Seven Tesla Evidence for Columnar and Rostral-Caudal Organization of the Human Periaqueductal Gray Response in the Absence of Threat: A Working Memory Study.

The periaqueductal gray (PAG) is a small midbrain structure that surrounds the cerebral aqueduct, regulates brain-body communication, and is often studied for its role in "fight-or-flight" and "freezing" responses to threat. We used ultra-high-field 7 T fMRI to resolve the PAG in humans and distinguish it from the cerebral aqueduct, examining its in vivo function during a working memory task (N = 87). Both mild and moderate cognitive demands elicited spatially similar patterns of whole-brain blood oxygenation level-dependent (BOLD) response, and moderate cognitive demand elicited widespread BOLD increases above baseline in the brainstem. Notably, these brainstem increases were not significantly greater than those in the mild demand condition, suggesting that a subthreshold brainstem BOLD increase occurred for mild cognitive demand as well. Subject-specific masks were group aligned to examine PAG response. In PAG, both mild and moderate demands elicited a well-defined response in ventrolateral PAG, a region thought to be functionally related to anticipated painful threat in humans and nonhuman animals-yet, the present task posed only the most minimal (if any) "threat," with the cognitive tasks used being approximately as challenging as remembering a phone number. These findings suggest that the PAG may play a more general role in visceromotor regulation, even in the absence of threat.

Imaging High-Risk Atherothrombosis Using a Novel Fibrin-Binding Positron Emission Tomography Probe.

BACKGROUND AND PURPOSE: High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. METHODS: Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. RESULTS: Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group (P<0.05). Ex vivo PET and autoradiography of the abdominal aorta correlated positively with in vivo PET measurements. Plaque disruption identified by 68Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. CONCLUSIONS: We demonstrated that molecular fibrin PET imaging using 68Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

Functional Involvement of Human Periaqueductal Gray and Other Midbrain Nuclei in Cognitive Control.

Recent theoretical advances have motivated the hypothesis that the periaqueductal gray (PAG) participates in behaviors that involve changes in the autonomic control of visceromotor activity, including during cognitively demanding tasks. We used ultra-high-field (7 tesla) fMRI to measure human brain activity at 1.1 mm resolution while participants completed a working memory task. Consistent with prior work, participants were less accurate and responded more slowly with increasing memory load-signs of increasing task difficulty. Whole-brain fMRI analysis revealed increased activity in multiple cortical areas with increasing working memory load, including frontal and parietal cortex, dorsal cingulate, supplementary motor area, and anterior insula. Several dopamine-rich midbrain nuclei, such as the substantia nigra and ventral tegmental area, also exhibited load-dependent increases in activation. To investigate PAG involvement during cognitive engagement, we developed an automated method for segmenting and spatially normalizing the PAG. Analyses using cross-validated linear support vector machines showed that the PAG discriminated high versus low working memory load conditions with 95% accuracy in individual subjects based on activity increases in lateral and ventrolateral PAG. Effect sizes in the PAG were comparable in magnitude to those in many of the cortical areas. These findings suggest that cognitive control is not only associated with cortical activity in the frontal and parietal lobes, but also with increased activity in the subcortical PAG and other midbrain regions involved in the regulation of autonomic nervous system function.SIGNIFICANCE STATEMENT Functional neuroimaging in humans has shown that cognitive control engages multiple corticostriatal networks and brainstem nuclei, but theoretical advances suggest that the periaqueductal gray (PAG) should also be engaged during cognitively demanding tasks. Recent advances in ultra-high-field fMRI provided an opportunity to obtain the first evidence that increased activation of intermediate and rostral portions of lateral and ventrolateral PAG columns in humans is modulated by cognitive load. These findings suggest that cognitive control is not solely mediated by activity in the cortex, but that midbrain structures important for autonomic regulation also play a crucial role in higher-order cognition.

Abstinence to chronic methamphetamine switches connectivity between striatal, hippocampal and sensorimotor regions and increases cerebral blood volume response.

