Aren van Waarde

Measuring drug-related receptor occupancy with positron emission tomography

Several techniques can be used to measure indirectly the effect of drugs (e.g., EEG, fMRI) in healthy volunteers and in patients. Although each technique has its merits, a direct link between drug efficacy and site of action in vivo usually cannot be established. In addition, when the specific mode of action of a drug has been determined from preclinical studies, it is often not known whether the administered dose is optimal for humans. Both industry and academia are becoming more and more interested in determining the dose-related occupancy of specific targets caused by administration of drugs under test. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are noninvasive imaging techniques that can give insight into the relationship between target occupancy and drug efficacy, provided a suitable radioligand is available. Although SPECT has certain advantages (e.g., a long half-life of the radionuclides), the spatial and temporal resolution as well as the labeling possibilities of this technique are limited. This review focuses on PET methodology for conducting drug occupancy studies in humans.

[11C]FMAU and [18F]FHPG as PET tracers for herpes simplex virus thymidine kinase enzyme activity and human cytomegalovirus infections

[(11)C]-2-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ([(11)C]FMAU) and [(18)F]-9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG), radiolabeled representatives of two classes of antiviral agents, were evaluated as tracers for measuring herpes simplex virus thymidine kinase (HSV-tk) enzyme activity after gene transfer and as tracers for localization of active human cytomegalovirus (HCMV) infections. In vitro accumulation experiments revealed that both [(11)C]FMAU and [(18)F]FHPG accumulated significantly more in HSV-tk expressing cells than they did in control cells. [(18)F]FHPG uptake in HSV-tk expressing cells, however, was found to depend strongly on the cell line used, which might be due to cell type dependent membrane transport or cell type dependent substrate specific susceptibility of the enzyme. In vitro, both tracers exhibited a good selectivity for accumulation in HCMV-infected human umbilical vein endothelial cells over uninfected cells. In contrast to [(18)F]FHPG, [(11)C]FMAU uptake in control cells was relatively high due to phosphorylation of the tracer by host kinases. Therefore, [(18)F]FHPG appears to be the more selective tracer not only to predict HSV-tk gene therapy outcome, but also to localize active HCMV infections with PET.

Influence of P-glycoprotein on brain uptake of [18F]MPPF in rats.

The aim of this study was to determine if the brain uptake of 4-(2-methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-[18F]fluorobenzamido ]ethylpiperazine ([18F]MPPF), a radioligand for the imaging of 5-HT1A receptors, is influenced by the action of P-glycoprotein. Anesthetized male Wistar rats were injected i.v. with the 5-HT1A receptor antagonist [18F]MPPF (2 MBq, S.A.>110 TBq/mmol) after treatment with saline (controls) or with the 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190) (2.5 mg/kg i.v.). After 60 min, the animals were sacrificed and 13 areas of the brain were dissected for ex vivo gamma counting. The regional distribution of radioactivity was also assessed in brain slices using a storage phosphor system. Modulation of P-glycoprotein was achieved by injection of cyclosporin A (50 mg/kg) 30 min prior to injection of [18F]MPPF.The distribution of 18F-derived radioactivity corresponded to regional 5-HT1A receptor density as known from autoradiography. Modulation of P-glycoprotein with cyclosporin A caused a 5- to 10-fold increase in the uptake of [18F]MPPF. Tissue/cerebellum ratios in the brain correlated with receptor densities determined by in vitro autoradiography. Measurements of plasma radioactivity showed that the increased brain uptake of [18F]MPPF is partially due to a rise in ligand delivery after treatment with cyclosporin A (area under the curve, AUC, increased by a factor of 1.8). Biodistribution experiments in wild type and mdr1a(-/-) knockout mice confirmed that [18F]MPPF is a substrate for P-glycoprotein.

Positron Emission Tomography Studies on Binding of Central Nervous System Drugs and P-Glycoprotein Function in the Rodent Brain

