Synthesis and preclinical evaluation of novel 18F-vancomycin-based tracers for the detection of bacterial infections using positron emission tomography.

INTRODUCTION: Bacterial infections are a major problem in medicine, and the rapid and accurate detection of such infections is essential for optimal patient outcome. Bacterial infections can be diagnosed by nuclear imaging, but most currently available modalities are unable to discriminate infection from sterile inflammation. Bacteria-targeted positron emission tomography (PET) tracers have the potential to overcome this hurdle. In the present study, we compared three 18F-labelled PET tracers based on the clinically applied antibiotic vancomycin for targeted imaging of Gram-positive bacteria. METHODS: [18F]FB-NHS and [18F]BODIPY-FL-NHS were conjugated to vancomycin. The resulting conjugates, together with our previously developed [18F]PQ-VE1-vancomycin, were tested for stability, lipophilicity, selective binding to Gram-positive bacteria, antimicrobial activity and biodistribution. For the first time, the pharmacokinetic properties of all three tracers were compared in healthy animals to identify potential binding sites. RESULTS: [18F]FB-vancomycin, [18F]BODIPY-FL-vancomycin, and [18F]PQ-VE1-vancomycin were successfully synthesized with radiochemical yields of 11.7%, 2.6%, and 0.8%, respectively. [18F]FB-vancomycin exhibited poor in vitro and in vivo stability and, accordingly, no bacterial binding. In contrast, [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin showed strong and specific binding to Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which was outcompeted by unlabeled vancomycin only at concentrations exceeding clinically relevant vancomycin blood levels. Biodistribution showed renal clearance of [18F]PQ-VE1-vancomycin and [18F]BODIPY-FL-vancomycin with low non-specific accumulation in muscles, fat and bones. CONCLUSION: Here we present the synthesis and first evaluation of the vancomycin-based PET tracers [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin for image-guided detection of Gram-positive bacteria. Our study paves the way towards real-time bacteria-targeted diagnosis of soft tissue and implant-associated infections that are oftentimes caused by Gram-positive bacteria, even after prophylactic treatment with vancomycin.

Will extended field-of-view PET/CT depopulate the graveyard of failed PET radiopharmaceuticals?

With the rapid emergence of extended Field-of-View PET-cameras several new applications for radiopharmaceuticals become within reach. Main reason is the significant increase of the sensitivity of the PET-camera so that much less radioactivity can be administered. Issues that that hampered development or use of PET-radiopharmaceuticals become realistic again. Molar activity requirements can become less strict. New low-yielding radiochemistry methods may become applicable. Carbon-11 labelled compounds can revive and potentially be shipped to nearby PET-facilities. PET-radiopharmaceuticals with slow kinetics in comparison to their half life can still be used. As additional infrastructure and equipment will likely remain unchanged and keep the same sensitivity therefore there will be issues with kinetic modelling requiring analysis of plasma or metabolites samples with lower count rate. Besides the potential revival of failed radiopharmaceuticals, novel challenges are ahead to develop novel radiochemistry based on thus far unsuitable (low yielding or time consuming) reactions.

New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals.

BACKGROUND: The introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 µg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES. RESULTS: HEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 µg/mL and a limit-of-detection (LOD) of 1 µg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11. CONCLUSION: The TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 µg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount.

Pharmacological Screening Identifies SHK242 and SHK277 as Novel Arginase Inhibitors with Efficacy against Allergen-Induced Airway Narrowing In Vitro and In Vivo.

Arginase is a potential target for asthma treatment. However, there are currently no arginase inhibitors available for clinical use. Here, a novel class of arginase inhibitors was synthesized, and their efficacy was pharmacologically evaluated. The reference compound 2(S)-amino-6-boronohexanoic acid (ABH) and >200 novel arginase inhibitors were tested for their ability to inhibit recombinant human arginase 1 and 2 in vitro. The most promising compounds were separated as enantiomers. Enantiomer pairs SHK242 and SHK243, and SHK277 and SHK278 were tested for functional efficacy by measuring their effect on allergen-induced airway narrowing in lung slices of ovalbumin-sensitized guinea pigs ex vivo. A guinea pig model of acute allergic asthma was used to examine the effect of the most efficacious enantiopure arginase inhibitors on allergen-induced airway hyper-responsiveness (AHR), early and late asthmatic reactions (EAR and LAR), and airway inflammation in vivo. The novel compounds were efficacious in inhibiting arginase 1 and 2 in vitro. The enantiopure SHK242 and SHK277 fully inhibited arginase activity, with IC50 values of 3.4 and 10.5 µM for arginase 1 and 2.9 and 4.0 µM for arginase 2, respectively. Treatment of slices with ABH or novel compounds resulted in decreased ovalbumin-induced airway narrowing compared with control, explained by increased local nitric oxide production in the airway. In vivo, ABH, SHK242, and SHK277 protected against allergen-induced EAR and LAR but not against AHR or lung inflammation. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. SIGNIFICANCE STATEMENT: Arginase is a potential drug target for asthma treatment, but currently there are no arginase inhibitors available for clinical use. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. Our new inhibitors show protective effects in reducing airway narrowing in response to allergens and reductions in the early and late asthmatic response.

