%), amygdala (-39%) and pituitary (-14%). Putamen, caudate and ventral striatum also exhibited significant decreases (-20%). The decrease in (18)F-fallypride binding in the extrastriatal regions points to the importance of dopaminergic neurotransmission in these brain regions. Furthermore, our findings support the use of (18)F-fallypride to measure extrastriatal dopamine release.

Metabolic alterations in muscle of thermally injured rabbits, measured by positron emission tomography

The hypermetabolic inflammatory state that occurs after major trauma has been extensively studied at the whole body level, however, there is only limited information on metabolic changes in individual tissues. In this study, the effect of thermal injury on metabolic function of uninjured hind limb muscle of rabbits was measured noninvasively by positron emission tomography (PET). Rabbits were subjected to full thickness burn on 25% of their body surface area. Two to three weeks after injury, PET and arterial blood sampling was performed during inhalation of 15O2, C15O2 and 11CO and after injection of 18FDG. The tissue and blood data were analyzed by standard kinetic models for blood flow, oxygen extraction fraction (OEF), oxygen utilization and glucose metabolism. A total of seven injured and five sham animals were studied. Total body oxygen consumption was measured by indirect calorimetry and plasma concentrations of glucose, insulin and IGF-1 were measured with standard assays. Compared to sham rabbits, blood flow to muscle of injured animals was unchanged. However, OEF, oxygen utilization and glucose metabolism were significantly reduced (p<0.01) in uninjured muscle of burned rabbits. These data demonstrate that thermal injury is associated with alterations in muscle metabolism, which are not related to change in blood flow.

Quantification of dopamine transporter density in monkeys by dynamic PET imaging of multiple injections of 11C-CFT.

Idiopathic Parkinsons disease (PD) is characterized by loss of dopaminergic terminals in the basal ganglia. The cocaine analog, CFT (WIN 35,428), has been shown to bind selectively to the pre‐synaptic dopamine transporters and thus represents an important probe for monitoring disease progression. In this study, we evaluated [11C] labeled CFT as a PET ligand for the quantitative in vivo assay of dopamine transporter density in three normal rhesus monkeys (Macaca mulatta). One of the animals was studied after treatment with the neurotoxin, MPTP.

In vitro and in vivo evaluation of the binding of the dopamine D2 receptor agonist11C-(R,S)-5-hydroxy-2-(di-n-propylamino)tetralin in rodents and nonhuman primate

The in vitro autoradiographic binding characteristics as well as in vivo imaging characteristics of a dopamine D2 receptor agonist, (R,S)‐2‐(N‐propyl‐N‐1′‐11C‐propyl)amino‐5‐hydroxytetralin (11C‐5‐OH‐DPAT), were studied. In 3H‐spiperone assays using rat striata, 5‐OH‐DPAT exhibited an affinity of IC50 = 2.5 nM. In vitro autoradiographs in rat brain slices with 11C‐5‐OH‐DPAT revealed selective binding to the dopaminergic regions in the striata which was displaceable by sulpiride. Varying concentrations of dopamine displaced this selective binding of 11C‐5‐OH‐DPAT to the striata in rat brain slices. This selective binding to the striata was also removed in the presence of the GTP analog, 5′‐guanylylimidodiphosphate, indicative of the binding of 11C‐5‐OH‐DPAT to the high‐affinity state of the D2 receptor. Ex vivo autoradiographic study in rats exhibited selective binding of 11C‐5‐OH‐DPAT to the striata. A PET study in a rhesus monkey showed selective localization of 11C‐5‐OH‐DPAT in the striata and the ratio between striata and cerebellum approached approximately 2 at 40 min postinjection. Synapse 37:64–70, 2000.

Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors

We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (+/-)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (+/-)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (+/-)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites (IC50 = 0.010 nM), PPHT was somewhat weaker (IC50 = 0.65 nM), and 5-OH-DPAT exhibited the weakest affinity (IC50 = 2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1-11C-propyl)amino-5-hydroxytetralin (11C-5-OH-DPAT), 2-(N-phenethyl-N-1-11C-propyl)amino-5-hydroxytetralin (11C-PPHT), and 2-(N-cyclohexylethyl-N-1-11C-propyl)amino-5-hydroxytetralin (11C-ZYY-339) was achieved by first synthesizing 11C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11CO2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11C-5-OH-DPAT, 11C-PPHT, and 11C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata.