Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP

We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[⁷⁶Br]BrQNP,E(-,-)-[⁷⁶Br]BrQNP andE(-,+)-[⁷⁶Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [⁷⁶Br]BrNH₄ and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M₂ selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M₁ selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., theE(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively.Z(-,-)-[⁷⁶Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for theE(-,+)-[⁷⁶Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors.Z(-,-)-[⁷⁶Br]BrQNP, which is displaced in higher amounts from M₂ mAChR-enriched structures, reveals an M₂ affinity. The two isomers having the (-,-)-configuration are potential probes for investigating central muscarinic receptors. The absolute configuration on the acetate chiral centre influences their muscarinic subtype selectivity and thecis-trans isomerism of the vinyl moiety affects their specific fixation.     

Post-mortem redistribution of three beta-blockers in the rabbit

To consider the role of the physico-chemical properties of drugs in their post-mortem redistribution, we designed the present study to investigate the influence of lipophilicity using an experimental rabbit model. Three beta-blockers (BB), atenolol, metoprolol and propranolol, with a similar dissociation constant (pK _a) and increasing partition coefficient (K _p) were administered intravenously to 18 rabbits. One hour after the last administration, the animals were killed by thiopental injection and placed in a supine position at room temperature. Autopsies were performed at 0, 2, 6, 12, 24 and 48 h post-mortem. Concentrations of the three BB were determined in fluids (right and left cardiac blood, peripheral blood, urine, bile, stomach content, vitreous humour) and tissues (cardiac muscle, lungs, liver, brain, diaphragm, iliopsoas muscle) using a previously published, validated liquid chromatography–electrospray–mass spectrometry method. Our results show that lipophilicity influences post-mortem redistribution of the molecules in a certain number of anatomical sites such as the stomach, lungs, cardiac muscle, cardiac blood or liver, but does not appear to intervene in other sites such as the brain or the vitreous humour.Electronic supplementary material Supplementary material is available in the online version of this article at and accessible for authorised users.This work was presented in part at the 40th International Meeting of The International Association of Forensic Toxicologists, 26–30 August 2002, Paris, France, and at the 11th Annual Meeting of the Société Française de Toxicologie Analytique, 11–13 June 2003, Dinard, France.     

Radioiodinated SB 207710 as a radioligand in vivo: imaging of brain 5-HT4 receptors with SPET

Single-photon emission tomography (SPET) and positron emission tomography (PET), when coupled to suitable radioligands, are uniquely powerful for investigating the status of neurotransmitter receptors in vivo. The serotonin subtype-4 (5-HT₄) receptor has discrete and very similar distributions in rodent and primate brain. This receptor population may play a role in normal cognition and memory and is perhaps perturbed in some neuropsychiatric disorders. SB 207710 [(1-butyl-4-piperidinylmethyl)-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate] is a selective high-affinity antagonist at 5-HT₄ receptors. We explored radioiodinated SB 207710 as a possible radioligand for imaging 5-HT₄ receptors in vivo. Rats were injected intravenously with iodine-125 labelled SB 207710, euthanised at known times and dissected to establish radioactivity content in brain tissues. Radioactivity entered brain but cleared rapidly and to a high extent from blood and plasma. Between 45 and 75 min after injection, the ratios of radioactivity concentration in each of 12 selected brain tissues to that in receptor-poor cerebellum correlated with previous measures of 5-HT₄ receptor density distribution in vitro. The highest ratio was about 3.4 in striatum. SB 207710 was labelled with iodine-123 by an iododestannylation procedure. A cynomolgus monkey was injected intravenously with [¹²³I]SB 207710 and examined by SPET. Maximal whole brain uptake of radioactivity was 2.3% of the injected dose at 18 min after radioligand injection. Brain images acquired between 9 and 90 min showed high radioactivity uptake in 5-HT₄ receptor-rich regions, such as striatum, and low uptake in receptor-poor cerebellum. At 169 min the ratio of radioactivity concentration in striatum to that in cerebellum was 4.0. In a second SPET experiment, the cynomolgus monkey was pretreated with a selective 5-HT₄ receptor antagonist, SB 204070, at 20 min before [¹²³I]SB 207710 injection. Radioactivity in all brain regions was reduced almost to the level in cerebellum by 176 min after radioligand injection. These findings show that [¹²³I]SB 207710 is an effective radioligand for imaging brain 5-HT₄ receptors in vivo.For preliminary accounts of this work, see Pike VW et al., J Nucl Med 1998; 39 (Suppl):185; Eur J Nucl Med 1999; 26:991.     

Initial evaluation of123|-5-|-R91150, a selective 5-HT2A ligand for single-photon emission tomography, in healthy human subjects

The mapping of 5-HT₂ receptors in the brain using functional imaging techniques has been limited by a relative lack of selective radioligands. Iodine-123 labelled 4-amino-N-[1-[3-(4-fluorophenoxy)propyl]-4-methyl-4-piperidinyl]-5-iodo-2-methoxybenzamide (¹²³I-5-I-R91150 or¹²³I-R93274) is a new ligand for single-photon emission tomography (SPET), with high affinity and selectivity for 5-HT_2A receptors. This study reports on preliminary¹²³I-5-I-R91150 SPET, wholebody and blood distribution findings in five healthy human volunteers. Maximal brain uptake was approximately 2% of total body counts at 180 min post injection (p.i.). Dynamic SPET sequences were acquired with the brain-dedicated, single-slice multi-detector system SEM-810 over 200 min p.i. Early peak uptake (at 5 min p.i.) was seen in the cerebellum, a region free from 5HT_2A receptors. In contrast, radioligand binding in the frontal cortex increased steadily over time, up to a peak at approximately 100–120 min p.i. Frontal cortex-cerebellum activity ratios reached values of 1.4, and remained stable from approximately 100 min p.i. onwards. Multi-slice SPET sequences showed a pattern of regional variation of binding compatible with the autoradiographic data on the distribution of 5-HT_2A receptors in (cerebral cortex>striatum>cerebellum). These findings suggest that¹²³I-5-I-R91150 may be used for the imaging of 5-HT_2A receptors in the living human brain with SPET.     

Distribution of the 5-HT<Subscript>1A</Subscript> receptor antagonist [<Superscript>18</Superscript>F]FPWAY in blood and brain of the rat with and without isoflurane anesthesia

Purpose To determine whether brain and plasma equilibrium of a proposed PET tracer for 5-HT_1A, [¹⁸F]FPWAY, can be achieved in a sufficiently short time for practical use of the brain to plasma equilibrium distribution ratio (DR) to monitor receptor availability with and without isoflurane anesthesia. Methods Awake (n=4) and isoflurane-anesthetized (n=4) rats were administered a continuous 60 min intravenous infusion of [¹⁸F]FPWAY with timed arterial blood sampling. Brains of the isoflurane-anesthetized rats were scanned with the ATLAS small animal PET scanner; awake rats were not. All rats were killed at 60 min and scanned postmortem for 15 min, followed by brain slicing for autoradiography. Several regions of interest (ROIs) were defined in the PET images as well as in the autoradiographic images. Regional DRs were calculated as total activity in the brain ROI divided by plasma [¹⁸F]FPWAY activity. Results DRs in the anesthetized animals were constant between 30 and 60 min, indicating that near equilibrium between brain and plasma had been achieved by ∼30 min. DRs determined from postmortem PET data were higher in the isoflurane-anesthetized rats by 24% (not significant) and 33% (p=0.065) in whole brain and hippocampus, respectively. DRs determined from autoradiographic data were greater in isoflurane-anesthetized rats in medial hippocampus, lateral hippocampus, and cerebellum by 33% (p=0.054), 63% (p<0.01), and 32% (p<0.05), respectively. Conclusion [¹⁸F]FPWAY could be an appropriate ligand for monitoring changes in receptor availability in the serotonergic system using a bolus/infusion paradigm. One possible explanation for higher DRs in anesthetized rats may be a reduction in endogenous 5-HT secretion under isoflurane anesthesia.     

Cyclosporine,a P-glycoprotein modulator,increases [<Superscript>18</Superscript>F]MPPF uptake in rat brain and peripheral tissues: microPET and ex vivo studies

Purpose  Pretreatment with cyclosporine, a P-glycoprotein (P-gp) modulator increases brain uptake of 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[¹⁸F]fluorobenzamido]ethylpiperazine ([¹⁸F]MPPF) for binding to hydroxytryptamine_1A (5-HT_1A) receptors. Those increases were quantified in rat brain with in vivo microPET and ex vivo tissue studies. Materials and methods  Each Sprague–Dawley rat (n = 4) received a baseline [¹⁸F]MPPF microPET scan followed by second scan 2–3 weeks later that included cyclosporine pretreatment (50 mg/kg, i.p.). Maximum a posteriori reconstructed images and volumetric ROIs were used to generate dynamic radioactivity concentration measurements for hippocampus, striatum, and cerebellum, with simplified reference tissue method (SRTM) analysis. Western blots were used to semiquantify P-gp regional distribution in brain. Results  MicroPET studies showed that hippocampus uptake of [¹⁸F]MPPF was increased after cyclosporine; ex vivo studies showed similar increases in hippocampus and frontal cortex at 30 min, and for heart and kidney at 2.5 and 5 min, without concomitant increases in [¹⁸F]MPPF plasma concentration. P-gp content in cerebellum was twofold higher than in hippocampus or frontal cortex. Conclusions  These studies confirm and extend prior ex vivo results (J. Passchier, et al., Eur J Pharmacol, 2000) that showed [¹⁸F]MPPF as a substrate for P-gp. Our microPET results showed that P-gp modulation of [¹⁸F]MPPF binding to 5-HT_1A receptors can be imaged in rat hippocampus. The heterogeneous brain distribution of P-gp appeared to invalidate the use of cerebellum as a nonspecific reference region for SRTM modeling. Regional quantitation of P-gp may be necessary for accurate PET assessment of 5-HT_1A receptor density when based on tracer uptake sensitive to P-gp modulation.     

Mass dose effects and in vivo affinity in brain PET receptor studies--a study of cerebral 5-HT4 receptor binding with [11C]SB207145

Attention to tracer dose principles is crucial in positron emission tomography (PET), and deviations can induce serious errors. In this study, we devise a method for determining receptor occupancy of the mass dose of the radioligand itself and the in vivo affinity.

Methods

The approach was used for [¹¹C]SB207145, a new PET radioligand for imaging the cerebral 5-HT₄ receptors in humans. Test–retest PET studies with varying specific activities of [¹¹C]SB207145 were conducted in seven healthy subjects, and the output parameter regional BP_ND was modeled. Individual occupancy plots were first computed to estimate the mass dose that saturates 50% of receptors (ID₅₀), and subsequently, the maximal mass dose that can be injected (arbitrarily set at an occupancy <5%) was calculated. Scatchard plots were computed to estimate the in vivo K_D.

Results

Increasing the mass dose resulted in a decrease in BP_ND, whilst the relative cerebellar uptake was unchanged. The ID₅₀ was 85.4±30.2 μg, and the upper mass dose limit was 4.5±1.6 μg, which does not require ultrahigh specific activity. The estimated in vivo K_D was 2.8 nM (range 1.0–4.8), without any regional differences.

Conclusion

The presented method for estimating the upper mass dose limit is suggested as part of validation of PET radioligands.     

Carbon-11 pb-12: an attempt to visualize the dopamine d4 receptor in the primate brain with positron emission tomography

The dopamine D₄ receptor (D₄R) is expressed in low density in various extrastriatal brain regions. This receptor subtype is discussed in relation to the pathophysiology and treatment of schizophrenia but no selective positron emission tomography (PET) ligand is available to date to study the distribution in vivo. The arylpiperazine derivative N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (PB-12) is a novel, high-affinity ( K_i=0.040 nM) and selective D₄R ligand. We radiolabeled PB-12 with carbon-11 (t_1/2 20.4 min) by O-methylation of the corresponding desmethyl analogue N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-hydroxybenzamide (LM-190) with [¹¹C]methyl triflate. Derivative LM-190 was prepared by condensing 3-hydroxybenzoic acid with the appropriate amine. For the radiolabeling, the incorporation yield was >90% and the total synthesis time including high performance liquid chromatography (HPLC) purification was about 35 min. The specific radioactivity of [¹¹C]PB-12 at time of injection was 67–118 GBq·μmol⁻¹. PET studies in a cynomolgus monkey showed a high uptake and widespread distribution of radioactivity in the brain, including the neocortex and thalamus. About 40% of total radioactivity in plasma represented unchanged radioligand at 60 min after injection as determined by HPLC. Pretreatment with the D₄R ligand 3-{[4-(4-chlorophenyl)piperazin-1-yl]methyl}-1H-pyrollo[2,3-b]pyridine (L-745,870) prior to radioligand injection failed to demonstrate receptor-specific binding in the monkey brain. Furthermore, the brain radioactivity distribution was left unaffected by pretreating with unlabeled PB-12. This failure to detect a D₄R-specific signal may be related to a very low density of the D₄R in primate brain, insufficient binding affinity of the radioligand, and a high background of nonspecific binding. It can be concluded from these findings that [¹¹C]PB-12 is not suitable to visualize the D₄R in the primate brain with PET.     

In vivo evaluation in rodents of [I]-3-I-CO as a potential SPECT tracer for the serotonin 5-HT2A receptor

Introduction[¹²³I]-(4-fluorophenyl)[1-(3-iodophenethyl)piperidin-4-yl]methanone ([¹²³I]-3-I-CO) is a potential single photon emission computed tomography tracer with high affinity for the serotonin 5-HT_2A receptor (K_i=0.51 nM) and good selectivity over other receptor (sub)types. To determine the potential of the radioligand as a 5-HT_2A tracer, regional brain biodistribution and displacement studies will be performed. The influence of P-glycoprotein blocking on the brain uptake of the radioligand will also be investigated.MethodsA regional brain biodistribution study and a displacement study with ketanserin were performed with [¹²³I]-3-I-CO. Also, the influence of cyclosporin A (50 mg/kg) on the brain distribution of the radioligand was investigated. For the displacement study, ketanserin (1 mg/kg) was administered 30 min after injection of [¹²³I]-3-I-CO.ResultsThe initial brain uptake of [¹²³I]-3-I-CO was quite high, but a rapid wash-out of radioactivity was observed. Cortex-to-cerebellum binding index ratios were low (1.1 – 1.7), indicating considerable aspecific binding and a low specific ‘signal’ of the radioligand. Tracer uptake was reduced to the levels in cerebellum (a 60% reduction) after ketanserin displacement. Administration of cyclosporin A resulted in a doubling of the brain radioactivity concentration.ConclusionsAlthough [¹²³I]-3-I-CO showed adequate brain uptake and could be displaced by ketanserin, high aspecific binding to brain tissue was responsible for very low cortex-to-cerebellum binding index ratios, possibly limiting the potential of the radioligand as a serotonin 5-HT_2A receptor tracer. We also demonstrated that [¹²³I]-3-I-CO is probably a weak substrate for the P-glycoprotein efflux transporter.     

Kinetics of radioiodinated species in subcellular fractions from rat hearts following administration of iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP)

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (¹²³IBMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1–120 min after intravenous injection of¹²³I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 1231-BMIPP was maximal at 5 min (6.81%±0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of¹²³I-BMIPP,¹²³I-p-iodophenylacetic acid (¹²³I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite,¹²³I-p-iodophenyldodecanoic acid (¹²³I-PIPC₁₂), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of¹²³I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial -oxidation followed by subsequent cycles of -oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.     

Multicompartmental study of fluorine-18 altanserin binding to brain 5HT2 receptors in humans using positron emission tomography

Serotoninergic type 2 (5HT₂) receptors have been implicated in the regulation of many brain functions in humans and may play a role in several neurological and psychiatric diseases. Fluorine-18 altanserin has been proposed as a new radiotracer for the study of 5HT₂ receptors by PET because of its high affinity for 5HT₂ receptors (Ki: 0.13 nM) and its good specificity in in vitro studies. Dynamic PET studies were carried out in 12 healthy volunteers after intravenous injection of 0.1 mCi/kg [¹⁸F]altanserin. Ninety minutes after injection, we observed mainly cortical binding. Basal ganglia and cerebellum showed very low uptake and the frontal cortex to cerebellum ratio was about 3. To evaluate the quantitative distribution of this ligand in the brain, we used two different methods of data analysis: a four-compartment model was used to achieve quantitative evaluation of rate constants (K ₁ and k ₂ through k ₆) by non-linear regression, and a multiple-time graphical analysis technique for reversible binding was employed for the measurement of k ₁/k ₂ and k ₃/k ₄ ratios. Using both methods, we found significant differences in binding capacity (estimated by k ₃/k ₄ = B _max/K _d) between regions, the values increasing as follows: occipital, limbic, parietal, frontal and temporal cortex. After correction of values obtained by the graphical method for the existence of non-specific binding, results generated by the two methods were consistent.     