Altered adenosine 2A and dopamine D2 receptor availability in the 6-hydroxydopamine-treated rats with and without levodopa-induced dyskinesia.

Several lines of evidence imply alterations in adenosine signaling in Parkinson's disease (PD). Here, we investigated cerebral changes in adenosine 2A receptor (A2AR) availability in 6-hydroxydopamine (6-OHDA)-lesioned rats with and without levodopa-induced dyskinesia (LID) using positron-emission tomography (PET) with [11C]preladenant. In parallel dopamine type 2 receptor (D2R) imaging with [11C]raclopride PET and behavioral tests for motor and cognitive function were performed. METHODS: Parametric A2AR and D2R binding potential (BPND) images were reconstructed using reference tissue models with midbrain and cerebellum as reference tissue, respectively. All images were anatomically standardized to Paxinos space and analyzed using volume-of-interest (VOI) and voxel-based approaches. The behavioral alternations were assessed with the open field test, Y-maze, novel object recognition test, cylinder test, and abnormal involuntary movement (AIM) score. In total, 28 female Wistar rats were included. RESULTS: On the behavioral level, 6-OHDA-lesioned rats showed asymmetry in forepaw use and deficits in spatial memory and explorative behavior as compared to the sham-operated animals. 15-Days of levodopa (L-DOPA) treatment induced dyskinesia but did not alleviate motor deficits in PD rats. Intranigral 6-OHDA injection significantly increased D2R binding in the lesioned striatum (BPND: 2.69 ± 0.40 6-OHDA vs. 2.31 ± 0.18 sham, + 16.6%; p = 0.03), whereas L-DOPA treatment did not affect the D2R binding in the ipsilateral striatum of the PD rats. In addition, intranigral 6-OHDA injection tended to decrease the A2AR availability in the lesioned striatum. The decrease became significant when data were normalized to the non-affected side (BPND: 4.32 ± 0.41 6-OHDA vs. 4.58 ± 0.89 sham; NS, ratio: 0.94 ± 0.03 6-OHDA vs. 1.00 ± 0.02 sham; - 6.1%; p = 0.01). L-DOPA treatment significantly increased A2AR binding in the affected striatum (BPND: 6.02 ± 0.91 L-DOPA vs. 4.90 ± 0.76 saline; + 23.4%; p = 0.02). In PD rats with LID, positive correlations were found between D2R and A2AR BPND values in the ipsilateral striatum (r = 0.88, ppeak = 8.56.10-4 uncorr), and between AIM score and the D2R BPND in the contralateral striatum (r = 0.98; ppeak = 9.55.10-5 uncorr). CONCLUSION: A2AR availability changed in drug-naïve and in L-DOPA-treated PD rats. The observed correlations of striatal D2R availability with A2AR availability and with AIM score may provide new knowledge on striatal physiology and new possibilities to further unravel the functions of these targets in the pathophysiology of PD.

Improved GMP-compliant multi-dose production and quality control of 6-[18F]fluoro-L-DOPA.

