A review of approaches to 18 F radiolabelling affinity peptides and proteins.

Affinity peptide and protein- (APP) based radiotracers are an increasingly popular class of radiotracer in positron emission tomography (PET), which was once dominated by the use of small molecule radiotracers. Radiolabelled monoclonal antibodies (mAbs) are important examples of APPs, yet a preference for smaller APPs, which exhibit fast pharmacokinetics and permit rapid PET aided diagnosis, has become apparent. 18 F exhibits favourable physical characteristics for APP radiolabelling and has been described as an ideal PET radionuclide. Notwithstanding, 18 F radiolabelling of APP is challenging, and this is echoed in the literature where a number of diverse approaches have been adopted. This review seeks to assess and compare the approaches taken to 18 F APP radiolabelling with the intention of highlighting trends within this expanding field. Generic themes have emerged in the literature, namely the use of mild radiolabelling conditions, a preference of site-specific methodologies with an impetus for short, automated procedures which produce high-yielding [18 F]APPs.

In vivo characterisation of a therapeutically relevant self-assembling 18 F-labelled ß-sheet forming peptide and its hydrogel using positron emission tomography.

Positron emission tomography (PET) and fluorescence labelling have been used to assess the pharmacokinetics, biodistribution and eventual fate of a hydrogel-forming nonapeptide, FEFKFEFKK (F9), in healthy mice, using 18 F-labelled and fluorescein isothiocyanate (FITC)-labelled F9 analogues. F9 was site-specifically radiolabelled with 2-[18 F]fluoro-3-pyridinecarboxaldehyde ([18 F]FPCA) via oxime bond formation. [18 F]FPCA-F9 in vivo fate was evaluated both as a solution, following intravenous administration, and as a hydrogel when subcutaneously injected. The behaviour of FITC-F9 hydrogel was assessed following subcutaneous injection. [18 F]FPCA-F9 demonstrated high plasma stability and primarily renal excretion; [18 F]FPCA-F9 when in solution and injected into the bloodstream displayed prompt bladder uptake (53.4 ± 16.6 SUV at 20 minutes postinjection) and rapid renal excretion, whereas [18 F]FPCA-F9 hydrogel, formed by co-assembly of [18 F]FPCA-F9 monomer with unfunctionalised F9 peptide and injected subcutaneously, showed gradual bladder accumulation of hydrogel fragments (3.8 ± 0.4 SUV at 20 minutes postinjection), resulting in slower renal excretion. Gradual disaggregation of the F9 hydrogel from the site of injection was monitored using FITC-F9 hydrogel in healthy mice (60 ± 3 over 96 hours), indicating a biological half-life between 1 and 4 days. The in vivo characterisation of F9, both as a gel and a solution, highlights its potential as a biomaterial.

A new technique for the radiolabelling of mixed leukocytes with zirconium-89 for inflammation imaging with positron emission tomography.

Mixed leukocyte (white blood cells [WBCs]) trafficking using positron emission tomography (PET) is receiving growing interest to diagnose and monitor inflammatory conditions. PET, a high sensitivity molecular imaging technique, allows precise quantification of the signal produced from radiolabelled moieties. We have evaluated a new method for radiolabelling WBCs with either zirconium-89 ((89) Zr) or copper-64 ((64) Cu) for PET imaging. Chitosan nanoparticles (CNs) were produced by a process of ionotropic gelation and used to deliver radiometals into WBCs. Experiments were carried out using mixed WBCs freshly isolated from whole human blood. WBCs radiolabelling efficiency was higher with [(89) Zr]-loaded CN (76.8 ± 9.6% (n = 12)) than with [(64) Cu]-loaded CN (26.3 ± 7.0 % (n = 7)). [(89) Zr]-WBCs showed an initial loss of 28.4 ± 5.8% (n = 2) of the radioactivity after 2 h. This loss was then followed by a plateau as (89) Zr remains stable in the cells. [(64) Cu]-WBCs showed a loss of 85 ± 6% (n = 3) of the radioactivity after 1 h, which increased to 96 ± 6% (n = 3) loss after 3 h. WBC labelling with [(89) Zr]-loaded CN showed a fast kinetic of leukocyte association, high labelling efficiency and a relatively good retention of the radioactivity. This method using (89) Zr has a potential application for PET imaging of inflammation.

Development of a method for the preparation of zirconium-89 radiolabelled chitosan nanoparticles as an application for leukocyte trafficking with positron emission tomography.

Positron Emission Tomography is an attractive imaging modality for monitoring the migration of cells to pathological tissue. We evaluated a new method for radiolabelling leukocytes with zirconium-89 (89Zr) using chitosan nanoparticles (CN, Z-average size 343 ± 210nm and zeta potential +46 ± 4mV) as the carrier. We propose that cell uptake of 89Zr-loaded CN occurred in a two-step process; cell membrane interaction with 89Zr-loaded CN was followed by a slower cell internalisation step.

