In vitro assessment of the binding and functional responses of ozanimod and its plasma metabolites across human sphingosine 1-phosphate receptors.

Ozanimod is approved in multiple countries for the treatment of adults with either relapsing multiple sclerosis or moderately to severely active ulcerative colitis. Ozanimod is metabolized in humans to form seven active plasma metabolites, including two major active metabolites CC112273 and CC1084037, and an inactive metabolite. Here, the binding and activity of ozanimod and its metabolites across human sphingosine 1-phosphate receptors were determined. Binding affinity was assessed in Chinese hamster ovary cell membranes expressing recombinant human sphingosine 1-phosphate receptors 1 and 5 via competitive radioligand binding using tritium-labeled ozanimod; selectivity via functional potency assessment was performed using [35S]-guanosine-5'-(γ-thio)-triphosphate binding assays. Receptor internalization was assessed in human embryonic kidney 293 cells overexpressing sphingosine 1-phosphate receptor 1-green fluorescent protein and Chinese hamster ovary cells overexpressing sphingosine 1-phosphate receptor 5-hemagglutinin via fluorescence activated cell sorting. Functional activity was assessed in primary cultures of human astrocytes via phosphorylation assays. Ozanimod and its functionally active metabolites bound to the same sites within sphingosine 1-phosphate receptors 1 and 5, with metabolites displaying the same selectivity profile as ozanimod. Agonism at sphingosine 1-phosphate receptor 1 induced receptor internalization, whereas sphingosine 1-phosphate receptor 5 did not. Ozanimod, CC112273, and CC1084037 elicited functional intracellular signaling in human astrocytes, pharmacologically characterized to be mediated by sphingosine 1-phosphate receptor 1. The active plasma metabolites of ozanimod bound to sphingosine 1-phosphate receptors 1 and 5 and displayed similar pharmacologic profiles as their parent compound, likely contributing to clinical efficacy in patients with relapsing multiple sclerosis or moderately to severely active ulcerative colitis.

Preclinical target validation for non-addictive therapeutics development for pain.

INTRODUCTION: The Helping to End Addiction Long-termSM Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly. While there are plentiful novel targets for pain treatment, successful clinical validation is rare. It is therefore crucial to develop processes whereby therapeutic targets can be reasonably 'de-risked' prior to substantial late-stage validation efforts. Such rigorous validation of novel therapeutic targets in the preclinical space will give potential private sector partners the confidence to pursue clinical validation of promising therapeutic concepts and compounds. AREAS COVERED: In 2020, the National Institutes of Health (NIH) held the Target Validation for Non-Addictive Therapeutics Development for Pain workshop to gather insights from key opinion leaders in academia, industry, and venture-financing. EXPERT OPINION: The result was a roadmap for pain target validation focusing on three modalities: 1) human evidence; 2) assay development in vitro; 3) assay development in vivo.

Widespread cell stress and mitochondrial dysfunction occur in patients with early Alzheimer's disease.

Cell stress and impaired oxidative phosphorylation are central to mechanisms of synaptic loss and neurodegeneration in the cellular pathology of Alzheimer's disease (AD). In this study, we quantified the in vivo expression of the endoplasmic reticulum stress marker, sigma 1 receptor (S1R), using [11C]SA4503 positron emission tomography (PET), the mitochondrial complex I (MC1) with [18F]BCPP-EF, and the presynaptic vesicular protein SV2A with [11C]UCB-J in 12 patients with early AD and in 16 cognitively normal controls. We integrated these molecular measures with assessments of regional brain volumes and cerebral blood flow (CBF) measured with magnetic resonance imaging arterial spin labeling. Eight patients with AD were followed longitudinally to estimate the rate of change of the physiological and structural pathology markers with disease progression. The patients showed widespread increases in S1R (≤ 27%) and regional reduction in MC1 (≥ -28%) and SV2A (≥ -25%) radioligand binding, brain volume (≥ -23%), and CBF (≥ -26%). [18F]BCPP-EF PET MC1 binding (≥ -12%) and brain volumes (≥ -5%) showed progressive reductions over 12 to 18 months, suggesting that they both could be used as pharmacodynamic indicators in early-stage therapeutics trials. Associations of reduced MC1 and SV2A and increased S1R radioligand binding with reduced cognitive performance in AD, although exploratory, suggested a loss of metabolic functional reserve with disease. Our study thus provides in vivo evidence for widespread, clinically relevant cellular stress and bioenergetic abnormalities in early AD.