BACKGROUND: 6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) is a frequently used radiopharmaceutical for detecting neuroendocrine and brain tumors and for the differential diagnosis of Parkinson's disease. To meet the demand for FDOPA, a high-yield GMP-compliant production method is required. Therefore, this study aimed to improve the FDOPA production and quality control procedures to enable distribution of the radiopharmaceutical over distances.FDOPA was prepared by electrophilic fluorination of the trimethylstannyl precursor with [18F]F2, produced from [18O]2 via the double-shoot approach, leading to FDOPA with higher specific activity as compared to FDOPA which was synthesized, using [18F]F2 produced from 20Ne, leading to FDOPA with a lower specific activity. The quality control of the product was performed using a validated UPLC system and compared with quality control with a conventional HPLC system. Impurities were identified using UPLC-MS. RESULTS: The [18O]2 double-shoot radionuclide production method yielded significantly more [18F]F2 with less carrier F2 than the conventional method starting from 20Ne. After adjustment of radiolabeling parameters substantially higher amounts of FDOPA with higher specific activity could be obtained. Quality control by UPLC was much faster and detected more side-products than HPLC. UPLC-MS showed that the most important side-product was FDOPA-quinone, rather than 6-hydroxydopa as suggested by the European Pharmacopoeia. CONCLUSION: The production and quality control of FDOPA were significantly improved by introducing the [18O]2 double-shoot radionuclide production method, and product analysis by UPLC, respectively. As a result, FDOPA is now routinely available for clinical practice and for distribution over distances.

GRPR-selective PET imaging of prostate cancer using [(18)F]-lanthionine-bombesin analogs.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of human malignancies, including prostate cancer. Bombesin (BBN) is a 14 amino acids peptide that selectively binds to GRPR. In this study, we developed two novel Al(18)F-labeled lanthionine-stabilized BBN analogs, designated Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN, for positron emission tomography (PET) imaging of GRPR expression using xenograft prostate cancer models. (Methyl)lanthionine-stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN analogs were conjugated with a NOTA chelator and radiolabeled with Al(18)F using the aluminum fluoride strategy. Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN was labeled with Al(18)F with good radiochemical yield and specific activity>30 GBq/µmol for both radiotracers. The logD values measured for Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN were -2.14 ± 0.14 and -2.34 ± 0.15, respectively. In athymic nude PC-3 xenografts, at 120 min post injection (p.i.), the uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in prostate cancer (PC-3) mouse models was 0.82 ± 0.23% ID/g and 1.40 ± 0.81% ID/g, respectively. An excess of unlabeled ɛ-aminocaproic acid-BBN(7-14) (300-fold) was co-injected to assess GRPR binding specificity. Tumor uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in PC-3 tumors was evaluated by microPET (µPET) imaging at 30, 60 and 120 min p.i. Blocking studies showed decreased uptake in PC-3 bearing mice. Stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN peptides were rapidly and successfully labeled with (18)F. Both tracers may have potential for GRPR-positive tumor imaging.

Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier.

ABC transporters protect the brain by transporting neurotoxic compounds from the brain back into the blood. P-glycoprotein (P-gp) is the most investigated ABC (efflux) transporter, as it is implicated in neurodegenerative diseases such as Alzheimer's disease. Altered function of P-gp can be studied in vivo, using Positron Emission Tomography (PET). To date, several radiopharmaceuticals have been developed to image P-gp function in vivo. So far, attempts to image expression levels of P-gp using radiolabeled P-gp inhibitors have not been successful. Improved knowledge of compound behavior toward P-gp from in vitro studies should increase predictability of in vivo outcome.

Adenosine A(2A) receptor antagonists as Positron Emission Tomography (PET) tracers.

The adenosine A(2A) receptor (A(2A)R) is highly concentrated in the striatum, and a therapeutic target for Parkinson's disorder (PD) and Huntington's disease. High affinity and selective radiolabeled A(2A)R antagonists can be important research and diagnostic tools for PD. Positron Emission Tomography (PET) can play an important role by measuring radiolabeled A(2A) antagonists non-invasively in the brain. However, till date no complete review on A(2A)R PET ligands is available. The present article has been therefore focused on available PET tracers for A(2A)R and their detailed biological evaluation in rodents, nonhuman primates and humans. Drug design and development by molecular modeling including new lead structures that are potential candidates for radiolabeling and mapping of cerebral A(2A)Rs is discussed in the present article. A brief overview of functions of adenosine in health and disease, including the relevance of A(2A)R for PD has also been presented.

Preclinical evaluation of a novel ¹¹¹In-labeled bombesin homodimer for improved imaging of GRPR-positive prostate cancer.

