Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development.

Alzheimer's disease (AD) is a large and increasing unmet medical need with no disease-modifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Single-nucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).

Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported. In this study, the kinetics of central KYNA-lowering effect in rats and nonhuman primates (NHPs, Cynomolgus macaques) was investigated using multiple KAT II irreversible inhibitors as pharmacologic probes. Mechanistic pharmacokinetic-pharmacodynamic analysis of in vivo responses to irreversible inhibition quantitatively revealed that 1) KAT II turnover is relatively slow [16-76 hours' half-life (t1/2)], whereas KYNA is cleared more rapidly from the brain (<1 hour t1/2) in both rats and NHPs, 2) KAT II turnover is slower in NHPs than in rats (76 hours vs. 16 hours t1/2, respectively), and 3) the percent contribution of KAT II to KYNA formation is constant (∼80%) across rats and NHPs. Additionally, modeling results enabled establishment of in vitro-in vivo correlation for both enzyme turnover rates and drug potencies. In summary, quantitative translational analysis confirmed the feasibility of central KYNA modulation in humans. Model-based analysis, where system-specific properties and drug-specific properties are mechanistically separated from in vivo responses, enabled quantitative understanding of the KAT II-KYNA pathway, as well as assisted development of promising candidates to test KYNA hypothesis in humans.

A Comparison of Both Motorized and Nonmotorized Treadmill Gait Kinematics to Overground Locomotion.

CONTEXT: Motorized treadmills (MTs) present an altered motor task compared to overground (OG) locomotion in that MT belt surfaces are motor-driven, whereas individuals walking/running OG must propel themselves. A possible solution may lie with novel nonmotorized treadmill (NMT) devices as the belt surface is propelled by the user. OBJECTIVE: The purpose of this study was to compare gait performance during both MT and NMT locomotion to OG. DESIGN: Crossover study. SETTING: A university research laboratory. PATIENTS: A total of 20 healthy adults (10 women) participated in the study. INTERVENTION: Each participant performed self-selected walking and running OG, and on both an MT and NMT. MAIN OUTCOME MEASURE: Shoulder, trunk, and lower-extremity kinematics were analyzed for each treadmill condition and compared to OG. RESULTS: The analyses demonstrated that there were no differences between MT and OG gait kinematics during either walking or running. However, NMT gait showed increased hip, knee, and ankle flexions in late swing and early stance compared to OG during both walking and running. For example, during walking, the NMT elicited hip-, knee-, and ankle-flexion/extension angles of 34.7°, 8.0°, and 3.6° at foot strike compared to 24.8°, -3.1°, and -5.8° in the OG condition (P < .05). There was also a significant reduction in trunk-flexion/extension range of motion during running compared to OG (7.7° in NMT vs 9.8° in OG). CONCLUSIONS: These differences may have implications for both training and rehabilitation on an NMT. Future studies should consider the influence of NMT familiarization on gait performance and should emphasize the assessment of neuromuscular performance.

Glial biomarkers in human central nervous system disease.

There is a growing understanding that aberrant GLIA function is an underlying factor in psychiatric and neurological disorders. As drug discovery efforts begin to focus on glia-related targets, a key gap in knowledge includes the availability of validated biomarkers to help determine which patients suffer from dysfunction of glial cells or who may best respond by targeting glia-related drug mechanisms. Biomarkers are biological variables with a significant relationship to parameters of disease states and can be used as surrogate markers of disease pathology, progression, and/or responses to drug treatment. For example, imaging studies of the CNS enable localization and characterization of anatomical lesions without the need to isolate tissue for biopsy. Many biomarkers of disease pathology in the CNS involve assays of glial cell function and/or response to injury. Each major glia subtype (oligodendroglia, astroglia and microglia) are connected to a number of important and useful biomarkers. Here, we describe current and emerging glial based biomarker approaches for acute CNS injury and the major categories of chronic nervous system dysfunction including neurodegenerative, neuropsychiatric, neoplastic, and autoimmune disorders of the CNS. These descriptions are highlighted in the context of how biomarkers are employed to better understand the role of glia in human CNS disease and in the development of novel therapeutic treatments. GLIA 2016;64:1755-1771.

Recent Developments in Group I Metabotropic Glutamate Receptor Allosteric Modulators for the Treatment of Psychiatric and Neurological Disorders (2014-May 2015).