Microfluidic technology for PET radiochemistry.

This paper describes the first application of a microfabricated reaction system to positron emission tomography (PET) radiochemistry. We have applied microfluidic technology to synthesise PET radiopharmaceuticals using (18)F and (124)I as labels for fluorodeoxyglucose (FDG) and Annexin-V, respectively. These reactions involved established methods of nucleophilic substitution on a mannose triflate precursor and direct iodination of the protein using iodogen as an oxidant. This has demonstrated a proof of principle of using microfluidic technology to radiochemical reactions involving low and high molecular weight compounds. Using microfluidic reactions, [(18)F]FDG was synthesised with a 50% incorporation of the available F-18 radioactivity in a very short time of 4s. The radiolabelling efficiency of (124)I Annexin-V was 40% after 1 min reaction time. Chromatographic analysis showed that such reaction yields are comparable to conventional methods, but in a much shorter time. The yields can be further improved with more optimisation of the microfluidic device itself and its fluid mixing profiles. This demonstrates the potential for this technology to have an impact on rapid and simpler radiopharmaceutical synthesis using short and medium half-life radionuclides.

Microfluidic reactor for the radiosynthesis of PET radiotracers.

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term "microfluidic PET". The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate "proof of principle" we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[(18)F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radiofluorination of 2-[(18)F]FDG (60% radiochemical yield within 4s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Regional cerebral L-[14C-methyl]methionine incorporation into proteins: evidence for methionine recycling in the rat brain.

The specific activity (SA) of free methionine was measured in plasma and in different regions of the rat brain at 15, 30, or 60 min after intravenous infusion of L-[14C-methyl]methionine. Within these time periods, an apparent steady state of labeled free methionine in plasma and in brain was reached. However, the brain-to-plasma free methionine SA ratio was found to be approximately 0.5, showing that an isotopic equilibrium between brain and plasma was not attained. This suggests the presence of an endogenous source of brain free methionine (likely originating from protein breakdown), in addition to the plasma source. The contribution of this endogenous source to the content of free methionine varies significantly among the different brain regions. Our results indicate that the regional rates of protein synthesis measured with L-[11C-methyl]methionine using positron emission tomography would be underestimated, since the local fraction of brain methionine derived from protein degradation would not be considered.

Regional specific binding of [11C]RO 15 1788 to central type benzodiazepine receptors in human brain: quantitative evaluation by PET.

The central type benzodiazepine receptors were studied in 17 healthy human subjects with 11C-RO 15 1788 and positron emission tomography (PET). The brain regional distribution of the tracer in eight control studies performed after injection of trace doses of 11C-RO 15 1788 was consistent with that of benzodiazepine receptors. Saturation studies with co-injected cold RO 15 1788 in the remaining subjects showed a dose-dependent decrease of brain radiotracer until full inhibition of specific binding was achieved with doses above 0.1 mg/kg (four studies). Based on the results, a simple method to estimate the specifically bound 11C-RO 15 1788 regionally in a single PET study is proposed, using the data from the full-saturation studies as a stable estimate of the nondisplaceable radioligand concentration. Using this method, it was found that quasiequilibrium between the estimated specifically bound and nondisplaceable components was achieved at times equal to or longer than 20 min after tracer administration. The validity of this method was partly supported by further results, showing a good agreement between the regional specific binding so calculated and postmortem data of receptor density.

Pentylenetetrazol-induced seizure is not mediated by benzodiazepine receptors in vivo.

The selective benzodiazepine antagonist RO 15-1788, labelled with carbon 11 [11C] RO 15-1788, as a specific marker, together with positron emission tomography, allows the in vivo study of benzodiazepine receptors in primates. In addition, when coupled with recordings of electroencephalographic activity, this method offers the feasibility of studying the correlation between occupancy of benzodiazepine receptors and the convulsant action of drugs acting at the benzodiazepine-GABA receptor complex in vivo. The present study showed that convulsant doses of pentylenetetrazol (PTZ) could affect the binding of [11C] RO 15-1788 in vivo in two ways, depending on the doses tested: at concentrations of 20 and 30 mg/kg, pentylenetetrazol increased the binding of [11C] RO 15-1788 whereas larger concentrations displaced the binding of [11C] RO 15-1788. The direct correlation between the occupancy of respective benzodiazepine receptors, afforded by increasing convulsant doses of pentylenetetrazol, revealed that competitive interaction with benzodiazepine receptors was not necessary for pentylenetetrazol to induce the appearance of seizures in vivo.

Simple production of [1-carbon-11]acetate.