Methamphetamine (meth), and other psychostimulants such as cocaine, present a persistent problem for society with chronic users being highly prone to relapse. We show, in a chronic methamphetamine administration model, that discontinuation of drug for more than a week produces much larger changes in overall meth-induced brain connectivity and cerebral blood volume (CBV) response than changes that occur immediately following meth administration. Areas showing the largest changes were hippocampal, limbic striatum and sensorimotor cortical regions as well as brain stem areas including the pedunculopontine tegmentum (PPTg) and pontine nuclei - regions known to be important in mediating reinstatement of drug-taking after abstinence. These changes occur concomitantly with behavioral sensitization and appear to be mediated through increases in dopamine D1 and D3 and decreases in D2 receptor protein and mRNA expression. We further identify a novel region of dorsal caudate/putamen, with a low density of calbindin neurons, that has an opposite hemodynamic response to meth than the rest of the caudate/putamen and accumbens and shows very strong correlation with dorsal CA1 and CA3 hippocampus. This correlation switches following meth abstinence from CA1/CA3 to strong connections with ventral hippocampus (ventral subiculum) and nucleus accumbens. These data provide novel evidence for temporal alterations in brain connectivity where chronic meth can subvert hippocampal - striatal interactions from cognitive control regions to regions that mediate drug reinstatement. Our results also demonstrate that the signs and magnitudes of the induced CBV changes following challenge with meth or a D3-preferring agonist are a complementary read out of the relative changes that occur in D1, D2 and D3 receptors using protein or mRNA levels.

Synthesis and evaluation of N-(methylthiophenyl)picolinamide derivatives as PET radioligands for metabotropic glutamate receptor subtype 4.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/µmol (n=6) and 463±263GBq/µmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.

Identification of discrete functional subregions of the human periaqueductal gray.

The midbrain periaqueductal gray (PAG) region is organized into distinct subregions that coordinate survival-related responses during threat and stress [Bandler R, Keay KA, Floyd N, Price J (2000) Brain Res 53 (1):95-104]. To examine PAG function in humans, researchers have relied primarily on functional MRI (fMRI), but technological and methodological limitations have prevented researchers from localizing responses to different PAG subregions. We used high-field strength (7-T) fMRI techniques to image the PAG at high resolution (0.75 mm isotropic), which was critical for dissociating the PAG from the greater signal variability in the aqueduct. Activation while participants were exposed to emotionally aversive images segregated into subregions of the PAG along both dorsal/ventral and rostral/caudal axes. In the rostral PAG, activity was localized to lateral and dorsomedial subregions. In caudal PAG, activity was localized to the ventrolateral region. This shifting pattern of activity from dorsal to ventral PAG along the rostrocaudal axis mirrors structural and functional neurobiological observations in nonhuman animals. Activity in lateral and ventrolateral subregions also grouped with distinct emotional experiences (e.g., anger and sadness) in a factor analysis, suggesting that each subregion participates in distinct functional circuitry. This study establishes the use of high-field strength fMRI as a promising technique for revealing the functional architecture of the PAG. The techniques developed here also may be extended to investigate the functional roles of other brainstem nuclei.

Re-exploring the N-phenylpicolinamide derivatives to develop mGlu4 ligands with improved affinity and in vitro microsomal stability.

In recent years, mGlu4 has received great attention and research effort because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). Many positive allosteric modulators of mGlu4 have been developed. To better understand the role of mGlu4 in healthy and disease conditions, we are interested in developing an mGlu4 selective radioligand for in vivo studies. Thus, we had synthesized and studied [(11)C]2 as a PET tracer for mGlu4, which demonstrated some promising features as a PET radioligand as well as the limitation need to be improved. In order to develop an mGlu4 ligand with enhanced affinity and improved metabolic stability, we have modified, synthesized and evaluated a series of new N-phenylpicolinamide derivatives. The SAR study has discovered a number of compounds with low nM affinity to mGlu4. The dideuteriumfluoromethoxy modified compound 24 is identified as a very promising mGlu4 ligand, which has demonstrated enhanced affinity, improved in vitro microsomal stability, good selectivity and good permeability.

Radiosynthesis of N-(4-chloro-3-[(11)C]methoxyphenyl)-2-picolinamide ([(11)C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4).