Competitive Binding of Ozanimod and Other Sphingosine 1-Phosphate Receptor Modulators at Receptor Subtypes 1 and 5.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator, binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), and is approved in multiple countries for treating adults with relapsing forms of multiple sclerosis (MS) or moderately to severely active ulcerative colitis (UC). Other S1P receptor modulators have been approved for the treatment of MS or are in clinical development for MS or UC, but it is unknown whether these compounds bind competitively with each other to S1P1 or S1P5. We developed a competitive radioligand binding assay using tritiated ozanimod and demonstrate full displacement of ozanimod by S1P (endogenous ligand), suggesting that ozanimod binds to the S1P1 and S1P5 orthosteric binding sites. S1P receptor modulators FTY720-p, siponimod, etrasimod, ponesimod, KRP-203-p, and amiselimod-p also completely displacing radiolabeled ozanimod; thus, on a macroscopic level, all bind to the same site. Molecular docking studies support these results and predict the binding of each molecule to the orthosteric site of the receptors, creating similar interactions within S1P1 and S1P5. The absolute free energy perturbation method further validated key proposed binding modes. Functional potency tightly aligned with binding affinities across S1P1 and S1P5 and all compounds elicited S1P1-mediated ß-arrestin recruitment. Since all the S1P modulators included in this study display similar receptor pharmacology and compete for binding at the same site, they can be considered interchangeable with one another. The choice of any one particular agent should therefore be made on the basis of overall therapeutic profile, and patients can be offered the opportunity to switch S1P medications without the potential concern of additive S1P pharmacology.

Deconstructing the Pharmacological Contribution of Sphingosine-1 Phosphate Receptors to Mouse Models of Multiple Sclerosis Using the Species Selectivity of Ozanimod, a Dual Modulator of Human Sphingosine 1-Phosphate Receptor Subtypes 1 and 5.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator that binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), is approved for the treatment of relapsing multiple sclerosis (MS) in multiple countries. Ozanimod profiling revealed a species difference in its potency for S1P5 in mouse, rat, and canine compared with that for human and monkey. Site-directed mutagenesis identified amino acid alanine at position 120 to be responsible for loss of activity for mouse, rat, and canine S1P5, and mutation back to threonine as in human/monkey S1P5 restored activity. Radioligand binding analysis performed with mouse S1P5 confirmed the potency loss is a consequence of a loss of affinity of ozanimod for mouse S1P5 and was restored with mutation of alanine 120 to threonine. Study of ozanimod in preclinical mouse models of MS can now determine the S1P receptor(s) responsible for observed efficacies with receptor engagement as measured using pharmacokinetic exposures of free drug. Hence, in the experimental autoimmune encephalomyelitis model, ozanimod exposures sufficient to engage S1P1, but not S1P5, resulted in reduced circulating lymphocytes, disease scores, and body weight loss; reduced inflammation, demyelination, and apoptotic cell counts in the spinal cord; and reduced circulating levels of the neuronal degeneration marker, neurofilament light. In the demyelinating cuprizone model, ozanimod prevented axonal degradation and myelin loss during toxin challenge but did not facilitate enhanced remyelination after intoxication. Since free drug levels in this model only engaged S1P1, we concluded that S1P1 activation is neuroprotective but does not appear to affect remyelination. SIGNIFICANCE STATEMENT: Ozanimod, a selective modulator of human sphingisone 1-phosphate receptor subtypes 1 and 5 (S1P1/5), displays reduced potency for rodent and dog S1P5 compared with human, which results from mutation of threonine to alanine at position 120. Ozanimod can thus be used as a selective S1P1 agonist in mouse models of multiple sclerosis to define efficacies driven by S1P1 but not S1P5. Based on readouts for experimental autoimmune encephalomyelitis and cuprizone intoxication, S1P1 modulation is neuroprotective, but S1P5 activity may be required for remyelination.

Absorption, Metabolism, and Excretion, In Vitro Pharmacology, and Clinical Pharmacokinetics of Ozanimod, a Novel Sphingosine 1-Phosphate Receptor Modulator.

