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Vitamin B12 is essential for cell function and only accessible in food for mammals. To monitor vitamin B12 deficiency, methylmalonic acid (MMA) is used. Since MMA in serum/plasma is a frequently requested analyte at clinical laboratories the analytical method was improved and validated on a 96 well plate. Using a Tecan robot a working solution of acetonitrile containing MMA-D3 was added to plasma/serum samples. The solution was shaken for 1 min and then centrifuged for 10min. The supernatant was transferred to another plate and evaporated with nitrogen gas. The residual was redissolved with 0.2% formic acid in MilliQ-water and the plate was shaken for 1 min prior to LC-MS/MS analysis. The total analysis time was 3 min, retention time for MMA was 1.1 min and it was well separated from the interfering succinic acid. The calibrator curve was 0.044 - 1.63 μmol/L, which was also the linear range and LLOQ was 0.044 μmol/L. The within- and between-run CV:s were 3-7%. Age dependent clinical cut-offs at 0.28 (age50 years) and 0.36 μmol/L (age ≥50 years) were applied. In 404 clinical routine samples 10% were0.28, 7%0.4, and only 1% were0.7 μmol/L. The method has been successfully implemented in the laboratory for routine MMA analysis.

Objectives To assess non-invasive imaging for detection and quantification of gland structure, inflammation and function in patients with primary Sjogren's syndrome (pSS) using PET-CT with 11C-Methionine (11C-MET; radiolabelled amino acid), and 18F-fluorodeoxyglucose (18F-FDG; glucose uptake marker), to assess protein synthesis and inflammation, respectively; multiparametric MRI evaluated salivary gland structural and physiological changes. Methods In this imaging/clinical/histology comparative study (GSK study 203818; NCT02899377) patients with pSS and age- and sex-matched healthy volunteers underwent MRI of the salivary glands and 11C-MET PET-CT. Patients also underwent 18F-FDG PET-CT and labial salivary gland biopsies. Clinical and biomarker assessments were performed. Primary endpoints were semi-quantitative parameters of 11C-MET and 18F-FDG uptake in submandibular and parotid salivary glands and quantitative MRI measures of structure and inflammation. Clinical and minor salivary gland histological parameter correlations were explored. Results Twelve patients with pSS and 13 healthy volunteers were included. Lower 11C-MET uptake in parotid, submandibular and lacrimal glands, lower submandibular gland volume, higher MRI fat fraction, and lower pure diffusion in parotid and submandibular glands were observed in patients vs healthy volunteer, consistent with reduced synthetic function. Disease duration correlated positively with fat fraction and negatively with 11C-MET and 18F-FDG uptake, consistent with impaired function, inflammation and fatty replacement over time. Lacrimal gland 11C-MET uptake positively correlated with tear flow in patients, and parotid gland 18F-FDG uptake positively correlated with salivary gland CD20+ B-cell infiltration. Conclusion Molecular imaging and MRI may be useful tools to non-invasively assess loss of glandular function, increased glandular inflammation and fat accumulation in pSS.

Neutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, [[[[

The use of oral fluid tests to detect drugs is of growing interest in various areas, including treatment centers, roadside and workplace testing. In this study, we investigated drug detection in oral fluid samples collected using a commercially available device, Oral Eze. Drug detection in oral fluid was compared to paired urine samples, which were simultaneously collected. We also evaluated the collection device by comparing A and B oral fluid samples. Finally, we studied the stability of various drugs in samples stored for at least 1 year. The drug profile was investigated by comparing the drugs detected in oral fluid samples with paired urine samples collected in a treatment center. A total of 113 paired oral fluid and urine samples were investigated for the presence of drugs in the following groups: amphetamines, benzodiazepines, opiates and opioids, cocaine and cannabis. A and B samples were collected from different workplaces through an uncontrolled sampling procedure (n = 76). The stability of drugs in A samples was assessed after storage at −20°C for 1 year. Generally, there was a good correlation between drugs detected in oral fluid samples and urine samples. The heroin metabolite, 6-MAM, was more frequently detected in oral fluid samples than in urine samples, while cannabis was better detected in urine samples. Drugs in oral fluid samples were stable when stored at −20°C for at least 1 year. However, in many positive A and B oral fluid samples, there was significant variation in the concentrations obtained. Hence, the collection device may need to be further standardized and improved.

Two-pore physiologically-based pharmacokinetics (PBPK) for biologics describes the tissue distribution and elimination kinetics of soluble proteins as a function of their hydrodynamic radius and the physiological properties of the organs. Whilst many studies have been performed in rodents to parameterize the PBPK framework in terms of organ-specific lymph flow rates, similar validation in humans has been limited. This is mainly due to the paucity of the tissue distribution time course data for biologics that is not distorted by target-related binding. Here, we demonstrate that a PBPK model based on rodent data provided good to satisfactory extrapolation to the tissue distribution time course of 89Zr-labeled albumin-binding domain antibody (AlbudAb™) GSK3128349 in healthy human volunteers, including correct prediction of albumin-like plasma half-life, volume of distribution, and extravasation half-life. The AlbudAb™ used only binds albumin, and hence it also provides information about the tissue distribution kinetics and turnover of that ubiquitous and multifunctional plasma protein.

Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of ‘kill-early-kill-cheap’ to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.

Oral fluid sampling offers several advantages compared to other methods of detecting drugs in biological matrices due to its easy sampling procedure and simple supervision. Hence, the use of oral fluid for drug testing is increasing in workplaces and in roadside testing. As a result, more laboratories are required to perform analyses of drugs in oral fluid. A multi-method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for tracing drugs in oral fluid was developed. Oral fluid was collected using the collection device Oral-Eze. Samples were prepared by dilution with acetonitrile and methanol followed by centrifugation prior to UPLC–MS/MS analysis. The UPLC separation was achieved by using a BEH-C18 column. Mass detection was performed in positive ion mode, and the most common drugs/metabolites were detected for amphetamines, benzodiazepines, cocaine, benzoylecgonine, opiates, opioids, phencyclidine, and Δ9- tetrahydrocannabinol. Thirty-seven analytes were validated using the developed UPLC–MS/MS multi-analyte method with a total run time of 5 minutes. Calibration curves were linear for all analytes over the concentration range 1.5–600 ng/mL. Within- and between-day CVs varied from 1.2% to 20% and from 0.7% to 20%, respectively, for most substances. Extraction recoveries from the oral fluid device were >70%, a few had a yield of 50%. The developed multi-method was successfully validated and applied in a clinical routine laboratory to detect drugs in oral fluid samples that were sent from different workplaces and clinics. High sensitivity, simple sample pretreatment and short analysis time are advantages of this method.