Rational-designed multimerization of targeting ligands can be used to improve kinetic and thermodynamic properties. Multimeric targeting ligands may be produced by tethering multiple identical or two or more monomeric ligands of different binding specificities. Consequently, multimeric ligands may simultaneously bind to multiple receptor molecules. Previously, multimerization has been successfully applied on radiolabeled RGD peptides, which resulted in an improved tumor targeting activity in animal models. Multimerization of peptide-based ligands may improve the binding characteristics by increasing local ligand concentration and by improving dissociation kinetics. Here, we present a preclinical study on a novel radiolabeled bombesin (BN) homodimer, designated (111)In-DOTA-[(Aca-BN(7-14)]2, that was designed for enhanced targeting of gastrin-releasing peptide receptor (GRPR)-positive prostate cancer cells. A BN homodimer was conjugated with DOTA-NHS and labeled with (111)In. After HPLC purification, the GRPR targeting ability of (111)In-DOTA-[Aca-BN(7-14)]2 was assessed by microSPECT imaging in SCID mice xenografted with the human prostate cancer cell line PC-3. (111)In labeling of DOTA-[(Aca-BN(7-14)]2 was achieved within 30 min at 85 °C with a labeling yield of >40%. High radiochemical purity (>95%) was achieved by HPLC purification. (111)InDOTA-[Aca-BN(7-14)]2 specifically bound to GRPR-positive PC-3 prostate cancer cells with favorable binding characteristics because uptake of 111In-DOTA-[Aca-BN(7-14)]2 in GRPR-positive PC-3 cells increased over time. A maximum peak with 30% radioactivity was observed after 2 h of incubation. The log D value was -1.8 ± 0.1. (111)In-DOTA-[Aca-BN(7-14)]2 was stable in vitro both in PBS and human serum for at least 4 days. In vivo biodistribution analysis and microSPECT/CT scans performed after 1, 4, and 24 h of injection showed favorable binding characteristics and tumor-to-normal tissue ratios. This study identifies (111)In-DOTA-[(Aca-BN(7-14)]2 as a promising radiotracer for nuclear imaging of GRPR in prostate cancer.

Adenosine A(1) receptors in the central nervous system: their functions in health and disease, and possible elucidation by PET imaging.

Adenosine is a neuromodulator with several functions in the central nervous system (CNS), such as inhibition of neuronal activity in many signaling pathways. Most of the sedating, anxiolytic, seizure-inhibiting and protective actions of adenosine are mediated by adenosine A(1) receptors (A(1)R) on the surface of neurons and glia. Positron Emission Tomography (PET) is a powerful in vivo imaging tool which can be applied to investigate the physiologic and pathologic roles of A(1)R in the human brain, and to elucidate the mechanism of action of therapeutic drugs targeting adenosine receptors, nucleoside transporters and adenosine-degrading enzymes. In this review article, we discuss (i) functions of adenosine and its receptors in cerebral metabolism; (ii) radioligands for A(1)R imaging: xanthine antagonists, non-xanthine antagonists, and agonists; (iii) roles of A(1)R in health and disease, viz. sleep-wake regulation, modulation of memory retention and retrieval, mediating the effects of alcohol consumption, protecting neurons during ischemia and reperfusion, suppression of seizures, modulating neuroinflammation and limiting brain damage in neurodegenerative disorders. The application of PET imaging could lead to novel insights in these areas. Finally (iv), we discuss the application of PET in pharmacodynamic studies and we examine therapeutic applications of adenosine kinase inhibitors, e.g. in the treatment of pain, inflammation, and epilepsy.

Fluorine-18 radiopharmaceuticals beyond [18F]FDG for use in oncology and neurosciences.

Positron emission tomography (PET) is a rapidly expanding clinical modality worldwide thanks to the availability of compact medical cyclotrons and automated chemistry for the production of radiopharmaceuticals. There is an armamentarium of fluorine-18 ((18)F) tracers that can be used for PET studies in the fields of oncology and neurosciences. However, most of the (18)F-tracers other than 2-deoxy-2-[18F]fluoro-D-glucose (FDG) are in less than optimum human use and there is considerable scope to bring potentially useful (18)F-tracers to clinical investigation stage. The International Atomic Energy Agency (IAEA) convened a consultants' group meeting to review the current status of (18)F-based radiotracers and to suggest means for accelerating their use for diagnostic applications. The consultants reviewed the developments including the synthetic approaches for the preparation of (18)F-tracers for oncology and neurosciences. A selection of three groups of (18)F-tracers that are useful either in oncology or in neurosciences was done based on well-defined criteria such as application, lack of toxicity, availability of precursors and ease of synthesis. Based on the recommendations of the consultants' group meeting, IAEA started a coordinated research project on "Development of (18)F radiopharmaceuticals (beyond [(18)F]FDG) for use in oncology and neurosciences" in which 14 countries are participating in a 3-year collaborative program. The outcomes of the coordinated research project are expected to catalyze the wider application of several more (18)F-radiopharmaceuticals beyond FDG for diagnostic applications in oncology and neurosciences.

