Multivariate analysis of PET pharmacokinetic parameters improves inferential efficiency

Purpose In positron emission tomography quantification, multiple pharmacokinetic parameters are typically estimated from each time activity curve. Conventionally all but the parameter of interest are discarded before performing subsequent statistical analysis. However, we assert that these discarded parameters also contain relevant information which can be exploited to improve the precision and power of statistical analyses on the parameter of interest. Properly taking this into account can thereby draw more informative conclusions without collecting more data. Methods By applying a hierarchical multifactor multivariate Bayesian approach, all estimated parameters from all regions can be analysed at once. We refer to this method as Parameters undergoing Multivariate Bayesian Analysis (PuMBA). We simulated patient–control studies with different radioligands, varying sample sizes and measurement error to explore its performance, comparing the precision, statistical power, false positive rate and bias of estimated group differences relative to univariate analysis methods. Results We show that PuMBA improves the statistical power for all examined applications relative to univariate methods without increasing the false positive rate. PuMBA improves the precision of effect size estimation, and reduces the variation of these estimates between simulated samples. Furthermore, we show that PuMBA yields performance improvements even in the presence of substantial measurement error. Remarkably, owing to its ability to leverage information shared between pharmacokinetic parameters, PuMBA even shows greater power than conventional univariate analysis of the true binding values from which the parameters were simulated. Across all applications, PuMBA exhibited a small degree of bias in the estimated outcomes; however, this was small relative to the variation in estimated outcomes between simulated datasets. Conclusion PuMBA improves the precision and power of statistical analysis of PET data without requiring the collection of additional measurements. This makes it possible to study new research questions in both new and previously collected data. PuMBA therefore holds great promise for the field of PET imaging

Validation and test-retest repeatability performance of parametric methods for [11C]UCB-J PET

[(11)C]UCB-J is a PET radioligand that binds to the presynaptic vesicle glycoprotein 2A. Therefore, [(11)C]UCB-J PET may serve as an in vivo marker of synaptic integrity. The main objective of this study was to evaluate the quantitative accuracy and the 28-day test–retest repeatability (TRT) of various parametric quantitative methods for dynamic [(11)C]UCB-J studies in Alzheimer’s disease (AD) patients and healthy controls (HC). Eight HCs and seven AD patients underwent two 60-min dynamic [(11)C]UCB-J PET scans with arterial sampling over a 28-day interval. Several plasma-input based and reference-region based parametric methods were used to generate parametric images using metabolite corrected plasma activity as input function or white matter semi-ovale as reference region. Different parametric outcomes were compared regionally with corresponding non-linear regression (NLR) estimates. Furthermore, the 28-day TRT was assessed for all parametric methods. Spectral analysis (SA) and Logan graphical analysis showed high correlations with NLR estimates. Receptor parametric mapping (RPM) and simplified reference tissue model 2 (SRTM2) BP(ND), and reference Logan (RLogan) distribution volume ratio (DVR) regional estimates correlated well with plasma-input derived DVR and SRTM BP(ND). Among the multilinear reference tissue model (MRTM) methods, MRTM1 had the best correspondence with DVR and SRTM BP(ND). Among the parametric methods evaluated, spectral analysis (SA) and SRTM2 were the best plasma-input and reference tissue methods, respectively, to obtain quantitatively accurate and repeatable parametric images for dynamic [(11)C]UCB-J PET. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13550-021-00874-8

The alpha2C-adrenoceptor as a neuropsychiatric drug tar-get - PET studies in human subjects

