Improving external beam radiotherapy by combination with internal irradiation.

The efficacy of external beam radiotherapy (EBRT) is dose dependent, but the dose that can be applied to solid tumour lesions is limited by the sensitivity of the surrounding tissue. The combination of EBRT with systemically applied radioimmunotherapy (RIT) is a promising approach to increase efficacy of radiotherapy. Toxicities of both treatment modalities of this combination of internal and external radiotherapy (CIERT) are not additive, as different organs at risk are in target. However, advantages of both single treatments are combined, for example, precise high dose delivery to the bulk tumour via standard EBRT, which can be increased by addition of RIT, and potential targeting of micrometastases by RIT. Eventually, theragnostic radionuclide pairs can be used to predict uptake of the radiotherapeutic drug prior to and during therapy and find individual patients who may benefit from this treatment. This review aims to highlight the outcome of pre-clinical studies on CIERT and resultant questions for translation into the clinic. Few clinical data are available until now and reasons as well as challenges for clinical implementation are discussed.

Cellular and molecular properties of (90)Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro.

PURPOSE: Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA) and radiolabeling with (90)Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI). METHODS: The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of (90)Y-cetuximab with EBI were evaluated. RESULTS: Control cetuximab and CHX-A″-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A″-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [(90)Y]Y-CHX-A″-DTPA-cetuximab ((90)Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of (90)Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A″-DTPA. Cetuximab and CHX-A″-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. (90)Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells. CONCLUSIONS: Conjugation of CHX-A″-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. (90)Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.

Evaluation of novel tropane analogues in comparison with the binding characteristics of [18F]FP-CIT and [131I]beta-CIT.

This study evaluated novel potential dopamine transporter (DAT) inhibitors as ligands for positron emission tomography. Five new tropane analogs were synthesized and compared with the known ligand 2beta-carbomethoxy-3beta-(4-iodophenyl)tropane (beta-CIT) and the recently characterized ligands N-(3-iodoprop-2E-enyl)-2beta-carbomethoxy-3beta-(4-methylphenyl)-nortropane (PE2I) and 2beta-carbofluoroethoxy-3beta-(4-methylphenyl)tropane (FETT). Evaluation with autoradiography measured the ability to antagonize the binding of [(131)I]iodine-labeled beta-CIT and [(18)F]fluorine-labeled N-(3-fluoropropyl)-2beta-carbomethoxy-3beta-(4-iodo-phenyl) nortropane in rat and pig brains. The standards for comparison (PE2I and FETT) competed strongly in all regions investigated (striatum, cortex, superior colliculus and cerebellum). Of the new compounds, 2alpha-amido-fluoroethyl-3beta-(4-iodophenyl)tropane (4) and 2beta-amido-fluoroethyl-3beta-(4-iodophenyl)tropane (4a) competed strongly with [(131)I]beta-CIT in DAT-rich striatum, but also in other brain regions suggesting poor DAT selectivity. Because [(131)I]beta-CIT binds unselectively both to DAT and serotonin transporters, no definite conclusion about the selectivity of the new compounds is possible. However, preclinical studies using the compounds and labeled with fluorine-18 or iodine-131 are encouraged.

Binding of tritiated and radioiodinated ZM241,385 to brain A2A adenosine receptors.

Autoradiography on rat brain using tritiated (1*), mono- (2*) and di-radioiodinated (3*) derivatives of the A(2A) adenosine receptor antagonist ZM241,385 showed high receptor density in striatum. K(D)s of 1*, 2* and 3* were 0.4, 2.2 and 15 nM and nonspecific binding was 5, 40 and 50% of total binding. Striatal uptake of 2* in mice was approximately 0.2% ID/g 60 min post-injection; blocking by 2 was insignificant. Poor penetration of the blood brain barrier and high nonspecific binding make 2* unsuitable for imaging striatal receptors.

Evaluation of radioiodinated 8-Cyclopentyl-3-[(E)-3-iodoprop-2-en-1-yl]-1-propylxanthine ([*I]CPIPX) as a new potential A1 adenosine receptor antagonist for SPECT.

