Imaging techniques to study drug transporter function in vivo

Transporter systems involved in the permeation of drugs and solutes across biological membranes are recognized as key determinants of pharmacokinetics. Typically, the action of membrane transporters on drug exposure to tissues in living organisms is inferred from invasive procedures, which cannot be applied in humans. In recent years, imaging methods have greatly progressed in terms of instruments, synthesis of novel imaging probes as well as tools for data analysis. Imaging allows pharmacokinetic parameters in different tissues and organs to be obtained in a non-invasive or minimally invasive way. The aim of this overview is to summarize the current status in the field of molecular imaging of drug transporters. The overview is focused on human studies, both for the characterization of transport systems for imaging agents as well as for the determination of drug pharmacokinetics, and makes reference to animal studies where necessary. We conclude that despite certain methodological limitations, imaging has a great potential to study transporters at work in humans and that imaging will become an important tool, not only in drug development but also in medicine. Imaging allows the mechanistic aspects of transport proteins to be studied, as well as elucidating the influence of genetic background, pathophysiological states and drug-drug interactions on the function of transporters involved in the disposition of drugs

Relevance of in vitro metabolism models for PET radiotracer development

The utility of in vitro metabolism models for the development of positron emission tomography (PET) radiotracers was evaluated using three xanthine-derived adenosine A1 receptor ligands, 8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), 8-cyclobutyl-3-(3-fluoropropyl)-1-propylxanthine (CBX) and 3-(3-fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine (MCBX), as model compounds. In vivo metabolic stability and metabolite patterns of the three compounds were investigated in the rat model and compared to in vitro data generated in rat liver microsomes. Following optimisation of the microsomal assay conditions, in vitro half-lives of the test compounds were determined and expressed as ratios for the purpose of in vitro-in vivo comparison. The half-life ratios (± SE) of CBX:CPFPX, MCBX:CPFPX and CBX:MCBX were 3.1 ± 0.11, 1.4 ± 0.029 and 2.2 ± 0.033. In vivo metabolic stability of the 18F-labelled compounds was assessed in anaesthetised rats via blood analysis. Plasma clearance values were calculated and expressed as inversed ratios to facilitate direct comparison with in vitro half-life ratios. The inversed clearance ratios (± SE) of [18F]CBX:[18F]CPFPX, [18F]MCBX:[18F]CPFPX and [18F]CBX:[18F]MCBX were 2.6 ± 0.12, 0.82 ± 0.019 and 3.1 ± 0.15. In vitro half-life ratios deviated between 19 and 71% from inversed clearance ratios. These deviations can be considered small in view of the reduced complexity of the microsomal model and the multitude of physiological parameters affecting in vivo pharmacokinetics of a substance. Visual comparison of metabolite profiles generated in vitro and in vivo revealed a high degree of similarity. In conclusion, both quantitative and qualitative aspects of radiotracer metabolism could be reasonably well predicted by microsomal data. This result encourages the implementation of in vitro metabolism studies as an integral part of PET radiotracer development

Measuring serotonin synthesis: from conventional methods to PET tracers and their (pre)clinical implications

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are α-[11C]methyltryptophan ([11C]AMT) and 5-hydroxy-L-[β-11C]tryptophan ([11C]5-HTP). Both tracers have advantages and disadvantages. [11C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [11C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain

Assessment of Serotonergic Function by Radioligands and Microdialysis:focus on stress-related behaviour and antidepressant efficacy

Serotonine is een chemische stof die betrokken is bij het doorgeven van signalen in de hersenen en speelt o.a. een rol bij het regelen van de gemoedstoestand, zoals in depressie. De meeste antidepressiva zijn erop gericht om serotonine concentraties in het menselijk brein te verhogen, maar deze geneesmiddelen lijken bij een deel van de patiënten niet erg goed te werken. De reden hiervan is niet duidelijk. Daarom is het van belang om te kunnen meten wat er met de signaaloverdracht door serotonine gebeurt onder invloed van stress en na behandeling met antidepressiva. Een methode om dit op een non-invasieve manier te meten is met positron emissie tomografie (PET). De studies die in dit poefschrift worden beschreven betreffen de validatie van radioactief gemerkte chemische stoffen, of PET tracers, die de aanmaak van serotonine ([11C]5-HTP) en de gevoeligheid van 5-HT2A receptoren voor serotonine ([11C]MDL 100907) in de hersenen kunnen meten. De laatstgenoemde stof bleek zeer geschikt. Hiermee hebben we aangetoond dat 5-HT2A receptoren waarschijnlijk geen essentiële rol spelen bij het omgaan met stress (coping style) en de reactie hierop. Daarnaast hebben we gekeken of de effectiviteit van antidepressiva verbeterd kan worden door aan serotonine heropname remmers (SSRIs) een specifieke 5-HT2C receptor blokker toe te voegen. Door deze geneesmiddelcombinatie lijken de niveaus van zowel serotonine als dopamine, een neurotransmitter betrokken bij motivatie, in de hersenen te kunnen worden verhoogd. PET lijkt een veelbelovende methode om serotonine signaaloverdracht te meten. Toekomstig onderzoek zal moeten uitwijzen of andere onderdelen betrokken bij serotonine signaaloverdracht belangrijk zijn voor stressgerelateerd gedrag. Daarnaast is het van belang om de effecten van antidepressiva op serotonine signaaloverdracht te meten en deze te relateren aan veranderingen in gedrag en gemoedstoestand

Monitoring the Function of the P-glycoprotein Transporter at the Blood Brain Barrier

