Sajinder K. Luthra

Benzodiazepine Receptor Quantification in vivo in Humans Using [11C]Flumazenil and PET: Application of the Steady-State Principle

Carbon-11-labeled flumazenil combined with positron emission tomography (PET) was used to measure the concentration (Bmax) of the benzodiazepine (Bz) receptor in the brain and its equilibrium dissociation constant (KD) for flumazenil in five normal subjects. The steady-state approach was used injecting the tracer as a bolus of high specific activity. In each subject two studies were carried out. The first study was performed at essentially zero receptor occupancy, the tracer alone study. The second study was performed at a steady-state receptor occupancy of about 50%, achieved by a prolonged constant infusion of nonlabeled (“cold”) flumazenil starting 2 h before the bolus tracer injection and continuing until the end of the scanning period. In this second study the free concentration of unmetabolized flumazenil in plasma water was measured in multiple blood samples. The observed tissue and plasma tracer curves, calibrated in the same units of radioactivity per millimeter, were analyzed in two ways: (a) by the noncompartmental (stochastic) approach making no assumptions regarding number of compartments in the tissue, and (b) by the single-compartment approach assuming rapid exchange (mixing) of tracer between all tissue compartments. The noncompartmental and the compartmental analyses gave essentially the same values for the distribution volume of the tracer, the parameter used for quantitation of the Bz receptor. As the compartmental approach could be applied to a shorter observation period (60 min instead of 120 min) it was preferred. The five subjects had a mean KD value of 12 nM/L of water and Bmax values of the grey matter ranging from 39 ± 11 in thalamus to 120 ± 14 nM/L of brain in occipital cortex. Most previous studies have been based on the pseudoequilibrium approach using the brain stem as a receptor-free reference region. This yields practically the same KD but lower Bmax values than the steady-state approach presented here.

Catalytic decarboxylative fluorination for the synthesis of tri- and difluoromethyl arenes.

Treatment of readily available α,α-difluoro- and α-fluoroarylacetic acids with Selectfluor under Ag(I) catalysis led to decarboxylative fluorination. This operationally simple reaction gave access to tri- and difluoromethylarenes applying a late-stage fluorination strategy. Translation to [(18)F]labeling is demonstrated using [(18)F]Selectfluor bis(triflate), a reagent affording [(18)F]tri- and [(18)F]difluoromethylarenes not within reach with [(18)F]F2.

Measurement of human cerebral monoamine oxidase type B (MAO-B) activity with positron emission tomography (PET): a dose ranging study with the reversible inhibitor Ro 19-6327

SummaryEight normal subjects (3 females and 5 males) were studied using intravenous L-11C] deprenyl and positron emission tomography. In a single blind study one subject received tracer alone, one subject received an oral pre-dose of 20 mg of L-deprenyl and 6 subjects received oral pre-doses of 10 to 50 mg of a novel reversible MAO-B inhibitor (Ro 19-6327). Dynamic PET scans beginning 12 h after the oral dose were collected over 90 min and arterial blood was continuously sampled. Data analysis was modelled for two tissue compartments and using an iterative curve fitting technique the value of the rate constant for irreversible binding of L-[11C] deprenyl to MAO-B (k3) in whole brain was obtained for each subject.The dose response curves obtained indicated that a dose of at least 0.48 mg·kg−1 of Ro 19-6327 was necessary for >90% decrease in whole brain k3. Inhibition of MAO-B in platelets isolated from blood samples taken at the time of scanning correlated strongly with decrease in whole brain k3 (r=0.949).The results indicate that PET can be used to determine the dose of Ro 19-6327 necessary to inhibit >90% of brain MAO-B. This technique is an attractive alternative to traditional large scale patient-based dose-finding studies. Moreover it is shown that inhibition of platelet MAO-B can be used as a marker for central MAO-B inhibition with Ro 19-6327.

