Multi-contrast, isotropic, single-slab 3D MR imaging in multiple sclerosis

To describe signal and contrast properties of an isotropic, single-slab 3D dataset [double inversion- recovery (DIR), fluid-attenuated inversion recovery (FLAIR), T2, and T1-weighted magnetization prepared rapid acquisition gradient-echo (MPRAGE)] and to evaluate its performance in detecting multiple sclerosis (MS) brain lesions compared to 2D T2-weighted spin-echo (T2SE). All single-slab 3D sequences and 2DT2SE were acquired in 16 MS patients and 9 age-matched healthy controls. Lesions were scored independently by two raters and characterized anatomically. Two-tailed Bonferroni-corrected Student’s t-tests were used to detect differences in lesion detection between the various sequences per anatomical area after logtransformation. In general, signal and contrast properties of the 3D sequences enabled improved detection of MS brain lesions compared to 2DT2SE. Specifically, 3D-DIR showed the highest detection of intracortical and mixed WM-GM lesions, whereas 3D-FLAIR showed the highest total number of WM lesions. Both 3D-DIR and 3D-FLAIR showed the highest number of infratentorial lesions. 3DT2 and 3D-MPRAGE did not improve lesion detection compared to 2DT2SE. Multi-contrast, isotropic, single-slab 3D MRI allowed an improved detection of both GM and WM lesions compared to 2D-T2SE. A selection of single-slab 3D contrasts, for example, 3D-FLAIR and 3D-DIR, could replace 2D sequences in the radiological practice

Cholinergic imbalance in the multiple sclerosis hippocampus

Hippocampal pathology was shown to be extensive in multiple sclerosis (MS) and is associated with memory impairment. In this post-mortem study, we investigated hippocampal tissue from MS and Alzheimer's disease (AD) patients and compared these to non-neurological controls. By means of biochemical assessment, (immuno)histochemistry and western blot analyses, we detected substantial alterations in the cholinergic neurotransmitter system in the MS hippocampus, which were different from those in AD hippocampus. In MS hippocampus, activity and protein expression of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was decreased, while the activity and protein expression of acetylcholinesterase (AChE), the acetylcholine degrading enzyme, was found to be unaltered. In contrast, in AD hippocampus both ChAT and AChE enzyme activity and protein expression was decreased. Our findings reveal an MS-specific cholinergic imbalance in the hippocampus, which may be instrumental in terms of future treatment options for memory problems in this diseas

Gray matter imaging in multiple sclerosis: what have we learned?

At the early onset of the 20th century, several studies already reported that the gray matter was implicated in the histopathology of multiple sclerosis (MS). However, as white matter pathology long received predominant attention in this disease, and histological staining techniques for detecting myelin in the gray matter were suboptimal, it was not until the beginning of the 21st century that the true extent and importance of gray matter pathology in MS was finally recognized. Gray matter damage was shown to be frequent and extensive, and more pronounced in the progressive disease phases. Several studies subsequently demonstrated that the histopathology of gray matter lesions differs from that of white matter lesions. Unfortunately, imaging of pathology in gray matter structures proved to be difficult, especially when using conventional magnetic resonance imaging (MRI) techniques. However, with the recent introduction of several more advanced MRI techniques, the detection of cortical and subcortical damage in MS has considerably improved. This has important consequences for studying the clinical correlates of gray matter damage. In this review, we provide an overview of what has been learned about imaging of gray matter damage in MS, and offer a brief perspective with regards to future developments in this field

Combining Isotopic Tracer Techniques to Increase Efficiency of Clinical Pharmacokinetic Trials in Oncology

With increasing numbers of drugs tested in oncology for smaller patient populations, fewer patients are available to answer important clinical pharmacological questions in the timeframe of clinical drug development. The quality and efficiency of trials to assess the pharmacokinetics of new drugs can be improved by making better use of available resources. One approach to do this is by making more effective use of isotopic tracer techniques. With increasing sensitivity of liquid chromatography-tandem mass spectrometry analyzing equipment over the years, it has now become possible to generate much more rich, high-quality pharmacokinetic data than before. In particular we want to make a plea here for a hybrid trial approach, where both radiolabeled drug and stable isotopically labeled drug are administered to patients to assess both the absolute bioavailability and absorption, distribution, metabolism and excretion in a single clinical trial experiment

Stable Isotopically Labeled Intravenous Microdose Pharmacokinetic Trials as a Tool to Assess Absolute Bioavailability: Feasibility and Paradigm to Apply for Protein Kinase Inhibitors in Oncology