Characterization of [123I]IDAM as a novel single-photon emission tomography tracer for serotonin transporters

Development of selective serotonin transporter (SERT) tracers for single-photon emission tomography (SPET) is important for studying the underlying pharmacology and interaction of specific serotonin reuptake site inhibitors, commonly used antidepressants, at the SERT sites in the human brain. In search of a new tracer for imaging SERT, IDAM (5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol) was developed. In vitro characterization of IDAM was carried out with binding studies in cell lines and rat tissue homogenates. In vivo binding of [¹²⁵I]IDAM was evaluated in rats by comparing the uptakes in different brain regions through tissue dissections and ex vivo autoradiography. In vitro binding studyshowed that IDAM displayed an excellent affinity to SERT sites (K _i=0.097 nM, using membrane preparations of LLC-PK₁ cells expressing the specific transporter) and showed more than 1000-fold of selectivity for SERT over norepinehrine and dopamine (expressed in the same LLC-PK₁ cells). Scatchard analysis of [¹²⁵I]IDAM binding to frontal cortical membrane homogenates prepared from control or p-chloroamphetamine (PCA)-treated rats was evaluated. As expected, the control membranes showed a K _d value of 0.25 nM±0.05 nM and a B _max value of 272±30 fmol/ mg protein, while the PCA-lesioned membranes displayed a similar K _d, but with a reduced B _max (20±7 fmol/ mg protein). Biodistribution of[¹²⁵I]IDAM (partition coefficient =473; 1-octanol/buffer) in the rat brainshowed a high initial uptake (2.44%dose at 2 min after i.v. injection) with the specific binding peaked at 60 min postinjection (hypothalamus-cerebellum/cerebellum =1.75). Ex vivo autoradiographs of rat brain sections (60 min after i.v. injection of [¹²⁵I]IDAM) showed intense labeling in several regions (olfactory tubercle, lateral septal nucleus, hypothalamic and thalamic nuclei, globus pallidus, central gray, superior colliculus, substantia nigra, interpeduncular nucleus, dorsal and median raphes and locus coeruleus), which parallel known SERT density. This novel tracer has excellent characteristics for in vivo SPET imaging of SERT in the brain. Received: 1 February / Revised: 11 March 1999     

Preparation and bioevaluation of a Tc-labeled chlorambucil analog as a tumor targeting agent

Chlorambucil belongs to a group of nitrogen mustards which are used for the treatment of variety of cancers. Hence, a chlorambucil derivative has been radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺ core and its efficacy as a tumor targeting agent has been evaluated. Radiochemical yield of the complex was >98% as observed by HPLC. The in vitro studies in MCF-7 breast cancer cells showed about 30% inhibition of the radiolabeled complex in presence of the cold chlorambucil derivative. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed an uptake of 3.2±0.3% ID/g at 3 h.p.i.     

Investigation of iodine-123-labelled amino acid derivatives for imaging cerebral gliomas: uptake in human glioma cells and evaluation in stereotactically implanted C6 glioma rats