The permeability of the blood–brain barrier (BBB) is one of the factors determining the bioavailability of drugs in the brain. The BBB only allows passage of lipophilic drugs by passive diffusion. However, some lipophilic drugs hardly enter the brain. The transmembrane protein P-glycoprotein (P-gp) is one of the carrier systems that is responsible for transportation of drugs out of the brain. P-Glycoprotein affects the pharmacokinetics of many drugs and can be inhibited by administration of modulators or competitive substrates. Identification and classification of central nervous system (CNS) drugs as P-gp substrates or inhibitors are of crucial importance in drug development. Positron emission tomography (PET) studies can play an important role in the screening process as a follow-up of high-throughput in vitro assays. Several rodent studies have shown the potential value of PET to measure the effect of P-gp on the pharmacokinetics and brain uptake of radiolabeled compounds. P-Glycoprotein-mediated effects were observed for two 5-HT1a receptor ligands, [18F]MPPF vs. [carbonyl-11C]WAY100635. Under control conditions, the specific brain uptake of [18F]MPPF is five- to eightfold lower than that of [11C]WAY100635. After cyclosporin A (CsA) modulation, [18F]MPPF uptake in the rat brain increased five- to tenfold. Cerebral uptake of [carbonyl-11C]WAY100635 was also increased by modulation, but in general the increase was lower than that observed for [18F]MPPF (two- to threefold). Brain uptake of the β-adrenergic receptor ligands [11C]carazolol and [18F]fluorocarazolol was increased in P-gp knockout mice and CsA-treated rats. Both the specific and nonspecific binding of [18F]fluorocarazolol were doubled by CsA. Cerebral uptake of [11C]carazolol in rats was much lower than that of [18F]fluorocarazolol and no specific binding was measured. After CsA modulation, the uptake of [11C]carazolol increased five- to sixfold, but this uptake was not receptor-mediated. Quantitative PET studies in rodents on P-gp functionality demonstrated a dose-dependent increase of radioligands after administration of CsA. Studies with [11C]verapamil and [11C]carvedilol showed that complete modulation was achieved at 50xa0mg/kg CsA. The distribution volume of [11C]carvedilol increased from 0.25 in the control study to 1.0 after full modulation with CsA. By quantitative PET measurement of P-gp function, the dose of modulators required to increase the concentration of CNS drugs may be determined, which may result in improved drug therapy.

Myocardial β-adrenoceptor downregulation in idiopathic dilated cardiomyopathy measured in vivo with PET using the new radioligand (S)-[11C]CGP12388

PurposeThe β-adrenoceptor (β-AR) plays an important role in heart failure. Recently, the new tracer (S)-[11C]CGP12388 has been developed. It displays excellent properties for investigation of the cardiac β-ARs in vivo with positron emission tomography (PET). Furthermore, the simple production method allows its use in a routine clinical setting. The aim of this study was to investigate whether decreased myocardial β-AR density in patients with idiopathic dilated cardiomyopathy (IDC) can be estimated using (S)-[11C]CGP12388 PET.MethodsMyocardial β-AR density was investigated in six patients with IDC and six age-matched healthy controls, using (S)-[11C]CGP12388 PET.Resultsβ-AR densities of 5.4±1.3xa0pmol/g (meanxa0±xa0SD) were observed in patients; these values were significantly lower than those observed in healthy controls (8.4±1.5xa0pmol/g, p<0.005).ConclusionThis study indicates that PET with (S)-[11C]CGP12388 is applicable for the measurement of myocardial β-AR density in patients. A highly significant reduction in β-AR density was found in patients with IDC compared with healthy controls.

Positron emission tomography: the conceptual idea using a multidisciplinary approach

Positron emission tomography (PET) is a method for quantitatively measuring biochemical and physiological processes in vivo by using radiopharmaceuticals labeled with positron-emitting radionuclides such as 11C, 13N, 15O, and 18F and by measuring the annihilation radiation using a coincidence technique. This technique is also used for measurement of the pharmacokinetics of labeled drugs and measurement of the effects of drugs on metabolism. Deviations from normal metabolism can be measured and insight into biological processes responsible for diseases can be obtained.

Quantitative imaging of 5-HT1A receptor binding in healthy volunteers with [18f]p-MPPF