Absolute bioavailability is defined as a measure of the extent to which the administered drug is absorbed systemically and becomes available in the general circulation compared with intravenously administered drug.1 For orally administered drugs, obtaining data on absolute bioavailability is an important component of clinical drug development. Low bioavailability may indicate poor solubility and/or permeability, membrane transport, and/or enzymatic metabolism.1, 2 Knowledge on absolute bioavailability in an early stage of clinical development is therefore considered essential to allow for the development of optimal drug formulations. Despite the clear usefulness, absolute bioavailability determination is not mandatory and therefore not a routine part of clinical drug development.1 For the group of orally administered tyrosine kinase inhibitors, an important oral drug class in oncology, it was identified that for more than half the drugs registered up to 2014, an absolute bioavailability trial was not performed during clinical drug development.2 The main reason for this might be that the assessment of absolute bioavailability requires the formulation and safety testing of an intravenous formulation at therapeutic strength, which serves as a reference to the oral formulation. Technical issues (eg, poor solubility) as well as costs associated with development and safety testing of an intravenous formulation make it often omitted. The microdose trial design may aid overcoming these problems by making use of an intravenous microdose formulation, defined as less than 1/100th of the therapeutic dose with a maximum of 100 μg. Because microdose studies involve exposure to very small amounts of drug, additional safety testing of the intravenous formulation is not required. Furthermore, drug solubility issues are most often no longer a problem, as only a 100‐μg amount needs to be dissolved into an intravenous formulation.3, 4 Originally, the major concern with microdosing has been the potential for nonlinear pharmacokinetics between the microdose and the therapeutic dose.5 The introduction of stable isotopically labeled microdosing has made it possible to overcome this problem.6 By allowing simultaneous administration of a labeled microdose next to a therapeutic unlabeled dose, this new approach has provided opportunity to further improve absolute bioavailability trial designs. In this review, we describe the way clinical absolute bioavailability trials are conducted using both a conventional trial design and a microdose trial design. The use of a stable isotopically labeled microdose (SILM) in combination with ultrasensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) as an analytical technique is described in more detail. For the group of orally administered small‐molecule protein kinase inhibitors (smPKIs), we investigated whether absolute bioavailability was determined during clinical drug development and if a SILM trial design in combination with LC‐MS/MS would have been feasible. We conclude by discussing how the use of SILM studies can affect the execution of absolute bioavailability trials in the future

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future

2010. Resting state networks change in clinically isolated syndrome

Task-functional magnetic resonance imaging studies have shown that early cortical recruitment exists in multiple sclerosis, which can partly explain the discrepancy between conventional magnetic resonance imaging and clinical disability. The study of the brain 'at rest' may provide additional information, because task-induced metabolic changes are relatively small compared to the energy use of the resting brain. We therefore questioned whether functional changes exist at rest in the early phase of multiple sclerosis, and addressed this question by a network analysis of no-task functional magnetic resonance imaging data. Fourteen patients with symptoms suggestive of multiple sclerosis (clinically isolated syndrome), 31 patients with relapsing remitting multiple sclerosis and 41 healthy controls were included. Resting state functional magnetic resonance imaging data were brought to standard space using non-linear registration, and further analysed using multi-subject independent component analysis and individual time-course regression. Eight meaningful resting state networks were identified in our subjects and compared between the three groups with non-parametric permutation testing, using threshold-free cluster enhancement to correct for multiple comparisons. Additionally, quantitative measures of structural damage were obtained. Grey and white matter volumes, normalized for head size, were measured for each subject. White matter integrity was investigated with diffusion tensor measures that were compared between groups voxel-wise using tract-based spatial statistics. Patients with clinically isolated syndrome showed increased synchronization in six of the eight resting state networks, including the default mode network and sensorimotor network, compared to controls or relapsing remitting patients. No significant decreases were found in patients with clinically isolated syndrome. No significant resting state synchronization differences were found between relapsing remitting patients and controls. Normalized grey matter volume was decreased and white matter diffusivity measures were abnormal in relapsing remitting patients compared to controls, whereas no atrophy or diffusivity changes were found for the clinically isolated syndrome group. Thus, early synchronization changes are found in patients with clinically isolated syndrome that are suggestive of cortical reorganization of resting state networks. These changes are lost in patients with relapsing remitting multiple sclerosis with increasing brain damage, indicating that cortical reorganization of resting state networks is an early and finite phenomenon in multiple sclerosis

Development, validation, and clinical application of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of total intracellular β-decitabine nucleotides and genomic DNA incorporated β-decitabine and 5-methyl-2'-deoxycytidine

DNA hypermethylation is an epigenetic event that is commonly found in malignant cells and is used as a therapeutic target for β-decitabine (β-DEC) containing hypomethylating agents (eg Dacogen® and guadecitabine). β-DEC requires cellular uptake and intracellular metabolic activation to β-DEC triphosphate before it can get incorporated into the DNA. Once incorporated in the DNA, β-DEC can exert its hypomethylating effect by trapping DNA methyltransferases (DNMTs), resulting in reduced 5-methyl-2'-deoxycytidine (5mdC) DNA content. β-DEC DNA incorporation and its effect on DNA methylation, however, have not yet been investigated in patients treated with β-DEC containing therapies. For this reason, we developed and validated a sensitive and selective LC-MS/MS method to determine total intracellular β-DEC nucleotide (β-DEC-XP) concentrations, as well as to quantify β-DEC and 5mdC DNA incorporation relative to 2'-deoxycytidine (2dC) DNA content. The assay was successfully validated according to FDA and EMA guidelines in a linear range from 0.5 to 100 ng/mL (β-DEC), 50 to 10,000 ng/mL (2dC), and 5 to 1,000 ng/mL (5mdC) in peripheral blood mononuclear cell (PBMC) lysate. An additional calibrator at a concentration of 0.1 ng/mL was added for β-DEC to serve as a limit of detection (LOD). Clinical applicability of the method was demonstrated in patients treated with guadecitabine. Our data support the use of the validated LC-MS/MS method to further explore the intracellular pharmacokinetics in patients treated with β-DEC containing hypomethylating agents