The effect of entacapone (OR‐611) on brain [18F]‐6‐L‐fluorodopa metabolism Implications for levodopa therapy of Parkinson's disease

We used PET and [18F]-6-L-fluorodopa ([18F]dopa) to measure the effect of a peripheral COMT inhibitor, entacapone, on the extracerebral metabolism and subsequent striatal uptake of [18F]dopa. Four parkinsonian patients and six age-matched normal controls were each scanned twice, once after carbidopa (150 mg) plus placebo and once after carbidopa (150 mg) plus entacapone (400 mg or 800 mg). Without entacapone premedication, by 90 minutes from injection, only 22% of the [18F] signal in plasma represented unmetabolized [18F]dopa (the balance being 3-0-methyl[18F]dopa). After entacapone medication, this fraction increased to 56% of the [18F] signal (p < 0.0001). We did not find any significant differences between the changes observed in patients versus controls or between those subjects who received 400 mg entacapone versus 800 mg in either this or any of the other reported measures. PET image contrast increased in all cases, reflecting an increase in the specific striatal signal ([striatum-oceipitalhoccipital ratio increased 38% [p < 0.0001]). Entacapone did not alter the rate of striatal uptake and decarboxylation of [18F]dopa as estimated using a graphic approach with metabolite-corrected plasma as input function to calculate the influx constant, Ki(p) (p = NS). This confirms that such an analytic approach adequately corrects for the effect of extracerebral [18F]dopa methylation. In contrast, the influx constant Ki(o) (calculated using occipital counts as the input function) increased 45% after entacapone (p < 0.0001). This demonstrates the sensitivity of this analytic approach to the presence of peripheral 3-O-methyl[18F]dopa and provides an estimate of the percentage increase in brain free [18F]dopa resulting from entacapone premedication.

Radiosynthesis and Evaluation of [18F]Selectfluor bis(triflate)†

Positron (b) emission tomography (PET) is a noninvasive molecular imaging technique that allows for the in vivo investigation of physiological processes. As a radioisotope, fluorine-18 benefits from an advantageous half-life (109.7 min), a clean decay process (97% b emission), and a short b trajectory, which is a property that enables the acquisition of high-resolution images. The global use of the radiotracer [F]-2-fluoro-2-deoxy-d-glucose has generated a vast amount of invaluable clinical data and has stimulated a worldwide interest in PET and F radiochemistry. For applications other than radiolabeling, nucleophilic and electrophilic fluorination are complementary processes that are used indiscriminately; the method of choice depends on the reactivity profile of the precursor to be fluorinated. A similar degree of synthetic flexibility would facilitate significantly the production and evaluation of new F radiotracers but to date, this is far from the reality because the range of reactions suitable for F labeling remains limited in comparison with the number of transformations available to access nonlabeled fluorinated material. Electrophilic F-fluorination suffers from well-recognized drawbacks. The carrieradded method employed for the production of [F]F2 gas gives labeled products with low specific activity (SA). In addition, [F]F2, a reagent that requires specialist equipment for its handling, can react unselectively and lead to a mixture of products. This complication lowers the radiochemical yield (RCY) and may lead to problematic and time-consuming purification processes. Despite these limitations, clinical doses of the radiotracers [F]-2-fluoro-l-tyrosine and [F]-6-fluoro3,4-dihydroxy-l-phenylalanine are currently prepared relying on an electrophilic fluorodestannylation reaction using [F]F2. [4]

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methl-11C]WAY-100635, in monkey and human plasma by HPLC: Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t1/2 = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-11C]WAY-100635 into humans, radioactivity was found to clear rapidly from blood and plasma. By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-11C]WAY-100635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-11C]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha 1-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-11C]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-11 in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake.

In vivo evaluation of [18F]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [18F]fluoroetanidazole ([18F]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [18F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [18F]FETA, with an octanol–water partition coefficient of 0.16±0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60–120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO2 values <5 mmHg (r=0.805, P=0.027) and carbogen breathing decreased [18F]FETA-derived radioactivity levels (P=0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30–60 min. Higher fractional retention of [18F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P=0.05) reflected higher percentage of pO2 values <1 mmHg (P=0.023), lower vessel density (P=0.026), and higher radiobiological hypoxic fraction (P=0.008) of the HT1080/1-3C tumours. In conclusion, [18F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.

Iodine-124 labelled annexin-V as a potential radiotracer to study apoptosis using positron emission tomography.