Nuclear imaging of prostate cancer with gastrin-releasing-peptide-receptor targeted radiopharmaceuticals.

Prostate cancer is one of the most common causes of cancer in men. Evaluating the different stages of prostate cancer with conventional imaging techniques still proves difficult. Nuclear imaging might provide a technique that is able to evaluate prostate cancer, but clinical application has been limited due to lack of accuracy of current radiopharmaceuticals. The development of radiopharmaceuticals that can be targeted to specific antigens, overexpressed in prostate cancer, but sparse in normal tissue, is crucial. Peptides are of particular interest because of their favourable characteristics, leading to increased attention for nuclear imaging of the gastrin-releasing-peptide-receptor (GRPR) with radiolabelled bombesin-like peptides. Several derivatives of bombesin and its truncated form have been prepared for imaging with single photon emission computed tomography (SPECT) or positron emission tomography (PET), thereby delivering potent candidates for further clinical evaluation. This article provides an overview of the development and preclinical evaluation of radiolabelled bombesin analogues for in vivo targeting of GRPR in prostate cancer. The effect of the radionuclide, chelator, spacer and unnatural amino acids on affinity, metabolic stability and image quality are discussed, as well as agonistic or antagonistic properties. Potent candidates are proposed based on these selection criteria: (I) high affinity for GRPR, with rapid and specific tumour uptake (II) high hydrophilicity resulting in the preferred renal-urinary mode of excretion and low hepatobiliary excretion, (III) high stability, but relatively rapid clearance from blood. Also, a summary is made of clinical studies that report on the detection of prostate cancer with GRPR targeted radiopharmaceuticals.

Positron emission tomography in patients with breast cancer using (18)F-3'-deoxy-3'-fluoro-l-thymidine ((18)F-FLT)-a pilot study.

BACKGROUND: This pilot study investigated the feasibility of (18)F-3'-deoxy-3'-fluoro-l-thymidine ((18)F-FLT) as a positron emission tomography (PET) tracer for the visualisation of breast cancer. METHODS: Patients with breast cancer underwent (18)F-FLT-PET prior to surgery. The uptake of (18)F-FLT was determined in the primary tumour and in the axilla. RESULTS: Eight tumours were visualized by (18)F-FLT-PET with a mean uptake value (SUV(mean)) of 1.7 and mean tumour-non-tumour ratio (TNT) of 5.0. In seven patients, axillary lymph-node metastases were found at pathological examinations, however, (18)F-FLT-PET showed uptake in only two large (and clinically evident) lymph-node metastases. CONCLUSIONS: (18)F-FLT shows uptake in most primary breast tumours and in large axillary lymph-node metastases.

P-glycoprotein activity and biological response.

P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators.

Development of an inexpensive, low attenuation styrofoam primate chair for use in a PET scanner.

Pharmacokinetic modelling of radiotracers for positron emission tomography (PET) imaging of neuroreceptors can be performed with time-activity data for brain and blood. We aimed to develop an alternative to withdrawal of arterial blood samples for acquisition of a blood curve. A supportive primate chair was constructed out of styrofoam and fixed to the head portion of the bed of a PET scanner. A lightly anaesthetised rhesus monkey was positioned in the chair in a sitting position and injected with the radiotracer. The styrofoam chair provided sufficient support for the monkey. The presence of the chair in the PET scanner caused negligible attenuation of radiation, allowing simultaneous acquisition of dynamic data from the subject's brain and heart. We conclude that a styrofoam primate chair is an ideal tool to measure blood and brain data from a rhesus monkey with PET. Invasiveness to the animal is reduced, as well as experimenter time.

Evaluation of [18F]fluorinated sigma receptor ligands in the conscious monkey brain.

