Dissolution dynamic nuclear polarisation (dDNP) of 13 C-labelled pyruvate in magnetic resonance spectroscopy/imaging (MRS/MRSI) has the potential for monitoring tumour progression and treatment response. Pyruvate delivery, its metabolism to lactate and efflux were investigated in rat P22 sarcomas following simultaneous intravenous administration of hyperpolarised 13 C-labelled pyruvate (13 C1 -pyruvate) and urea (13 C-urea), a nonmetabolised marker. A general mathematical model of pyruvate-lactate exchange, incorporating an arterial input function (AIF), enabled the losses of pyruvate and lactate from tumour to be estimated, in addition to the clearance rate of pyruvate signal from blood into tumour, Kip , and the forward and reverse fractional rate constants for pyruvate-lactate signal exchange, kpl and klp . An analogous model was developed for urea, enabling estimation of urea tumour losses and the blood clearance parameter, Kiu . A spectral fitting procedure to blood time-course data proved superior to assuming a gamma-variate form for the AIFs. Mean arterial blood pressure marginally correlated with clearance rates. Kiu equalled Kip , indicating equivalent permeability of the tumour vasculature to urea and pyruvate. Fractional loss rate constants due to effluxes of pyruvate, lactate and urea from tumour tissue into blood (kpo , klo and kuo , respectively) indicated that T1 s and the average flip angle, θ, obtained from arterial blood were poor surrogates for these parameters in tumour tissue. A precursor-product model, using the tumour pyruvate signal time-course as the input for the corresponding lactate signal time-course, was modified to account for the observed delay between them. The corresponding fractional rate constant, kavail , most likely reflected heterogeneous tumour microcirculation. Loss parameters, estimated from this model with different TRs, provided a lower limit on the estimates of tumour T1 for lactate and urea. The results do not support use of hyperpolarised urea for providing information on the tumour microcirculation over and above what can be obtained from pyruvate alone. The results also highlight the need for rigorous processes controlling signal quantitation, if absolute estimations of biological parameters are required.

word count: 261 Text word count: 3927 Number of Tables: 2 Number of Figures: 4 Supplemental Information: 0

The rate of blood flow through a tissue (F) is a critical parameter for assessing the functional efficiency of a blood vessel network following angiogenesis. This chapter aims to provide the principles behind the estimation of F, how F relates to other commonly used measures of tissue perfusion, and a practical approach for estimating F in laboratory animals, using small readily diffusible and metabolically inert radio-tracers. The methods described require relatively nonspecialized equipment. However, the analytical descriptions apply equally to complementary techniques involving more sophisticated noninvasive imaging.Two techniques are described for the quantitative estimation of F based on measuring the rate of tissue uptake following intravenous administration of radioactive iodo-antipyrine (or other suitable tracer). The Tissue Equilibration Technique is the classical approach and the Indicator Fractionation Technique, which is simpler to perform, is a practical alternative in many cases. The experimental procedures and analytical methods for both techniques are given, as well as guidelines for choosing the most appropriate method.

Antagonism of 5-hydroxytrypamine-6 (5HT6) receptors is associated with procognitive effects in preclinical species, suggesting a therapeutic potential for this mechanism in Alzheimer disease (AD) and other cognitive diseases. In a phase 2 dose study, SB742457, a novel 5HT6 antagonist, showed increasing procognitive effects in patients with AD as the dose increased, with a procognitive signal in AD patients at a dose of 35 mg/d superior to the other doses tested (5 and 15 mg/d). Methods: In this article, we describe the quantification and pharmacologic selectivity of a new 5HT6 PET ligand (11C-GSK215083) in healthy volunteers and its use to measure occupancies achieved at various doses of SB742457. Results: Kinetic analysis of 11C-GSK215083 uptake in the human brain demonstrated the multilinear model, MA2, to represent the method of choice when a blood input was available and the full tissue reference method when no input was available. Pharmacologic dissection of the in vivo 11C-GSK215083–specific binding showed the ligand bound mostly the 5HT6 in the striatum (blocked by SB742457 but not by the selective 5-hydroxytryptamine-2A (5HT2A) antagonist ketanserin) and the 5HT2A in the frontal cortex (blocked by both ketanserin and SB742457). Repeated administration of SB742457 (3, 15, and 35 mg/d) saturated the 5HT6 receptors at all doses. In the cortex, 5HT2A receptor occupancy was 24% ± 6% (3 mg/d), 35% ± 4% (15 mg/d), and 58% ± 19% (35 mg/d; mean ± SD), suggesting a progressive engagement of 5HT2A as the dose increased. Conclusion: Collectively, these data support the use of 11C-GSK215083 as a 5HT6 clinical imaging tool and suggest that blocking both the 5HT6 and the 5HT2A receptors may be required for the optimal therapeutic action of SB742457 in AD.