Annexin-V is a calcium-dependent protein that binds with high affinity to phosphaditylserine exposed during apoptosis. The aim of this study was to radiolabel annexin-V with iodine-124 for use as a potential probe of apoptosis by positron emission tomography. Annexin-V was radioiodinated directly using the cyclotron-produced positron emitter iodine-124 by the chloramine-T (CAT) method and indirectly by the pre-labelled reagent N-succinimidyl 3-[124I]iodobenzoate ([124I]m-SIB). Some reaction parameters of the CAT method such as reaction time and pH were optimised to give radiochemical yields of 22.3 +/- 2.6%(n = 3, gel-filtration). After incubation with [124I]m-SIB, radiolabelled annexin-V was obtained in 14% and 25% yield by FPLC and gel-filtration, respectively. The radiochemical purities from direct and indirect labelling were 97.7 +/- 1.0%(n = 3) and 96.7 +/- 2.1%(n = 3), respectively. The new radiotracers could be stored for up to four days without significant de-iodination. The biological activity of radiolabelled annexin-V was tested in control and camptothecin-treated (i.e. apoptotic) human leukaemic HL60 cells. A significantly higher (21%) binding in treated cells was observed with [125I]m-SIB-annexin-V. The binding of [125I]m-SIB labelled annexin-V to camptothecin treated cells was blocked (68%) by a 100-fold excess of unlabelled annexin-V.

Regional cerebral opioid receptor studies with [11C]diprenorphine in normal volunteers

The results are described of the cerebral uptake and heterogeneous retention of [11C]diprenorphine after intravenous injection in 4 normal volunteers. This potent opioid antagonist (Kd = 0.2 nM) was chosen because of its safety, lack of side-effects at trace doses in human pilot studies, rapid cerebral uptake and high percentage (80-90%) specific binding in animal in vivo studies. High uptake of [11C]diprenorphine was demonstrated in regions such as the thalamus, caudate nucleus, temporal, frontal and parietal cortices, which are known from postmortem studies to have high concentrations of opioid receptors. A stable level of activity or a very slow decline in activity was observed between 20 and 50 min after injection in areas such as the caudate nucleus and thalamus. Conversely, rapid washout of activity was observed in the occipital cortex, which is known to have low opioid receptor concentrations. Some 80-90% of maximum binding was naloxone reversible. These results with a ligand that is safe and without side-effects, suggest that this technique is suitable for studies of opioid physiology in man.

Pharmacokinetic Evaluation of N-[2-(Dimethylamino)Ethyl]Acridine-4-Carboxamide in Patients by Positron Emission Tomography

PURPOSE To evaluate tumor, normal tissue, and plasma pharmacokinetics of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA). The study aimed to determine the pharmacokinetics of carbon-11-labeled DACA ([11C]DACA) and evaluate the effect of pharmacologic doses of DACA on radiotracer kinetics. PATIENTS AND METHODS [11C]DACA (at 1/1,000 phase I starting dose) was administered to 24 patients with advanced cancer (pre-phase I) or during a phase I trial of DACA in five patients. Positron emission tomography (PET) was performed to assess pharmacokinetics and tumor blood flow. Plasma samples were analyzed for metabolite profile of [11C]DACA. RESULTS There was rapid systemic clearance of [11C]DACA over 60 minutes (1.57 and 1.46 L x min(-1) x m(-2) in pre-phase I and phase I studies, respectively) with the production of several radiolabeled plasma metabolites. Tumor, brain, myocardium, vertebra, spleen, liver, lung, and kidneys showed appreciable uptake of 11C radioactivity. The area under the time-versus-radioactivity curves (AUC) showed the highest variability in tumors. Of interest to potential toxicity, maximum radiotracer concentrations (Cmax) in brain and vertebra were low (0.67 and 0.54 m(2) x mL(-1), respectively) compared with other tissues. A moderate but significant correlation was observed for tumor blood flow with AUC (r = 0.76; P =.02) and standardized uptake value (SUV) at 55 minutes (r = 0.79; P =.01). A decrease in myocardial AUC ( P =.03) and splenic and myocardial SUV ( P =.01 and.004, respectively) was seen in phase I studies. Significantly higher AUC, SUV, and Cmax were observed in tumors in phase I studies. CONCLUSION The distribution of [11C]DACA and its radiolabeled metabolites was observed in a variety of tumors and normal tissues. In the presence of unlabeled DACA, pharmacokinetics were altered in myocardium, spleen, and tumors. These data have implications for predicting activity and toxicity of DACA and support the use of PET early in drug development.