UNLABELLED: We report an attractive approach for the preparation of [1-11C]acetate. METHODS: The procedure involved the instantaneous hydrolysis of [1-11C]acetyl chloride back to [1-11C]acetic acid by simply trapping the volatile acid chloride in physiological saline. This delivered [1-11C]acetate immediately in pharmaceutical quality. RESULTS: An easy and quantitative gas phase separation of the radiopharmaceutical from any inorganic residue and organic contamination could be achieved. The preparation required a minimum of automation and afforded only 5 min for an amount of 15 GBq of [1-11C]acetate which was yet ready for injection. Multiple preparations could be performed within 1 day. CONCLUSION: The use of [1-11C]acetyl chloride as a precursor to [1-11C]acetate is of considerable practical importance lending itself to automation with ease and giving the target compound directly in sterile solution without the need for further care and purification.

PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: a case report.

The utility of the peripheral type benzodiazepine site ligand 11C-PK 11195, for imaging human glioma in conjunction with Positron Emission Tomography, relies on a high specific binding of the tracer to tumoral peripheral type benzodiazepines sites. In a patient with glioblastoma, we found that 11C-PK 11195 binding was two-fold higher in the tumor than in normal gray matter and that 30% of tumoral binding could be displaced by a large excess of unlabeled drug. These findings suggest that tumoral retention of the ligand is due, in part, to specific binding.

Positron emission tomography study of [11C]methyl-tetrahydroaminoacridine (methyl-tacrine) in baboon brain.

THA (1,2,3,4-tetrahydro-9-amino-acridine, tacrine), a potential therapeutic agent for patients suffering from Alzheimer's disease, has multiple pharmacological sites of action in the brain. In order to study the cerebral binding sites of THA in vivo, we labeled a close derivative of THA with carbon 11 for positron emission tomography (PET) analysis. We report the biodistribution of this compound, 1,2,3,4-tetrahydro-9-[11C]methylaminoacridine ([11C]MTHA), in the rodent and describe the first PET experiments in non-human primates. The distribution of [11C]MTHA in baboon brain, although rather diffuse in the gray matter, showed a higher concentration in the cortex and basal ganglia than in the cerebellum and binding could be displaced (50%) by cold THA. These results suggest that [11C]MTHA is a promising PET ligand for the study of the cerebral binding of THA.

Benzodiazepine receptors studied in living primates by positron emission tomography: antagonist interactions.

After labelling the brain benzodiazepine receptors of sub-human primates with [11C]RO15-1788, the interactions of different benzodiazepine receptor antagonist ligands were studied by positron emission tomography (PET). Various doses of either RO15-1788, RO15-3505 or propyl beta-carboline-3-carboxylate were injected intravenously 20 min after the radiotracer, and induced an immediate and specific dose-dependent displacement of the brain radioactivity. However, a comparison of the dose-receptor occupancy patterns of these three antagonists established from the displacement experiments revealed that only propyl beta-carboline-3-carboxylate displayed clear biphasic dose-receptor occupancy curves. This indicates that, in the living primate brain, there are two different benzodiazepine receptor subpopulations (which can be either different benzodiazepine receptor subtypes or distinct conformational states of a single receptor).

In vivo visualization of central muscarinic receptors using [11C]quinuclidinyl benzilate and positron emission tomography in baboons.

The muscarinic antagonist, quinuclidinyl benzilate (QNB), labeled with carbon 11 was used as a radioligand to visualize in vivo by positron emission tomography (PET) the central muscarinic acetylcholine receptors (mAChR) in baboons (Papio papio). The binding characteristics of [11C]QNB showed its specific binding to central mAChR. [11C]QNB brain uptake was high in cerebral cortex and striatum, areas that are rich in mAChR, whereas it decreased rapidly in cerebellum, evidencing non-specific binding in this structure that is almost devoid of mAChR. These results are consistent with the known cerebral distribution of mAChR in primates. [11C]QNB specific cerebral binding was enhanced by pretreatment with methyl-QNB, a peripherally acting muscarinic antagonist. Specifically labeled binding sites alone were blocked by prior administration of dexetimide, a muscarinic antagonist. Specific radioactivity was driven out from mAChR-rich regions by atropine and dexetimide, drugs with high affinity for mAChR. This competition was stereospecific since only dexetimide, the pharmacologically active isomer of benzetimide, was able to compete with the radioligand on its binding sites. A relationship between the occupancy of [11C]QNB-labeled receptors by atropine or dexetimide and the concomitant induction of a pharmacological effect was also detected by simultaneous PET scanning and electroencephalographic recording. Since mAChR form an important part of choline receptors in the central nervous system, [11C]QNB appears to be a suitable radiotracer to monitor cerebral physiological or pathological phenomena linked to the cholinergic system in living subjects.