Purpose While the aetiology of rheumatoid arthritis (RA) remains unclear, many of the inflammatory components are well characterised. For diagnosis and therapy evaluation, in vivo insight into these processes would be valuable. Various imaging probes have shown value including dynamic contrast-enhanced (DCE) MRI and PET/CT using 18F-fluorodeoxyglucose (18F-FDG) or tracers targeting the translocator protein (TSPO). To evaluate 18F-GE-180, a novel TSPO PET tracer, for detecting and quantifying disease activity in RA, we compared 18F-GE-180 uptake with that of 18F-FDG and DCE-MRI measures of inflammation. Methods Eight RA patients with moderate-to-high, stable disease activity and active disease in at least one wrist were included in this study (NCT02350426). Participants underwent PET/CT examinations with 18F-GE-180 and 18F-FDG on separate visits, covering the shoulders and from the pelvis to the feet, including hands and wrists. DCE-MRI was performed on one affected hand. Uptake was compared visually between tracers as judged by an experienced radiologist and quantitatively using the maximum standardised uptake value (SUVmax). Uptake for both tracers was correlated with DCE-MRI parameters of inflammation, including the volume transfer coefficient Ktrans using Pearson correlation (r). Results PET/CT imaging with 18F-GE-180 in RA patients showed marked extra-synovial uptake around the affected joints. Overall sensitivity for detecting clinically affected joints was low (14%). 18F-GE-180 uptake did not or only weakly correlate with DCE-MRI parameters in the wrist (r = 0.09–0.31). 18F-FDG showed higher sensitivity for detecting symptomatic joints (34%), as well as strong positive correlation with DCE-MRI parameters (SUVmax vs. Ktrans: r = 0.92 for wrist; r = 0.68 for metacarpophalangeal joints). Conclusions The correlations between DCE-MRI parameters and 18F-FDG uptake support use of this PET tracer for quantification of inflammatory burden in RA. The TSPO tracer 18F-GE-180, however, has shown limited use for the investigation of RA due to its poor sensitivity and ability to quantify disease activity in RA.

Background: Saliva and tear flow rates and minor salivary gland biopsy are typically used to assess gland function and disease activity in primary Sjogren’s syndrome (pSS); however, these have limitations. Imaging methods can directly visualize and generate quantitative values in individual glands. 18F-FDG-PET/CT (18F-FDG) measures glucose metabolism, a potential surrogate for inflammation; 11C-methionine-PET/CT (11C-met) is an amino acid PET tracer that assesses protein synthesis, a potential surrogate for gland synthetic function. Objectives: Explore the potential of molecular imaging and multi-parametric MRI to characterize and quantify pSS disease manifestations. Methods: In this pilot imaging study (203818), patients with pSS diagnosed per AECG criteria, with a EULAR Sjogren’s syndrome disease activity index score ≥5, and basal salivary flow >0.0 mL/min or stimulated salivary flow rate ≥0.05 mL/min, underwent 18F-FDG and 11C–met and dynamic contrast-enhanced and diffusion-weighted MRI, followed by minor salivary gland biopsy for histological analysis. Age- and sex-matched healthy volunteers (HV) underwent MRI and 11C-met. HV-pSS mean differences (m-diff; 95% confidence intervals [CI]) were calculated and Pearson’s correlation coefficients (r) estimated. Peak PET standard uptake values (SUVpeak) were used in the correlations and SUVmax values were recorded. All methods were compared with routine clinical and laboratory tests. Results: 12 patients had MRI, 18F-FDG and 11C–met; while HV (n=12) had MRI (n=12) and 11C–met (n=8). A lower 11C-met SUVpeak was seen in the parotid (m-diff: 1.4 g/mL [0.4, 2.3]) and submandibular (m-diff: 2.0 g/mL [0.9, 3.2]) glands in pSS versus HV, as was a trend for lower lacrimal gland uptake (m-diff: 0.5 g/mL [-0.2, 1.3]) in patients with pSS. On structural MRI, the fat fraction (mean%) was higher in pSS vs HV in the submandibular glands (m-diff: -14.8 [CI: -29.3, -0.4]), with similar trends observed in the parotid glands (-11.2 [-24.3, 1.9]). There was a negative correlation between 11C-met uptake and fat fraction (r: -0.7 [-0.9, -0.4]) in the combined parotid glands. There was positive correlation between 11C-met uptake and stimulated salivary flow (r: 0.5 [0.03, 0.8]) and negative correlation for stimulated salivary flow and fat fraction (r: -0.5 (-0.8, -0.1)] in the parotid glands; similar correlations were also seen in the submandibular glands. There was negative correlation between the global lymphoid aggregation score on minor salivary gland biopsy and the combined salivary gland fat fraction (r: -0.7 [-0.9, -0.2]). Parotid gland SUVmax 18F-FDG uptake was higher than historical control values (mean: 1.9 g/ml, [SD: 0.5]1) in some patients (pSS mean SUVmax: 2.8 g/mL [SD: 0.8, range 1.7–4.6]), with positive correlation between 18F–FDG and 11C-met uptake (r: 0.7 [0.2, 0.9]) in the combined salivary glands. Conclusion: Imaging showed clear differences between pSS and HV and correlated with clinical endpoints. Low 11C-met uptake and high fat fraction on MRI may indicate poor residual gland function, while high 18F-FDG and stable 11C-met uptake may define a subpopulation that responds well to anti-inflammatory therapies. References [1] Basu S, et al. Nucl Med Commun 2008; 29:367–73. Acknowledgement: Study/editorial support by Fishawack Indicia Ltd, UK funded by GSK. Disclosure of Interests: Michele Bombardieri Grant/research support from: Celgene, Consultant for: Medimmune, Coziana Ciurtin: None declared, Michalis Kostapanos Consultant for: Is an NHS consultant seconded to the GSK Clinical Cambridge Unit (50%) and has nothing to disclose., Elisa Astorri: None declared, Anwar Tappuni: None declared, Natasha Jordan: None declared, Saleem Azeem Shareholder of: GSK, Teresa Fuller Shareholder of: GSK, Employee of: GSK, Kathleen Port Shareholder of: GSK, Employee of: GSK, Nirav Ratia Shareholder of: GSK, Employee of: GSK, Andre van Maurik Shareholder of: GSK, Employee of: GSK, Calum Gray: None declared, Lucy Kershaw: None declared, Rob Janiczek Shareholder of: GSK, Employee of: GSK, Graham Searle: None declared, Paul Galette Shareholder of: GSK, Employee of: GSK, Marius de Groot Shareholder of: GSK, Employee of: GSK, Neel Patel Shareholder of: GSK, Employee of: GSK, Nicolas Wisniacki Shareholder of: GSK, Employee of: GSK, Mats Bergstrom Shareholder of: GSK, Consultant for: acted as external consultant to GSK, Employee of: GSK, Pilar Jimenez-Royo Shareholder of: GSK, Employee of: GSK, Ruth Tarzi Shareholder of: GSK, Employee of: GSK

Microdosing as a regulatory concept was introduced to facilitate exploratory studies in humans. The concept involves the use of very low doses of a radionuclide-labeled compound for imaging studies or for assessing plasma pharmacokinetics using equipment that has a highly sensitive readout. The supporting principle is that use of these low doses for a limited time in well-controlled, small populations will limit exposure and have a low risk of adverse effects. Microdosing regulations specify a reduced preclinical toxicology-assessment package in order to shorten the route to human studies and reduce its cost. However, for extrapolation to therapeutically relevant doses and plasma concentrations, there are specific aspects of the use of these low doses and low plasma concentrations that require special attention. These specific aspects are reviewed in this article, with separate attention being paid to small organic molecules and protein therapeutics. The indications for microdosing in drug development are discussed in terms of the 3 pillars of survival in drug development, the first of which is characterization of tissue distribution and access to the site of action; the second, engagement of the target; and the third, induction of tissue responses relevant to a therapeutic response.

