Kinetic analysis of central [76Br]bromolisuride binding to dopamine D2 receptors studied by PET.

The in vivo kinetic analysis of dopamine D2 receptors was obtained in baboon brain using positron emission tomography (PET) and [76Br]bromolisuride [( 76Br]BLIS) as radioligand. An injection of a trace amount of [76Br]BLIS was followed 3 h later by an injection of a mixture of [76Br]BLIS and BLIS in the same syringe (coinjection experiment). A third injection performed at 6 h was either an excess of unlabeled ligand (displacement experiment) or a second coinjection. This protocol allowed us to evaluate in the striatum of each animal and after a single experiment the quantity of available receptors (B'max) and the kinetic parameters including the association and dissociation rate constants (k + 1VR and k-1, respectively, where VR is the volume of reaction). The cerebellum data were fitted using a model without specific binding. All the parameters were estimated using nonlinear mathematical models of the ligand-receptor interactions including or not including nonspecific binding. The plasma time-concentration curve was used as an input function after correction for the metabolites. An estimate of standard errors was obtained for each PET study and for each identified parameter using the covariance matrix. The average values of B'max and KdVR were 73 +/- 11 pmol/ml tissue and 1.9 +/- 0.9 pmol/ml, respectively. The nonspecific binding was identifiable in the experiment where the last injection corresponded to a second coinjection. We found that approximately 6% of the striatal binding was nonspecific after a tracer injection of [76Br]BLIS. The nonspecific binding appeared to be reversible in the striatum but irreversible in the cerebellum.

Anticonvulsant activity of the diaryltriazine, LY81067: studies using electroencephalographic recording and positron emission tomography.

It is reported that LY81067, a new diaryltriazine, possesses anticonvulsant properties against grand mal status epilepticus induced by intravenous administration of picrotoxin binding site ligands (Ro 5-4864 and pentylenetetrazole) in the baboon. Intravenous administration of LY81067 during the seizures blocked grand mal type electroencephalographic (EEG) paroxysmal discharges and led to a long electrical silence, progressively replaced by spike-and-wave discharges of low frequency (2 c/sec). A transient blocking effect was also observed when LY81067 was injected during grand mal status epilepticus induced by the benzodiazepine inverse agonist methyl beta-carboline-3-carboxylate; however, the long electrical silence observed after administration of LY81067 was rapidly followed by grand mal type paroxysmal discharges in the EEG, which could be stopped by a subsequent injection of Ro 15-1788. However, LY81067 also displayed intrinsic epileptogenic properties. Administration of this drug alone led to the appearance of rhythmic EEG (2-3 c/sec) associated with myoclonia. Concomitantly with the EEG studies, interactions of all these drugs with benzodiazepine receptors were observed in vivo using [11C]Ro 15-1788 as radioligand and positron emission tomography (PET) as a non-invasive technique to measure the binding of the [11C]benzodiazepine antagonist in brain, in vivo. The [11C]Ro 15-1788 bound in the brain could not be displaced by the administration of LY81067 but rather, the [11C]antagonist binding in the brain was somewhat enhanced. Administration of pentylenetetrazole or Ro 5-4864 decreased the rate of wash-out of the radioligand. This fast effect of these two convulsant drugs was partially inhibited by the subsequent administration of LY81067. The concomitant blocking of the grand mal status epilepticus was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo bidirectional modulatory effect of benzodiazepine receptor ligands on GABAergic transmission evaluated by positron emission tomography in non-human primates.