In developing iodine-123-labelled amino acid derivatives for imaging cerebral gliomas by single-photon emission tomography (SPET), we compared p-[¹²³I]iodo-l-phenylalanine (IPA), l-[¹²³I]iodo-1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (ITIC) and l-3-[¹²³I]iodo-!-methyltyrosine (IMT) with regard to their uptake in human glioblastoma T99 and T3868 cells, and thereafter studied the mechanisms promoting the cellular uptake. The potential of the ¹²³I-iodinated agents for use as SPET radiopharmaceuticals was evaluated in healthy experimental rats as well as in rats with stereotactically implanted C6 gliomas. The radiopharmaceutical uptake into glioblastoma cells was rapid, temperature and pH dependent, and linear during the first 5 min. Equilibrium was reached after 15-20 min, except in the case of ITIC, the initial uptake of which gradually decreased from 15 min onwards. The radioactivity concentration in glioma cells following 30-min incubation at 37°C (pH 7.4) varied from 11% to 35% of the total activity per million cells (ITIC < IMT h IPA). Competitive inhibition experiments using !-(methylamino)-isobutyric acid and 2-amino-2-norbornane-carboxylic acid, known as specific substrates for systems A and L, respectively, as well as representative amino acids preferentially transported by system ASC, indicated that IPA, like IMT, is predominantly mediated by the L and ASC transport systems, while no significant involvement of the A transport system could be demonstrated. By contrast, none of the three principal neutral amino acid transport systems (A, L and ASC) appear to be substantially involved in the uptake of ITIC into glioblastoma cells. Analysis of uptake under conditions that change the cell membrane potential, i.e. in high K⁺ medium, showed that the membrane potential plays an important role in ITIC uptake. Alteration of the mitochondrial activity by means of valinomycin or nigericin induces a slight increase or decrease in the radiopharmaceutical uptake, suggesting a minor contribution of the mitochondria in the uptake. IPA, IMT and ITIC passed the blood-brain barrier, and thereafter showed efflux from the brain. The radioactivity concentration in healthy rat brain 15 min following intravenous injection varied from 0.07% (ITIC) to 0.27% ID/g (IPA). In comparison, the brain uptake in the stereotactically implanted C6 glioma rats was substantially higher (up to 1.10% ID/g 15 min p.i.), with tumour-to-background ratios greater than 4. These data indicate that IPA and ITIC, like IMT, exhibit interesting biological characteristics which hold promise for in vivo brain tumour investigations by SPET.     

不同标准X射线RQR辐射场刻度眼晶状体当量剂量Hp(3)比较研究

目的通过在不同标准X射线RQR辐射场对Hp(3)进行刻度,并对刻度结果进行比较,探究国内标准X射线RQR辐射场刻度Hp(3)的可行性。方法选择直径20 cm、高20 cm的柱模,分别选取国内外标准X射线RQR辐射场对同一TLD进行Hp(3)的刻度,选择射线包括RQR4(60 kV)、RQR7(90 kV)、RQR9(120 kV),刻度内容包括能量响应、角度响应和线性响应。结果在能量响应方面,TLD对国内外标准X射线RQR辐射场响应均较好,响应值与照射值差异均在10%以内。在角度响应方面,TLD在国外辐射场响应值较好,响应值与照射值差异均在6%以内。而在国内辐射场,TLD在20°响应值偏低,响应值与照射值差异>10%。在线性响应方面,TLD在国内和国外标准X射线RQR辐射场拟合程度均较好。结论本研究的各项检测结果表明,国内标准X射线RQR辐射场可以对TLD进行Hp(3)的刻度。     

In vivo and in vitro evaluation of [<Superscript>18</Superscript>F]FETO with respect to the adrenocortical and GABAergic system in rats