Zolmitriptan is a serotonin5-HT1B/1Dreceptor agonist that is an effective and well-tolerated drug for migraine treatment. In a human positron emission tomography study, [11C]zolmitriptan crossed the blood-brain barrier but no clear pattern of regional uptake was discernable. The objective of this study was to map the binding of [11C]zolmitriptan in Rhesus monkey brain using whole hemispherein vitroautoradiography with [11C]zolmitriptan as a radioligand. In saturation studies, [11C]zolmitriptan showed specific (90%) binding to a population of high-affinity binding sites (Kd 0.95–5.06 nM). There was regional distribution of binding sites with the highest density in the ventral pallidum, followed by the external globus pallidus, substantia nigra, visual cortex, and nucleus accumbens. In competitive binding studies with 5-HT1receptor antagonists, [11C]zolmitriptan binding was blocked by selective 5-HT1Band 5-HT1Dligands in all target areas. There was no appreciable change in binding with the addition of a 5-HT1Areceptor antagonist.

To examine temporal trends in the incidence of hydatidiform mole (HM) in relation to maternal age, the occurrence of choriocarcinoma in women with a history of HM and the extent of underreporting of HM to the Swedish Cancer Register (SCR).Women registered with a diagnosis of HM were identified in the Swedish Cancer Register and the Swedish Inpatient Register (IPR). Record linkage to the Medical Birth Register provided information to assess the incidence of HM.We identified 3 844 unique cases of HM in the SCR and in the IPR combined between 1973 and 2004, yielding an incidence of 1.2 per 1 000 deliveries. The incidence of HM increased during the period under study. The highest incidence was observed in women below 20 and above 39 years of age. Of all registered cases of choriocarcinoma, 37% occurred in women with a previous history of HM. The risk of choriocarcinoma following HM was 1.3%, compared to 0.005% in women without a previous molar diagnosis. The records of the Cancer Register included 83.2% of all identified cases of HM.The incidence of HM in Sweden has increased over time, and is characterized by a bimodal pattern with distinctive peaks in the youngest and oldest women of reproductive age. More than one third of all women registered with choriocarcinoma had a previous diagnosis of HM. Despite mandatory reporting, there was evidence of underreporting of HM to the SCR that remained virtually unchanged over calendar time.

Our understanding of altered patterns of gene expression being responsible for many diseases has been growing thanks to modern molecular biological methods. Today, these changes can only be identified when tissue samples are available. Therefore, a noninvasive method allowing us to monitor gene expression in vivo would be valuable, not only as a research tool, but also for patient stratification before treatment and for treatment follow-up. Antisense oligonucleotides (ODN) have been considered to be suitable molecules to trace active genes in vivo, as well as to treat diseases by hybridising to its complementary messenger RNA (mRNA) sequence in the cells thereby preventing the synthesis of the peptide. However, the use of ODNs in the organisms are endangered by many hurdles such as physical barriers to pass and enzyme attack to be avoided. Positron emission tomography (PET) provides a most advanced in vivo imaging technology that allows the exploration of the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. This review provides a general summary about the antisense concept and displays the present status of the antisense imaging field with the major achievements and remaining challenges on the long journey towards accomplishing in vivo monitoring of gene expression using PET.

This study introduces a new approach for the application of principal component analysis (PCA) with pre-normalization on dynamic positron emission tomography (PET) images. These images are generated using the amyloid imaging agent N-methyl [(11)C]2-(4'-methylaminophenyl)-6-hydroxy-benzothiazole ([(11)C]PIB) in patients with Alzheimer's disease (AD) and healthy volunteers (HVs). The aim was to introduce a method which, by using the whole dataset and without assuming a specific kinetic model, could generate images with improved signal-to-noise and detect, extract and illustrate changes in kinetic behavior between different regions in the brain. Eight AD patients and eight HVs from a previously published study with [(11)C]PIB were used. The approach includes enhancement of brain regions where the kinetics of the radiotracer are different from what is seen in the reference region, pre-normalization for differences in noise levels and removal of negative values. This is followed by slice-wise application of PCA (SW-PCA) on the dynamic PET images. Results obtained using the new approach were compared with results obtained using reference Patlak and summed images. The new approach generated images with good quality in which cortical brain regions in AD patients showed high uptake, compared to cerebellum and white matter. Cortical structures in HVs showed low uptake as expected and in good agreement with data generated using kinetic modeling. The introduced approach generated images with enhanced contrast and improved signal-to-noise ratio (SNR) and discrimination power (DP) compared to summed images and parametric images. This method is expected to be an important clinical tool in the diagnosis and differential diagnosis of dementia.

Multivariate image analysis tools are used for analyzing dynamic or multidimensional Positron Emission To- mography, PET data with the aim of noise reduction, dimension reduction and signal separation. Principal Component Analysis is one of the most commonly used multivariate image analysis tools, applied on dynamic PET data. Independent Component Analysis is another multivariate image analysis tool used to extract and separate signals. Because of the pres- ence of high and variable noise levels and correlation in the different PET images which may confound the multivariate analysis, it is essential to explore and investigate different types of pre-normalization (transformation) methods that need to be applied, prior to application of these tools. In this study, we explored the performance of Principal Component Analysis (PCA) and Independent Component Analysis (ICA) to extract signals and reduce noise, thereby increasing the Signal to Noise Ratio (SNR) in a dynamic sequence of PET images, where the features of the noise are different compared with some other medical imaging techniques. Applications on computer simulated PET images were explored and com- pared. Application of PCA generated relatively similar results, with some minor differences, on the images with different noise characteristics. However, clear differences were seen with respect to the type of pre-normalization. ICA on images normalized using two types of normalization methods also seemed to perform relatively well but did not reach the im- provement in SNR as PCA. Furthermore ICA seems to have a tendency under some conditions to shift over information from IC1 to other independent components and to be more sensitive to the level of noise. PCA is a more stable technique than ICA and creates better results both qualitatively and quantitatively in the simulated PET images. PCA can extract the signals from the noise rather well and is not sensitive to type of noise, magnitude and correlation, when the input data are correctly handled by a proper pre-normalization. It is important to note that PCA as inherently a method to separate signal information into different components could still generate PC1 images with improved SNR as compared to mean images.

This article describes the experimental set‐up and pharmacokinetic modeling of P‐glycoprotein function in the rat blood‐brain barrier using [ 11 C]verapamil as the substrate and cyclosporin A as an inhibitor of P‐gp. [ 11 C]verapamil was administered to rats as an i.v. bolus dose followed by graded infusions to obtain steady‐state concentrations in the brain during 70 min. CsA was administered as a bolus followed by a constant infusion 20 min after the start of the [ 11 C]verapamil infusion. The brain uptake of [ 11 C]verapamil over 2 h was portrayed in a sequence of PET scans in parallel with measurement of [ 11 C]verapamil concentrations in blood and plasma and CsA concentrations in blood. Mixed effects modeling in NONMEM was used to build a pharmacokinetic model of CsA‐induced P‐gp inhibition. The brain pharmacokinetics of [ 11 C]verapamil was well described by a two‐compartment model. The effect of CsA on the uptake of [ 11 C]verapamil in the brain was best described by an inhibitory indirect effect model with an effect on the transport of [ 11 C]verapamil out of the brain. The CsA concentration required to obtain 50% of the maximal inhibition was 4.9 µg/mL (4.1 µM). The model parameters indicated that 93% of the outward transport of [ 11 C]verapamil was P‐gp mediated. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5386–5400, 2008