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu4 positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu4 PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu4 expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([(11)C]3) as a PET radiotracer for mGlu4, and characterized its biological properties in Sprague Dawley rats. [(11)C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [(11)C]CH3I. Total synthesis time was 38±2.2min (n=7) from the end of bombardment to the formulation. The radioligand [(11)C]3 was obtained in 27.7±5.3% (n=5) decay corrected radiochemical yield based on the radioactivity of [(11)C]CO2. The radiochemical purity of [(11)C]3 was >99%. Specific activity was 188.7±88.8GBq/mol (n=4) at the end of synthesis (EOS). PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu4 modulator (4) to investigate specificity and 3 studies blocking with an mGlu5 modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [(11)C]3 (peak activity between 1-3min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu4 modulator 4 showed 22-28% decrease of [(11)C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu5. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu4, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.

Pharmacologic neuroimaging of the ontogeny of dopamine receptor function.

Characterization of the ontogeny of the cerebral dopaminergic system is crucial for gaining a greater understanding of normal brain development and its alterations in response to drugs of abuse or conditions such as attention-deficit hyperactivity disorder. Pharmacological MRI (phMRI) was used to determine the response to dopamine transporter (DAT) blockers cocaine and methylphenidate (MPH), the dopamine releaser D-amphetamine (AMPH), the selective D1 agonist dihydrexidine, and the D2/D3 agonist quinpirole in young (<30 days old) and adult (>60 days old) rats. In adult rats, cocaine (0.5 mg/kg i.v.) or MPH (2 mg/kg) induced primarily positive cerebral blood volume (rCBV) changes in the dopaminergic circuitry, but negative rCBV changes in the young animals. Microdialysis measurements in the striatum showed that young rats have a smaller increase in extracellular dopamine in response to cocaine than adults. The young rats showed little rCBV response to the selective D1 agonist dihydrexidine in contrast to robust rCBV increases observed in the adults, whereas there was a similar negative rCBV response in the young and adult rats to the D2 agonist quinpirole. We also performed a meta-analysis of literature data on the development of D1 and D2 receptors and the DAT. These data suggest a predominance of D2-like over D1-like function between 20 and 30 days of age. These combined results suggested that the dopamine D1 receptor is functionally inhibited at young age.

Synthesis and Characterization of Fluorine-18-Labeled N-(4-Chloro-3-((fluoromethyl-d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain.

We have synthesized and characterized [18F]-N-(4-chloro-3-((fluoromethyl-d2)thio)phenyl)-picolinamide ([18F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [18F]15 displays central nervous system drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity, and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [18F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol. Ex vivo biodistribution studies showed reversible binding of [18F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague Dawley rats showed that [18F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analogue) and 15 showed significant dose-dependent blocking effects. These results suggest that [18F]15 is a promising radioligand for PET imaging mGluR4 in the brain.

The blood-brain barrier is intact after levodopa-induced dyskinesias in parkinsonian primates--evidence from in vivo neuroimaging studies.

It has been suggested, based on rodent studies, that levodopa (L-dopa) induced dyskinesia is associated with a disrupted blood-brain barrier (BBB). We have investigated BBB integrity with in vivo neuroimaging techniques in six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned primates exhibiting L-dopa-induced dyskinesia. Magnetic resonance imaging (MRI) performed before and after injection of Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) revealed an intact BBB in the basal ganglia showing that l-dopa-induced dyskinesia is not associated with a disrupted BBB in this model.

Magnetic resonance spectroscopy of regional brain metabolite markers in FALS mice and the effects of dietary creatine supplementation.

We investigated the effects of disease progression on brain regional neurochemistry in a mutant mouse model of familial amyotrophic lateral sclerosis (FALS; the G93A model) using in vivo and in vitro magnetic resonance spectroscopy (MRS). There were numerous changes in the brain spectra that were brain region dependent. At early time points starting around 80 days of age there were increases in brain glutamate. At later time points there were more extensive changes including decreased N-acetyl aspartate and glutamate and increased glutamine, taurine and myo-inositol. The effects of the disease were most severe in spinal cord followed by medulla and then sensorimotor cortex. There were no changes noted in cerebellum as a control region. The effects of creatine supplementation in the diet (2%) were measured in wild-type and FALS animals in medulla, cerebellum and cortex. The increase in brain creatine was largest in cerebellum (25%) followed by medulla (11%) and then cortex (4%), reflecting the ordering of creatine kinase activity. There was a protective effect of creatine on N-acetyl aspartate loss in the medulla at late stages. Creatine supplementation had a positive effect on weight retention, leading to a 13% increase in weight between 120 and 130 days. MRS shows promise in monitoring multiple facets of neuroprotective strategies in ALS and ALS models.