Background The ability to quantify the capacity of a central nervous system (CNS) drug to cross the human blood-brain barrier (BBB) provides valuable information for de-risking drug development of new molecules. Here, we present a study, where a suitable positron emission tomography (PET) ligand was not available for the evaluation of a potent muscarinic acetylcholine receptor type-1 (M 1 ) allosteric agonist (GSK1034702) in the primate and human brain. Hence, direct radiolabelling of the novel molecule was performed and PET measurements were obtained and combined with in vitro equilibrium dialysis assays to enable assessment of BBB transport and estimation of the free brain concentration of GSK1034702 in vivo . Methods GSK1034702 was radiolabelled with 11 C, and the brain distribution of [ 11 C]GSK1034702 was investigated in two anaesthetised baboons and four healthy male humans. In humans, PET scans were performed (following intravenous injection of [ 11 C]GSK1034702) at baseline and after a single oral 5-mg dose of GSK1034702. The in vitro brain and plasma protein binding of GSK1034702 was determined across a range of species using equilibrium dialysis. Results The distribution of [ 11 C]GSK1034702 in the primate brain was homogenous and the whole brain partition coefficient ( V T ) was 3.97. In contrast, there was mild regional heterogeneity for GSK1034702 in the human brain. Human whole brain V T estimates (4.9) were in broad agreement with primate V T and the f P / f ND ratio (3.97 and 2.63, respectively), consistent with transport by passive diffusion across the BBB. Conclusion In primate and human PET studies designed to evaluate the transport of a novel M 1 allosteric agonist (GSK1034702) across the BBB, we have demonstrated good brain uptake and BBB passage consistent with passive diffusion or active influx. These studies discharged some of the perceived development risks for GSK1034702 and provided information to progress the molecule into the next stage of clinical development. Trial registration Clinical trial details: ‘Brain Uptake of GSK1034702: a Positron Emission Tomography (PET) Scan Study.’; clinicaltrial.gov identifier: NCT00937846 .

Abnormalities of the tumour vasculature and their consequences on the microenvironment of tumour cells impact on tumour progression and response to both blood-borne anti-cancer agents and radio-therapy, as well as making tumour blood vessels a target for therapy in their own right. Intravital microscopy of experimental tumours, most commonly grown in ‘window’ chambers, such as the dorsal skin fold chamber in mice and rats, enables investigations of tumour microcirculatory function. This is needed both to understand the molecular control of tumour vascular function and to measure the response of the vasculature to treatment. In particular, intravital microscopy enables parameters associated with blood supply, vascular permeability and oxygenation to be estimated, at high spatial and temporal resolution. In this chapter, methods used for measuring a range of these parameters, specific examples of their applications, the significance of findings and some of the limitations of the techniques are described.

word count: 261 Text word count: 3927 Number of Tables: 2 Number of Figures: 4 Supplemental Information: 0

The inhibitory γ-aminobutyric acid (GABA) neurotransmitter system is associated with the regulation of normal cognitive functions and dysregulation has been reported in a number of neuropsychiatric disorders including anxiety disorders, schizophrenia and addictions. Investigating the role of GABA in both health and disease has been constrained by difficulties in measuring acute changes in synaptic GABA using neurochemical imaging. The aim of this study was to investigate whether acute increases in synaptic GABA are detectable in the living human brain using the inverse agonist GABA-benzodiazepine receptor (GABA-BZR) positron emission tomography (PET) tracer, [11C]Ro15-4513. We examined the effect of 15mg oral tiagabine, which increases synaptic GABA by inhibiting the GAT1 GABA uptake transporter, on [11C]Ro15-4513 binding in 12 male participants using a paired, double blind, placebo-controlled protocol. Spectral analysis was used to examine synaptic α1 and extrasynaptic α5 GABA-BZR subtype availability in brain regions with high levels of [11C]Ro15-4513 binding. We also examined the test–retest reliability of α1 and a5-specific [11C]Ro15-4513 binding in a separate cohort of 4 participants using the same spectral analysis protocol. Tiagabine administration produced significant reductions in hippocampal, parahippocampal, amygdala and anterior cingulate synaptic α1 [11C]Ro15-4513 binding, and a trend significance reduction in the nucleus accumbens. These reductions were greater than test–retest reliability, indicating that they are not the result of chance observations. Our results suggest that acute increases in endogenous synaptic GABA are detectable in the living human brain using [11C]Ro15-4513 PET. These findings have potentially major implications for the investigation of GABA function in brain disorders and in the development of new treatments targeting this neurotransmitter system.