For a PET agent to be successful as a biomarker in early clinical trials of new anticancer agents, some conditions need to be fulfilled: the selected tracer should show a response that is related to the antitumoral effects, the quantitative value of this response should be interpretable to the antitumoral action, and the timing of the PET scan should be optimized to action of the drug. These conditions are not necessarily known at the start of a drugdevelopment program and need to be explored. We proposed atranslational imaging activity in whichexperiments in spheroids and later in xenografts are coupled to modeling of growth inhibition and to the related changes in the kinetics of PET tracers and other biomarkers. In addition, we demonstrated how this information can be used for planning clinical trials. Methods:Thefirst part of this concept is illustrated in a spheroid model with BT474 breast cancer cells treated with the heat shock protein 90 (Hsp90) inhibitor NVP-AUY922. The growth-inhibitory effect after a pulse treatment with the drug was measured with digital image analysis to determine effects on volume with high accuracy. The growthinhibitory effect was described mathematically by a combined Emax and time course model fitted to the data. The model was then used to simulate a once-per-week treatment; in these experiments the uptake of the PET tracers 18 F-FDG and 39-deoxy39- 18 F-fluorothymidine ( 18 F-FLT) was determined at different doses and different time points. Results: A drug exposure of 2 h followed by washout of the drug from the culture medium generated growth inhibition that was maximal at the earliest time point of 1 d and decreased exponentially with time during 10‐12 d. The uptake of 18 F-FDG per viable tumor volume was minimally affected by the treatment, whereas the 18F-FLT uptake decreased in correlation with the growth inhibition. Conclusion: The study suggests a prolonged action of the Hsp90 inhibitor that supports a once-per-week schedule. 18 F-FLT is a suitable tracer for the monitoring of effect, andthe 18 F-FLTPETstudymightbeperformedwithin3dafterdosing.

Introduction There are several instances when it is desirable to control brain concentration of pharmaceuticals, e.g., to modulate the concentration of anesthetic agents to different desired levels fitting to different needs during the course of surgery. This has so far only been possible using indirect estimates of drug concentration such as assuming constant relation between tissue and blood including extrapolations from animals. Methods A system for controlling target tissue concentration (UIPump) was used to regulate whole-brain concentrations of a central benzodiazepine receptor antagonist at therapeutic levels with input from brain kinetics as determined with PET. The system was tested by using pharmacological doses of flumazenil mixed with tracer amounts of [ 11 C]flumazenil. Flumazenil was used as a model compound for anesthesia. An infusion scheme to produce three different steady-state levels in sequence was designed based on kinetic curves obtained after bolus injection. The subjects (Sprague-Dawley rats, n =6) were monitored in a microPET scanner during the whole experiment to verify resulting brain kinetic curves. Results A steady-state brain concentration was rapidly achieved corresponding to a whole-brain concentration of 118±6 ng/ml. As the infusion rate decreased to lower the exposure by a factor of 2, the brain concentration decreased to 56±4 ng/ml. A third increased steady-state level of anesthesia corresponding to a whole-brain concentration of 107±7 ng/ml was rapidly achieved. Conclusion The experimental setup with computerized pump infusion and PET supervision enables accurate setting of target tissue drug concentration.

In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with 68Ga and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2--differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched--differing only in two nucleotides from the two Antisenses--was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after 68Ga-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.

The transport of hydroxyurea, a ribonucleoside reductase inhibitor, over biological membranes is slow and it has therefore been suggested that the substance could interact with an active efflux transporter. The transport of [(11)C]hydroxyurea into the rat brain was therefore studied after administration of the multidrug resistance protein inhibitor probenecid (50 and 150 mg/kg), the P-glycoprotein inhibitor cyclosporin A (25 mg/kg), hydroxyurea (50, 150 and 450 mg/kg) and mannitol (25%). None of the intervention drugs affected the brain uptake of [(11)C]hydroxyurea. The brain-to-plasma concentration ratios (K(p)), with or without intervention drug, were in the range 0.12-0.25 after 60 min of [(11)C]hydroxyurea infusion. [(11)C]Verapamil, a P-glycoprotein substrate with low brain penetration, was used to study the ability of hydroxyurea to inhibit P-glycoprotein. Administration of hydroxyurea (150 and 450 mg/kg) did not increase brain concentrations of [(11)C]verapamil. It is therefore unlikely that hydroxyurea is a substrate for or an inhibitor of P-glycoprotein or a substrate for a probenecid sensitive transport system. The low brain concentrations may instead be the result of slow uptake due to the hydrophilic nature of hydroxyurea.

Background: Animal studies demonstrate that stress and negative affect enhance the release of the neuropeptide substance P (SP), which binds to the neurokinin 1 (NK1) receptor. This positron emission tomography (PET) study evaluated how the activity in the SP-NK1 receptor system in the amygdala was affected by fear provocation in subjects with specific phobia. Methods: Sixteen adult women with DSM-IV-defined specific phobia for either snakes or spiders but not both viewed pictures of feared and non-feared animals while being PET-scanned for 60 min with the highly specific NK1 receptor antagonist [11C]GR205171 as the labeled PET tracer. Results: The uptake of the labeled NK1 receptor antagonist was significantly reduced in the right amygdala during phobic stimulation. In the left amygdala no significant differences were found between phobic and non-phobic conditions. There was a negative correlation in the right, but not left, amygdala between subjective anxiety ratings and NK1 tracer binding. Conclusions: Fear provocation affects the SP-NK1 receptor system in the right amygdala. This reflects reduced NK1 receptor availability during fear and could mirror an increased release of endogenous substance P.

Background BNP and NT-proBNP are widely used as rule-out tests for heart failure (HF) and they are frequently requested by primary care doctors. A point-of-care (POC) test would reduce the time to diagnosis for patients with suspected HF. The aim of the study was to evaluate a POC BNP test. Methods Plasma BNP results obtained with the Meritas® POC instrument (n = 82) were compared with the corresponding plasma BNP results analyzed on an Advia Centaur analyzer (Siemens Healthcare, Erlangen, Germany). Results The two methods showed concordant results with a Passing-Bablok correlation between the two methods: BNP(Meritas) = 1.00 x BNP(Siemens) + 1.09; r = 0.9773. Conclusions The study show that the Meritas® BNP assay could be used in primary care permitting rapid BNP testing to rule out heart failure.

Most of the methods used for analyzing PET data are applied in the spatial domain (image domain), in which reconstructed images contain all different types of effects and errors caused by the reconstruction algorithm such as correlation in-between pixels, correlations in-between frames, and streak-artifacts. In this paper, we have investigated a new, pixel wise, noise prenormalization method used for transformation of input data followed by volume-wise application of principal component analysis (PCA) on masked dynamic PET data in the sinogram domain. We are aiming to improve the performance of PCA and to provide images with improved quality and signal extraction. We compare the performance of PCA and the image quality obtained with the new method with previously published approaches. The results show improvement of performance of PCA with respect to image quality, signal extraction, precision, and visualization

Aromatase is an enzyme that converts androgens to estrogens and may play a role in mood and mental status. The aim of this study was to demonstrate that brain aromatase distribution could be evaluated with a novel positron emission tomography (PET) tracer [(11)C]vorozole. Vorozole is a nonsteroidal aromatase inhibitor that reversibly binds to the heme domain of aromatase. In vitro experiments in rat brain, using frozen section autoradiography, illustrated specific binding in the medial amygdala (MA), the bed nucleus of stria terminalis (BST) and the preoptic area (POA) of male rat brain. Specific binding in female rat brain was found in the MA and the BST; however, the signals were lower than those of males. The K(d) of [(11)C]vorozole binding to aromatase in MA was determined to be 0.60+/-0.06 nM by Scatchard plot analysis using homogenates. An in vivo PET study in female rhesus monkey brain demonstrated the uptake of [(11)C]vorozole in the amygdala, where the uptake was blocked by the presence of excess amounts of unlabeled vorozole. Thus, this tracer has a high affinity for brain aromatase and could have a potential for in vivo aromatase imaging. This technique might enable the investigation of human brain aromatase in healthy and diseased persons.