The central type benzodiazepine receptor (BDZr), an allosteric modulatory site of the GABAA receptor-anion channel, has been shown in vitro to respond to drugs with positive efficacy (agonists), zero efficacy (competitive antagonists) and drugs with negative efficacy (inverse agonists). However, this general concept of the function of BDZr drugs has rarely been assessed in intact living brain. We report here in on a non-invasive in vivo assessment of the intrinsic efficacies of BDZr drugs in the brain of non-human primates. We have performed an in vivo simultaneous determination of fractional BDZr occupancy and the resulting pharmacological efficacies of the full agonist diazepam, the partial agonist bretazenil, the antagonist flumazenil (Ro15-1788), the partial inverse agonist Ro15-4513 and the full inverse agonist methyl beta-carboline-3-carboxylate (beta-CCM). Positron emission tomography (PET) was used to estimate fractional BDZr occupancy measured as the in vivo displacement in the brain of the positron emitter radioligand, [11C]flumazenil. Simultaneously, the proconvulsant or anticonvulsant efficacies of the BDZr drugs were measured as their abilities to facilitate or counteract the central effects of an infusion of pentylenetetrazol, a non-competitive GABA antagonist acting on the picrotoxin site of the receptor complex. This was measured using electroencephalographic recording (EEG). Our results show that, in vivo, the fractional receptor occupancy by a given drug is perfectly correlated with its resulting graded pharmacological effects, as predicted from the competitive drug receptor interaction theory. Furthermore, the slope of the relationship between fractional receptor occupancies and the resulting pharmacological effects (an index of intrinsic efficacy) strictly depends on the BDZr ligand considered. Diazepam displayed a strong positive intrinsic efficacy, and, in contrast, beta-CCM a marked negative one. Between these two extremes, the partially active drugs bretazenil and Ro15-4513, which required a large fractional receptor occupancy to produce significant anti- or proconvulsant effects, respectively, displayed only a weak intrinsic efficacy. Flumazenil did not produce any significant pharmacological effect. We observed that the in vivo intrinsic efficacies of diazepam, flumazenil and beta-CCM correlate with their intrinsic efficacies as measured by their modulatory effects on the GABA-dependent membrane chloride conductance in vitro. Thus, the intrinsic efficacies measured using PET and EEG are likely to reflect the different in vivo abilities of BDZr drugs to induce or stabilize the GABAA-benzodiazepine chloride channel in a given conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of suriclone with central type benzodiazepine receptors in living baboons.

The interaction of suriclone and two of its main metabolites with central type benzodiazepine receptors, which had been labeled in vivo with the radioligand [11C]RO 15-1788, was investigated in living baboons. The concentration of radioligand bound to the receptors, as measured in brain transverse sections by positron emission tomography, decreased rapidly after the i.v. administration of suriclone at doses known to induce pharmacological effects. The rate and extent to which [11C]RO 15-1788 binding was displaced increased with increasing doses of suriclone. The half-inhibitory dose (ID50) was determined to be 0.08 mg/kg in vivo. The rapid inhibitory effect of suriclone on the in vivo binding of [11C]RO 15-1788 in the brain seems to reflect its ability to act at the GABA-benzodiazepine receptor complex, at or near to the benzodiazepine binding site, to induce its pharmacological activity. The i.v. injection of the demethylated metabolite of suriclone, RP 35,489, only caused a slight displacement of [11C]RO 15-1788 binding even at a dose of 2 mg/kg. Thus, suriclone appears to be more potent than RP 35,489 to displace the benzodiazepine 11C antagonist in vivo. The sulfoxide metabolite, RP 46,166, did not significantly change the kinetics of [11C]RO 15-1788 binding in the brain. The slight effects produced by high doses of RP 35,489 and RP 46,166 on [11C]RO 15-1788 binding in the brain suggest that these metabolites are probably not responsible for the expression of biological activity of suriclone mediated by benzodiazepine receptors.

Relationships between benzodiazepine receptors, impairment of GABAergic transmission and convulsant activity of beta-CCM: a PET study in the baboon Papio papio.