11-Hydroxylase (CYP11B1, P450₁₁) plays an important role in the biosynthesis of cortisol and aldosterone and has been shown to be a good target for the in vivo imaging of adrenocortical incidentalomas in nuclear medicine. [¹¹C]Metomidate (MTO), a potent inhibitor of this enzyme, is used for positron emission tomography (PET) imaging of adrenocortical pathology. The synthesis of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 2-[¹⁸F]fluoroethylester (FETO), a close analogue to MTO and etomidate (ETO), has been presented recently, and the present investigation aimed to characterise the in vivo distribution of FETO. Since ETO is a well-known anaesthetic drug acting via the GABAergic system, the interaction of FETO with GABA_A receptors was also evaluated. Eighteen male Sprague-Dawley rats were injected with 1.73–3.06 MBq of FETO into a tail vein after venodilatation in a 40°C water bath. Rats were sacrificed by exsanguination from the abdominal aorta under deep ether anaesthesia after 10 (n=6), 30 (n=6) or 60 min (n=6); organs were removed, weighed and counted. For binding experiments, rat cerebellar membranes were incubated for 90 min at 4°C in TC-50 buffer, 150 mM NaCl and 2 nM of [³H]flunitrazepam in the absence or presence of 10 µM diazepam or various concentrations of ETO, MTO and FETO. In vivo evaluation evinced very high uptake in the adrenal glands (7.52%±1.19% ID/g at 30 min), followed by lung (1.18%±0.19% ID/g, 10 min), liver (0.59%±0.13% ID/g, 10 min) and duodenum (0.7%±0.29% ID/g, 60 min). No defluorination nor fluoroethyl-ester cleavage was observed. When brain regions were compared with the thalamus (the reference region), highest relative uptake was seen in the cortex (2.34), followed by "rest brain" (2.13) and cerebellum (1.96). FETO and ETO were able to increase the binding of [³H]flunitrazepam with similar potencies and to a comparable extent. It is concluded that FETO shows characteristics suitable for the imaging of adrenocortical pathology with PET. Binding experiments on GABA receptors demonstrate a comparable effect of FETO and ETO. Hence, FETO possibly could also be used to elucidate the function, dynamics and kinetics of narcotic drugs with PET.     

FDG—a marker of tumour hypoxia? A comparison with [18F]fluoromisonidazole and pO2-polarography in metastatic head and neck cancer

Purpose Experimental data suggest that the accumulation of [¹⁸F]fluorodeoxyglucose (FDG) in malignant tumours is related to regional hypoxia. The aim of this study was to evaluate the clinical potential of FDG positron emission tomography (PET) to assess tumour hypoxia in comparison with [¹⁸F]fluoromisonidazole (FMISO) PET and pO₂-polarography. Methods Twenty-four patients with head and neck malignancies underwent FDG PET, FMISO PET, and pO₂-polarography within 1 week. Parameters of pO₂-polarography were the relative frequency of pO₂ readings ≤2.5 mmHg, ≤5 mmHg and ≤10 mmHg, respectively, as well as the mean and median pO₂. Results We observed a moderate correlation of the maximum standardised uptake value (SUV) of FDG with the tumour to blood ratio of FMISO at 2 h (R=0.53, p<0.05). However, SUV of FDG was similar in hypoxic and normoxic tumours as defined by pO₂-polarography (6.9±3.2 vs 6.2±3.0, NS), and the FDG uptake was not correlated with the results of pO₂-polarography. The retention of FMISO was significantly higher in hypoxic tumours than in normoxic tumours (tumour to muscle ratio at 2 h: 1.8±0.4 vs 1.4±0.1, p<0.05), and the FMISO tumour to muscle ratio showed a strong correlation with the frequency of pO₂ readings ≤5 mmHg (R=0.80, p<0.001). Conclusion These results support the hypothesis that tumour hypoxia has an effect on glucose metabolism. However, other factors affecting FDG uptake may be more predominant in chronic hypoxia, and thus FDG PET cannot reliably differentiate hypoxic from normoxic tumours.     

Imaging the norepinephrine transporter with positron emission tomography: initial human studies with (<Emphasis Type="Italic">S,S</Emphasis>)-[<Superscript>18</Superscript>F]FMeNER-D<Subscript>2</Subscript>

Introduction (S,S)-[¹⁸F]FMeNER-D₂ is a recently developed positron emission tomography (PET) ligand for in vivo quantification of norepinephrine transporter. A monkey occupancy study with the radioligand indicated that (S,S)-[¹⁸F]FMeNER-D₂ can be useful for quantitative PET analysis. In this preliminary study, regional distributions in the living human brain were evaluated. Materials and methods Brain PET measurements were performed for a total of 255 min after the injection of 188.3 ± 5.7 MBq of (S,S)-[¹⁸F]FMeNER-D₂ in four healthy male subjects. Regions of interests were drawn on the thalamus and the caudate in the coregistered MRI/PET images. Results (S,S)-[¹⁸F]FMeNER-D₂ displayed good brain penetration and selective retention in regions rich in norepinephrine reuptake sites. The transient peak equilibrium was reached during the PET measurements. The ratios of radioactivity uptake in the thalamus to that in the caudate were 1.50 ± 0.06 for the time period of 90–255 min. Conclusion The present preliminary investigation indicates that (S,S)-[¹⁸F]FMeNER-D₂ has suitable characteristics for probing the norepinephrine reuptake system with PET in the human brain.     

