Frontiers in Clinical Drug Research - CNS and Neurological Disorders: Volume 9 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Frontiers in Clinical Drug Research - CNS and Neurological Disorders
- Sprache: Englisch
Frontiers in Clinical Drug Research - CNS and Neurological Disorders is a book series that brings updated reviews to readers interested in advances in the development of pharmaceutical agents for the treatment of central nervous system (CNS) and other nerve disorders. The scope of the book series covers a range of topics including the medicinal chemistry, pharmacology, molecular biology and biochemistry of contemporary molecular targets involved in neurological and CNS disorders. Reviews presented in the series are mainly focused on clinical and therapeutic aspects of novel drugs intended for these targets. Frontiers in Clinical Drug Research - CNS and Neurological Disorders is a valuable resource for pharmaceutical scientists and postgraduate students seeking updated and critical information for developing clinical trials and devising research plans in the field of neurology.
The ninth volume of this series features reviews that cover the following topics related to the treatment of a different CNS disorders, related diseases and basic neuropharmacology research:
- Integrating imaging and microdialysis into systems neuropharmacology
- Depression heterogeneity and the potential of a transdiagnostic and dimensional approach to identify biologically relevant phenotypes
- CAR-T cells in brain tumors and autoimmune diseases – from basics to the clinic
- Revaluation of thyrotropin-releasing hormone and its mimetics as candidates for treating a wide range of neurological and psychiatric disorders
- Natural BACE1 inhibitors: promising drugs for the management of Alzheimer’s disease
- The possibilities of safe lithium therapy in the treatment of neurological and psychoemotional disorders
- Pharmacotherapy of multiple sclerosis and treatment strategies
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Seitenzahl: 507
Veröffentlichungsjahr: 2021
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PREFACE
The progressive death of brain neuronal cells is the root cause of several neurodegenerative pathophysiological processes. Once dead, these brain cells cannot then be regenerated. There is an urgent need to dig deeper into the neurodegenerative pathology, identify unexplored markers, and investigate novel therapeutic approaches to identify drugs targets for therapeutics that might improve brain functions and outcomes in the longer run
Volume 9 of our book series Frontiers in Clinical Drug Research - CNS and Neurological Disorders showcases another set of state-of-the-art and innovative research ventures produced by the eminent as well as budding scientists in the field of neurodegeneration. They have reviewed, evaluated, and commented to provide a creative futuristic outlook to some of the most exciting latest research findings happening in the field of CNS and Neurological Disorders. This could lead to a better insight into various brain ailments with ground-breaking therapeutic advances and serve as an impetus for future drug development.
Thus chapter 1 explores the possibility of integrated use of microdialysis with expanded use of imaging modalities to better understand and treat Alzheimer’s Disease. Chapter 2 highlights the therapeutics targeting immunometabolic dysregulations to benefit patients with atypical depression. It also proposes the use of a transdiagnostic dimensional approach to capture the complexity of mood disorders by incorporating the pathophysiological and clinical data and considers the influence of neurodevelopmental and environmental factors. Chapter 3 summarizes the basics of chimeric antigen receptor (CAR)-T therapy and discusses its current pre-clinical and clinical progress and applications in brain tumors and autoimmune diseases. Chapter 4 discusses the efficacy of thyrotropin-releasing hormone (TRH) and its various mimetics to treat various neurological and psychiatric disorders, such as spinocerebellar degeneration (SCD), cognitive impairment, and Alzheimer’s disease given by non-oral routes. Chapter 5 reviews the role of beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1) in cognitive decline associated with Alzheimer’s disease, and investigates the use of natural plant extracts and phytoconstituents as BACE1 inhibitors. Chapter 6 analyses the anxiolytic and adaptogenic effects of a new drug, a complex of lithium citrate and a sorbent (aluminum oxide and polydimethylsiloxane or lithium complex) to target cognitive impairment in experimental animals via a course of preclinical studies. Chapter7 evaluates the therapeutic potential of approved disease-modifying therapies (DMTs) to address the acute relapse of multiple sclerosis (MS).
In short, the current volume presents a scholarly collection of review articles to advance the field further. It is anticipated that the compiled views and reviews as well as the critical analysis will drive further research in the area to provide avenues for future drug exploration not only in the field of neuroscience but also in a vast majority of other science disciplines.
We are grateful for the timely efforts made by the editorial personnel, especially Mr. Mahmood Alam (Director Publications), and Mrs. Salma Sarfaraz, Miss Asma Ahmed (Senior Manager Publications) at Bentham Science Publishers.
Atta-ur-Rahman, FRS Honorary Life Fellow Kings College University of Cambridge Cambridge UKList of Contributors
Integrating Imaging and Microdialysis into Systems Neuropharmacology
Carla Biesdorf1,Robert E. Stratford1,*Abstract
Microdialysis sampling has been coupled with several imaging modalities over the past two decades to either support the development of imaging approaches as diagnostic, prognostic or treatment response biomarkers, or to use this temporally rich sampling approach of brain tissue in parallel with one or more imaging modalities to provide an integrated, systems neuropharmacology, perspective of normal and diseased brain physiology. This chapter provides a comprehensive review of the scientific literature that encompasses several imaging modalities (including PET, MRI, EEG, CT) that relied on microdialysis sampling for its supportive and/or parallel use in systems neuropharmacology research. A review of the important role microdialysis has played in supporting several PET imaging applications used in neuropharmacology research is provided. Integrated with PET, various MRI modalities, EEG and CT, microdialysis has deepened understanding of various neurotransmitter systems and their temporal and spatial integration as an in-tune, “normal” or dysynchronous, “diseased” system. Parallel use of microdialysis in humans suffering from traumatic brain injury or chronic epilepsy has been coupled with PET, MRI, EEG and CT approaches to develop systems-level understanding at the cellular, regional, and whole brain levels. Throughout the chapter, several publications are discussed that exemplify the results of this research. The chapter concludes with a presentation of the integrated use of microdialysis with imaging in Alzheimer’s Disease research, ending with the hope for expanded use of imaging modalities that can even be used in an ambulatory capacity, and how microdialysis can continue to play its established role to support their development and use in understanding and treating this disease.
