Changning Wang

Design, Synthesis and Evaluation of Coumarin-based Molecular Probes for Imaging of Myelination

Myelination represents one of the most fundamental biological processes in the vertebrate nervous system. Abnormalities and changes in myelination in the central nervous system (CNS) are seen in many neurodegenerative disorders, such as multiple sclerosis (MS). A long-standing goal has been to directly detect and quantify myelin content in order to facilitate diagnosis and therapeutic treatments of myelin-related diseases. In the course of our studies, we have developed a series of small-molecule probes (SMP) as myelin-imaging agents. Among them are coumarin derivatives, which exhibit promising brain permeability and myelin-binding properties. Herein we report a full account of the design and synthesis of coumarin-based SMPs as myelin-imaging agents. Systematic evaluation of these SMPs in both the CNS and peripheral nervous system (PNS) allowed us to identify some lead agents for potential use as fluorescent dyes for intraoperative nerve mapping in surgical operations or as radiotracers for positron emission tomography (PET) imaging of myelination.

In Vivo Quantification of Myelin Changes in the Vertebrate Nervous System

Destruction or changes associated with myelin membranes in the CNS play a key role in the pathogenesis of multiple sclerosis and other related neurodegenerative disorders. A long-standing goal has been to detect and quantify myelin content in vivo. For this reason, we have developed a myelin-imaging technique based on positron emission tomography (PET). PET is a quantitative imaging modality that has been widely used in clinical settings for direct assessment of biological processes at the molecular level. However, lack of myelin-imaging probes has hampered the use of PET for imaging of myelination in the CNS. Here, we report a myelin-imaging agent, termed Case Imaging Compound (CIC) that readily penetrates the blood–brain barrier and preferentially localizes to myelinated regions of the brain. After radiolabeling with positron-emitting carbon-11, [11C]CIC–PET was conducted in longitudinal studies using a lysolethicin-induced rat model of focal demyelination and subsequent remyelination. Quantitative analysis showed that the retention of [11C]CIC correlates with the level of demyelination/remyelination. These studies indicate that, for the first time, [11C]CIC–PET can be used as an imaging marker of myelination, which has the potential to be translated into clinical studies in multiple sclerosis and other myelin-related diseases for early diagnosis, subtyping, and efficacy evaluation of therapeutic treatments aimed at myelin repair.

Longitudinal near-infrared imaging of myelination.

Myelination is one of the fundamental biological processes in the development of vertebrate nervous system. Disturbance of myelination is found to be associated with progression in many neurological diseases such as multiple sclerosis. Tremendous efforts have been made to develop novel therapeutic agents that prevent demyelination and/or promote remyelination. These efforts need to be accompanied by the development of imaging tools that permit direct quantification of myelination in vivo. In this work, we describe a novel near-infrared fluorescence imaging technique that is capable of direct quantification of myelination in vivo. This technique is developed based on a near-infrared fluorescent probe, 3,3′-diethylthiatricarbocyanine iodide (DBT) that readily enters the brain and specifically binds to myelinated fibers. In vivo imaging studies were first conducted in two animal models of hypermyelination and hypomyelination followed by longitudinal studies in the cuprizone-induced demyelination/remyelination mouse model. Quantitative analysis suggests that DBT is a sensitive and specific imaging probe of myelination, which complements other current myelin-imaging modalities and is of low cost.

In Vivo Imaging of Histone Deacetylases (HDACs) in the Central Nervous System and Major Peripheral Organs

Epigenetic enzymes are now targeted to treat the underlying gene expression dysregulation that contribute to disease pathogenesis. Histone deacetylases (HDACs) have shown broad potential in treatments against cancer and emerging data supports their targeting in the context of cardiovascular disease and central nervous system dysfunction. Development of a molecular agent for non-invasive imaging to elucidate the distribution and functional roles of HDACs in humans will accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and validation of an HDAC imaging agent, [11C]6. Our imaging results demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [11C]6 for human epigenetic imaging.

In Situ Fluorescence Imaging of Myelination

We describe a novel fluorescent dye, 3-(4-aminophenyl)-2H-chromen-2-one (termed case myelin compound or CMC), that can be used for in situ fluorescent imaging of myelin in the vertebrate nervous system. When administered via intravenous injection into the tail vein, CMC selectively stained large bundles of myelinated fibers in both the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS, CMC readily entered the brain and selectively localized in myelinated regions such as the corpus callosum and cerebellum. CMC also selectively stained myelinated nerves in the PNS. The staining patterns of CMC in a hypermyelinated mouse model were consistent with immunohistochemical staining. Similar to immunohistochemical staining, CMC selectively bound to myelin sheaths present in the white matter tracts. Unlike CMC, conventional antibody staining for myelin basic protein also stained oligodendrocyte cytoplasm in the striatum as well as granule layers in the cerebellum. In vivo application of CMC was also demonstrated by fluorescence imaging of myelinated nerves in the PNS.