8-Cyclopentyl-3-[(E)-3-[(131)I]iodoprop-2-en-1-yl]-1-propylxanthine (2*) was generated by iododestannylation of the tributyl-stannyl-precursor with [(131)I]NaI and chloramine T. The radiochemical yield of 2* was 82 +/- 4%, and the purity exceeded 98%. The specific activity was 33 +/- 19 GBq/micromol. Affinities for rat, pig and human A(1) adenosine receptors (A(1)ARs) were in the low nanomolar range, but poor selectivity for the human A(1)AR over the A(2A)AR was found. Additionally, in vitro and ex vivo autoradiographic studies revealed high unspecific binding which makes this ligand unsuitable for SPECT imaging.

Development of ligands for in vivo imaging of cerebral nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) mediate a variety of brain functions. Findings from postmortem studies and clinical investigations have implicated them in the pathophysiology and treatment of Alzheimer's and Parkinson's diseases and other CNS disorders (e.g. Tourette's syndrome, epilepsy, nicotine dependence). Therefore, it ultimately might be useful to image nAChRs noninvasively for diagnosis, for studies on how changes in nAChRs might contribute to cerebral disorders, for development of therapies targeted at nAChRs, and to monitor the effects of such treatments. To date, only (S)-(-)-nicotine, radiolabeled with 11C, has been used for external imaging of nAChRs in human subjects. Since this radiotracer presents drawbacks, new ligands, with more favorable properties, have been synthesized and tested. Three general classes of compounds, namely, nicotine and its analogs, epibatidine and related compounds, and 3-pyridyl ether compounds, including A-85380, have been evaluated. Analogs of A-85380 appear to be the most promising candidates because of their low toxicity and high selectivity for the alpha4beta2 subtype of nAChRs.

Ligands for in vivo imaging of nicotinic receptor subtypes in Alzheimer brain.

The neuronal nicotinic acetylcholine receptors (nAChR) are involved in functional processes in brain including cognitive function and memory. A severe loss of the nAChRs has been detected in brain of patients with Alzheimer's disease (AD). There is a great interest to image nAChRs noninvasive for detection of receptor impairments even at a presymptomatic stage of AD as well for monitoring outcome of drug treatment. (S) [11C]Nicotine, has so far been the only nAChR ligand used in positron emission tomography (PET) studies for visualizing nAChRs in human brain. In order to develop PET/SPECT nAChRs ligands for detection of subtypes of nAChRs nicotine analogues, epibatidine and A-85380 compounds have been characterized in vitro and investigated in vivo. Epibatidine and A-85380 have been found to have higher specific signals and more favorable kinetic parameters than nicotine and its analogues. The epibatidine and A-85380 compounds can also be radiolabeled with high specific radioactivity, show affinities for the nAChRs in the pM range and readily cross the blood-brain barrier. In addition they reversibly bind to the nAChRs and show low non-specific binding and moderately fast metabolism. Due to a probably high alpha4beta2 nAChR selectivity combined with low toxicity, the A-85380 analogs presently seem to be the most promising nAChR ligand imaging of subtypes of nAChRs in human brain.

In vivo positron emission tomography studies on the novel nicotinic receptor agonist [11C]MPA compared with [11C]ABT-418 and (S)(-)[11C]nicotine in rhesus monkeys.

The novel 11C-labeled nicotinic agonist (R,S)-1-[11C]methyl-2(3-pyridyl)azetidine ([11C]MPA) was evaluated as a positron emission tomography (PET) ligand for in vivo characterization of nicotinic acetylcholine receptors in the brain of Rhesus monkeys in comparison with the nicotinic ligands (S)-3-methyl-5-(1-[11C]methyl-2-pyrrolidinyl)isoxazol ([11C]ABT-418) and (S)(-)[11C]nicotine. The nicotinic receptor agonist [11C]MPA demonstrated rapid uptake into the brain to a similar extent as (S)(-) [11C]nicotine and [11C]ABT-418. When unlabeled (S)(-)nicotine (0.02 mg/kg) was administered 5 min before the radioactive tracers, the uptake of [11C]MPA was decreased by 25% in the thalamus, 19% in the temporal cortex, and 11% in the cerebellum, whereas an increase was found for the uptake of (S)(-)[11C]nicotine and [11C]ABT-418. This finding indicates specific binding of [11C]MPA to nicotinic receptors in the brain in a simple classical displacement study. [11C]MPA seems to be a more promising radiotracer than (S)(-)[11C]nicotine or [11C]ABT-418 for PET studies to characterize nicotinic receptors in the brain.