INTRODUCTION
It is truly remarkable when one considers the brain’s ability to coordinate its myriad activities, such as, to code dynamic visual cues into behavior, or to retrieve information at a moment’s notice and build upon it to create new learning, or to instantly recognize a familiar face or voice. Perhaps it is even more remarkable that these integrated activities are a consequence of a system that operates through electrochemical and chemical mechanisms that encompass spatial and temporal continuums from the subcellular and microsecond domains to circuits composed of circuits that can remain constant over a lifetime. At both the anatomic and functional levels, the healthy brain is a highly integrated system that exhibits remarkable adaptability over decades of life. Our understanding of this system at these two levels is arguably rudimentary, thus dedication to continuous development and refinement of experimental and computational tools that can describe circuit anatomy at local and regional levels, and then relate these in a cause-effect way to circuit function in healthy and diseased brain is worthy. Positron emission tomography (PET) and magnetic resonance imaging (MRI) have demonstrated power as non-clinical and clinical approaches to evaluate non-invasively the anatomic and functional circuitry of the brain. While use of these tools in living animals and humans has continued to improve over the last 20 years, the need for advances remains. An objective of this chapter is to describe how microdialysis, as an in vivo sampling method, has advanced the application of these imaging approaches. The chapter will present microdialysis as a supportive tool enabling application of imaging modalities as biomarkers to inform disease diagnosis and prognosis, and support the development of new treatments for human brain diseases. In addition, microdialysis sampling is a proven and important independent technique in preclinical neuropharmacology research; accordingly, this chapter will present examples of its parallel use with various imaging modalities in a pre- or non-clinical environment to inform systems neuropharmacology.
The chapter will first provide an overview of the microdialysis sampling method and its use in neuropharmacology, including general support of drug discovery and development, and lastly, its use in humans in a specific way to inform treatment of traumatic brain injury. A PubMed survey of the literature coupled ‘microdialysis’ with various imaging modality keywords. These included computed tomography (CT), electroencephalography (EEG), PET, single-photon emission computed spectroscopy (SPECT), MRI, optical imaging, fluorescence, near-infrared spectroscopy (NIRS), and mass spectrometry imaging (MSI). The survey identified several examples using microdialysis with PET, MRI or EEG, thus separate sections devoted to the use of microdialysis alongside these imaging modalities will follow the microdialysis overview. There will then be a brief section on integration of microdialysis with other, less frequently used, imaging modalities. The chapter will conclude with a section devoted to Alzheimer’s disease research. This last section represents a change in focus from one of microdialysis use with specific imaging modalities to a discussion of how all modalities and microdialysis have been and conceivably could be used to inform research whose overarching objective is to discover therapies to address this devastating disease.
MICRODIALYSIS OVERVIEW
More than 100 billion neurons and non-neuronal cells comprise the human brain [1], and are bathed by an interstitial fluid commonly referred to as the brain extracellular fluid (ECF). Through this fluid, cells communicate via the release of neurotransmitters and neuromodulators. Microdialysis enables direct sampling of ECF in a living organism; when coupled to an analytical technique, it provides a means to identify and measure these released chemicals and associated metabolites. Fig. (1) is a diagram of a concentric microdialysis probe commonly used in CNS research. The dialysis membrane is a key component of the probe, composed of a porous membrane, cellulose or polyether-based, and varying in pore size. Commercially available membrane pore sizes commonly span molecular weight cut-offs ranging from 6 – 100 kilodaltons. Typical perfusion flow rates range from 0.5 – 2.0 µL/min, with typical collection times of 10 – 30 minutes.
Fig. (1))Diagram of a concentric microdialysis probe design commonly used in CNS microdialysis. The term “concentric” refers to two cylinders: a smaller cylinder delivering fluid into the probe tip (“inlet”) fitting inside a larger cylinder that carries fluid (dialysate) away from the tip (outlet) for subsequent analysis of solutes. Fig. obtained by permission from publisher of Fig. 6 in OuYang C, Liang, Z and Li L. 2015. Mass spectrometric analysis of spatio-temporal dynamics of crustacean neuropeptides. Biochim Biophys Acta 1854 (7): 798-811.One of the earliest applications of microdialysis in the brain involved measurement of dopamine and two of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in rat striatum using high performance liquid chromatography (HPLC) with fluorescence detection [2]. Over the ensuing 35-plus years, refinement in probe design and analysis methods have expanded the versatility of microdialysis. In particular, coupling microdialysis sampling with the sensitivity and selectivity afforded by HPLC-tandem mass spectroscopy (LC-MS/MS) in the past ten years has enabled detection of numerous energy metabolites, and the identification and analysis of neurotransmitters and neuromodulators, the latter group which have been estimated at over 100 [3]. Over the past 20 years, the annual number of microdialysis publications has been in the hundreds, peaking in 2000 at nearly 800 and leveling off to just under 500 in the years 2014 – 2016 [4].