The characterisation of a pharmacokinetic-receptor occupancy (PK-RO) relationship derived from a PET study is typically modelled in a conventional non-linear least squares (NLLS) framework. In the present work, we explore the application of a non-linear mixed effects approach (NLME) and compare this with NLLS estimation (using both naive pooled data and two-stage approaches) in the context of a direct PK-RO relationship described by an Emax model, using simulated data sets. Target and reference tissue time-activity curves were simulated using a two-tissue compartmental model and an arterial plasma input function for a typical PET study (12 subjects in 3 dose groups with 3 scans each). A range of different PET scenarios was considered to evaluate the impact of between-subject variability and reference region availability. The PET outcome measures derived from the simulations were then used to estimate the parameters of the PK-RO model. The performance of the two approaches was compared in terms of parameters estimates (square mean error SME, root mean square error RMSE) and prediction of the exposure-occupancy relationship. In general, both NLME and NLLS estimation methods provided unbiassed and precise population estimates for the Emax model parameters, although a slight bias was observed for the individual-NLLS method due to a few outliers. The increased value of NLME over NLLS was most notable in the estimation of the between-subject variability (BSV), especially in the case of a more complex PK-RO model when no reference region was available (maximum SME and RMSE values related to BSV of EC₅₀ of 27.6% and 86.5% from NLME versus 264.6% and 689.5% from NLLS). Overall, the NLME approach provided a more robust estimation and produced less-biassed estimates of the population means and variances than either the NLLS approach for the simulations considered.

Mathematical models are required to estimate kinetic parameters of [1-(13)C] pyruvate-lactate interconversion from magnetic resonance spectroscopy data. One- or two-way exchange models utilizing a hypothetical approximation to the true arterial input function (AIF), (e.g. an ideal 'box-car' function) have been used previously. We present a method for direct measurement of the AIF in the rat. The hyperpolarized [1-(13)C] pyruvate signal was measured in arterial blood as it was continuously withdrawn through a small chamber. The measured signal was corrected for T1 relaxation of pyruvate, RF pulses and dispersion of blood in the chamber to allow for the estimation of the direct AIF. Using direct AIF, rather than the commonly used box-car AIF, provided realistic estimates of the rate constant of conversion of pyruvate to lactate, kpl, the rate constant of conversion of lactate to pyruvate klp, the clearance rate constant of pyruvate from blood to tissue, Kip, and the relaxation rate of lactate T1la. Since no lactate signal was present in blood, it was possible to use a simple precursor-product relationship, with the tumor tissue pyruvate time-course as the input for the lactate time-course. This provided a robust estimate of kpl, similar to that obtained using a directly measured AIF.

The development of a PET radioligand for imaging 5-hydroxytryptamine (5-HT) 6 receptors in the brain would, for the first time, enable in vivo imaging of this target along with assessment of its involvement in disease pathophysiology. In addition, such a tool would assist in the development of novel drugs targeting the 5-HT6 receptor. Methods: On the basis of in vitro data, GSK215083 was identified as a promising 5-HT6 radioligand candidate and was radiolabeled with 11C via methylation. The in vivo properties of 11C-GSK215083 were evaluated first in pigs (to investigate brain penetration and specific binding), second in nonhuman primates (to confirm brain penetration, specific binding, selectivity, and kinetics), and third in human subjects (to confirm brain penetration and biodistribution). Results: 11C-GSK215083 readily entered the brain in all 3 species, leading to a heterogeneous distribution (striatum > cortex > cerebellum) consistent with reported 5-HT6 receptor densities and distribution determined by tissue-section autoradiography in preclinical species and humans. In vivo saturation studies using escalating doses of GSK215083 in primates demonstrated saturable, dose-dependent binding to the 5-HT6 receptor in the striatum. Importantly, 11C-GSK215083 also exhibited affinity for the 5-HT2A receptor; however, given the differential localization of these 2 receptors in the central nervous system, the discrete 5-HT6 binding properties of this radioligand were able to be determined. Conclusion: These data demonstrate the utility of 11C-GSK215083 as a promising PET radioligand for probing the 5-HT6 receptor in vivo in both preclinical and clinical species.