Ozanimod is approved for the treatment of relapsing forms of multiple sclerosis. Absorption, metabolism, and excretion of ozanimod were investigated after a single oral dose of 1.0 mg [14C]ozanimod hydrochloride to six healthy subjects. In vitro experiments were conducted to understand the metabolic pathways and enzymes involved in the metabolism of ozanimod and its active metabolites. The total mean recovery of the administered radioactivity was ∼63%, with ∼26% and ∼37% recovered from urine and feces, respectively. Based on exposure, the major circulating components were active metabolite CC112273 and inactive metabolite RP101124, which together accounted for 50% of the circulating total radioactivity exposure, whereas ozanimod accounted for 6.7% of the total radioactive exposure. Ozanimod was extensively metabolized, with 14 metabolites identified, including two major active metabolites (CC112273 and CC1084037) and one major inactive metabolite (RP101124) in circulation. Ozanimod is metabolized by three primary pathways, including aldehyde dehydrogenase and alcohol dehydrogenase, cytochrome P450 isoforms 3A4 and 1A1, and reductive metabolism by gut microflora. The primary metabolite RP101075 is further metabolized to form major active metabolite CC112273 by monoamine oxidase B, which further undergoes reduction by carbonyl reductases to form CC1084037 or CYP2C8-mediated oxidation to form RP101509. CC1084037 is oxidized rapidly to form CC112273 by aldo-keto reductase 1C1/1C2 and/or 3ß- and 11ß-hydroxysteroid dehydrogenase, and this reversible oxidoreduction between two active metabolites favors CC112273. The ozanimod example illustrates the need for conducting timely radiolabeled human absorption, distribution, metabolism, and excretion studies for characterization of disproportionate metabolites and assessment of exposure coverage during drug development. SIGNIFICANCE STATEMENT: Absorption, metabolism, and excretion of ozanimod were characterized in humans, and the enzymes involved in complex metabolism were elucidated. Disproportionate metabolites were identified, and the activity of these metabolites was determined.

Enhancing Choice and Outcomes for Therapeutic Trials in Chronic Pain: N-of-1 + Imaging (+ i).

The attrition of novel analgesic drugs in the clinic can be attributed in the main to two factors: failure of preclinical research findings translating into human pain conditions, and a drop-off of efficacy between proof-of-concept (i.e., Phase II trials) and pivotal, confirmatory (Phase III trials) testing. In order to enhance the efficiency of the clinical drug evaluation process and determine rapidly whether a potential therapeutic candidate gives pain relief, by modulating central pain neurobiology, we propose a 'pre-proof-of-concept' approach, in which an efficacy assessment is performed in a single individual (N-of-1) using subjective clinical pain assessments supported by objective validated functional neuroimaging measures. Using an N-of-1 + i methodology, clinical- and neuroimaging-based metrics can be quantified under conditions of drug versus placebo or drug versus current standard of care conditions.

Calcitonin Gene-Related Peptide Modulators - The History and Renaissance of a New Migraine Drug Class.

Several lines of evidence pointed to an important role for CGRP in migraine. These included the anatomic colocalization of CGRP and its receptor in sensory fibers innervating pain-producing meningeal blood vessels, its release by trigeminal stimulation, the observation of elevated CGRP in the cranial circulation during migraine with normalization concomitant with headache relief by sumatriptan, and translational studies with intravenous (IV) CGRP that evoked migraine only in migraineurs. The development of small molecule CGRP receptor antagonists (CGRP-RAs) that showed clinical antimigraine efficacy acutely and prophylactically in randomized placebo-controlled clinical trials subsequently gave definitive pharmacological proof of the importance of CGRP in migraine. More recently, CGRP target engagement imaging studies using a CGRP receptor PET ligand [11 C]MK-4232 demonstrated that there was no brain CGRP receptor occupancy at clinically effective antimigraine doses of telcagepant, a prototypic CGRP-RA. Taken together, these data indicated that (1) the therapeutic site of action of the CGRP-RAs was peripheral not central; (2) that IV CGRP had most likely evoked migraine through an action at sites outside the blood-brain barrier; and (3) that migraine pain was therefore, at least in part, peripheral in origin. The evolution of CGRP migraine science gave impetus to the development of peripherally acting drugs that could modulate CGRP chronically to prevent frequent episodic and chronic migraine. Large molecule biologic antibody (mAb) approaches that are given subcutaneously to neutralize circulating CGRP peptide (fremanezumab, galcanezumab) or block CGRP receptors (erenumab) have shown consistent efficacy and tolerability in multicenter migraine prevention trials and are now approved for clinical use. Eptinezumab, a CGRP neutralizing antibody given IV, shows promise in late stage clinical development. Recently, orally administered next-generation small molecule CGRP-RAs have been shown to have safety and efficacy in acute treatment (ubrogepant and rimegepant) and prevention (atogepant) of migraine, giving additional CGRP-based therapeutic options for migraine patients.