Primary application of microdialysis has been to measure neurotransmitter changes in response to a pharmacologic challenge. However, beginning in the 1990s, quantification of drug concentrations in the brain, and more specifically, unbound concentrations in brain ECF, has developed. Probe implantation into brain tissue is obviously an invasive technique, so application in animals, especially rodents, but also non-human primates (NHPs), has predominated. Measurement of dopamine change in striatum and prefrontal cortex of rhesus monkeys in response to a D-amphetamine challenge is an excellent example of the latter [5].
Because of its ability to directly measure neurotransmitter response in a living animal prior to and following a pharmacologic challenge, this pre-clinical application has served to confirm and/or expand understanding of a candidate drug’s mechanism and causality of behavioral effects in animal models. In contrast to this discovery-phase application, use of microdialysis to quantify drug concentration in brain ECF is a sequentially later, development-phase, application that can be used to describe the brain pharmacokinetics (PK) of a candidate drug. This application is technically more demanding because drug recovery from ECF across the dialysis membrane into the collected and analyzed dialysate is commonly < 100%. It is necessary, therefore, to measure this recovery. The reader is referred to Hammarlund-Udenaes for an excellent recent review of the various approaches used to measure probe recovery [4]. Three important consequences derive from measuring drug concentration in ECF. For one, when coupled with knowledge of whole brain drug concentrations, ECF measures indicate the extent that drug distributes between ECF and brain tissue. This information provides insight regarding the burden of drug required to achieve an ECF concentration, perhaps a concentration necessary to engage a neuronal membrane receptor in a pharmacologically relevant manner. Secondly, linking these concentrations to unbound plasma concentrations provides insight regarding drug transport across the blood-brain barrier (BBB), including evidence for the role of transporter-mediated transport, such as when post-distributional equilibrium drug concentrations are > 1, suggestive of net carrier-mediated uptake transport from plasma to ECF, or < 1, suggestive of net efflux carrier-mediated transport from ECF to plasma. Lastly, combining brain ECF PK in animals with species-specific brain physiologic parameters such as blood flow and ECF volume supports development of physiologic-based PK (PBPK) models that have preclinical to clinical translational capability. Coupled with in vitro studies that determine transporter identity (if appropriate), knowledge of transporter involvement and approximations of transporter expression between preclinical species and human, supports model translation to predict drug exposure in human brain. Yamamoto, et al. [6] provides an excellent recent review of this application of preclinical microdialysis. For a CNS drug in clinical development, the ability to infer CNS drug exposure from plasma exposure and dose creates a higher level of efficiency and confidence in clinical trial investment. Relating plasma drug concentrations measured during clinical trials to unbound, pharmacologically relevant, human brain concentrations is no simple matter. This is largely due to the substantial paracellular resistance of the blood-brain barrier (BBB) and complex within-brain drug distribution, both of which collaborate to regulate brain physiology and ensuing behavior by preventing xenobiotic effects.
European regulatory approval to use microdialysis in humans came in 1995; in 2002, the FDA granted approval. Its use to support treatment of traumatic brain injury (TBI) is where clinical microdialysis has made its largest impact. Thelin, et al. [7] present an excellent review of the history and current perspectives regarding microdialysis use in TBI. In that review, the authors describe the usefulness of following glucose, lactate and pyruvate concentrations in ECF taken over several days in hourly increments to inform injury severity and treatment approach. Glucose levels provide an indication of the ability of this preferred energy source to enter the brain, while the ratio of lactate-to-pyruvate, the so-called lactate-pyruvate ratio (LPR), is a surrogate measure of cell health, as it provides an index of anaerobic metabolism, which is an indicator of cellular derangement via deviation from the preferred aerobic pathway. Through their experience and that of other clinicians, the authors have found that a LPR < 25 correlates with improved outcome, and signifies that aerobic metabolism is functional; whereas, a ratio > 25 is a positive predictor of mortality [8]. In contrast to imaging modalities that assess brain structure and function, a general limitation of microdialysis is its spatially confined nature, which seems to be a key reason limiting its broader uptake in TBI treatment [9]. This limitation has led to uncertainty regarding probe placement in TBI patients, that is, should it be adjacent to the site of a contusion, or is it sufficient to place it in healthy tissue? The ability of imaging modalities to provide information at a regional level, specifically computerized tomography (CT) images in the case of TBI, however, has provided guidance by demonstrating that probes placed in radiologically normal-appearing tissue, which has a lower LPR than probes placed peri-contusionally, nonetheless presents LPRs that also correlate with long-term outcome [10]. From a systems perspective, combined use of microdialysis and PET imaging is encouraged to understand more fully the pathophysiology of TBI [7, 11]. This is because the two approaches are complimentary, with microdialysis providing focal information of glucose utilization over several days, and PET, using [18F]-fluorodeoxyglucose (FDG) as an index of metabolic consumption of glucose, providing a regional and even whole brain readout over an approximate one-hour period. In addition, while microdialysis measures reflect extracellular levels, FDG-PET reflects the intracellular environment.