Central type benzodiazepine receptors were studied in vivo by positron emission tomography in brain areas of 2 different groups of the baboon Papio papio: non-photosensitive (group 1) and those with an allylglycine-induced decrease in GABA-mediated inhibition (group 2). Further, a naturally photosensitive Papio papio (+3 level of photosensitive response) was compared to both groups. Regional brain binding of the specific benzodiazepine receptor ligand, [11C]Ro 15-1788, was not significantly different between groups 1 and 2. In addition, the data from the naturally photosensitive Papio papio did not seem to differ markedly from groups 1 and 2 either. Pharmacological effects of increasing doses of beta-CCM (0.05-3 mg/kg i.v.) and regional benzodiazepine receptor occupancy by the drug were simultaneously studied using electroencephalographic activity recording and positron emission tomography. A positive correlation was observed between the degree of photosensitivity of the baboon and sensitivity to the action of beta-CCM, with increasing convulsant efficacy of beta-CCM in going from group 1 to the naturally photosensitive baboon, then to group 2. Dose-related displacement curves of [11C]Ro 15-1788 binding by beta-CCM revealed that reduction in brain GABA concentration did not modify the inhibitory potency of beta-CCM on [11C]Ro 15-1788 binding in cerebral cortex. This suggests a lack of detectable in vivo allosteric effects of GABA on beta-CCM binding during beta-CCM-induced seizures. Thus, a given dose of beta-CCM displayed increasing pharmacological potency in going from baboons with the lowest photosensitivity to those with the highest, whereas benzodiazepine receptor occupancy by beta-CCM was similar in the cerebral cortex of the different baboons. Conversely, a given level of convulsant activity of beta-CCM was related to a different benzodiazepine receptor occupancy by the drug, depending on the photosensitivity of Papio papio. A given dose of a drug may, thus, have a different pharmacological potency when occupying the same number of receptors, depending on the physiopathological state of the subject.

Synthesis, tissue distribution in rats and PET studies in baboon brain of no-carrier-added [18F]RU 52461: in vivo evaluation as a brain glucocorticoid receptor radioligand.

11,17 beta-Dihydroxy-6-methyl-17 alpha-(3-[18F]fluoro-prop-1- ynyl)androsta-1,4,6-trien-3-one [( 18F]RU 52461), an 18F-analog of RU 28362, was synthesized by bromide displacement with [18F]fluoride in 12-30% overall radiochemical yield (decay-corrected) within 140 min from end of bombardment (EOB). The specific activity was 900-1500 mCi/mumol (33.3-55.5 GBq/mumol) at the end of synthesis (EOS). Biodistribution studies indicated high adrenal and pituitary retention, and uniformly low uptake of [18F]RU 52461 in all other brain regions of the rat. Except for the pituitary, no specific receptor-mediated uptake of [18F]RU 52461 could be demonstrated using saturating doses of unlabeled RU 52461 in rat brain. While no change was observed throughout the brain areas in adrenalectomized rats and in animals coinjected with dexamethasone, when compared to controls. PET studies revealed extremely low levels of radioactivity in baboon brain. Therefore, [18F]RU 52461 does not appear to cross the blood-brain barrier, suggesting that this radiopharmaceutical is not suitable to visualize the brain glucocorticoid binding sites by PET.

Quantitative evaluation of benzodiazepine receptors in live Papio papio baboons using positron emission tomography.

The binding of the 11C-labeled benzodiazepine antagonist Ro 15-1788 (flumazenil) was measured in the neocortex of live Papio papio baboons by positron emission tomography. This allowed us to calculate in vivo (i.e., at physiological temperature, neurotransmitters concentrations, and ionic environment) the apparent density of available benzodiazepine receptors (B'max) and the dissociation constant of Ro 15-1788 (Kd). By coadministering increasing doses of unlabeled Ro 15-1788 with [11C]Ro 15-1788 and assuming that nonsaturable radioactivity indicated the free ligand concentration, we were able to obtain saturation isotherms. We showed that a state of quasiequilibrium was reached 50 min after the administration of the radioligand. Linear Scatchard plots allowed us to calculate B'max at 78 and 50 pmol/ml of cerebral tissue in the occipital and frontal cortices, respectively. In both these areas, Kd is on the order of 6 nM, with a Hill number very close to unity. This indicates that Ro 15-1788 binds in vivo with high affinity to an homogeneous population of saturable sites. A similar measurement was carried out on a naturally photosensitive P. papio baboon. Absolute values of B'max, Kd, and Hill number were similar to those of the control baboons. Although results concerning this baboon can only be considered as a case report, this similarity may suggest that its epileptic syndrome is not related to a large change in B'max or Kd, at least in occipital and frontal cortices. Our results showed that quantitative estimation by positron emission tomography of some characteristics of benzodiazepine receptors is possible in live baboons and may represent a supplementary tool for investigating further the molecular mechanisms of benzodiazepine receptor function in physiological and physiopathological conditions. We suggest that a similar method of quantification of classic in vivo [3H]Ro 15-1788 binding could be usefully adapted when studying rodent models of epilepsy, stress, and other neuropsychological disorders. On the other hand, the similarity between the B'max and Kd values we obtained in baboons and those recently reported in humans using similar methods emphasizes that most of the in vivo characteristics of the benzodiazepine receptors of baboons are very close to those of human benzodiazepine receptors. This confirms that P. papio baboons are a suitable animal model for studying the pharmacology of benzodiazepine receptor ligands before clinical applications in humans.