The P-glycoprotein (P-gp) transporter located at the blood-brain barrier (BBB) is an efflux transporter that pumps neurotoxic compounds out of the brain. Its main function is to protect the brain to ensure an appropriate neural function. Decreases in the P-gp function can result in increased accumulation of toxic compounds inside the brain such as beta-amyloid and this may cause the development of Alzheimer´s or other neurological disorders. By contrast, increases in the P-gp function can decrease the therapeutic drug concentration inside the brain and influence the efficacy of the treatment (drug resistance) as occurred in patients with intractable epilepsy. Thus, it is of interest to monitor the P-gp function in vivo to facilitate the early diagnosis of brain disorders and to monitor drug resistance. To this aim, we used Positron Emission Tomography (PET) imaging, a non-invasive technique that allows the quantification of biological processes in vivo, and the novel radiotracer [18F]MC225 which measures the P-gp function. The aim was to study the kinetic properties of the radiotracer in different species and prove its efficacy to measure increases and decrease in the P-gp function before its clinical evaluation. We conclude that the obtained results have broadened the knowledge of the P-gp function at the BBB. Moreover, the results highlight that [18F]MC225 may become the first radiofluorinated P-gp PET tracer able to measure both decreases and increases in the P-gp function in vivo. The radiolabeling with fluorine-18 would allow its distribution to other PET centers and improve the image quality

Quantification methods for brain imaging with novel and repurposed PET tracers

The number of people suffering from brain disorders is annually increasing. Knowledge about the molecular processes in the healthy and diseased brain is essential for a better understanding of disease conditions, treatment selection, and drug development. Positron emission tomography (PET) is a noninvasive imaging technique that can be used to acquire information about processes that are essential for normal brain functioning, but are altered in neurodegenerative diseases. Quantitative information about specific targets inside the brain, such as the density, activity, or occupancy of particular enzymes, transporters, or receptors, can be obtained by pharmacokinetic modeling of PET data. In the present study, we assessed quantification methods for brain imaging with novel and repurposed PET tracers. A PET tracer for inflammation in the brain, called [11C]SC-560, was evaluated, but overexpression of the inflammatory marker COX-1, could not be detected in the inflamed rat brain. Thus, more efforts to find an appropriate tracer are required. Next, we determined the optimal method for quantification of histamine H3 receptors in the rat brain, using PET and the radiotracer [11C]GSK-189254. Blockade of these receptors may improve cognition in patients with dementia. [11C]GSK-189254 PET and [11C]raclopride PET were subsequently used to measure the dose-dependent occupancy of histamine H3 and dopamine D2 receptors in the brain of living rats by the investigational drug AG-0029. D2 receptors play an important role in motor control. Since AG-0029 blocks histamine H3 receptors and stimulates dopamine D2 receptors, AG-0029 is a candidate drug for treatment of Parkinson disease. Finally, we evaluated the feasibility of quantifying the expression of estrogen receptors in the brains of post-menopausal women with [18F]FES PET. We were able to detect estrogen receptors in brain regions with a high density of the receptor (i.e., the pituitary). The methods described in this study may be used to enhance knowledge about the brain, the treatment of brain diseases and the development of novel drugs

Current Aspects of Radiopharmaceutical Chemistry

Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are in vivo molecular imaging techniques which are widely used in nuclear medicine for the diagnosis and treatment follow-up of many major diseases. They use biomolecules as probes, which are labeled with radionuclides of short half-lives, synthesized prior to the imaging studies. These probes are called radiopharmaceuticals. Their design and development require a rather interdisciplinary process involving many different disciplines of natural and health sciences. In addition to their diagnostic and therapeutic purposes in the field of nuclear medicine, radiopharmaceuticals provide powerful tools for in vivo pharmacology during the process of pre-clinical drug development to identify new drug targets, investigate the pathophysiology of diseases, discover potential drug candidates, and evaluate the pharmacokinetics and pharmacodynamics of drugs in vivo. Furthermore, they allow molecular imaging studies in various small-animal models of disease, including genetically engineered animals. The current collection of articles provides unique examples covering all major aspects in the field

Minimally Invasive Pharmacokinetic and Pharmacodynamic Technologies in Hypothesis-Testing Clinical Trials of Innovative Therapies

Clinical trials of new cancer drugs should ideally include measurements of parameters such as molecular target expression, pharmacokinetic (PK) behavior, and pharmacodynamic (PD) endpoints that can be linked to measures of clinical effect. Appropriate PK/PD biomarkers facilitate proof-of-concept demonstrations for target modulation; enhance the rational selection of an optimal drug dose and schedule; aid decision-making, such as whether to continue or close a drug development project; and may explain or predict clinical outcomes. In addition, measurement of PK/PD biomarkers can minimize uncertainty associated with predicting drug safety and efficacy, reduce the high levels of drug attrition during development, accelerate drug approval, and decrease the overall costs of drug development. However, there are many challenges in the development and implementation of biomarkers that probably explain their disappointingly low implementation in phase I trials. The Pharmacodynamic/Pharmacokinetic Technologies Advisory committee of Cancer Research UK has found that submissions for phase I trials of new cancer drugs in the United Kingdom often lack detailed information about PK and/or PD endpoints, which leads to suboptimal information being obtained in those trials or to delays in starting the trials while PK/PD methods are developed and validated. Minimally invasive PK/PD technologies have logistic and ethical advantages over more invasive technologies. Here we review these technologies, emphasizing magnetic resonance spectroscopy and positron emission tomography, which provide detailed functional and metabolic information. Assays that measure effects of drugs on important biologic pathways and processes are likely to be more cost-effective than those that measure specific molecular targets. Development, validation, and implementation of minimally invasive PK/PD methods are encourage

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aβ) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aβ plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aβ at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aβ plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aβ accumulation, suggesting that BChE is a promising early biomarker for incipient AD