In summary, as a stand-alone approach, microdialysis’ broad impact in neuropharmacology is largely because of its ability to directly measure neurotransmitter and drug levels in living organisms. Used pre-clinically, present application of the method is robust because of the ability to couple its intensive sampling capability with numerous analytical methods to measure a wide-array of neurochemicals, and now drug candidates with the development of LC-MS/MS. This wide application capability in a pre-clinical drug discovery environment is advantageous relative to PET imaging given the oftentimes lack of tracer availability for novel pharmacologic mechanisms at such an early stage. However, arguably, its focal and invasive aspects are limiting, and its invasive nature most certainly limits its use in clinical development of CNS drugs, although recent strides have been made to expand its use in humans [12, 13]. Fortunately, many of the strengths and limitations of microdialysis are in respective opposition to those inherent to PET imaging, which is largely non-invasive and provides regional output, but indirect regarding the functional information it seeks to provide and ambiguity regarding signal source (intact drug: bound and free, and the possibility of drug-related metabolites). Viewed in this complementary way, judicious application of microdialysis is a powerful means to support PET imaging, particularly in the latter’s application to support clinical drug development, but also when combining PET with microdialysis in a preclinical domain to support a systems-level understanding of disease, animal models of disease and drug mechanisms.
INTEGRATION OF MICRODIALYSIS WITH PET IMAGING
Generally, there are five ways PET imaging can support CNS research and clinical diagnostics. It can provide an indirect measurement of oxygen and glucose consumption by the brain. Secondly, it can provide evidence that a substance, usually a drug or drug candidate, present in the systemic circulation can get into the brain. Thirdly, a dose-related displacement of a PET tracer that is specific for a therapeutically intended target provides evidence that a drug candidate can bind to this target. Fourthly, alteration of the distribution of tracer binding to a target in a disease model or actual diseased brain relative to control brain, or in response to a treatment hypothesized to cause a downstream alteration of PET-tracer target expression is another application of PET imaging. Lastly, use of a PET tracer as an indirect measure of synaptic neurotransmitter concentration. In this final application, a treatment (pharmacologic or non-pharmacologic) alters the release/uptake dynamics of a neurotransmitter, which then competes with tracer binding to that neurotransmitter’s receptor or transporter. Microdialysis has played a role in the development and/or application of all five of these uses of PET imaging. Following in turn will be examples of each of these roles. Table 1 provides an overview of noteworthy examples.
Measurement of Brain Metabolic Activity
The LPR obtained from microdialysis samples of victims of TBI currently provides the most consistent indicator of prognosis for recovery [8]. Its limitation, however, is its reliance upon a ratio of two glucose metabolites as a general indicator of brain function. TBI is increasingly recognized as a complex and multidimensional condition involving brain network dysfunction [14]. While LPR is considered indicative of anaerobic (glycolytic) metabolism, and thereby an index of mitochondrial dysfunction, PET-derived measures of cerebral metabolic rates for oxygen (CMRO2), using C15O, 15O-O2 and H215O, and glucose (CMRglc) using 18FDG, suggest a more complicated picture. Some PET studies using these so-called ‘triple oxygen’ protocols combined with 18FDG have found evidence supporting a shift from aerobic (tricarboxylic acid, ‘TCA’) to glycolytic metabolism at locations of injury, thus agreeing with LPR [15, 16]. These studies observed disproportionally greater glucose consumption than that of oxygen, so termed a ‘hyperglycolysis state’ [16]. However, another study observed an increase in glucose metabolism that was associated with higher production of both lactate and pyruvate, and thereby no association with LPR, suggesting a generalized increase in glucose utilization instead of a shift to glycolytic metabolism in TBI [11]. An alternative use of microdialysis applied in TBI has provided additional insight. One of the advantages of the technique is its ability to deliver substances locally to the brain at the point of probe insertion. Used in this way, delivery of stable-label 13C-glucose or 13C-metabolites of glucose (acetate and lactate) with subsequent NMR analysis of microdialysates suggests that metabolism of glucose by alternative pathways may be occurring in injured brain [17]. Using this powerful combination of stable-label delivery via microdialysis with subsequent NMR analysis of microdialysates, clinicians demonstrated that lactate also feeds back into the TCA cycle [17, 18]. One idea demonstrating the integrated or systems-level functioning of the brain in TBI is the astrocyte-neuron lactate shuttle hypothesis proposed over 20 years ago [19]. It suggests that neurons can use lactate produced by astrocytes as an energy source via the TCA cycle. This idea is consistent with the association of low lactate with improved outcomes [20], and high lactate with poor outcome [17] in TBI. Interestingly, a recent study delivered lactate intravenously to TBI patients and demonstrated a beneficial effect [21]. Taken together, clinical research in TBI indicates the importance of preserving and/or restoring mitochondrial function in TBI because of its robust ability to produce energy by the TCA cycle, utilizing pyruvate derived from glucose or derived from lactate. PET and microdialysis studies in human brain played key roles establishing this understanding.
Another pathway, the pentose phosphate pathway (PPP) operating in the cytosol, can also use glucose as a source of energy. Primarily, in healthy tissue it is responsible for the production of ribose sugars that support DNA synthesis and repair via production of nucleotides. Importantly, the PPP produces NADPH, which is necessary to maintain glutathione in a reduced state so that it can function as an antioxidant [22]. The PPP is upregulated in animal models of TBI [23, 24]. In TBI patients, delivery of 13C-glucose by microdialysis with NMR analysis of collected microdialysates demonstrated PPP functionality [8]. Building on this observation, a recent study reported neuroprotective effects in TBI following administration of N-acetylcysteine amide, which supplied N-acetylcysteine used in the production of mitochondrial glutathione [25]. Thus for two reasons, as an alternative pathway for glucose utilization to supply energy to cells, and for its purported neuroprotective effects via glutathione, the capacity for upregulation of the PPP in TBI offers new treatment approaches. Overall, integrated use of microdialysis and PET imaging played important roles in identifying the complex systems of energy production in the brain, and, via the PPP, the beneficial effects of increasing antioxidant capacity in injured brain [8].