Positron emission tomography (PET) with the drug radiolabelled allows a direct measurement of brain or other organ kinetics, information which can be essential in drug development. Usually, however, a PET-tracer is administered intravenously (i.v.), whereas the therapeutic drug is mostly given orally or by a different route to the PET-tracer. In such cases, a recalculation is needed to make the PET data representative for the alternative administration route. To investigate the blood-brain barrier penetration of a drug (zolmitriptan) using dynamic PET and by PK modelling quantify the brain concentration of the drug after the nasal administration of a therapeutic dose. [11C]Zolmitriptan at tracer dose was administered as a short i.v. infusion and the brain tissue and venous blood kinetics of [11C]zolmitriptan was measured by PET in 7 healthy volunteers. One PET study was performed before and one 30 min after the administration of 5 mg zolmitriptan as nasal spray. At each of the instances, the brain radioactivity concentration after subtraction of the vascular component was determined up to 90 min after administration and compared to venous plasma radioactivity concentration after correction for radiolabelled metabolites. Convolution methods were used to describe the relationship between arterial and venous tracer concentrations, respectively between brain and arterial tracer concentration. Finally, the impulse response functions derived from the PET studies were applied on plasma PK data to estimate the brain zolmitriptan concentration after a nasal administration of a therapeutic dose. The studies shows that the PET data on brain kinetics could well be described as the convolution of venous tracer kinetics with an impulse response including terms for arterial-to-venous plasma and arterial-to-brain impulse responses. Application of the PET derived impulse responses on the plasma PK from nasal administration demonstrated that brain PK of zolmitriptan increased with time, achieving about 0.5 mg/ml at 30 min and close to a maximum of 1.5 mg/ml after 2 hr. A significant brain concentration was observed already after 5 min. The data support the notation of a rapid brain availability of zolmitriptan after nasal administration.

The local regulation of ovarian aromatase enzyme in polycystic ovary syndrome (PCOS) was studied with aromatase conversion and [C-11]vorozole-binding assays to analyze aromatase activity, substrate-enzyme affinity and number of aromatase binding sites in non-cultured human granulosa cells (GC) incubated with different sources and preparations of follicular fluid (FF). Incubation with FF from women stimulated in in vitro fertilization cycles with follicle-stimulating hormone yielded higher conversion activity than with FF from healthy women and PCOS patients, paralleled with higher substrate affinity (lower K-d) than with FF from healthy women. In PCOS women, charcoal-pretreated FF yielded higher conversion, whereas the ether-pretreated FF yielded lower conversion activity, than with untreated PCOS FF. Both preparations of FF yielded higher affinity to substrate (lower Kd values) and the ether-pretreated FF a lower number of binding sites (B-max). It seems that steroids with the presence of proteins in PCOS FF reduced aromatase conversion activity through decreased substrate affinity, whereas FF preparations devoid of proteins reduced the aromatase conversion activity mainly through blocking of aromatase active sites. Identification of specific agents responsible for this rapid regulation of aromatase function might help to understand normal regulation of the menstrual cycle and supposed imbalances of inhibitors/activators in PCOS.

Nine healthy volunteers aged 18-28 years were recruited into this open, single-centre, two-phase trial. In phase 1, two volunteers received a single dose of 11C-zolmitriptan 2.5 mg administered as a nasal spray and then underwent positron emission tomography (PET) scanning to determine the most appropriate times for scanning in phase 2. In phase 2, six volunteers received two doses and an additional volunteer one dose of 11C-zolmitriptan 2.5 mg intranasally. Volunteers underwent PET scanning over sectors covering one of the nasopharynx, lungs or abdomen, for up to 1.5 h postdose. The brain was also scanned and plasma zolmitriptan levels were measured. Almost 100% of the administered dose was detected in the nasopharynx immediately after dosing. This declined thereafter to about 50% at 20 min and to 35% at 80 min after dosing. Radioactivity appeared slowly in the upper abdomen, with 25% of given radioactivity detected at 20 min and persisting until 80 min after dosing. Minimal radioactivity was detected in the lungs. Radioactivity was detectable within brain tissue suggesting central penetration of zolmitriptan. Zolmitriptan in plasma had approached its maximum concentration by 15 min postdose. The data indicate initial absorption across the nasal mucosa contributing to an early systemic availability. 11C-Zolmitriptan administered intranasally was well tolerated.

PURPOSE: Positron emission tomography (PET) with 11C-5-hydroxytryptophan (5-HTP) as tracer is a promising imaging instrument in the management of patients with neuroendocrine tumours (NETs). However, high radioactivity concentrations in the urinary collecting system sometimes produce image reconstruction artefacts that can make detection of small NETs difficult. As a means to decrease urinary excretion of radioactivity and thereby improve image quality, we examined the effect of pretreatment with carbidopa (CD), a peripheral inhibitor of aromatic amino acid decarboxylase (AADC), which converts 5-HTP to serotonin (5-hydroxytryptamine, 5-HT). METHODS: Six patients with midgut carcinoid metastases were examined with 11C-5-HTP PET before and 1 h after oral administration of 100 or 200 mg of CD. RESULTS: There was a fourfold significant reduction of tracer uptake in the urinary collecting system after CD administration (p=0.0277, n=6), with a mean standard uptake value (SUV) of 155+/-195 before CD and 39+/-14 after CD. In tumour lesions there was a significant increase in SUV after CD administration (p

Background. Traditionally, the vagina is cleansed, before a curettage is performed. A previous study, comparing cleansing with chlorhexidine solution and cleansing with saline solution before vacuum aspiration in the first trimester, did not show any difference in the frequency of postoperative pelvic inflammatory disease. We wanted to investigate whether this was true also for vaginal cleansing with chlorhexidine, compared to no vaginal cleansing at all. Methods. Consecutive women having surgical first trimester legal abortions were randomized to vulvar and vaginal cleansing with chlorhexidine or vulvar cleansing only. The frequency of postabortion pelvic inflammatory disease was evaluated with patient questionnaires and study of medical records. Results. Of the 486 patients included in the study, vaginal cleansing was performed on 246 and no vaginal cleansing on 240. The frequency of probable pelvic inflammatory disease was 2·4% with cleansing and 2·1% without cleansing (no significant difference). Conc...

Positron emission tomography (PET) supplies a range of labelled compounds to be used for the characterization of tumour biochemistry. Some of these have proved to be of value for clinical diagnosis, treatment follow-up, and clinical research. (18)F-fluorodeoxyglucose PET scanning is now a widely accepted imaging approach in clinical oncology, reflecting increased expression of glucose transporters in cancerous tissue. This tracer, however, does not show sufficient uptake in well-differentiated tumours such as neuroendocrine tumours. Endocrine tumours have the unique characteristics of taking up and decarboxylating amine precursors. These so-called APUD characteristics offer highly specific targets for PET tracers. Using this approach, radiopharmaceuticals such as [(11)C]-5-hydroxytryptophan and [(11)C]-L-dihydroxyphenylalanine for localization of carcinoid and endocrine pancreatic tumours, 6-[(18)F]-fluorodopamine and [(11)C]-hydroxyephedrine for phaeochromocytomas, and [(11)C]-metomidate for adrenal cortical tumours have been developed. Functional imaging with PET using these compounds is now being employed to complement rather than replace other imaging modalities. Development of new PET radiopharmaceuticals may in the future allow in vivo detection of tumour biological properties, such as malignant potential and responsiveness to treatment.