PET-imaging of the sigma receptors is very helpful to understand processes, e.g., several central nervous system (CNS)-diseases in which the sigma receptors are involved. The [(18)F]fluoroethylated analogs of SA4503 and SA5845 ([(18)F]FE-SA4503 and [(18)F]FE-SA5845) were evaluated in conscious monkeys to estimate its suitability for human application for PET. Conscious monkeys (Macaca Mulatta) were either scanned with [(18)F]FE-SA4503 or [(18)F]FE-SA5845 (n = 3 for both groups, 220-802 MBq). After a dynamic study of 120 min, radioactivity was displaced by intravenous (i.v.) injection of haloperidol (1 mg/kg). One month later the same set of three monkeys were scanned with [(18)F]FE-SA4503 for 120 min and "cold" SA4503 (1 mg/kg) was infused to displace the radioactivity, and the other three monkeys were pretreated with haloperidol (1 mg/kg) before the 120-min PET-scan with [(18)F]FE-SA5845. Cortical areas (cingulate, frontal, occipital, parietal, temporal), striatum, and thalamus showed high radioactivity uptake. Infusion of haloperidol displaced the radioactivity levels of the two radioligands. The same effect was found for [(18)F]FE-SA4503 after SA4503 displacement. Pretreatment with haloperidol blocked the [(18)F]FE-SA5845 binding to give PET-images with low and uniform uptake in the brain. The findings demonstrated the reversible binding of the two radioligands. Metabolite analysis showed that 14% and 23% parent compound of [(18)F]FE-SA5845 and [(18)F]FE-SA4503, respectively, at 120 min postinjection was present in plasma. Kinetic analysis showed that the binding potential of [(18)F]FE-SA5845 was higher in all brain regions than that of [(18)F]FE-SA4503 (4.75-8.79 vs. 1.65-4.04). The highest binding potential was found in the hippocampus, followed by the cortical regions, thalamus, cerebellar hemisphere, striatum and vermis. Both [(18)F]FE-SA compounds bound specifically to cerebral sigma receptors of the monkey and have potential for mapping sigma receptors in the human brain.

11C-choline positron emission tomography for the evaluation after treatment of localized prostate cancer.

RATIONALE: The evaluation of the efficacy of the treatment of men with prostate cancer is largely based on post treatment levels of PSA. An increase in PSA or biochemical recurrence is the first sign of recurrent disease and precedes a clinically detectable recurrence by months to years. Digital rectal examination and conventional imaging techniques are not sensitive to detect a local recurrence. A metabolic imaging technique, which is not dependent on anatomical distortions, could be of use. In this study we investigated 11C-choline positron emission tomography (PET) for the evaluation after treatment of localized prostate cancer. METHODS: Thirty-six patients with localized prostate cancer, treated by either radical prostatectomy (n=20) or by external beam radiotherapy (n=16) were studied with 11C-choline PET. The results of PET were compared with the results of histology and with clinical follow up. RESULTS: Fourteen patients had no biochemical failure after therapy. 11C-choline PET was true negative in 14/14 patients. Twenty-two patients had a biochemical failure. In the radical prostatectomy patients 11C-choline PET was true positive in 5/13 (38%) cases. In the external beam radiotherapy patients 11C-choline PET was true positive in 7/9 (78%). The recurrent tumor was confirmed by biopsy or by bone scan in eleven of the twelve true positive patients. In ten patients with a negative 11C-choline PET scan, no recurrent tumor could be proven yet clinically, by biopsy or during follow up. CONCLUSION: 11C-choline PET is a feasible technique for evaluation of treatment for localized prostate cancer. The site of recurrence was detected correctly in 78% of the patients after external beam radiotherapy compared to 38% of the patients after radical prostatectomy. No positive PET scans were observed sofar in patients with a serum PSA <5ng/ml. Confirmatory studies and longer follow up are needed to determine the efficacy of 11C-choline PET compared to other imaging techniques.

New diagnostic techniques in staging in the surgical treatment of cutaneous malignant melanoma.

The emphasis of the research on the surgical treatment of melanoma has been on the resection margins, the role of elective lymph node dissection in high risk patients and the value of adjuvant regional treatment with hyperthermic isolated lymph perfusion with melphalan. Parallel to this research, new diagnostic techniques, such as Positron Emission Tomography and the introduction of the sentinel lymph node biopsy with advanced laboratory methods such as immuno-histochemical markers, and reverse transcriptase polymerase chain reaction, have been developed to facilitate early detection of metastatic melanoma. The role of these new techniques on the staging and surgical treatment of melanoma is discussed in this paper.

An improved method for the preparation of [11C]verapamil.

This paper describes an improved preparation of [11C]verapamil by reaction of [11C]methyl triflate with desmethylverapamil. The optimal reaction temperature, amount of precursor and reaction time were assessed. With this method [11C]verapamil can be prepared with a reproducible radiochemical yield of 66 +/- 4% (EOB, based on [11C]methyltriflate). Total synthesis time was 60 min. Radiochemical purity was >99% and specific activities varied between 5 and 30TBq/mmol.