Positron emission tomography imaging has both academic and applied uses in revealing the distribution and density of different molecular targets in the central nervous system. Following the significant progress made with the dopamine D2 receptor, advances have been made in developing PET tracers to allow analysis of receptor occupancy of many other receptor types as well as evaluating changes in endogenous synaptic transmitter concentrations of transmitters e.g. serotonin and noradrenaline. Noradrenergic receptors are divided into α1-, α2- and β-adrenoceptor subfamilies, in humans each of which is composed of three receptor subtypes. The α2-adrenoceptors have an important presynaptic auto-inhibitory function on noradrenaline release but they also have postsynaptic roles in modulating the release of other neurotransmitters, such as serotonin and dopamine. One of the subtypes, the α2C-adrenoceptor, has been detected at distinct locations in the central nervous system, most notably the dorsal striatum. Several serious neurological conditions causing dementia, Alzheimer’s disease and Parkinson’s disease have been linked to disturbed noradrenergic signaling. Furthermore, altered noradrenergic signaling has also been implicated in conditions like ADHD, depression, anxiety and schizophrenia. In order to benefit future research into these central nervous system disorders as well as being useful in the clinical development of drugs affecting brain noradrenergic neurotransmission, validation work of a novel tracer for positron emission tomography studies in humans was performed. Altogether 85 PET imaging experiments were performed during four separate clinical trials. The repeatability of [11C]ORM-13070 binding was tested in healthy individuals, followed by a study to evaluate the dose-dependent displacement of [11C]ORM-13070 from α2C-adrenoceptors by a competing ligand, and the final two studies examined the sensitivity of [11C]ORM-13070 binding to reflect changes in endogenous noradrenaline levels. The repeatability of [11C]ORM-13070 binding was very high. The binding properties of the tracer allowed for a reliable estimation of α2C-AR occupancy by using the reference tissue ratio method with low test-retest variability. [11C]ORM-13070 was dose-dependently displaced from its specific binding sites by the subtype-nonselective α2-adrenoceptor antagonist atipamezole, and thus it proved suitable for use in clinical drug development of novel α2C-adrenoceptor ligands e.g. to determine the best doses and dosing intervals for clinical trials. Convincing experimental evidence was gained to support the suitability of [11C]ORM-13070 for detecting an increase in endogenous synaptic noradrenaline in the human brain. Tracer binding in the thalamus tended to increase in accordance with reduced activity of noradrenergic projections from the locus coeruleus, although statistical significance was not reached. Thus, the investigation was unable to fully validate [11C]ORM-13070 for the detection of pharmacologically evoked reductions in noradrenaline levels.Siirretty Doriast