In the last ~30 years, scientists have made great strides in understanding the biological function of group I metabotropic glutamate receptors (mGlu) in health and disease, as well as developing a broad array of potent and selective agents able to activate or inhibit these receptors. This article provides a comprehensive review of the most recent group I mGlu modulators published in patent and non-patent literatures from 2014 to May, 2015, including design, structure-activity relationship, in silico, in vitro and in vivo properties of key compounds. The current status of clinical mGlu5 negative allosteric modulators (NAMs) and the development of mGlu1 and mGlu5 PET ligands are also highlighted. While the therapeutic potential for group I mGlu modulating agents appears high, significant challenges remain. Strategies to reduce clinical development risks and mitigate important side effects, including psychotomimetic events observed with several mGlu5 NAMs and cellular toxicity associated with mGlu5 positive allosteric modulators (PAMs), while retaining therapeutic efficacy through approaches such as biased ligand signaling are discussed.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-ß-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

The Contribution of Trunk Axial Kinematics to Poststrike Ball Velocity During Maximal Instep Soccer Kicking.

To date, biomechanical analyses of soccer kicking have focused predominantly on lower-extremity motions, with little emphasis on the trunk and upper body. The purpose of this study was to evaluate differences in trunk axial kinematics between novice (n = 10) and skilled (n = 10) participants, as well as to establish the relationship of trunk axial motion and sagittal plane thigh rotation to poststrike ball velocity. Three-dimensional body segmental motion data were captured using high-resolution motion analysis (120 Hz) while each participant completed 5 maximal instep soccer-style kicks. The results demonstrate that skilled participants use 53% greater axial trunk range of motion compared with novice participants (P < .01), as well as 62% greater peak trunk rotation velocity (P < .01). The results also show a moderate, positive correlation of peak trunk rotation velocity with poststrike ball velocity (r = .57; P < .01), and peak hip flexion velocity with poststrike ball velocity (r = .63; P < .01). The current study highlights the potential for trunk rotation-specific training to improve maximum instep kick velocity in developing soccer athletes.

Automated gait temporal-spatial assessment from non-motorized treadmill belt speed data.

Non-motorized treadmills (NMT) provide belt speed data that can be used to estimate work output, and potentially, gait temporal-spatial parameters that provide an improved understanding of gait performance. The purpose of this study was to determine the validity of an automated technique that uses belt speed data from an NMT to estimate temporal-spatial gait parameters. Seventeen injury-free adult participants performed a series of 20-s, metronome-guided walking and running trials for each of eight predetermined cadence conditions (72-200 steps/min). Two NMT-based cadence algorithms [PSD estimated cadence (PEC) and threshold estimated cadence (TEC)], and one NMT-based step length algorithm (NMT_SL) were evaluated for their ability to predict traditional motion analysis-based measures of cadence and step length (MAC and MA_SL, respectively). The results of this study demonstrate that both the PEC and TEC algorithms were capable of predicting MAC with a standard error of the estimate (SEE) less than four steps/min (R(2) = 0.997 and R(2) = 0.993, respectively). Predictions of MA_SL from NMT_SL were separated by gait type (walking vs. running) to account for an obvious separation in the step length data with a qualitative gait change. When applied to walking data, NMT_SL was capable of predicting MA_SL with an SEE of 23 mm (R(2) = 0.96). When applied to running data, NMT_SL was capable of predicting MA_SL with an SEE of 44 mm (R(2) = 0.80). The assessment of the novel technique suggests that it is feasible to use non-motorized treadmill belt speed data to predict gait events and analyze simple gait metrics. Future research should evaluate the applicability of these algorithms for use with participants/patients presenting with pathological gait.

Reduction of brain kynurenic acid improves cognitive function.

The elevation of kynurenic acid (KYNA) observed in schizophrenic patients may contribute to core symptoms arising from glutamate hypofunction, including cognitive impairments. Although increased KYNA levels reduce excitatory neurotransmission, KYNA has been proposed to act as an endogenous antagonist at the glycine site of the glutamate NMDA receptor (NMDAR) and as a negative allosteric modulator at the α7 nicotinic acetylcholine receptor. Levels of KYNA are elevated in CSF and the postmortem brain of schizophrenia patients, and these elevated levels of KYNA could contribute to NMDAR hypofunction and the cognitive deficits and negative symptoms associated with this disease. However, the impact of endogenously produced KYNA on brain function and behavior is less well understood due to a paucity of pharmacological tools. To address this issue, we identified PF-04859989, a brain-penetrable inhibitor of kynurenine aminotransferase II (KAT II), the enzyme responsible for most brain KYNA synthesis. In rats, systemic administration of PF-04859989 dose-dependently reduced brain KYNA to as little as 28% of basal levels, and prevented amphetamine- and ketamine-induced disruption of auditory gating and improved performance in a sustained attention task. It also prevented ketamine-induced disruption of performance in a working memory task and a spatial memory task in rodents and nonhuman primates, respectively. Together, these findings support the hypotheses that endogenous KYNA impacts cognitive function and that inhibition of KAT II, and consequent lowering of endogenous brain KYNA levels, improves cognitive performance under conditions considered relevant for schizophrenia.