6-[18F]fluoro-L-dopa uptake and [76Br]bromolisuride binding in the excitotoxically lesioned caudate-putamen of nonhuman primates studied using positron emission tomography.

The functional status of the dopaminergic system following striatal excitotoxic lesions was studied in living baboons by positron emission tomography (PET) using 6-[18F]fluoro-L-dopa as specific tracer for the presynaptic dopaminergic terminals and [76Br]bromolisuride as selective dopamine D2-receptor marker. The glutamate receptor agonist ibotenic acid (IA) was injected into the right caudate-putamen of six baboons to induce a neuropathological and behavioral model of Huntington's disease (HD). In vivo PET studies performed 3 to 6 months after the IA injections showed that subtotal excitotoxic lesions of the CP were accompanied by changes in the kinetic of [76Br]bromolisuride binding indicating a dose-dependent reduction in binding sites in the lesioned striatum of all IA-injected animals. In the most severely lesioned animals, there was also a decrease in the uptake of the nigrostriatal dopaminergic marker. The loss of D2-receptors and decrease in striatal dopamine uptake are consistent with clinical and postmortem findings in HD. In addition, the decrease in 6-[18F]fluoro-L-dopa uptake confirms previous studies performed in a rat model of HD suggesting a continuous decline of nigral dopamine cell function following destruction of their intrinsic striatal target neurons. The results of our experience to date in PET studies of 6-[18F]fluoro-L-dopa and [76Br]bromolisuride binding in IA-lesioned primates indicate that PET can identify effects of cell loss on markers of pre- and postsynaptic function in the striatum of living subjects.

In vivo benzodiazepine receptor occupancy by CL 218,872 visualized by positron emission tomography in the brain of the living baboon: modulation by GABAergic transmission and relation with anticonvulsant activity.

In vivo benzodiazepine receptor occupancy by increasing doses of CL 218,872 has been evaluated in the baboon Papio papio, using (11C) RO 15-1788 as specific radioligand and positron emission tomography as external detection system. Although BZR heterogeneity has been previously demonstrated in the brain of the living baboon using PET, we did not observe in our studies that CL 218,872 interacts preferentially with one of the BZR subtypes. The monophasic pattern of the dose dependent CL 218,872 displacement curve and the corresponding "in vivo Hill coefficient" near unity suggest that CL 218,872 binds in cerebral baboon cortex with a similar affinity with BZ1 as well as BZ2 subtypes. The anticonvulsant properties of CL 218,872 against bicuculline and allylglycine-induced seizures were correlated with benzodiazepine receptor occupancy by assessment of electroencephalographic activity during positron emission tomography studies. Our data confirmed in vivo the hypothesis of a partial agonist anticonvulsant activity of CL 218,872. At the same time, the use of a GABA-antagonist (bicuculline) or an inhibitor of the GABA synthesis (allylglycine) suggested the existence of an allosteric interaction between benzodiazepine receptors and GABA receptors.