Demonstration of Drug Delivery to the Brain
Two principles of brain physiology conspire to challenge the development of CNS therapeutics: (1) presence of a well-developed barrier, the blood-brain barrier (BBB), which can limit the rate and extent of distribution of drug candidates from the systemic circulation to brain parenchyma, and (2) intra-brain distribution of drug candidates, which can severely curtail availability of the unbound, pharmacologically relevant, form of a candidate to bind to its intended target. Not surprisingly, the time taken to register CNS therapeutics is longer, the success rate lower, and failure frequency higher deeper into clinical trial investment (often in Phase III) relative to other therapeutic domains [26]. Because of its non-invasive capability, PET imaging in clinical trials has been used to provide some assurance that compound labelled with a PET tracer is getting into the brain. This approach can be useful to demonstrate that increases in brain exposure correlate with increasing systemic exposure, as well as to evidence pharmacologically relevant exposure following tolerated doses, in turn providing evidence of an adequate safety margin. As previously mentioned, however, limitations of PET imaging are its inability to distinguish bound from free compound, and intact compound from total radionuclide signal, which could also represent metabolites. Combined use of in vitro approaches to measure free drug concentrations, such as brain homogenates [27] or brain slices [28, 29], coupled with in vivo measurement of whole brain levels, or direct measurement of unbound concentrations in brain ECF by microdialysis, both of which are preclinical animal-based, are used to estimate the time course of unbound concentrations in brain. Incorporation of these various approaches into translational PBPK models is one means to estimate unbound concentrations in human brain [6]. Recently, a modification of this multi-staged approach that is more direct was advocated [30]; in effect, this involved integration of microdialysis with PET imaging. The brain slice method provided an estimate of unbound volume of distribution (Vu, brain) of oxycodone, in this case in rat brain, but the technique also applies to human brain. This measure enabled conversion of the PET time course in rat brain to an unbound PET trace of the model drug. Microdialysis, applied to a separate group of rats assessed the accuracy of this PET-derived estimate of unbound oxycodone. Results demonstrated excellent concordance of the two measures of unbound oxycodone during infusion, but divergence of the acquired PET signal from microdialysis during the elimination phase, presumably due to the accumulation of radionuclide metabolites in the brain. The authors concluded that, provided there is adjustment for radiolabeled metabolites and in conjunction with in vitro assessment of Vu, PET can provide a non-invasive measure of unbound drug in the brain. Combining this outcome in the same experiment with a PET-based measure of target binding would provide the ability to evaluate in an in vivo setting the kinetic relationship between concentration and receptor binding, including receptor dissociation constant and potential change in the number of receptors over time. Such information would be valuable in developing pharmacokinetic-pharmacodynamic models of drug action in the brain with reduced assumptions regarding drug concentration, binding and effect relationships.
Demonstration of Drug Binding to Target by Displacement of Tracer Binding
Perhaps the most common use of PET imaging is to administer in close association with a candidate drug a tracer dose of a positron-emitting compound possessing high binding specificity to a target of interest. Effectively, this approach relies on competitive inhibition of PET ligand binding by the candidate drug. Administration of the candidate covers a range of doses that have been determined to be safe. As candidate exposure increases, it progressively displaces tracer from the target site in accordance with tracer vs. candidate binding affinities and receptor number, thus resulting in a smaller PET signal. Suffice it to say, outcome is an estimate of the receptor occupancy of the candidate in relation to measured systemic exposure, optimally reported as EC50 and Emax, the concentration at which candidate binding is 50% of maximum and the maximum occupancy attainable (or at the highest dose administered), respectively. This clinical experiment establishes that the candidate compound and/or metabolite(s) are able to enter the brain and bind to the intended target.
A host of preclinical pharmacology studies that demonstrate, for one, candidate binding to the human target expressed in a cell line, as well as in vitro functional studies demonstrating an effect associated with binding, precede the clinical experiment. If the desired effect is alteration of neurotransmitter levels, then microdialysis studies in animals, typically rats, can improve confidence by bridging in vitro pharmacology with behavioral measures in an animal model(s) of a disease. Viewed in this way, microdialysis associates target binding with a functional response that culminates in a behavioral effect. For drugs in which binding to a receptor or transporter is responsible for a behavioral effect, there are two sequential steps in this mechanistic association, the first being that binding alters neurotransmitter concentration, and the second that altered neurotransmitter levels result in a behavioral response. In both steps, demonstration of dose dependency is essential. PET imaging is a powerful technique, providing a non-invasive measurement of target engagement in living human brain, but it is also expensive and applied only to a limited number of subjects. Consequently, preclinical investment to identify and qualify a PET ligand for human use that is BBB permeable and of high specific activity makes sense. Integration of microdialysis into this process addresses the first of the two sequential mechanistic steps and supports PET tracer development for use in humans to provide evidence of CNS target engagement of a drug candidate.