Autoradiographic comparison of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine binding in relation to vesicular acetylcholine transport sites in the temporal cortex in Alzheimer's disease.

The laminar binding distribution of three nicotinic receptor agonists, [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine, and their relation to the [3H]vesamicol binding, which is known to represent the vesicular acetylcholine transport sites, was performed employing in vitro autoradiography on the medial temporal cortex (Brodmann area 21). Autopsied brain tissue from nine Alzheimer patients and seven age-matched controls were used. The binding pattern of the three nicotinic ligands in the normal cortex was in general similar, showing binding maxima in the cortical layers I, III and V. The binding of [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine was lower in the older controls and more uniform throughout the layers as compared with younger controls. There was a significant age-related decrease in the binding of the three nicotinic ligands within the controls (age range: 58 to 89 years; P[3H](-)nicotine = 0.002, P[3H]epibatidine = 0.010, P[3H]cytisine = 0.037). In the older controls, the [3H]epibatidine binding was much decreased as compared with that of [3H](-)nicotine and [3H]cytisine. This may indicate a higher selectivity of [3H]epibatidine for a nicotinic receptor subtype that is particularly affected by aging. The laminar binding pattern of [3H]vesamicol showed one maximum in the outer cortical layers II/III. The [3H]vesamicol binding did not change with aging. The binding of all ligands was significantly decreased in all layers of the temporal cortex in Alzheimer's disease, but the [3H]vesamicol binding decreased only half as much as the nicotinic receptors. Also, choline acetyltransferase activity was percentually more reduced than [3H]vesamicol binding in Alzheimer's disease. The cortical laminar binding pattern of all 3H-ligands was largely absent in the Alzheimer's disease cases. The less severe loss of vesicular acetylcholine transport sites as compared with the loss of the nicotinic receptors and choline acetyltransferase activity may suggest that vesamicol binding sites might be more preserved in presynaptic terminals still existing and thereby expressing compensatory capacity to maintain cholinergic activity.

Synthesis and characterization of binding of 5-[76Br]bromo-3-[[2(S)-azetidinyl]methoxy]pyridine, a novel nicotinic acetylcholine receptor ligand, in rat brain.

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.

Quantitative autoradiography with short-lived positron emission tomography tracers: a study on muscarinic acetylcholine receptors with N-[(11)C]methyl-4-piperidylbenzilate.

The present work demonstrates quantitative autoradiography by using positron emission tomography tracers and storage phosphorimaging plates. The uptake and association of [(11)C]N-methyl-4-piperidylbenzilate was measured in rat brain tissue cryosections of various thicknesses. The signal increased with increasing section thickness, but only in 10-micrometer-thick sections did the binding reach the steady state during a 50-min observation time. This violation of the equilibrium condition, potentially combined with perfusion limitations, leads to erroneous increased binding-site density and decreased affinity in the 25- and 50-micrometer-thick sections. For better imaging of receptor distribution it is reasonable to use thicker sections. For quantitative analysis of receptor-binding parameters, the specific properties of ligands at different thicknesses of cryosections need to be considered. Evidence is provided that the nonselective muscarinic antagonist N-methyl-4-piperidylbenzilate binds preferentially to the M(4) subtype of muscarinic acetylcholine receptors.

In vitro evaluation of 11C-labeled (S)-nicotine, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (R,S)-1-methyl-2-(3-pyridyl)azetidine as nicotinic receptor ligands for positron emission tomography studies.