Benzodiazepine receptors studied in living primates by positron emission tomography: inverse agonist interactions.

The convulsant actions of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and of methyl beta-carboline-3-carboxylate (beta-CCM) were evaluated in the baboon (Papio papio). DMCM, 0.6-4 mg/kg, induced epileptic seizures with short latency. DMCM convulsive seizures could be blocked by i.v. administration of the benzodiazepine agonist diazepam (10 mg). Similarly, beta-CCM, 0.3-3 mg/kg i.v., provoked generalized seizures in the baboons. These seizures were also reversed by the administration of propyl beta-carboline-3-carboxylate (3 mg/kg) or of diazepam (5 mg/kg). Combining the results from Positron Emission Tomography and the EEG assessments, benzodiazepine receptor occupancy by beta-CCM and DMCM was directly correlated with their convulsant actions in the living baboon. beta-CCM exerted its convulsant action in the living baboon at 76 and 74% benzodiazepine receptor occupancy in, respectively, occipital and temporal cortices whereas DMCM displayed a similar convulsive activity when only 58 and 65% of these receptors in the above regions were occupied.

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

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

Central type benzodiazepine binding sites: a positron emission tomography study in the baboon's brain.

An in vivo characterization of specific central type benzodiazepine (BZD) binding sites, labelled with [11C]Ro 15-1788 was performed, using positron emission tomography. After i.v. injection of 10 mCi [11C]Ro 15-1788 (corresponding to 1 nmol/kg), sequential quantitative tomographic slices of the brain were obtained during 80 min. In some experiments various doses of different cold drugs (BZD agonist or antagonist) were injected i.v. subsequently in order to explore the specificity of the binding of the radioligand in brain structures. The main criteria usually utilized in vitro to demonstrate a specific binding to receptors, such as regional distribution, stereospecificity and saturability of the binding and pharmacological effect linked to the receptor's occupancy, were demonstrated in the brain of a living baboon.

Feasibility of labeled alpha-acetamido-aminoisobutyric acid as new tracer compound for kinetic labeling of neutral amino acid transport: preparation of alpha-(N-[1-11C]acetyl)- and alpha-(N-[1-14C]acetyl)-aminoisobutyric acid.

The nonphysiological, nonracemic, branched-chain alpha-acetamido-aminoisobutyric acid was labeled with the carbon isotope 11C with the intention to use it in conjunction with positron emission tomography (PET) to measure the kinetics of amino acid transport in vivo. It was produced by the reaction of the novel 11C-precursor N-[1-11C]acetylpyridinium chloride with alpha-aminoisobutyric acid. Typically, 2 GBq of alpha-(N-[1-11C]acetyl)-aminoisobutyric acid were isolated with a specific activity of 12 to 20 GBq. mumol-1 at the time of application, and with a radiochemical purity of > 98%. The chemical identity of alpha-(N-[1-11C]acetyl)-aminoisobutyric acid was confirmed by comparison with alpha-(N-[1-14C]acetyl)-aminoisobutyric acid that was independently prepared by a standard acetylation procedure of alpha-aminoisobutyric acid using [1-14C]acetic anhydride. In vivo, both labeled substrates were not metabolized. In cell-culture experiments, 84% of the substrate entered the cells by the sodium-dependent amino acid transport system A, whereas 16% was taken up by the sodium-independent system. The uptake of the radiotracer was measured 20 min and 40 min postinjection in tumor-bearing male Copenhagen rats for assessment of its in vivo biodistribution.

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

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

Visualization by positron emission tomography of the apparent regional heterogeneity of central type benzodiazepine receptors in the brain of living baboons.

The feasibility of visualizing the heterogeneity of benzodiazepine (BDZ) receptors in the brain of living baboons was investigated using Positron Emission Tomography. Ethyl 8-fluoro-5,6-dihydro-5-methyl 6-oxo-4H-imidazo (1,5-a) (1, 4) benzodiazepine-3-carboxylate (RO 15 1788) labelled by carbon 11 (11C-RO 15 1788) was I.V. injected for the "in vivo" labelling of the central type BDZ receptors. Displacement experiments were performed 20 minutes after the administration of the radioligand by two different cold drugs: RO 15 1788 which has an equal affinity for central type BDZ receptors, and propyl B-Carboline-3-carboxylate (B-CCP) which favours the sites located in the cerebellum. Different sensitivities to these two drugs displacement of 11C-RO 15 1788 binding "in vivo" were observed: on the one hand in the regional localization of the displacement, and on the other hand, in the amount of the radioactivity displaced. The apparent interregional heterogeneity of the displacement seen in the cerebellum and in the temporal cortex are discussed in terms of discrepancies observed "in vitro" at physiological temperature, between cerebellar and non-cerebellar BDZ central type binding sites.