co-injection of hyperpolarized C pyruvate and urea in combination with measurement of arterial input functions Samira Kazan, Steven Reynolds, Tooba Alizadeh, Adriana Bucur, Aneurin Kennerley, Vincent Cunningham, Martyn Paley, and Gillian Tozer CR-UK/YCR Sheffield Cancer Research Centre, University of Sheffield, Sheffield, South Yorkshire, United Kingdom, Academic Unit of Radiology, University of Sheffield, Sheffield, South Yorkshire, United Kingdom, Signal Processing in Neuroimaging and Systems Neuroscience, University of Sheffield, Sheffield, South Yorkshire, United Kingdom, Aberdeen Biomedical Imaging Centre, University of Aberdeen, Aberdeen, Scotland, United Kingdom

The evaluation of drug pharmacodynamics and early tumour response are integral to current clinical trials of novel cancer therapeutics to explain or predict long term clinical benefit or to confirm dose selection. Tumour vascularity assessment by positron emission tomography could be viewed as a generic pharmacodynamic endpoint or tool for monitoring response to treatment. This review discusses methods for semi-quantitative and quantitative assessment of tumour vascularity. The radioligands and radiotracers range from direct physiological functional tracers like [(15)O]-water to macromolecular probes targeting integrin receptors expressed on neovasculature. Finally we make recommendations on ways to incorporate such measurements of tumour vascularity into early clinical trials of novel therapeutics. Key Points • [ ( 15 ) O]-water is the gold standard for blood flow/tissue perfusion with PET • In some instances dynamic [ ( 18 ) F]-FDG uptake may be used to estimate perfusion • Radiopharmaceuticals that target integrins are now being evaluated for measuring tumour vascularity.

There is growing recognition of the importance of integrating drug occupancy data acquired by positron emission tomography (PET) with the plasma pharmacokinetics of the drug, in order to establish proper dose selection in subsequent clinical trials. Here we present a study in human subjects of the occupancy of NK1 receptors achieved following different doses of casopitant, a selective NK1 antagonist.

Positron emission tomography (PET) is used in drug development to assist dose selection and to establish the relationship between blood and tissue pharmacokinetics (PKs). We present a new biomathematical approach that allows prediction of repeat-dose (RD) brain target occupancy (TO) using occupancy data obtained after administration of a single dose (SD). A PET study incorporating a sequential adaptive design was conducted in 10 healthy male adults who underwent 4 PET scans with [(11)C]DASB ([(11)C]N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine): 1 at baseline, 2 after 20 mg SD of the 5-hydroxytryptamine transporter (5-HTT) inhibitor duloxetine, and 1 after 4 days daily administration of 20 mg duloxetine. An adaptive design was used to select optimal times after SD for measurement of occupancy. Both direct and indirect PK/TO models were fitted to the SD data to characterise the model parameters and then applied to a predicted RD duloxetine plasma time course to predict the 5-HTT occupancy after RD. Repeat-dose prediction from the indirect model (OC(50)=2.62±0.93 ng/mL) was significantly better (P<0.05) than that from the direct model (OC(50)=2.29±1.11 ng/mL). This approach increases the value of SD occupancy studies that are performed as part of first time in human drug development programmes by providing an estimate of the dose required to achieve the desired TO at RD.

Here we describe methods for application of quantitative fluorodeoxyglucose positron emission tomography ([F-18] FDG-PET) measures of brain glucose metabolism in multi-centre clinical trials for Alzheimer's disease. We validated methods and demonstrated their use in the context of a treatment trial with the PPAR gamma agonist Rosiglitazone XR versus placebo in mild to moderate AD patients. Novel quantitative indices related to the combined forward rate constant for [F-18] FDG uptake (K-i(index)) and to the rate of cerebral glucose utilization (CMRgluindex) were applied. Active treatment was associated with a sustained but not statistically significant trend from the first month for higher mean values in both. However, neither these nor another analytical approach recently validated using data from the Alzheimer's Disease Neuroimaging Initiative suggested that active treatment decreased the progression of decline in brain glucose metabolism. Rates of brain atrophy were similar between activeand placebo groups and measures of cognition also did not demonstrate clear group differences. Our study demonstrates the feasibility of using [F-18]FDG-PET as part of a multi-centre therapeutics trial and describes new measures that can be employed. It suggests that Rosiglitazone is associated with an early increase in whole brain glucose utilisation, but not with any biological or clinical evidence for slowing progression over the period of study in the selected patient group.