The binding characteristics of the novel 11C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[11C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [11C]MPA in comparison with (S)-[11C]nicotine and [11C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K(D) values) of 2.4 and 560 nM and binding site densities (Bmax values) of 65.5 and 223 fmol/mg of protein for (S)-[11C]nicotine, K(D) values of 0.011 and 2.2 nM and Bmax values of 4.4 and 70.7 fmol/mg of protein for [11C]MPA, and K(D) values of 1.3 and 33.4 nM and Bmax values of 8.8 and 69.2 fmol/mg of protein for [11C]ABT-418. In competing with the 11C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (-)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the 11C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [11C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three 11C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [11C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [11C] MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[11C]nicotine and [11C]ABT-418 raise the level of interest in [11C]MPAfor application in positron emission tomography.

Laminar distribution of nicotinic receptor subtypes in human cerebral cortex as determined by [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in vitro autoradiography.

The subregional localization of different nicotinic acetylcholine receptor subtypes in human cerebral cortex was estimated by quantitative in vitro autoradiography using the nicotinic ligands [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in large whole human forebrain hemispheres. Saturation experiments in frontal cortex revealed for [3H](-)nicotine two binding sites with affinity constants (Kd) of 0.45 and 6.3 nM and binding site densities (Bmax) of 3.0 and 14.2 pmol/g, for [3H]cytisine one binding site with Kd of 0.19 nM and Bmax of 21.8 pmol/g, and for [3H]epibatidine one binding site with Kd of 0.011 nM and Bmax of 20.0 pmol/g. The laminar binding distributions of the three ligands were compared in different cortical areas by creating binding profiles perpendicular to the entire cortical depth. The regional autoradiographic binding patterns of the three ligands were essentially similar, with higher receptor binding in cortical layers I, III and V. In the primary sensory cortex and inferior frontal sulcus, marked binding of all ligands was observed in layer III. [3H]Cytisine showed the lowest difference between maximal and minimal binding within the gray tissue in all other areas. In the primary motor cortex, [3H]epibatidine and [3H](-)nicotine showed high binding in layers III and V. The [3H](-)nicotine binding was higher than that of the other ligands in layers I and VI of the primary motor cortex, the deeper layer V of the primary sensory cortex, layer III of the superior temporal sulcus and layer VI of the parietal cortex. A distinct band of binding of [3H](-)nicotine and [3H]epibatidine but not of [3H]cytisine was found in layer IIlb of the occipital cortex and layer V of the superior temporal sulcus. [3H]Epibatidine showed higher binding than the other ligands in all layers of the medial frontal, superior frontal and superior temporal sulcus. The findings with the three nicotinic ligands suggest three binding sites in the cortex with different laminar distributions. All three ligands bound to an identical receptor site, most likely the alpha4 nicotinic receptor subunit. The morphological distribution of [3H]epibatidine and [3H](-)nicotine binding indicate that they bind to an additional site, especially in the primary motor cortex, in layer IIIb of the occipital cortex and layer V of the superior temporal sulcus. High binding of [3H](-)nicotine in layers I and VI of the primary motor cortex, the deeper layer V of the primary sensory cortex, layer III of the superior temporal sulcus and layer VI of the parietal cortex may indicate a third binding site.

In vitro and in vivo characterization of (+)-3-[11C]cyano-dizocilpine.

(+)-3-[11C]Cyano-5-methyl-10,11 -dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine ([11C]MKC) was successfully synthesized as a potential radiotracer for PET studies on the NMDA receptor channel complex. In vitro binding properties of [11C]MKC were investigated with newly developed techniques for efficient evaluation of 11C-labeled compounds. The association curve of [11C]MKC binding in rat forebrain membranes showed that the specific binding reached an equilibrium within 30 min. Specific binding was saturable with affinity constant KD=8.2+/-0.4nM and Bmax=1.62+/-0.04 pmol/mg protein with glutamate and glycine included in the incubation medium. The binding of [11C]MKC was decreased by extensive washing of the membrane preparation. (+)- and (-)-Dizocilpine, 3-cyano-dizocilpine, and ketamine inhibited the specific binding of [11C]MKC with IC50 values of 37.3, 445.0, 65.8nM and 3.91 microM, respectively. High specific binding in in vitro autoradiography was distributed predominantly in telencephalic regions (the hippocampus, cerebral cortex, and striatum) followed by thalamus. PET studies using rhesus monkeys under anesthesia showed high uptake of [11C]MKC in the temporoparietal and frontal cerebral cortices, striatum, and thalamic regions, although it is problematic to verify the specific binding in vivo by PET.