Development of novel radiotracers as tools for imaging the human brain

Introduction: Brain imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the processes underlying neurological and psychiatric disorders. In addition, in vivo brain imaging using SPECT or PET may provide new approaches for drug target identification, pre-clinical testing and occupancy studies, and therefore improve drug discovery. The utility of in vivo brain imaging using SPECT or PET relies on the ability of different radiotracers (typically organic compounds labelled with radionuclides) to bind to a wide variety of targets, including receptors, transporters and enzymes. Therefore the development of novel radiotracers for in vivo brain imaging using SPECT of PET is of vital importance. This thesis is focused on the process of developing novel radiotracers as tools for imaging the human brain, where the radiotracer discovery and development pipeline is discussed and each step prior to clinical trials investigated. Radiotracer discovery: Previously, discovery of novel brain radiotracers has largely relied on simplistic screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. This thesis investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. In this study, ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (Pm); percentage of plasma protein binding (%PPB); and membrane partition coefficient (Km). Relationships between brain entry measurements (Log P, Pm and %PPB) and in vivo brain percentage injected dose (%ID); and Km and specific binding in vivo (BPND) were investigated. Results showed that HPLC measurements of Pm, %PPB and Km were potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery. The HPLC tool developed provides information on in vivo non-specific binding and binding potential that is not possible using conventional screening methods. Another important finding reported in this thesis is that Log P should not be relied on as a predictor of brain entry. The HPLC tool developed, together with competition binding assays, was used to characterise a newly synthesised library of compounds for imaging of the translocator protein (TSPO) in brain using SPECT. Results showed that compound LS 1 was the most likely to succeed within the library investigated, but the high %PPB observed for LS 1 suggested novel compounds with improved %PPB were needed. Thus, a novel library of compounds for imaging of TSPO in brain using SPECT is currently been developed for future testing using the HPLC tool developed here and competition binding assays. Pre-clinical research: radiotracers for imaging the noradrenaline transporter (NAT) in brain using SPECT. In this thesis, NKJ64, a novel iodinated analogue of reboxetine, was successfully radiolabelled via electrophilic iododestannylation and evaluated as a potential SPECT radiotracer for imaging the NAT in brain using rodents and non-human primates. Biological evaluation of the novel radiotracer, 123/125I-NKJ64, in rodents included: in vitro ligand binding assays; in vitro and ex vivo autoradiography; in vivo biodistribution studies and ex vivo pharmacological blocking studies. In rats, 123/125I-NKJ64 displayed saturable binding with nanomolar affinity for the NAT in cortical homogenates, regional distribution consistent with the known density of NAT in the rodent brain and high maximum brain uptake of around 2.93 % of the injected dose. The specific: non-specific ratio (locus coeruleus:caudate putamen) of 123I-NKJ64 uptake was 2.8 at 30 minutes post intravenous injection and prior administration of reboxetine significantly reduced the accumulation of 123I-NKJ64 in the locus coeruleus (> 50% reduction). Data obtained using rodents indicated that further evaluation of 123I-NKJ64 in non-human primates was needed to determine its utility as a SPECT radiotracer for imaging of NAT in brain. Consequently, in vivo kinetic modelling studies using SPECT imaging with 123I-NKJ64 and two baboons were carried out to determine 123I-NKJ64 brain binding kinetics, brain distribution and plasma metabolism in non-human primates. Even though a high brain uptake of around 3.0% of the injected dose was determined, the high non-specific binding observed throughout the brain, a low binding potential (BPND<2) in NAT rich regions and a brain distribution that was inconsistent with the known NAT distribution in non-human primate brain precludes the translation of 123I-NKJ64 into humans. Another NAT radiotracer, 123I-INER, developed by Tamagnan and colleagues at Yale University and Institute for Degenerative Disorders, New Haven, USA, was also investigated as part of this thesis. Kinetic modelling analysis of 123I-INER in baboon brain was investigated for different models, namely invasive and reference tissue models. Bolus plus constant infusion experiments with displacement at equilibrium using six different doses of atomoxetine and four different doses of reboxetine were carried out in several baboons to obtain occupancy measurements as a function of injected dose (mg/kg) for the two NAT selective drugs. Results showed that reference tissue models were able to determine BPND values of 123I-INER in different brain regions. In addition the volume of distribution could be determined by dividing concentration in tissue by the concentration in venous blood at 3 hours post-injection. After administration of atomoxetine or reboxetine, dose-dependent occupancy was observed in brain regions known to contain high densities of NATs. Results supported the translation of 123I-INER into humans studies, despite the slow kinetics determined over the imaging period. Pharmacokinetic properties of 123I-INER described in this thesis may be used to simplify future data acquisition and image processing. Conclusion In conclusion, this thesis reported: (1) the development of novel radiotracers for brain imaging, namely NAT and TSPO; and (2) the development of a new methodology for aiding lead molecule identification at early stages of radiotracer discovery (i.e. prior to radiolabelling). In addition, an overview of radiotracer discovery and development process is provided in a single document, with a focus on brain radiotracers

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

Human pharmacology of current and new treatments for schizophrenia

The studies in this thesis together show different ways of studying human pharmacology, give an impression of the current drug development in schizophrenia, and provide examples how human pharmacology can be applied in an early stage of drug development in healthy volunteers. The investigated compounds show that the main pharmacological focus in this area has shifted from psychosis to improvement of individual negative or cognitive symptom complexes, from direct receptor inhibition to indirect receptor modulation, and from single drug strategies to combination therapies, each targeted at different symptoms. We have tried to create a pharmacological fingerprint of the investigated compounds by making use of an intensive CNS test battery to measure effects in different functional domains of the brain and additional 'tools' (i.e. positive controls, dose escalation, PK-PD modeling and pharmacological challenge tests) to improve the reliability of the tests. This diversity of drug development strategies and range of neurotransmitters in schizophrenia reflects the increasing complexity of neuropharmacological hypotheses in this field. Despite these difficulties, incremental changes in drug characteristics and treatment strategies may well lead to the introduction of new classes or combinations of drugs in the future.The publication of this thesis was financially supported by the foundation Centre for Human Drug Research (CHDR), Leiden, the NetherlandsUBL - phd migration 201