In the past 10 years, there are a few published examples that used microdialysis in conjunction with PET imaging in this capacity. A study published in 2009 evaluated the effects of ecstasy (3,4-methylenedioxymethamphetamine, MDMA) on the dopaminergic system in non-human primates (NHPs) [31]. Observed increases in extracellular dopamine were small, and associated with minor alteration of binding of the PET-tracer [18F]-FECNT that is selective for the DAT [32, 33]. These small changes also translated to absence of a motor-stimulant effect. Connectivity between weak MDMA displacement of PET tracer binding, a weak dopamine response measured by microdialysis and the absence of a stimulatory effect indicated that other behavioral effects observed in NHPs, namely, the ability of MDMA to substitute for amphetamine and for monkeys to self-administer MDMA are due to a non-dopaminergic mechanism(s). The authors suggested an important role for serotonergic pharmacology for these other behaviors elicited by MDMA in NHPs. Importantly, this internal consistency of PET imaging and microdialysis, compared to measurement of transporter occupancy in isolation, provided a definitive conclusion regarding a weak dopamine mechanism in NHPs. This conclusion stands in contrast to demonstration of a strong dopamine response (as measured by microdialysis) to MDMA in rats [34], indicating important differences between rodents and primates regarding mechanism of MDMA effects.
Another study conducted in NHPs examined the abuse liability of modafinil [35], a drug approved for the treatment of narcolepsy and somnolence. Cocaine and methylphenidate have high abuse potential because of their ability to block DAT and consequently increase extracellular dopamine [36]. In the modafinil study, blockade of DAT, as measured using [18F] FECNT, at a behaviorally relevant modafinil dose was similar to that observed following doses of cocaine associated with abuse behavior. Interestingly, increases in extracellular dopamine observed at this level of modafinil blockade of DAT were smaller relative to a similar alteration of DAT occupancy by cocaine and associated with abuse. Combined use of PET imaging with microdialysis was similar to the work described in the preceding paragraph demonstrating connectivity between DAT occupancy and resultant increase in dopamine; however, these modafinil studies were important also in showing quantitative differences in this association compared to cocaine and, thus, alleviating the abuse liability concern with modafinil. Another NHP study by this same group reaffirmed the value of measuring DAT occupancy and extracellular dopamine simultaneously [37]. The authors evaluated the time course of dopamine and DAT occupancy using [18F]-FECNT for the DAT following single dose administration of cocaine and three novel DAT inhibitors. While all agents increased extracellular dopamine, onset and duration of the effect did not correspond with DAT-occupancy time course onset and duration. The authors concluded that DAT occupancy alone does not determine dopamine response pharmacodynamics, and suggested that pharmacokinetic differences between the compounds as well as counter-regulatory measures, such as dopamine-2 (D2) receptor-mediated downregulation of dopamine release, contribute to the dopamine response magnitude and duration. In this example, integrated use of microdiaysis and PET imaging provided deeper mechanistic understanding of DAT inhibition effects on dopaminergic system activity. Integration of PET imaging and microdialysis has also provided mechanistic insight regarding treatment of major depression and treatment-refractory depression using electroconvulsive therapy (ECT). A meta-analysis of several preclinical microdialysis studies and PET-imaging studies, along with clinical PET imaging studies indicated the important roles played by serotonin and dopamine systems in response to ECT [38].
A recent study conducted in rats used microdialysis to confirm the mechanism of a novel serotonin transporter (SERT) reuptake inhibitor for the treatment of premature ejaculation [39]. Administration of the candidate compound, DA-8031, dose-dependently blocked occupancy of SERT by the PET tracer, [11C]-DASB, in several brain regions. Elevation of serotonin in the dorsal raphe nucleus as measured by microdialysis also increased in a dose dependent manner, thus definitively linking inhibition of SERT with increased extracellular serotonin.
Alteration of Tracer Binding in Response to Drug Treatment and in Disease
Cocaine increases extracellular dopamine by blocking DAT-mediated dopamine reuptake into dopamine nerve terminals in brain areas associated with feelings of pleasure, reward, motivation and cognition. Important regions responsible for these effects include the amygdala, striatum, ventral tegmental area, prefrontal cortex and cortex. Currently, there is no effective pharmacotherapy for cocaine addiction. Among possible treatment strategies, modulation of serotonin neurotransmitter circuits in the brain has been considered because of their known ability to alter dopaminergic tone [40]. More specifically, selective serotonin reuptake inhibitors (SSRIs) have been investigated. Acute administration of these agents to rodents and NHPs attenuates behaviors associated with cocaine addiction [41-43], and decreased cocaine’s positive subjective effects in humans [44]. In NHPs, acute administration of an SSRI attenuated cocaine induced increases in extracellular dopamine as measured by microdialysis [42]. Unfortunately, clinical trials of SSRIs administered chronically failed to reduce cocaine abuse [45-47]. A study conducted in NHPs used PET imaging and microdialysis to improve understanding of the effects of chronic SSRI treatment in the context of daily cocaine use [48]. As with clinical trials, chronic administration of fluoxetine, a prototypical SSRI, failed to reduce cocaine self-administration in this study; whereas, a single dose did attenuate this behavior. The authors postulated that the loss of effect upon chronic dosing may have been due in part to neurobiological changes, as are thought to be responsible for the delay in demonstration of the therapeutic effects of SSRIs in depression [49]. Interestingly, the study did find that chronic fluoxetine treatment attenuated cocaine-primed reinstatement, suggesting that SSRIs may be useful in preventing relapse of cocaine abuse. This attenuation was associated with a decrease in cocaine-induced extracellular dopamine in the striatum, and this effect persisted 6 weeks after discontinuing fluoxetine administration. On the other hand, PET imaging of the 5HT2A receptor, using [11C]M100907, demonstrated receptor up-regulation in the frontal cortex, but no alteration in the striatum relative to baseline immediately following termination of fluoxetine treatment and for 6 weeks following termination. Expectation was this up-regulation would increase extracellular dopamine in the striatum via the corticostriatal pathway; however, this contrasts with the decrease actually observed. Concomitant with this effect, prolactin secretion, which is a measure of 5HT2A receptor function [50], decreased. Based on this loss of 5HT2A function, the authors suggested that chronic fluoxetine treatment led to cortical 5HT2A receptor desensitization, a phenomenon that imaging alone could not detect, and this may have been responsible for loss of SSRI ability to reduce cocaine self-administration, but also support its potential to prevent cocaine relapse. Application of microdialysis in parallel with PET imaging improved the ability to capture regional specific alterations (through imaging of multiple brain regions) and time-dependent changes (receptor desensitization) in the integrated serotonin-dopamine system in response to SSRI treatment. Based on review of the literature over a 20-year period, combining microdialysis with PET imaging to understand how the brain responds to chronic drug treatment, as this case with SSRI treatment of cocaine addiction illustrates, is rather unique, as it was the only such case identified. The following examples, which were also few, relate to the use of PET imaging as a disease-biomarker, either in clinical application, or in development of animal models of various diseases. Integration of microdialysis into these efforts helped to support these goals.