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique.

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.

Repeated administration of tacrine to normal rats: effects on cholinergic, glutamatergic, and GABAergic receptor subtypes in rat brain using receptor autoradiography.

Tacrine, a potent acetylcholinesterase inhibitor, has been reported to improve cognitive function in patients with Alzheimer's disease. The present investigation was conducted to elucidate in vivo any interaction between tacrine-induced cortical cholinergic hyperactivity and glutamatergic and GABAergic neurotransmission, which might influence the therapeutic potential of tacrine. Seven days after a daily dosage of 10 mg/kg tacrine i.p. quantitative receptor autoradiography was performed in coronal sections throughout the brain. Repeated administration of tacrine resulted in decreased binding to high-affinity choline uptake, nicotinic and M2-muscarinic acetylcholine receptor sites in a number of cortical regions, while reductions in M1-muscarinic receptor binding were restricted to the cingulate and entorhinal cortex as well as caudate-putamen. Moreover, tacrine injections decreased cortical AMPA receptor binding throughout the brain, while NMDA, kainate, and GABAA receptor binding remained unchanged. Tacrine administration alters cortical AMPA receptor binding in the opposite direction to that observed in patients with Alzheimer's disease, suggesting that tacrine may exert a reversal in up/down-regulation of cortical glutamate receptor subtypes in Alzheimer patients. However, the drug-induced reductions in cortical high-affinity choline uptake sites as well as in nicotinic and in muscarinic acetylcholine receptor binding might partially counteract the cognition-enhancing effects of tacrine produced by acetylcholinesterase inhibition.

Methodological aspects for in vitro characterization of receptor binding using 11C-labeled receptor ligands: a detailed study with the benzodiazepine receptor antagonist [11C]Ro 15-1788.

As a complement to in vivo studies with positron emission tomography (PET), it is desirable to perform in vitro characterization of newly developed 11C tracers. In this report we describe the technique for determination of receptor-ligand kinetics utilizing ligands labeled with the short-lived radionuclide 11C. The limitations and advantages are discussed. The benzodiazepine antagonist [11C]Ro 15-1788 was used as a model substance, and the use of storage phosphor plates for quantification of radioactivity was validated. Storage phosphor plates showed an excellent linear range (approximately 10[3]) and acceptable resolution (approximately 0.5 mm). Receptor-ligand kinetics, including depletion, association and dissociation, saturation and displacement were evaluated with good results through the use of short-lived radiotracers and storage phosphor plates.

Receptor imaging technique with 11C-labeled receptor ligands in living brain slices: its application to time-resolved imaging and saturation analysis of benzodiazepine receptor using [11C]Ro15-1788.

Recently we developed a novel imaging technique using positron emitter-labeled compounds as probes and a storage phosphor screen as a detector. This approach makes it possible to follow a variety of biochemical processes with spatial information in living brain slices. Further technical development is reported here in terms of time-resolved imaging and receptor characterization in a real equilibrium state. The method was validated by use of [11C]Ro15-1788, a benzodiazepine receptor antagonist. Fresh brain slices were incubated with [11C]Ro15-1788 in oxygenated Krebs-Ringer solution at 37 degrees C, in a specially designed chamber. By placing the chamber on a storage phosphor screen, we could obtain two-dimensional images of radioactivity in the slices. Time-resolved imaging was made at 5 min intervals, revealing that it took 60 min to reach equilibrium binding. The dissociation process was observed by adding an excess amount of unlabeled Ro15-1788 to the chamber, 25 min was required for the full dissociation. In the equilibrium state, i.e. in the presence of free radio-ligand, Scatchard plot analysis was performed on the cerebral cortex (Kd = 7.4 nM, Bmax = 146 fmol/mg tissue) and striatum (Kd = 7.5 nM, Bmax = 107 fmol/mg tissue), suggesting the presence of a single component of binding site in these two regions. The present method, for the first time, made it possible to study a ligand-receptor interaction in living brain slices with temporal and spatial resolutions. This technique should prove useful for studies of receptor function under physiological conditions.