The serotonin transporter radioligand [11C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, or [11C]DASB, was examined in order to assess its potential for measuring fluctuations in endogenous serotonin concentrations with positron emission tomography. Binding characteristics of [11C]DASB and the propensity for serotonin to displace the tracer were explored in rat brain homogenates. Experiments showed that serotonin displaced [11C]DASB in vitro. Ex vivo experiments performed after tranylcypromine injection (3 or 15 mg/kg) showed a dose-dependent trend in radioactivity uptake and suggested that serotonin may compete with [11C]DASB for transporter binding.

Objective: Triptans are highly effective in the treatment of migraine. Both central and peripheral mechanisms of action have been suggested. Until now, firm data about the passage of triptans into the CNS in humans have been lacking. The aim of the current study was to evaluate, using positron emission tomography (PET), the uptake and distribution of zolmitriptan into the CNS after intranasal administration. Subjects and methods: Eight healthy volunteers, five males and three females (mean ages 23 and 26 years, respectively), were included. Radioactive [carbonyl-11C]zolmitriptan was infused intravenously for 5 minutes on two occasions: once alone, and once 30–40 minutes after intranasal administration of unlabelled zolmitriptan 5mg. PET was used to measure the concentration of labelled zolmitriptan in the brain, from the start of the tracer infusion for 90 minutes. Regional cerebral blood volume was determined with [15O]carbon monoxide. In addition, an MRI scan was performed to obtain anatomical information. The PET images were analysed quantitatively for different areas of the brain, generating [11C]zolmitriptan time-activity data corrected for circulating tracer activity. The rate of uptake of intranasal zolmitriptan into the CNS was estimated by kinetic modelling using the PET data. Results: PET data from this study demonstrate a rapid dose-proportional uptake of [11C]zolmitriptan into the brain. Significant concentrations of [11C]zolmitriptan were found in all brain regions studied. Calculated CNS concentrations after intranasal zolmitriptan administration showed a gradual increase, reaching about 2nM (0.5 μg/L) 30 minutes after administration and 3.5nM (1.0 μg/L), or one-fifth of the plasma concentration, 1 hour after administration. Five minutes after zolmitriptan administration, the mean CNS concentration had already reached 0.5nM, which is higher than in vitro values for initiation of the agonistic action on 5-HT1B/1D receptors. Conclusion: This study demonstrates by direct measurements that zolmitriptan enters the brain parenchyma in humans, achieving an uptake rate and concentration compatible with a central mode of action.

Methods have been developed to label oligonucleotides (ODNs) in the 5′-position with 76 Br via a prosthetic group on a hexylamino-linker. The purpose of the study was to explore whether the labelling procedure would prevent specific hybridisation by using reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing of the PCR product. Antisense ODNs (30 mer, specific for rat Chromogranin A [CgA] mRNA) with phosphodiester (O-ODN) or phosphothioate (S-ODN) backbone, either unlabelled or labelled with 76 Br, served as one of the primers in individual PCR reactions. Using O-ODN as a primer, irrespective of being labelled or not, a selected 225-bp PCR fragment was successfully amplified. However, no amplification was obtained using S-ODN as a primer. The proper PCR products were only detected in the sample prepared from the adrenal gland, but not in that from the heart, liver or kidney. Autoradiographic recording of the gel, after gel electrophoresis, revealed radioactive signals corresponding to the amplified PCR products. The sequence of the PCR product matched the rat CgA mRNA sequence obtained from the EMBL database. RT-PCR is an attractive method to identify the selective binding of modified ODNs to target mRNA. This method confirmed that the labelling with 76 Br did not change the hybridisation ability of antisense O-ODN.

The purpose of this study was to investigate the potential use of PET in vivo to record cytotoxic effects of 2-methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol. The anti-proliferative and pro-apoptotic effects of 2-ME on human prostate cancer cell (PC3) aggregates in vitro, were correlated with the uptake of fluoro-deoxy-D-glucose, FMAU and choline labelled with 18F, 11C, or 3H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the uptake of the putative proliferation markers 11C-FMAU or 3H-choline failed to record the growth inhibitory effects of 2-ME on PC3 cell aggregates. The uptake of 18F-FDG was used as a marker for effects on cellular metabolism and also failed to show any dose-dependent effects in PC3 aggregates. The use of these PET-tracers in vivo is therefore not recommended in order to evaluate the cytotoxic effects of 2-ME on human prostate cancer cells.

For general oncological imaging, positron emission tomography (PET) using [18F]fluoro-deoxy-glucose (FDG) has evolved as a powerful functional imaging modality. Unfortunately, FDG-PET has not been as advantageous for imaging gastropancreatic neuroendocrine tumors, and only tumors with high proliferative activity and low differentiation have shown an increased FDG uptake. Therefore, the 11C-labeled amine precursors L-dihydroxyphenylalanine and 5-hydroxy-L-tryptophan (5-HTP) were developed for PET imaging of these tumors. Because of the higher tumor uptake of the latter tracer in a study of patients with endocrine pancreatic tumors, 11C-5-HTP was chosen for further evaluation. In comparative studies of patients with carcinoids and endocrine pancreatic tumors, 5-HTP-PET proved better than CT and somatostatin receptor scintigraphy for tumor visualization, and many small, previously overlooked lesions were diagnosed by 11C-5-HTP-PET. The strong correlation found during medical treatment between the changes in the transport rate constant at repeated PET and those of U-HIAA indicates the possible use of 11C-5-HTP-PET also for therapy monitoring. By premedication of patients with Carbidopa orally before PET examination, in order to block the aromatic amino acid decarboxylase enzyme, the decarboxylation rate of 11C-5-HTP was decreased, leading to a higher tumor uptake and a considerably lower urinary radioactivity concentration.

A spatially variant convolution subtraction scatter correction was developed for a Hamamatsu SHR-7700 animal PET scanner. This scanner, with retractable septa and a gantry that can be tilted 90 degrees, was designed for studies of conscious monkeys. The implemented dual-exponential scatter kernel takes into account both radiation scattered inside the object and radiation scattered in gantry and detectors. This is necessary because of the relatively large contribution of gantry and detector scatter in this scanner. The correction is used for scatter correction of emission as well as transmission data. Transmission scatter correction using the dual-exponential kernel leads to a measured attenuation coefficient of 0.096 cm(-1) in water, compared to 0.089 cm(-1) without scatter correction. Scatter correction on both emission and transmission data resulted in a residual correction error of 2.1% in water, as well as improved image contrast and hot spot quantification.