An evaluation of upper-body muscle activation during coupled and uncoupled instability resistance training.

Recently, there has been a growth in the popularity of resistance exercises performed on unstable surfaces. However, the relationship between unstable surface training and load coupling on muscle activation is unclear. The purpose of this study was to evaluate changes in muscle activation during a barbell (BB) (coupled) and dumbbell (DB) (uncoupled) chest press exercise performed on an unstable surface. The 3 specific chest press conditions included 50% 1 repetition maximum (RM) with BB (50% BB), 50% 1RM with DBs (50% DB), and 25% 1RM with DBs (25% DB). Ten male subjects participated in the study (age, 23.9 ± 2.6 years; body weight, 82.8 ± 10.2 kg). During testing, mean electromyographic activity was assessed for pectoralis major (PM), triceps brachii, anterior deltoid (AD), and rectus abdominis (RA) and was presented as a percent change across the lifting conditions. It was observed that muscle activation increased by 15% in both the PM and RA from the 50% BB condition to the 50% DB condition. Also, the greatest percent difference in muscle activation between the 50 and 25% DB conditions occurred for PM and AD (+54% during 50% DB). These results suggest that demands on the core musculature to provide stability are increased with the use of DBs (uncoupled) as opposed to a BB (coupled). Where instability training provides a sufficient hypertrophy stimulus in prime mover muscle groups, there may be the added benefit of core stability training. Specifically, this type of training may benefit both untrained persons and those engaged in active rehabilitation.

Kynurenines in CNS disease: regulation by inflammatory cytokines.

The kynurenine pathway (KP) metabolizes the essential amino acid tryptophan and generates a number of neuroactive metabolites collectively called the kynurenines. Segregated into at least two distinct branches, often termed the "neurotoxic" and "neuroprotective" arms of the KP, they are regulated by the two enzymes kynurenine 3-monooxygenase and kynurenine aminotransferase, respectively. Interestingly, several enzymes in the pathway are under tight control of inflammatory mediators. Recent years have seen a tremendous increase in our understanding of neuroinflammation in CNS disease. This review will focus on the regulation of the KP by inflammatory mediators as it pertains to neurodegenerative and psychiatric disorders.

Metabotropic glutamate receptor 5-negative allosteric modulators for the treatment of psychiatric and neurological disorders (2009-July 2013).

Negative allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) have been actively pursued for over a decade as a potential treatment for anxiety, depression, substance abuse, pain, levodopa-induced dyskinesia in Parkinson's disease, fragile X Syndrome, autism, gastroesophageal reflux disease and lower-urinary-tract disorders. This article begins with an introduction of preclinical validation of potential therapies for psychiatric and neurological disorders, and of clinical results, followed by a comprehensive overview of the mGlu5-negative allosteric modulator patent applications published between 2009 and July 2013, with a focus on the analysis of structure and in silico CNS drug-like properties of example compounds and disclosed data. Given positive results in proof-of-concept studies in humans for certain indications such as levodopa-induced dyskinesia in Parkinson's disease, fragile X Syndrome, gastroesophageal reflux disease, migraine and anxiety, and the soaring chemical diversity among the mGlu5-negative allosteric modulators, there is reason to believe that a drug will emerge from this therapeutic class in the near future.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions.

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.

Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the UK.

To compare the diagnostic sensitivity and specificity of seven Cryptosporidium diagnostic assays used in the UK, results from 259 stool samples from patients with acute gastrointestinal symptoms were compared against a nominated gold standard (real-time PCR and oocyst detection). Of the 152 'true positives', 80 were Cryptosporidium hominis, 68 Cryptosporidium parvum, two Cryptosporidium felis, one Cryptosporidium ubiquitum and one Cryptosporidium meleagridis. The Cryptosporidium spp. diagnostic sensitivities of three Cryptosporidium and Giardia combination enzyme immunoassays (EIA) coupled with confirmation of positive reactions were 91.4-93.4 %, whilst the sensitivity of auramine phenol microscopy was 92.1 % and that of immunofluorescence microscopy (IFM) was 97.4 %, all with overlapping 95 % confidence intervals. However, IFM was significantly more sensitive (P = 0.01, paired test of proportions). The sensitivity of modified Ziehl-Neelsen microscopy was 75.4 %, significantly lower than those for the other tests investigated, including an immunochromatographic lateral flow assay (ICLF) (84.9 %) (P = 0.0016). Specificities were 100 % when the ICLF and EIA test algorithms included confirmation of positive reactions; however, four positive EIA reactions were not confirmed for either parasite. There was no significant difference in the detection of C. parvum and C. hominis by each assay, but the detection of other Cryptosporidium spp. requires further investigation, as the numbers of samples were small. EIAs may be considered for diagnostic testing, subject to local validation, and diagnostic algorithms must include confirmation of positive reactions.