Neuropharmacology of novel dopamine modulators

De neurotransmitter dopamine speelt een essenti_le rol in diverse neurofysiologische functies en is betrokken bij de pathofysiologie van diverse neuropsychiatrische aandoeningen, waaronder de ziekte van Parkinson, schizofrenie, drugsverslaving en hyperprolactinemie. De huidige farmacotherapeutische methoden om dopaminerge neurotransmissie te be_nvloeden, hebben slechts een beperkt effect op de symptomen, terwijl hinderlijke bijwerkingen kunnen optreden. Derhalve heeft verbetering van de farmacotherapie van deze ziekten een hoge prioriteit. De bevindingen van studies in dit proefschrift en followup studies tonen aan dat verbetering van de kinetiek van het geneesmiddel ter plaatse van de receptor en verbetering van de selectiviteit van het geneesmiddel veelbelovende strategie_n zijn. De resultaten van be_nvloeding van de controle mechanismen door tachykinines en gaba lijken vooralsnog minder therapeutisch nut op te leveren, maar geven wel indicaties voor biologische effecten die verder onderzoek verdienen. Deze onderzoekslijnen geven aan dat, ondanks de grote verscheidenheid aan beschikbare dopamine agonisten en antagonisten, de therapeutische mogelijkheden om dopamine neurotransmissie te be_nvloeden nog lang niet verzadigd zijnUBL - phd migration 201

Pharmacotherapy for pain: what to measure, how to measure?

In this thesis novel techniques are described which are developed to analyze or predict the efficacy of pharmacotherapy for pain. In Chapter 2 an overview is presented on recent pharmacokinetic pharmacodynamic (PKPD) modeling in acute and chronic pain. Chapter 3 describes the analgesic effect of a capsaicin patch in diabetic painful neuropathy (DPN) pain patients. A longitudinal population PD analysis is performed. In Chapter 4 a subgroup analysis is performed on pooled data from 4 multicenter trials on the effect of capsaicin 8% patch in post herpetic neuralgia (PHN). Apart from longitudinal and mixture analyses, covariates are examined to identify predictors of efficacy. In Chapter 5 the analgesic effect of orodispersible oxycodone versus orodispersible paracetamol for the management of breakthrough pain is quantified using a novel pharmacodynamic model. ________________________________Finally, Chapter 6 describes the effect of tapentadol versus morphine on conditional pain modulation (CPM) in healthy volunteers. A difference in CPM engagement between these two __opioids__ might proof the difference in mechanisms of action between the classical mu opioid receptor (MOR) agonist and the combined MOR-noradrenalin reuptake inhibitor (NRI) compound.UBL - phd migration 201

Neuroimaging of fetal cell therapy in Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disease characterised by the elevated formation of α-synuclein-immunopositive intraneuronal proteinaceous inclusions (Lewy pathology) and the progressive loss of neuromelanin-laden dopaminergic cells of the substantia nigra pars compacta, resulting in the loss of striatal dopaminergic terminals and emergence of cardinal motor features including bradykinesia, rigidity, tremor and postural instability. Dopaminomimetic agents provide effective symptomatic relief in the early stages of illness, yet due to the inherently progressive nature of the disease and the induction of debilitating side effects their efficacy is eventually lost. Cellular restorative strategies involving intrastriatal transplantation of human fetal ventral mesencephalic (hfVM) tissue gained traction from the early 1990’s, when several multi-disciplinary teams reported drastic motoric improvements concomitant with graft-derived dopaminergic re-innervation. However, outcomes of double-blind randomised controlled trials and the presentation of novel dyskinetic movements persisting in the “off-state” called for substantial revision of cell delivery strategies. The current thesis utilises positron emission tomography to examine the effects of hfVM implantation under the Transeuro protocol on dopaminergic ([18F]FDOPA, [11C]PE2I) and serotonergic ([11C]DASB) systems in patients with Parkinson’s disease and elucidate the neural underpinnings of its clinical impact. The main findings are; 1) implanted hfVM tissue led to increases in putamenal dopamine synthesis and storage capacity, dopamine and serotonin transporter density as compared to non-transplanted patients; 2) modification to surgical procedures provided inhomogenous and inconsistent re-innervation; 3) hfVM transplantation was associated with clinical improvements in measures of bradykinesia, rigidity and tremor; 4) graft-related changes in posterior putamenal dopamine and serotonin transporter density predicted symptomatic relief of bradykinesia and tremor; 5) heterogeneity of posterior putamenal re-innervation may impact upon potential clinical benefit; 6) graft-induced dyskinesia was associated with greater post-operative increases in dopamine transporter expression in the anterior putamen; 7) there was no evidence that graft-induced dyskinesia was related to serotonergic hyperinnervation. The novel findings presented in this thesis have major implications for cell-based restorative strategies beyond the hfVM era and will likely foster informed [re]consideration of many aspects of therapeutic delivery and trial design. For its ability to provide mechanistic insight in vivo, neuroimaging may continue to play a central role in the optimisation of future interventions.Open Acces