Animal experiments have shown that 4-(2-methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-[(18)F]fluorobenzamido+ ++] ethylpiperazine ([(18)F]p-MPPF) can be used for 5-hydroxytryptamine(1A) (5-HT(1A)) receptor imaging. The aim of this study was to develop a method for the quantitative imaging of 5-HT(1A) receptors in healthy volunteers with [(18)F]p-MPPF. After injection of [(18)F]p-MPPF radioactivity was rapidly taken up in the brain, with the highest accumulation in the medial temporal cortex. Low levels of radioactivity were found in cerebellum and basal ganglia. Plasma clearance and metabolism of [(18)F]p-MPPF resulted in only about 1% of the radioactivity in plasma as parent radioligand after 10 min. Using a linear graphical method (Logan-Patlak), binding potentials were calculated in several brain areas. A good correlation (r = 0.95) was found between the obtained binding potentials and literature values for 5-HT(1A) receptor densities. A good correlation (r = 0.96) was also found between the body weight-corrected region/cerebellum ratios and the respective binding potentials. Moreover, a blocking experiment with pindolol (n = 3) showed a decrease of 40% in the region/cerebellum ratios of the target areas. Compared to those of [carbonyl-(11)C]WAY-100635, the binding potentials were four to six times lower, indicating that [(18)F]p-MPPF has a lower in vivo affinity for 5-HT(1A) receptors. In conclusion, [(18)F]p-MPPF can be used for the quantitative analysis of 5-HT(1A) receptor distribution in human brain.

Quantification of β-adrenoceptor density in the human heart with (S)-[11C]CGP 12388 and a tracer kinetic model

Abstract. The aim of this study was to determine whether the β-adrenoceptor receptor density (Bmax) and the ligand affinity (KD) of (S)-[11C]CGP 12388 for the β-adrenoceptor receptor could be determined using full tracer kinetic modelling of the transport of the ligand and its interaction with the receptor. This approach minimises the a priori assumptions and may thus serve as a gold standard to validate other simplified methods. Dynamic positron emission tomography (PET) data were acquired in six healthy subjects during 60xa0min. Three different injection protocols were applied, each consisting of three injections with varying SAs: high specific activity (SA), low SA or unlabelled ligand only. Arterial blood samples were collected via a cannula in the radial artery. Time-activity data in myocardial tissue were obtained using regions of interest (ROIs) on short-axis planes. All data were analysed with a two-tissue compartment, six-parameter (K1, k2, kon, koff, Bmax, Fbv) model that relies on explicit compartments for describing the kinetics of both labelled and unlabelled radioligand. Time-activity curves showed that unlabelled ligand could displace the radioligand from the receptor. This resulted in increased radioactivity levels in plasma. Modelling results yielded Bmax values of 9.74±1.80xa0nM and a KD of 0.58±0.22xa0nM, assuming a reaction volume of 0.15. In addition, parametric polar images of Bmax could be calculated. The protocol with injections of high SA, low SA, and unlabelled ligand, respectively, was found to be the most sensitive to parameter changes. We conclude that with tracer kinetic modelling of (S)-[11C]CGP 12388, the β-adrenoceptor density in the human heart can accurately be obtained in vivo. This approach may thus serve as a gold standard.

Steroid hormones affect binding of the sigma ligand 11C-SA4503 in tumour cells and tumour-bearing rats

PurposeSigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist 11C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels.Methods11C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1xa0h or 5xa0min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone.ResultsBinding of 11C-SA4503 to C6 cells was increased (~50%) upon removal and decreased (~60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC50 progesterone 33xa0nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of 11C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected.ConclusionsThe binding of 11C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids.

Validation of S-1 '-[F-18]fluorocarazolol for in vivo imaging and quantification of cerebral beta-adrenoceptors

S-1-[18F]fluorocarazolol (S-(-)-4-(2-hydroxy-3-(1-[18F]fluoroisopropyl)-aminopropoxy)carba zole, a non-subtype-selective beta-adrenoceptor antagonist) has been investigated for in vivo studies of beta-adrenoceptors. Previous results indicated that uptake of this radioligand in heart and lung can be inhibited by beta-adrenoceptor agonists and antagonists. In the present study, blocking, displacement and saturation experiments were performed in rats, in combination with metabolite analysis to investigate the suitability of this radioligand for in vivo positron emission tomography (PET) imaging and quantification of beta-adrenoceptors in the brain. The results demonstrate that, (i) the uptake of S-1-[18F]fluorocarazolol reflects specific binding to beta-adrenoceptors, (ii) binding of S-1-[18F]fluorocarazolol to atypical or non-beta-adrenergic sites is negligible, (iii) uptake of radioactive metabolites in the brain is less than 25% of total radioactivity, 60 min after injection, (iv) in vivo measurements of receptor densities (Bmax) in cortex, cerebellum, heart, lung and erythrocytes are within range of densities determined from in vitro assays, (v) binding of S-1-[18F]fluorocarazolol can be displaced. In conclusion, S-1-[18F]fluorocarazolol seems to possess the appropriate characteristics to visualize and quantify beta-adrenoceptors in vivo in the central nervous system using PET.