The positron emitting radionuclide 68Ga (T1/2 = 68 min) might become of practical interest for clinical positron emission tomography (PET). The metallic cation, 68Ga(III), is suitable for complexation with chelators, either naked or conjugated with biological macromolecules. Such labelling procedures require pure and concentrated preparations of 68Ga(III), which cannot be sufficiently fulfilled by the presently available 68Ge/68Ga generator eluate. This thesis presents methods to increase the concentration and purity of 68Ga obtained from a commercial 68Ge/68Ga generator. The use of the preconcentrated and purified 68Ga eluate along with microwave heating allowed quantitative 68Ga-labelling of peptide conjugates within 15 min. The specific radioactivity of the radiolabelled peptides was improved considerably compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial 68Ge/68Ga generator in combination with the method for preconcentration/purification and microwave heated labelling might result in an automated device for 68Ga-based radiopharmaceutical kit production with quantitative incorporation of 68Ga(III).Macromolecules were labelled with 68Ga(III) either directly or via a chelator. The bifunctional chelator, DOTA, was conjugated in solution to peptides, an antibody and oligonucleotides. The peptides had varied pI values, constitution, and length ranging from 8 to 53 amino acid residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a molecular weight up to 9.8 kDa. The bioconjugates were labeled with 68Ga(III), and the resulting tracers were characterised chemically and biologically. The identity of the 68Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity maintained. Specific radioactivity was shown to be an important parameter influencing the feasibility of accurate imaging data quantification.Furthermore, 68Ga-labelled peptide imaging was shown to be a useful tool to study peptide adsorption to microstructures in a chemical analysis device.

The steep rise in the incidence of Alzheimer's disease (AD) has further added to the considerable public health burden caused by aging of the United States population. Among the most characteristic pathologic hallmarks of AD are neuritic plaques and neurofibrillary tangles. The capability to use positron emission tomography and selective markers for amyloid protein deposition promises to substantially alter the way we diagnosis and manage patients who have AD.

The realisation that new chemical entities under development as drug candidates fail in three of four cases in clinical trials, together with increased costs and increased demands of reducing preclinical animal experiments, have promoted concepts for improvement of early screening procedures in humans. Positron emission tomography (PET) is a non-invasive imaging technology, which makes it possible to determine drug distribution and concentration in vivo in man with the drug labelled with a positron-emitting radionuclide that does not change the biochemical properties. Recently, developments in the field of rapid synthesis of organic compounds labelled with positron-emitting radionuclides have allowed a substantial number of new drug candidates to be labelled and potentially used as probes in PET studies. Together, these factors led to the logical conclusion that early PET studies, performed with very low drug doses-PET-microdosing-could be included in the drug development process as one means for selection or rejection of compounds based on performance in vivo in man. Another important option of PET, to evaluate drug interaction with a target, utilising a PET tracer specific for this target, necessitates a more rapid development of such PET methodology and validations in humans. Since only very low amounts of drugs are used in PET-microdosing studies, the safety requirements should be reduced relative to the safety requirements needed for therapeutic doses. In the following, a methodological scrutinising of the concept is presented. A complete pre-clinical package including limited toxicity assessment is proposed as a base for the regulatory framework of the PET-microdosing concept.

Background. Locally produced androgens and estrogens are important in the hormonal regulation of follicular development. The present study aimed to further elucidate the mechanism through which androgens exert their ambivalent effects on aromatization. Methods. Non-cultured human granulosa-luteal cells (GC) were treated with different concentrations of androstenedione (A 4 ), testosterone (T), 5α-androstane-3,17-dione (5α-A) and dihydrotestosterone (DHT). The effects on aromatase activity were evaluated in a tritiated water assay (incubation time 2 h) and the availability of aromatase active sites was measured in a radiotracer-binding assay using the non-steroidal competitive aromatase inhibitor [ 11 C]-vorozole (incubation time 15 min). Results. A 4 , T and 5α-A caused dose-dependent inhibition of both aromatase activity and [ 11 C]-vorozole binding; IC 50 -values for both inhibition processes were calculated for these three steroids, revealing A 4 as the most potent inhibitor and T and 5α-A as moderate ...

During the period February 1997 to April 2000, 15 patients with clinical symptoms of Creutzfeldt-Jakob disease (CJD) were referred to Uppsala University PET Centre. Positron emission tomography (PET) was performed to detect characteristic signs of the disease, e.g. neuronal death and/or astrocytosis in the brain. The examinations were performed in one session starting with oxygen-15 labelled water scan to measure regional cerebral blood flow, followed by imaging with the monoamine oxidase B inhibitor N-[(11)C-methyl]- L-deuterodeprenyl (DED) to assess astrocytosis in the brain and finally imaging with fluor-18 2-fluorodeoxyglucose (FDG) to assess regional cerebral glucose metabolism (rCMR(glu)) [corrected]. Nine of the patients fulfilled the clinical criteria of probable CJD. In eight of them, FDG and DED imaging revealed, in comparison with normal controls, a typical pattern characterized by a pronounced regional decrease (2SD) in glucose brain metabolism, indicative of neuronal dysfunction; this was accompanied by a similar increase (2SD) in DED binding, indicating astrocytosis. These changes were most pronounced in the cerebellum and the frontal, occipital and parietal cortices, whereas the pons, the thalamus and the putamen were less affected and the temporal cortex appeared unaffected. The cerebral blood flow showed a pattern similar to that observed with FDG. In the ninth patient, analysis with DED was not possible. The diagnosis of definite CJD according to international consensus criteria was confirmed in six of these patients. In one patient with probable CJD, protease-resistant prion protein (PrPres) could not be demonstrated. In two patients with probable CJD, autopsy was not allowed. Computed tomography and magnetic resonance imaging, performed in four and seven of these nine patients respectively, showed unspecific, mainly atrophic changes. In six other patients, the PET examinations gave a different pattern. In three of them, high rCMR(glu) was noticed in parts of the brain, particularly in the temporal lobes and basal ganglia, which could suggest encephalitis. One of the patients had Sjögren's syndrome, one had paraneoplastic limbic encephalitis and the third recovered spontaneously. In the other three patients, the DED binding was normal despite a hypometabolic glucose pattern. In conclusion, the PET findings obtained using DED and FDG paralleled neuropathological findings indicating neuronal dysfunction and astrocytosis, changes that are found in CJD.

Anabolic-androgenic steroids (AAS) have recently been shown to induce neurochemical alterations in areas of the male rat CNS related to behavioural changes that have been observed among AAS misusers. In the present study, positron emission tomography (PET) is suggested as a suitable in vivo method in order to visualize the density of the dopamine transporter ([11C]-FE-beta-CIT) as well as the dopamine D1-like ([11C]-(+)-SCH23390) and the D2-like receptors ([11C]-raclopride) in the male rat brain. Chronic treatment with the AAS nandrolone decanoate (15 mg/kg/day for 14 days) caused an up-regulation of the binding potential of the dopamine transporter in the striatum.

The binding characteristics of the novel 11C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[11C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [11C]MPA in comparison with (S)-[11C]nicotine and [11C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K(D) values) of 2.4 and 560 nM and binding site densities (Bmax values) of 65.5 and 223 fmol/mg of protein for (S)-[11C]nicotine, K(D) values of 0.011 and 2.2 nM and Bmax values of 4.4 and 70.7 fmol/mg of protein for [11C]MPA, and K(D) values of 1.3 and 33.4 nM and Bmax values of 8.8 and 69.2 fmol/mg of protein for [11C]ABT-418. In competing with the 11C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (-)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the 11C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [11C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three 11C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [11C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [11C] MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[11C]nicotine and [11C]ABT-418 raise the level of interest in [11C]MPAfor application in positron emission tomography.