Development, validation, and clinical application of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of total intracellular β-decitabine nucleotides and genomic DNA incorporated β-decitabine and 5-methyl-2'-deoxycytidine

DNA hypermethylation is an epigenetic event that is commonly found in malignant cells and is used as a therapeutic target for β-decitabine (β-DEC) containing hypomethylating agents (eg Dacogen® and guadecitabine). β-DEC requires cellular uptake and intracellular metabolic activation to β-DEC triphosphate before it can get incorporated into the DNA. Once incorporated in the DNA, β-DEC can exert its hypomethylating effect by trapping DNA methyltransferases (DNMTs), resulting in reduced 5-methyl-2'-deoxycytidine (5mdC) DNA content. β-DEC DNA incorporation and its effect on DNA methylation, however, have not yet been investigated in patients treated with β-DEC containing therapies. For this reason, we developed and validated a sensitive and selective LC-MS/MS method to determine total intracellular β-DEC nucleotide (β-DEC-XP) concentrations, as well as to quantify β-DEC and 5mdC DNA incorporation relative to 2'-deoxycytidine (2dC) DNA content. The assay was successfully validated according to FDA and EMA guidelines in a linear range from 0.5 to 100 ng/mL (β-DEC), 50 to 10,000 ng/mL (2dC), and 5 to 1,000 ng/mL (5mdC) in peripheral blood mononuclear cell (PBMC) lysate. An additional calibrator at a concentration of 0.1 ng/mL was added for β-DEC to serve as a limit of detection (LOD). Clinical applicability of the method was demonstrated in patients treated with guadecitabine. Our data support the use of the validated LC-MS/MS method to further explore the intracellular pharmacokinetics in patients treated with β-DEC containing hypomethylating agents

Stable Isotopically Labeled Intravenous Microdose Pharmacokinetic Trials as a Tool to Assess Absolute Bioavailability: Feasibility and Paradigm to Apply for Protein Kinase Inhibitors in Oncology

Absolute bioavailability is defined as a measure of the extent to which the administered drug is absorbed systemically and becomes available in the general circulation compared with intravenously administered drug.1 For orally administered drugs, obtaining data on absolute bioavailability is an important component of clinical drug development. Low bioavailability may indicate poor solubility and/or permeability, membrane transport, and/or enzymatic metabolism.1, 2 Knowledge on absolute bioavailability in an early stage of clinical development is therefore considered essential to allow for the development of optimal drug formulations. Despite the clear usefulness, absolute bioavailability determination is not mandatory and therefore not a routine part of clinical drug development.1 For the group of orally administered tyrosine kinase inhibitors, an important oral drug class in oncology, it was identified that for more than half the drugs registered up to 2014, an absolute bioavailability trial was not performed during clinical drug development.2 The main reason for this might be that the assessment of absolute bioavailability requires the formulation and safety testing of an intravenous formulation at therapeutic strength, which serves as a reference to the oral formulation. Technical issues (eg, poor solubility) as well as costs associated with development and safety testing of an intravenous formulation make it often omitted. The microdose trial design may aid overcoming these problems by making use of an intravenous microdose formulation, defined as less than 1/100th of the therapeutic dose with a maximum of 100 μg. Because microdose studies involve exposure to very small amounts of drug, additional safety testing of the intravenous formulation is not required. Furthermore, drug solubility issues are most often no longer a problem, as only a 100‐μg amount needs to be dissolved into an intravenous formulation.3, 4 Originally, the major concern with microdosing has been the potential for nonlinear pharmacokinetics between the microdose and the therapeutic dose.5 The introduction of stable isotopically labeled microdosing has made it possible to overcome this problem.6 By allowing simultaneous administration of a labeled microdose next to a therapeutic unlabeled dose, this new approach has provided opportunity to further improve absolute bioavailability trial designs. In this review, we describe the way clinical absolute bioavailability trials are conducted using both a conventional trial design and a microdose trial design. The use of a stable isotopically labeled microdose (SILM) in combination with ultrasensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) as an analytical technique is described in more detail. For the group of orally administered small‐molecule protein kinase inhibitors (smPKIs), we investigated whether absolute bioavailability was determined during clinical drug development and if a SILM trial design in combination with LC‐MS/MS would have been feasible. We conclude by discussing how the use of SILM studies can affect the execution of absolute bioavailability trials in the future