Brain neurokinin-1 receptor availability in never-medicated patients with major depression - A pilot study.

BACKGROUND: Neurotransmitter substance P (SP) and its preferred neurokinin-1 receptor (NK1R) have been implicated in the treatment of affective and addiction disorders. Despite promising preclinical data on antidepressant action, the clinical trials of NK1R antagonists in major depression have been disappointing. There are no direct in vivo imaging studies on NK1R characteristics in patients with a major depressive disorder (MDD). METHODS: In this cross-sectional case-control study, we recruited nine never-medicated patients with moderate to severe MDD and nine matched healthy controls. NK1R availability (NK1R binding potential, BPND) was measured with in vivo 3-D positron emission tomography and a specific NK1 receptor tracer [18F]SPA-RQ. Clinical symptoms were assessed with the 17-item Hamilton Rating Scale for Depression (HAM-D17). RESULTS: NK1R-BPND did not differ statistically significantly between patients with MDD and healthy controls. HAM-D17 total scores (range 21-32) correlated positively with NK1R-BPND in cortical and limbic areas. HAM-D17 subscale score for anxiety symptoms correlated positively with NK1R-BPND in specific brain areas implicated in fear and anxiety. LIMITATIONS: Small sample size. Low variability in the clinical HAM-D subscale ratings may affect the observed correlations. CONCLUSIONS: Our preliminary results do not support a different baseline expression of NK1Rs in a representative sample of never-medicated patients with MDD during a current moderate/severe depressive episode. The modulatory effect of NK1Rs on affective symptoms is in line with early positive results on antidepressant action of NK1 antagonists. However, the effect is likely to be too weak for treatment of MDD with NK1R antagonists alone in clinical practice.

Amyloid beta-positive subjects exhibit longitudinal network-specific reductions in spontaneous brain activity.

Amyloid beta (Aß) deposition and cognitive decline are key features of Alzheimer's disease. The relationship between Aß status and changes in neuronal function over time, however, remains unclear. We evaluated the effect of baseline Aß status on reference region spontaneous brain activity (SBA-rr) using resting-state functional magnetic resonance imaging and fluorodeoxyglucose positron emission tomography in patients with mild cognitive impairment. Patients (N = 62, [43 Aß-positive]) from the Alzheimer's Disease Neuroimaging Initiative were divided into Aß-positive and Aß-negative groups via prespecified cerebrospinal fluid Aß42 or 18F-florbetapir positron emission tomography standardized uptake value ratio cutoffs measured at baseline. We analyzed interaction of biomarker-confirmed Aß status with SBA-rr change over a 2-year period using mixed-effects modeling. SBA-rr differences between Aß-positive and Aß-negative subjects increased significantly over time within subsystems of the default and visual networks. Changes exhibit an interaction with memory performance over time but were independent of glucose metabolism. Results reinforce the value of resting-state functional magnetic resonance imaging in evaluating Alzheimer''s disease progression and suggest spontaneous neuronal activity changes are concomitant with cognitive decline.

From LBR-101 to Fremanezumab for Migraine.

Calcitonin gene-related peptide (CGRP) is a neuropeptide of importance in migraine pathogenesis. Its central role in migraine was proven pharmacologically by the development of CGRP receptor antagonists. Monoclonal antibodies targeting CGRP or its receptor are effective in the preventive treatment of episodic and chronic migraine and are considered potential breakthroughs in their treatment. Fremanezumab (previously known as TEV-48125, LBR-101, or RN-307) is a humanized IgG2a monoclonal antibody that binds to CGRP. The development of this antibody validated the role of CGRP in chronic migraine and the drug has been recently approved in the US by the FDA, while it continues to be reviewed by other regulatory agencies. Herein we provide an in-depth review of its development. We start by summarizing its in vitro and in vivo pharmacology, and the phase I studies. We then review the late-stage clinical development, with a focus on its efficacy, safety, similarities, and uniqueness relative to other CGRP antibodies. We close by discussing lessons learned on the mechanisms of migraine and areas for future development and exploration.