Changes in regional cerebral blood flow (rCBF) and glucose metabolism are commonly associated with traumatic brain injury (TBI). Reactive oxygen species (ROS) have been implicated as key contributors to the secondary injury process after TBI. Here, pretreatment with the nitrone radical scavengers (alpha-phenyl-N-tert-butyl nitrone (PBN) or its sulfonated analogue sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) were used as tools to study the effects of ROS on rCBF and glucose metabolism after moderate (2.4-2.6 atm) lateral fluid percussion injury (FPI) in rats. S-PBN has a half-life in plasma of 9 min and does not penetrate the blood-brain barrier (BBB). In contrast, PBN has a half-life of 3 h and readily penetrates the BBB. Regional cerebral blood flow (rCBF) and glucose metabolism was estimated by using (99m)Tc-HMPAO and [(18)F]Fluoro-2-deoxyglucose (FDG) autoradiography, respectively, at 42 min (n = 37) and 12 h (n = 34) after the injury. Regions of interest were the parietal cortex and hippocampus bilaterally. As expected, FPI produced an early (42-min) hypoperfusion in ipsilateral cortex and an increase in glucose metabolism in both cortex and hippocampus, giving way to a state of hypoperfusion and decreased glucose metabolism at 12 h postinjury. On the contralateral side, a hypoperfusion in the cortex and hippocampus was seen at 12 h only, but no significant changes in glucose metabolism. Both S-PBN and PBN attenuated the trauma-induced changes in rCBF and glucose metabolism. Thus, the early improvement in rCBF and glucose metabolism correlates with and may partly mediate the improved functional and morphological outcome after TBI in nitrone-treated rats.

Positron emission tomography (PET) supplies a range of labeled compounds to be used for the characterization of tumor biochemistry. Some of these have proved to be of value for clinical diagnosis, treatment follow up, and clinical research. The first routinely used PET tracer in oncology, (18)F-labeled deoxyglucose (FDG), was successfully used for diagnosis of cancer, reflecting increased expression of glucose transporter in cancerous tissue. This tracer, however, usually does not show sufficient uptake in well-differentiated tumors such as neuroendocrine tumors. We developed a tracer more specific to neuroendocrine tumors-the serotonin precursor 5-hydroxytryptophan (5-HTP) labeled with (11)C-and demonstrated increased uptake and irreversible trapping of this tracer in carcinoid tumors. The uptake was so selective and the resolution was so high that we could detect more liver and lymph node metastases with PET than with CT or octreotide scintigraphy. To further improve the method, especially to reduce the high renal excretion of the tracer producing streaky artifacts in the area of interest, we introduced premedication by the decarboxylase inhibitor carbidopa, leading to a six-fold decreased renal excretion while the tumor uptake increased three-fold, hence improving the visualization of the tumors. (11)C-labeled l-DOPA was evaluated as an alternative tracer, especially for endocrine pancreatic tumors, which usually do not demonstrate enhanced urinary serotonin metabolites. However, only half of the EPTs, mainly functioning tumors, could be detected with l-DOPA. Instead 5-HTP seems to be a universal tracer for EPT and foregut carcinoids. With new, more sensitive PET cameras, larger field of view and procedures for whole-body coverage, the PET examination with 5-HTP is now routinely performed as reduced whole-body PET examinations with coverage of the thorax and abdomen. With this method we have been able to visualize small neuroendocrine lesions in the pancreas and thorax (e.g., ACTH-producing bronchial carcinoids) not detectable by any other method, including octreotide scintigraphy, MRI, and CT. Another tracer, the 11beta-hydroxylase inhibitor, metomidate labeled with (11)C, was developed to simplify diagnosis and follow-up of patients with incidentalomas. A large series of patients with incidentally found adrenal masses have been investigated and so far all lesions of adrenocortical origin have been easily identified because of exceedingly high uptake of (11)C-metomidate, whereas noncortical lesions showed very low uptake. In addition, adrenocortical cancer shows high uptake, suggesting that this PET tracer can be used for staging purposes.

Uncontrolled cell proliferation is one of the prominent features in cancer development. Precise tools are needed for determination of the proliferation rate before, during and after treatment, thereby permitting assessment of treatment efficacy. The purpose of this study was to validate the use of 5-[(76)Br]bromo-2'-fluoro-2'-deoxyuridine ((76)Br-BFU) as a proliferation marker in an animal tumour model. Comparison was made with 2-[(14)C]thymidine ((14)C-TdR) incorporation and the labelling index assessed by bromodeoxyuridine (BrdUrd-LI). Fibrosarcoma (NFSA)-bearing mice were used for all experiments. Gemcitabine (dFdC), a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. dFdC was injected intraperitoneally at a dose of 0.5 mg/kg or 40 mg/kg to induce partial ( approximately 50%) or complete inhibition of DNA synthesis, respectively. (76)Br-BFU (0.5-3 MBq per animal), (14)C-TdR (37-74 kBq per animal) and cold BrdUrd (60 mg/kg) were injected intraperitoneally in combination or alone. Animals were sacrificed at various times after tracer administration, and tumour and small intestine were removed for determination of radioactivity in whole tissue and the DNA fraction, as well as for LI assessment by flow cytometry. Cimetidine (6 mg/kg) was used to decrease (76)Br-BFU elimination and increase its bioavailability. The fraction of radioactivity associated with DNA increased with the time interval between tracer injection and tissue removal. At 6 h after injection, for both tracers, more than 95% of the radioactivity in the tumours was associated with the DNA fraction and an excellent correlation was observed with the LI. Similar findings were observed in the small intestine. Under all experimental conditions, (76)Br-BFU uptake was 4-10 times lower than (14)C-TdR uptake. Co-injection of cimetidine resulted in a three- to fourfold increase in (76)Br-BFU incorporation without affecting the effect of dFdC on DNA synthesis. (76)Br-BFU is a potentially good tracer for the assessment of tumour proliferation. It has all the specifications required of a PET tracer for clinical use. One limitation to its use is the necessity of co-injecting cimetidine to increase its bioavailability and hence its sensitivity for PET detection.

Inhibition of the V600E mutated BRAF kinase gene (BRAF(V600E) ) is an important and effective approach to treating melanomas. A new specific small molecule inhibitor of BRAF(V600E) , PLX3603, showed potent melanoma growth-inhibiting characteristics in preclinical studies and is currently under clinical investigation. In this study we investigated the feasibility of (18) F-FDG and (18) F-FLT-PET to monitor the early effects of the BRAF(V600E) inhibitor in mice with melanoma xenografts. SCID/beige mice with subcutaneous (s.c.) A375 melanoma xenografts, expressing BRAF(V600E) , received the BRAF(V600E) inhibitor twice daily orally (0, 25, 50 and 75 mg/kg). At 1, 3 and 7 days after start of therapy, the uptake of (18) F-FDG and (18) F-FLT in the tumor and normal tissues was determined in ex vivo tissue samples. Serial (18) F-FDG and (18) F-FLT-PET scans were acquired of animals at 1 day before and 1, 3 and 7 days after start of treatment with 75 mg/kg BRAF(V600E) inhibitor. A dose-dependent decrease in (18) F-FDG uptake in the A375 tumors was observed by ex vivo biodistribution analysis. Administration of 75 mg/kg BRAF inhibitor for 1, 3 and 7 days resulted in a significantly decreased (18) F-FDG uptake in A375 tumors (41, 35 and 51%, respectively). (18) F-FLT uptake in the A375 tumors was low at baseline and no significant changes in (18) F-FLT uptake were observed at any of the doses administered. These effects were corroborated by serial in vivo (18) F-FDG and (18) F-FLT-PET imaging. These data demonstrate that (18) F-FDG-PET can be used as an imaging biomarker to noninvasively evaluate the early effects of PLX3603.