Dopaminergic response to graded dopamine concentration elicited by four amphetamine doses.

UNLABELLED: We studied the metabolic responses to different DA concentrations elicited by four doses of D-amphetamine (AMPH, 0, 0.25, 0.5, 1.0, or 3.0 mg/kg). We compared the degree of DA release (via microdialysis) with striatal cAMP activity and whole brain maps of cerebral blood volume (rCBV) changes (via pharmacological MRI, phMRI). RESULTS: AMPH increased DA release in the caudate/putamen (CPu) and cAMP activity in the CPu, nucleus accumbens (NAc), and medial prefrontal cortex (mPFC) in a linear dose-dependent manner (P < 0.0001). The cAMP data suggest that, postsynaptically, signal transduction induced by D1 receptor is stronger than that of D2 receptor at the higher doses (1-3 mg/kg). phMRI showed that, while higher doses of AMPH (3 mg/kg (n = 7) and 1 mg/kg (n = 6)) induced significant rCBV increases in the CPu and NAc, the degree of rCBV increase was much smaller with AMPH of 0.5 mg/kg (n = 6). In contrast, AMPH of 0.25 mg/kg (n = 8) induced significant rCBV decreases in the anteromedial CPu and NAc. The sign switch of rCBV in response to AMPH from low to high doses likely reflects the switching in the balance of D2/D3 stimulation vs. D1/D5 stimulation. In conclusion, degree of postsynaptic signal transduction is linearly correlated to the extracellular DA concentration. However, the presynaptic binding may dominate the overall DA innervation at the lower range of DA concentration.

Dual dose-dependent effects of fingolimod in a mouse model of Alzheimer's disease.

Lipid metabolism is abnormal in Alzheimer's disease (AD) brain leading to ceramide and sphingosine accumulation and reduced levels of brain sphingosine-1-phosphate (S1P). We hypothesize that changes in S1P signaling are central to the inflammatory and immune-pathogenesis of AD and the therapeutic benefits of fingolimod, a structural analog of sphingosine that is FDA approved for the treatment of multiple sclerosis. We recently reported that the neuroprotective effects of fingolimod in 5xFAD transgenic AD mice treated from 1-3 months of age were greater at 1 mg/kg/day than at 5 mg/kg/day. Here we performed a dose-response study using fingolimod from 0.03 to 1 mg/kg/day in 5xFAD mice treated from 1-8 months of age. At 1 mg/kg/day, fingolimod decreased both peripheral blood lymphocyte counts and brain Aß levels, but at the lowest dose tested (0.03 mg/kg/day), we detected improved memory, decreased activation of brain microglia and astrocytes, and restored hippocampal levels of GABA and glycerophosphocholine with no effect on circulating lymphocyte counts. These findings suggests that, unlike the case in multiple sclerosis, fingolimod may potentially have therapeutic benefits in AD at low doses that do not affect peripheral lymphocyte function.

Protective effects of 7,8-dihydroxyflavone on neuropathological and neurochemical changes in a mouse model of Alzheimer's disease.