Association of variation in the DISC1 gene with schizophrenia is an active area of research [51]. Mice carrying mutated forms of the gene exhibit behavioral deficits that are consistent with schizophrenia [52]; these transgenic animals also display altered dopaminergic neurotransmission [53], consistent with the dopaminergic hypothesis of schizophrenia [54]. Jaaro-Peled, et al. [55] used PET imaging and microdialysis to mechanistically characterize these alterations and more fully validate the model for use in schizophrenia research. PET imaging of the D2 receptor in schizophrenic patients vs. controls has revealed elevated receptor expression in the striatum of non-medicated patients [56]. Effectively, the Jaaro-Peled, et al. study amounted to a reverse (backward, as opposed to forward) translational experiment. Employing [11C]-raclopride, the authors found elevated D2 receptor expression in DISC1 transgenic adult mice relative to age-matched controls, thus replicating differences seen in humans. One component of our present understanding of schizophrenia is that elevated D2 receptor expression in patients relative to control subjects is a consequence of exaggerated dopamine release upon stimulation. Microdialysis of extracellular dopamine in the striatum revealed lower baseline (non-stimulated) levels in the DISC1 mice and a larger increase in dopamine response upon a methamphetamine challenge. The latter results are also consistent with heightened behavioral sensitivity to an amphetamine challenge in schizophrenics vs. control subjects [57].
In another reverse-translational exercise, Walker, et al. [58] used the PET tracer, [18F]-fluorodopa (FDOPA) in conjunction with [11C]-dihydrotetrabenazine (DTBZ) and microdialysis measures of dopamine and its metabolites to support use of FDOPA in animal models of Parkinson’s Disease (PD). Application of FDOPA for more than 20 years has provided a non-invasive assessment of dopaminergic system integrity in PD patients [59]. As with L-DOPA used for the treatment of PD, the PET tracer undergoes several steps leading ultimately to elimination of radiotracer from the brain primarily as dopamine metabolites. Sequentially, these are active transport across the BBB, transport into dopamine nerve terminals, metabolism to dopamine, and subsequent metabolism of dopamine and metabolite clearance from brain. Because PET cannot distinguish between FDOPA, F-dopamine and F-dopamine metabolites, kinetic analyses have been developed and found to discriminate between health and disease states [60]. The Walker, et al. [58] study related FDOPA kinetics of striatal images in 6-hydroxydopamine-lesioned rats to parallel measures of striatal dopamine and striatal vesicular monoamine transporter 2 (VMAT2) content, the latter assessed with DTBZ and used as a measure of dopamine terminal integrity [61]. The work established correlations between dopamine metabolite levels measured via microdialysis sampling to DTBZ binding potential as well as the effective distribution volume derived from FDOPA scans that reflects F-dopamine distribution volume. The strength of the correlations between indirect FDOPA imaging with kinetic analysis and direct measures of dopamine system integrity through microdialysis provided additional insight and confidence regarding use of FDOPA in support of preclinical PD research or clinical diagnostics of PD severity.
Evidence implicates impairment of dopaminergic neurotransmission in the striatal region of the basal forebrain in major depression [62-64]. Studies suggest that chronic inflammation mediated by inflammatory cytokines contribute to this impairment [65]. PET studies with FDG, to evaluate metabolic function [66], and with FDOPA, to evaluate dopamine neuron integrity [67], reveal declines in dopaminergic function in the striatum associated with interferon-α (IFN-α) treatment. In a study conducted in NHPs administered IFN-α for 4 weeks, Felger, et al. [68] used PET imaging with [11C]-raclopride to image the D2 receptor, [18F]-FECNT to image the DAT and striatal microdialysis to measure dopamine. Microdialysis measures obtained after two and four weeks of IFN-α administration vs. vehicle control showed increased dopamine in response to an amphetamine challenge following two weeks of the drug, but a decrease at four weeks compared to vehicle. PET imaging revealed a decline in D2 receptor binding and no change in DAT binding following IFN-α for four weeks relative to vehicle. Downregulation of the D2 autoreceptor in the face of declined dopamine release at four weeks was unexpected. The authors suggested that decline in D2 binding potential was a consequence of the initial increase in dopamine output observed at two weeks. To summarize, incorporation of microdialysis into the study design revealed a time-dependent system response to this IFN-α model of inflammation, with an initial increase in dopaminergic sensitivity followed by a decline. Integration of PET imaging and microdialysis increased mechanistic insight into this complex system response to an inflammatory stimulus.