DISTRIBUTION OF ANTIRETROVIRALS WITHIN THE BRAIN TISSUE AND RELATIONSHIP WITH NEUROCOGNITIVE IMPAIRMENT DUE TO HIV

As of the year 2015, 36.7 million people worldwide were living with HIV infection. While the introduction of highly active antiretroviral therapy (HAART) has greatly reduced the morbidity and mortality of HIV infection, there is still no cure for this disease. In the central nervous system (CNS), HIV RNA in the cerebrospinal fluid (CSF) has been found even in patients who otherwise have viral suppression in the plasma. Further, HIV infection in the brain may lead to the development of HIV-associated neurocognitive disorders (HAND). Milder forms of cognitive decline in HAND remain highly prevalent in people taking HAART and this may be a function of ineffective antiretroviral (ARV) distribution in the brain tissue. However, existing methods only measure ARV pharmacokinetics (PK) in the CSF and are insufficient to explain brain distribution of ARVs. Therefore, the goal of this project was to conduct a comprehensive analysis of ARV penetration into the brain tissue in preclinical models, evaluate the role of drug transporters in modulating ARV brain tissue disposition across species, and develop a model to predict disposition of one ARV (efavirenz [EFV]) in human brain tissue using PK data from preclinical models and determine the relationship between model-predicted drug exposure in the brain and neurocognitive impairment in a cohort of HIV-positive participants. In the first aim of the study, the brain tissue concentration and brain tissue:plasma penetration ratio of six ARVs were determined across two humanized mouse models and one nonhuman primate (NHP) model by LC-MS/MS. ARV brain tissue:plasma concentrations were only preserved across all three species for raltegravir, and showed no differences based on infection status or sex (in the NHPs). In the NHPs, ARV concentrations in the CSF were >6-fold lower than brain tissue. The CSF concentrations were poorly predictive of the brain tissue concentrations for all ARVs except EFV (r=0.91, pIC90 in the brain tissue across all NHPs, however, MSI showed that IC50 of viral replication. In the second aim of the study, we noted significant differences in the gene expression of drug-transporters in the brain tissue across all three species. For example, the gene expression of Abcb1 was 10-fold higher in the hu-HSC-RAG mice compared to the BLT mice. Only BCRP and P-gp proteins were quantified in the majority of the brain tissue samples and there was 16-fold higher BCRP protein in the NHPs relative to the humanized mouse models. There were no differences in the expression of drug transporters due to infection status, but female macaques showed >two-fold higher protein expression of BCRP and P-gp compared to male animals. The protein concentration of transporters in the brain tissue did not predict the brain tissue:plasma concentration of any of the ARVs. In the final aim of the study, we developed an eight-compartment PK model to describe the distribution of EFV into the CSF and brain tissue in rhesus macaques. Using the preclinical model structure and human PK data from a small clinical trial, the brain tissue distribution of EFV was predicted in our cohort of HIV positive participants. At steady state, EFV profile in the brain tissue was predicted to be flat with a median concentration of 8,000 ng/mL. Model-predicted brain tissue exposure showed good agreement with plasma (r=0.99, p0.05). Through these experiments, it was determined that ARV penetration into the brain tissue is highly variable across preclinical models. With the limited ARV concentration data that are available from the human brain tissue, drug concentrations achieved in the brain tissue of NHPs closely approximate what is seen clinically. The CSF is not an appropriate surrogate for brain tissue PK of all ARVs investigated except EFV, and our surrogate measures of efficacy for EFV indicate that although the drug achieves high concentrations in the brain tissue, a lack of adequate spatial coverage over HIV-target cells may lead to reduced efficacy. There are several inter-species differences in drug transporter expression in the brain tissue; however, brain tissue transporter expression was not predictive of ARV brain tissue penetration. Finally, our data demonstrate that sparse preclinical and clinical data can be leveraged to predict human brain tissue exposure of ARVs by the use of novel Bayesian models. Our small study suggests that factors other than ARV brain tissue PK may influence HAND persistence.Doctor of Philosoph