Interest in brain-derived neurotrophic factor (BDNF) was greatly enhanced when it was recognized that its expression is reduced in neurodegenerative disorders, especially in Alzheimer's disease (AD). BDNF signaling through the TrkB receptor has a central role in promoting synaptic transmission, synaptogenesis, and facilitating synaptic plasticity making the BDNF-TrkB signaling pathway an attractive candidate for targeted therapies. Here we investigated the early effect of the small molecule TrkB agonist, 7,8 dihydroxyflavone (7,8-DHF), on AD-related pathology, dendritic arborization, synaptic density, and neurochemical changes in the 5xFAD mouse model of AD. We treated 5xFAD mice with 7,8-DHF for 2 months beginning at 1 month of age. We found that, in this model of AD, 7,8-DHF treatment decreased cortical Aß plaque deposition and protected cortical neurons against reduced dendritic arbor complexity but had no significant impact on the density of dendritic spines. In addition 7,8-DHF treatment protected against hippocampal increase in the level of choline-containing compounds and glutamate loss, but had no significant impact on hippocampal neurogenesis.

Anxiety, neuroinflammation, cholinergic and GABAergic abnormalities are early markers of Gulf War illness in a mouse model of the disease.

Gulf War Illness (GWI) is a chronic disease that affects the 1991 Gulf War (GW) veterans for which treatment is lacking. It has been hypothesized that drugs used to protect military personnel from chemical attacks and insects during the war: pyridostigmine bromide (PB),N, N-diethyl-m-toluamide (DEET), and permethrin (PER) together with stress may have contributed collectively and synergistically to generate GWI. There is a need to find markers of pathology to be used in pre-clinical trials. For this purpose we employed a previously validated mouse model of GWI evoked by daily exposure to PB (1.3 mg/kg), DEET (40 mg/kg), PER (0.13 mg/kg), and 5 min of restraint stress for 28 days to analyze behavior, brain pathology and neurochemical outcomes three months later. GWI-model mice were characterized by increased anxiety, decreased hippocampal levels of N-acetyl aspartate, GABA, the GABA-producing enzyme GAD-67 and microglial activation. We also observed that GWI model was sexually dimorphic on some measures: males had increased while females had decreased protein levels of the acetylcholine-synthesizing enzyme, choline acetyltransferase, in the septum and hippocampus and decreased levels of the receptor for brain-derived neurotrophic factor, TrkB140, in the hippocampus. Increased hippocampal levels of nerve growth factor were detected in males only. Together the data show behavioral and neuropathological abnormalities detected at 3 months post-exposure and that some of them are sexually dimorphic. Future preclinical studies for GWI may take advantage of this short latency model and should include both males and females as their response to treatment may differ.

ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs.

INTRODUCTION: Antigen-specific immunotherapy could provide a targeted approach for the treatment of multiple sclerosis that removes the need for broad-acting immunomodulatory drugs. ATX-MS-1467 is a mixture of four peptides identified as the main immune-dominant disease-associated T-cell epitopes in myelin basic protein (MBP), an autoimmune target for activated autoreactive T cells in multiple sclerosis. Previous animal studies have shown that ATX-MS-1467 treatment prevented the worsening of signs of disease in experimental autoimmune encephalitis (EAE) in the humanized (DR2 × Ob1)F1 mouse in a dose-dependent fashion. METHODS AND RESULTS: Our study extends these observations to show that subcutaneous treatment with 100 µg of ATX-MS-1467 after induction of EAE in the same mouse model reversed established clinical disability (p < 0.0001) and histological markers of inflammation and demyelination (p < 0.001) compared with vehicle-treated animals; furthermore, in longitudinal magnetic resonance imaging analyses, disruption of blood-brain barrier integrity was reversed, compared with vehicle-treated animals (p < 0.05). Chronic treatment with ATX-MS-1467 was associated with an enduring shift from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in interleukin 10 secretion, relative to interleukin 2, interleukin 17 and interferon γ, a decrease in splenocyte proliferation and an increase in interleukin 10+ Foxp3- T cells in the spleen. CONCLUSION: Our results suggest that ATX-MS-1467 can induce splenic iTregs and long-term tolerance to MBP with the potential to partially reverse the pathology of multiple sclerosis, particularly during the early stages of the disease. FUNDING: EMD Serono, Inc., a business of Merck KGaA.

Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.