A final example integrating PET imaging with microdialysis to support the use of PET imaging as a disease-biomarker relates to a medicinal chemistry effort to identify a superior PET imaging ligand in support of the preclinical and clinical application of this modality in diseases of cholingergic deficiency, such as Alzheimer’s disease (AD) [69]. Several fluorine-substituted N-benzylpiperidine derivatives possessing nM potency (based on in vitro IC50) to inhibit acetylcholinesterase (AChE) were prepared. Earlier work found that incorporation of a meta-substituted fluorine into donezepil, an approved AChE inhibitor, demonstrated an IC50 < 10 nM, which was approximately an order of magnitude lower than ortho- or para-substituted derivatives, but the meta-substitution synthesis was problematic [70]. Aromatic fluorine substitution of the novel compounds demonstrated similar potency trends, with the meta-substituted isomer possessing an IC50 of 1.4 nM and the para-substituted form 10.8 nM. The ortho-substituted derivative possessed a slightly higher IC50 (3.2 nM), but its specific uptake based on PET into an AChE rich region (striatum) relative to poor region (cerebellum) was poor relative to the meta-substituted compound. The difference translated to a faster decline in striatal acetylcholine measured with microdialysis for the ortho-substituted compound. Correlations between in vitro inhibitory potency, and in vivo relative uptake into striatum and resultant acetylcholine increase demonstrated that the [18F]-meta-substituted compound would be an excellent imaging ligand for assessment of cholinergic activity in the brain.
Assessment of Neurotransmitter Concentrations
This final application of PET imaging provides an indirect measure of neurotransmitter release and uptake over time in response to pharmacologic or non-pharmacologic (for example, direct brain stimulation) interventions. By contrast, microdialysis provides a direct measure of neurotransmitter levels (either relative to baseline or absolute concentrations) when it is associated with an assay of high specificity capable of differentiating neurotransmitter from its metabolites. Integration of microdialysis into this application of PET imaging verifies that change over time in PET ligand binding potential reflects neurotransmitter concentration change. Using microdialysis to qualify a PET ligand for this use is important because of the potential for tracer metabolism, which may produce metabolites with altered ability to compete with neurotransmitter binding to the ligand target, or the ligand may bind to internalized receptors, a process that would obviate competition with extracellular neurotransmitter [71, 72].
Dopamine was the first neurotransmitter monitored in this way in humans [73-75]; not surprisingly, application of this approach to the dopaminergic system has received the most attention. A competition model between neurotransmitter and PET-ligand for binding to the target of interest represents the basic tenet of this approach: as neurotransmitter levels increase, PET-ligand binding decreases in accordance with different reversible binding affinities between neurotransmitter and tracer. Microdialysis has served as the standard for this application, enabling development of correlations between magnitude and temporal changes in binding potential with directly measured change in dopamine concentrations [76, 77]. For reasons that are not entirely clear, the method is limited to PET ligands to the D2 receptor, and more specifically, antagonists to this receptor. [11C]-Raclopride has been the mainstay tracer used in this application. Although the D1 receptor is abundant in the CNS, either antagonist or agonist ligands to it are not sensitive to changes in dopamine concentration measured by microdialysis [78, 79]. Lower affinity of this receptor to dopamine compared to the D2 receptor is the postulated cause of this insensitivity [80]. Similarly, while D2 receptor agonists demonstrate increased sensitivity to change in extracellular dopamine concentrations [81-83], uncertainty around the cause of this and, therefore, ability to interpret the change and infer its significance in disease and treatment response have limited their use. Microdialysis studies have also supported the development of other D2 receptor antagonist tracers, these with higher binding affinity than raclopride to support method application to cortical regions, which have lower D2 receptor numbers than in the basal forebrain [84-86].
This capability to monitor in living human brain concentrations of neurotransmitter over time is a powerful application of PET imaging. As stated, microdialysis has played an important role in developing this application [76, 77], but more recently microdialysis has supported its increasing sophistication, such as in the development of kinetic models that predict temporal changes in dopamine in response to a treatment, such as methamphetamine [87], or smoking [88, 89]. Integration of this application of PET imaging with microdialysis has also supported a systems analysis of brain circuitry by demonstrating attenuation of D-amphetamine stimulation of striatal dopamine release by 5HT-2A and 5HT-2C antagonists [90], and the effect of cortical dopamine depletion enhancing striatal dopamine release, such as may occur in schizophrenia [84]. There is promise for extension of this influential application of PET imaging to other neurotransmitters, such as serotonin, norepinephrine, acetylcholine, γ-aminobutyric acid (GABA) and glutamate [91]. Specific to serotonin, microdialysis has supported development of PET tracers to 5HT-1B and 5HT-2A receptors as a means of monitoring treatment effects on 5HT concentrations [92-94].
INTEGRATION OF MICRODIALYSIS WITH MRI
Three primary applications of MRI are used to support systems neuropharmacology inquiries. These are structural (or anatomical) MRI, magnetic resonance spectroscopy (MRS) and functional MRI (fMRI). There are several examples associating microdialysis with each of these applications in the neurosciences literature over the past 10 – 15 years. Following in turn will be examples representative of each of these MRI modalities. Table 2 also provides an overview of noteworthy examples for each.