Design, synthesis, and pharmacological evaluation of azetedine and pyrrolidine derivatives as dual norepinephrine reuptake inhibitors and 5-HT(1A) partial agonists.

Compounds with combined norepinephrine reuptake inhibitor (NRI) and serotonin 1A (5-HT(1A)) partial agonist pharmacology may offer a new therapeutic approach for treating symptoms of neuropsychiatric disorders including ADHD, depression, and anxiety. Herein we describe the design and optimization of novel chemical matter that exhibits favorable dual NRI and 5-HT(1A) partial agonist activity. Lead compounds in this series were found to be devoid of activity at the dopamine transporter and were shown to be brain penetrant with high receptor occupancy.

Lower extremity muscle activation during baseball pitching.

The purpose of this study was to investigate muscle activation levels of select lower extremity muscles during the pitching motion. Bilateral surface electromyography data on 5 lower extremity muscles (biceps femoris, rectus femoris, gluteus maximus, vastus medialis, and gastrocnemius) were collected on 11 highly skilled baseball pitchers and compared with individual maximal voluntary isometric contraction (MVIC) data. The pitching motion was divided into 4 distinct phases: phase 1, initiation of pitching motion to maximum stride leg knee height; phase 2, maximum stride leg knee height to stride foot contact (SFC); phase 3, SFC to ball release; and phase 4, ball release to 0.5 seconds after ball release (follow-through). Results indicated that trail leg musculature elicited moderate to high activity levels during phases 2 and 3 (38-172% of MVIC). Muscle activity levels of the stride leg were moderate to high during phases 2-4 (23-170% of MVIC). These data indicate a high demand for lower extremity strength and endurance. Specifically, coaches should incorporate unilateral and bilateral lower extremity exercises for strength improvement or maintenance and to facilitate dynamic stabilization of the lower extremities during the pitching motion.

Design, synthesis, and pharmacological evaluation of phenoxy pyridyl derivatives as dual norepinephrine reuptake inhibitors and 5-HT1A partial agonists.

Preclinical studies suggest that compounds with dual norepinephrine reuptake inhibitor (NRI) and 5-HT(1A) partial agonist properties may provide an important new therapeutic approach to ADHD, depression, and anxiety. Reported herein is the discovery of a novel chemical series with a favorable NRI and 5-HT(1A) partial agonist pharmacological profile as well as excellent selectivity for the norepinephrine transporter over the dopamine transporter.

Discovery and pharmacological characterization of aryl piperazine and piperidine ethers as dual acting norepinephrine reuptake inhibitors and 5-HT1A partial agonists.

Compounds that are both norepinephrine reuptake inhibitors (NRI) and 5-HT1(A) partial agonists may have the potential to treat neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD) and depression. Targeted screening of NRI-active compounds for binding to the 5-HT(1A) receptor provided a series of thiomorpholinone hits with this dual activity profile. Several iterations of design, synthesis, and testing led to substituted piperidine diphenyl ethers which are potent NRIs with 5-HT1(A) partial agonist properties. In addition, optimization of these molecules provided compounds which exhibit selectivity for NRI over the dopamine (DAT) and serotonin (SERT) reuptake transporters. Monoamine and 5-HT(1A) in vitro functional activities for select compounds from the developed piperidine diphenyl ether series are also presented.

Synthesis and pharmacological evaluation of aminopyrimidine series of 5-HT1A partial agonists.

Aminopyrimidine 2 (4-(1-(2-(1H-indol-3-yl)ethyl)piperidin-3-yl)-N-cyclopropylpyrimidin-2-amine) emerged from a high throughput screen as a novel 5-HT(1A) agonist. This compound showed moderate potency for 5-HT(1A) in binding and functional assays, as well as moderate metabolic stability. Implementation of a strategy for improving metabolic stability by lowering the lipophilicity (cLogD) led to identification of methyl ether 31 (4-(1-(2-(1H-indol-3-yl)ethyl)piperidin-3-yl)-N-(2-methoxyethyl)pyrimidin-2-amine) as a substantially improved compound within the series.