In vivo imaging of adenosine A2A receptors in rat and primate brain using [11C]SCH442416

Purpose The aim of this study was to evaluate the suitability of [¹¹C]SCH442416 for the in vivo imaging of adenosine A_2A receptors.Methods In rats and Macaca nemestrina, we evaluated the time course of the cerebral distribution of [¹¹C]SCH442416. Furthermore, in rats we investigated the rate of metabolic degradation, the inhibitory effects of different drugs acting on adenosine or dopamine receptors and the modification induced by the intrastriatal administration of quinolinic acid (QA).Results The rate of metabolic degradation of [¹¹C]SCH442416 in rats was slow; 60 min after tracer injection, more than 40% of total plasma activity was due to unmetabolised [¹¹C]SCH442416. At the time of maximum uptake, radioactive metabolites represented only 6% of total extractable activity in the cerebellum and less than 1% in the striatum. In the striatum, the region with the highest expression of A_2A receptors, the in vivo uptake of [¹¹C]SCH442416 was significantly reduced only by drugs acting on A_2A receptors or by QA, a neurotoxin that selectively reduces the number of intrastriatal GABAergic neurons. Position emission tomography (PET) studies in monkeys indicated that the tracer rapidly accumulates in brain, reaching maximum uptake between 5 and 10 min. Twenty minutes after the injection, radioactivity concentration in the striatum was two times that in the cerebellum.Conclusion The specificity of binding, the rank order of regional distribution in the brain of rats and M. nemestrina, the good signal to noise ratios and the low amount of radioactive metabolites in brain and periphery indicate that [¹¹C]SCH442416 is a promising tracer for the in vivo imaging of A_2A adenosine receptors using PET.     

In-vivo imaging characteristics of two fluorinated flumazenil radiotracers in the rat

Purpose  [¹¹C]Flumazenil shows promise as a clinical and research PET radiotracer to image changes in GABA_A central benzodiazepine receptor (cBZR), but its widespread use has been limited by practical limitations of [¹¹C]. This study evaluated the imaging characteristics of two fluorinated PET radiotracers in rats in vivo: [¹⁸F]fluoroflumazenil ([¹⁸F]FFMZ) and [¹⁸F]flumazenil ([¹⁸F]FMZ). Methods  PET acquisitions were performed on a small-animal scanner following injection of [¹⁸F]FFMZ in nine rats and [¹⁸F]FMZ in eight rats. The following treatments were investigated: (1) injection of the tracer dose, (2) presaturation then injection of the tracer dose, and (3) injection of the tracer dose followed by a displacement injection. Unchanged tracer was measured in plasma and brain structures in four animals 10 and 30 min after injection, and ex-vivo autoradiography was also performed. Results  For both [¹⁸F]FFMZ and [¹⁸F]FMZ maximal brain activity peaked rapidly, and was highest in the hippocampus (1.12±0.06 SUV, 1.24±0.10 SUV, respectively), and lowest in the pons (1.00±0.07 SUV, 1.03±0.09 SUV, respectively). By 50 min after injection, maximal uptake for [¹⁸F]FFMZ and [¹⁸F]FMZ had decreased in the hippocampus to 18±3% and 80±1% (p<0.01), respectively. The presaturation and displacement studies showed a higher nonspecific component for [¹⁸F]FFMZ than for [¹⁸F]FMZ. Metabolite studies showed that at 30 min only 10% of the signal was from [¹⁸F]FFMZ in the brain. This nonspecific binding was apparent on autoradiography. In contrast, [¹⁸F]FMZ accounted for >70% of the signal in the brain, which resulted in well-defined regional binding on autoradiography. Conclusion  These results demonstrate that [¹⁸F]FMZ is a superior radiotracer to [¹⁸F]FFMZ for in-vivo PET imaging of the GABA_A/cBZR, having slower metabolism and leading to lower concentrations of metabolites in the brain that results in a substantially better signal-to-noise ratio.