Imaging Evaluation of 5HT2C Agonists, [11C]WAY-163909 and [11C]Vabicaserin, Formed by Pictet–Spengler Cyclization

The serotonin subtype 2C (5HT2C) receptor is an emerging and promising drug target to treat several disorders of the human central nervous system. In this current report, two potent and selective 5HT2C full agonists, WAY-163909 (2) and vabicaserin (3), were radiolabeled with carbon-11 via Pictet–Spengler cyclization with [11C]formaldehyde and used in positron emission tomography (PET) imaging. Reaction conditions were optimized to exclude the major source of isotope dilution caused by the previously unknown breakdown of N,N-dimethylformamide (DMF) to formaldehyde at high temperature under mildly acid conditions. In vivo PET imaging was utilized to evaluate the pharmacokinetics and distribution of the carbon-11 labeled 5HT2C agonists. Both radiolabeled molecules exhibit high blood–brain barrier (BBB) penetration and nonspecific binding, which was unaltered by preadministration of the unlabeled agonist. Our work demonstrates that Pictet–Spengler cyclization can be used to label drugs with carbon-11 to study their pharmacokinetics and for evaluation as PET radiotracers.

A myelin-specific contrast agent for magnetic resonance imaging of myelination.

Myelination is one of the most fundamental biological processes in the development of vertebrate nervous systems. Abnormal or disrupted myelination occurs in many acquired or inherited neurodegenerative diseases, including multiple sclerosis (MS) and various leukodystrophies. To date, magnetic resonance imaging (MRI) has been the primary tool for diagnosing and monitoring the progression of MS and related diseases; however, any change in signal intensity of conventional MRI reflects a change only in tissue water content, which is a nonspecific measure of the overall changes in macroscopic tissue injury. Thus, the use of MRI as a primary measure of disease activity was shown to be disassociated from the clinical outcome due to the lack of specificity for myelination. In order to increase the MRI specificity for myelin pathologies, we designed and synthesized the first Gd-based T(1) MR contrast agent (MIC) that binds to myelin with high specificity. In this Communication, we demonstrate that MIC localizes in brain regions in proportion to the extent of myelination. In addition, MIC possesses promising MR contrast properties, which allow for direct detection of myelin distribution through T(1) mapping in the mouse brain.

Visualizing epigenetics: Current advances and advantages in HDAC PET imaging techniques

Abnormal gene regulation as a consequence of flawed epigenetic mechanisms may be central to the initiation and persistence of many human diseases. However, the association of epigenetic dysfunction with disease and the development of therapeutic agents for treatment are slow. Developing new methodologies used to visualize chromatin-modifying enzymes and their function in the human brain would be valuable for the diagnosis of brain disorders and drug discovery. We provide an overview of current invasive and noninvasive techniques for measuring expression and functions of chromatin-modifying enzymes in the brain, emphasizing tools applicable to histone deacetylase (HDAC) enzymes as a leading example. The majority of current techniques are invasive and difficult to translate to what is happening within a human brain in vivo. However, recent progress in molecular imaging provides new, noninvasive ways to visualize epigenetics in the human brain. Neuroimaging tool development presents a unique set of challenges in order to identify and validate CNS radiotracers for HDACs and other histone-modifying enzymes. We summarize advances in the effort to image HDACs and HDAC inhibitory effects in the brain using positron emission tomography (PET) and highlight generalizable techniques that can be adapted to investigate other specific components of epigenetic machinery. Translational tools like neuroimaging by PET and magnetic resonance imaging provide the best way to link our current understanding of epigenetic changes with in vivo function in normal and diseased brains. These tools will be a critical addition to ex vivo methods to evaluate - and intervene - in CNS dysfunction.

Myelin Imaging Compound (MIC) Enhanced Magnetic Resonance Imaging of Myelination

The vertebrate nervous system is characterized by myelination, a fundamental biological process that protects the axons and facilitates electric pulse transduction. Damage to myelin is considered a major effect of autoimmune diseases such as multiple sclerosis (MS). Currently, therapeutic interventions are focused on protecting myelin integrity and promoting myelin repair. These efforts need to be accompanied by an effective imaging tool that correlates the disease progression with the extent of myelination. To date, magnetic resonance imaging (MRI) is the primary imaging technique to detect brain lesions in MS. However, conventional MRI cannot differentiate demyelinated lesions from other inflammatory lesions and therefore cannot predict disease progression in MS. To address this problem, we have prepared a Gd-based contrast agent, termed MIC (myelin imaging compound), which binds to myelin with high specificity. In this work, we demonstrate that MIC exhibits a high kinetic stability toward transmetalation with promising relaxometric properties. MIC was used for in vivo imaging of myelination following intracerebroventricular infusion in the rat brain. MIC was found to distribute preferentially in highly myelinated regions and was able to detect regions of focally induced demyelination.

In vivo positron emission tomography (PET) imaging of mesenchymal-epithelial transition (MET) receptor.

We report the radiosynthesis and evaluation of 3-[3,5-dimethyl-4-(4-[11C]methylpiperazinecarbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chlorophenyl)methylamide, termed [11C]SU11274 ([11C]14) for in vivo imaging of mesenchymal-epithelial transition (MET) receptor by positron emission tomography (PET). Following the synthesis of the precursor (13) that was achieved in 10 steps with a total yield of 9.7%, [11C]14 was obtained through radiomethylation in a range of 5-10% radiochemical yield and over 95% radiochemical purity. For in vivo PET studies, two human lung cancer xenograft models were established using MET-positive NCI-H1975 and MET-negative NCI-H520 cell lines. Quantitative [11C]14-PET studies showed that the tumor uptake of [11C]14 in the NCI-H1975 xenografts was significantly higher than that in the NCI-H520 xenografts, which is consistent with their corresponding immunohistochemical tissue staining patterns of MET receptors from the same animals. These studies demonstrated that [11C]14-PET is an appropriate imaging marker for quantification of MET receptor in vivo, which can facilitate efficacy evaluation in the clinical development of MET-targeted cancer therapeutics.