We examined a novel farnesoid X receptor agonist, EDP-305, for its antifibrotic effect in bile duct ligation (BDL) and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) models of hepatic injury. We used molecular magnetic resonance imaging with the type 1 collagen-binding probe EP-3533 and the oxidized collagen-specific probe gadolinium hydrazide to noninvasively measure treatment response. BDL rats (n = 8 for each group) were treated with either low or high doses of EDP-305 starting on day 4 after BDL and were imaged on day 18. CDAHFD mice (n = 8 for each group) were treated starting at 6 weeks after the diet and were imaged at 12 weeks. Liver tissue was subjected to pathologic and morphometric scoring of fibrosis, hydroxyproline quantitation, and determination of fibrogenic messenger RNA expression. High-dose EDP-305 (30 mg/kg) reduced liver fibrosis in both the BDL and CDAHFD models as measured by collagen proportional area, hydroxyproline analysis, and fibrogenic gene expression (all P < 0.05). Magnetic resonance signal intensity with both EP-3533 in the BDL model and gadolinium hydrazide in the CDAHFD model was reduced with EDP-305 30 mg/kg treatment (P < 0.01). Histologically, EDP-305 30 mg/kg halted fibrosis progression in the CDAHFD model. Conclusion: EDP-305 reduced fibrosis progression in rat BDL and mouse CDAHFD models. Molecular imaging of collagen and oxidized collagen is sensitive to changes in fibrosis and could be used to noninvasively measure treatment response in clinical trials. (Hepatology Communications 2018;2:821-835).

Heterocyclic analogues of N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)arylcarboxamides with functionalized linking chains as novel dopamine D3 receptor ligands: potential substance abuse therapeutic agents.

Dopamine D3 receptor antagonists and partial agonists have been shown to modulate drug-seeking effects induced by cocaine and other abused substances. Compound 6 [PG01037, (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-pyridine-2-ylbenzamide)] and related analogues are currently being evaluated in animal models of drug addiction. In these studies, a discrepancy between in vitro binding affinity, in vivo occupancy, and behavioral potency has been observed. The purpose of this study was to examine (1) modifications of the 2-pyridylphenyl moiety of 6 and (2) hydroxyl, acetyl, and cyclopropyl substitutions on the butylamide linking chain systematically coupled with 2-fluorenylamide or 2-pyridylphenylamide and 2-methoxy- or 2,3-dichloro-substituted phenylpiperazines to measure the impact on binding affinity, D2/D3 selectivity, lipophilicity, and function. In general, these modifications were well tolerated at the human dopamine D3 (hD3) receptor (Ki = 1-5 nM) as measured in competition binding assays. Several analogues showed >100-fold selectivity for dopamine D3 over D2 and D4 receptors. In addition, while all the derivatives with an olefinic linker were antagonists, in quinpirole-stimulated mitogenesis at hD3 receptors, several of the hydroxybutyl-linked analogues (16, 17, 21) showed partial agonist activity. Finally, several structural modifications reduced lipophilicities while retaining the desired binding profile.

Fingolimod modulates multiple neuroinflammatory markers in a mouse model of Alzheimer's disease.

Sphingosine 1-phosphate (SP1) receptors may be attractive targets for modulation of inflammatory processes in neurodegenerative diseases. Recently fingolimod, a functional S1P1 receptor antagonist, was introduced for treatment of multiple sclerosis. We postulated that anti-inflammatory mechanisms of fingolimod might also be protective in Alzheimer's disease (AD). Therefore, we treated a mouse model of AD, the 5xFAD model, with two doses of fingolimod (1 and 5 mg/kg/day) and measured the response of numerous markers of Aß pathology as well as inflammatory markers and neurochemistry using biochemical, immunohistochemistry and high resolution magic angle spinning magnetic resonance spectroscopy (MRS). In mice at 3 months of age, we found that fingolimod decreased plaque density as well as soluble plus insoluble Aß measured by ELISA. Fingolimod also decreased GFAP staining and the number of activated microglia. Taurine has been demonstrated to play a role as an endogenous anti-inflammatory molecule. Taurine levels, measured using MRS, showed a very strong inverse correlation with GFAP levels and ELISA measurements of Aß, but not with plaque density or activated microglia levels. MRS also showed an effect of fingolimod on glutamate levels. Fingolimod at 1 mg/kg/day provided better neuroprotection than 5 mg/kg/day. Together, these data suggest a potential therapeutic role for fingolimod in AD.