A critical evaluation of validity and utility of translational imaging in pain and analgesia: Utilizing functional imaging to enhance the process.

Assessing clinical pain and metrics related to function or quality of life predominantly relies on patient reported subjective measures. These outcome measures are generally not applicable to the preclinical setting where early signs pointing to analgesic value of a therapy are sought, thus introducing difficulties in animal to human translation in pain research. Evaluating brain function in patients and respective animal model(s) has the potential to characterize mechanisms associated with pain or pain-related phenotypes and thereby provide a means of laboratory to clinic translation. This review summarizes the progress made towards understanding of brain function in clinical and preclinical pain states elucidated using an imaging approach as well as the current level of validity of translational pain imaging. We hypothesize that neuroimaging can describe the central representation of pain or pain phenotypes and yields a basis for the development and selection of clinically relevant animal assays. This approach may increase the probability of finding meaningful new analgesics that can help satisfy the significant unmet medical needs of patients.

Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice.

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Challenges in developing drugs for primary headaches.

This review considers the history of drug development in primary headaches and discusses challenges to the discovery of innovative headache therapeutics. Advances in headache genetics have yet to translate to new classes of therapeutics and there are currently no clear predictive human biomarkers for any of the primary headaches that can guide preventative drug discovery and development. Primary headache disorder subtypes despite common phenotypic presentation are undoubtedly heterogeneous in their pathophysiology as judged by the variability of response to headache medicines. Sub-classification of headache subtypes into more homogenous and specific phenotypes groups may facilitate genotyping and provide sentinel patient groups that can be used in a mechanism specific manner to test new and more personalized treatment strategies in headache medicine. The development of the triptan class of serotonin 5-HT1B/1D/1F receptor agonists has advanced our understanding of the neurobiology of migraine pain, which subsequently resulted in the development of calcitonin gene-related peptide (CGRP) modulators that are now showing promise as acute and preventative anti-migraine agents. Despite these successes, there have been many near misses and failures in the discovery and development of headache therapeutics. Glutamate receptor antagonism whilst efficacious has central side effects and some approaches such as nitric oxide synthase inhibition, substance P antagonism and cortical spreading depression blockade, despite having promising effects in basic pain models, have not delivered efficacy in the clinic. Future efforts may triage novel physiological mediators using human experimental models of headache pain to support drug discovery strategies that target active pathways pharmacologically.

Inhibition of Orexin Signaling Promotes Sleep Yet Preserves Salient Arousability in Monkeys.

STUDY OBJECTIVES: In addition to enhancing sleep onset and maintenance, a desirable insomnia therapeutic agent would preserve healthy sleep's ability to wake and respond to salient situations while maintaining sleep during irrelevant noise. Dual orexin receptor antagonists (DORAs) promote sleep by selectively inhibiting wake-promoting neuropeptide signaling, unlike global inhibition of central nervous system excitation by gamma-aminobutyric acid (GABA)-A receptor (GABAaR) modulators. We evaluated the effect of DORA versus GABAaR modulators on underlying sleep architecture, ability to waken to emotionally relevant stimuli versus neutral auditory cues, and performance on a sleepiness-sensitive cognitive task upon awakening. METHODS: DORA-22 and GABAaR modulators (eszopiclone, diazepam) were evaluated in adult male rhesus monkeys (n = 34) with continuous polysomnography recordings in crossover studies of sleep architecture, arousability to a classically conditioned salient versus neutral acoustical stimulus, and psychomotor vigilance task (PVT) performance if awakened. RESULTS: All compounds decreased wakefulness, but only DORA-22 sleep resembled unmedicated sleep in terms of underlying sleep architecture, preserved ability to awaken to salient-conditioned acoustic stimuli while maintaining sleep during neutral acoustic stimuli, and no congnitive impairment in PVT performance. Although GABAaR modulators induced lighter sleep, monkeys rarely woke to salient stimuli and PVT performance was impaired if monkeys were awakened. CONCLUSIONS: In nonhuman primates, DORAs' targeted mechanism for promoting sleep protects the ability to selectively arouse to salient stimuli and perform attentional tasks unimpaired, suggesting meaningful differentiation between a hypnotic agent that works through antagonizing orexin wake signaling versus the sedative hypnotic effects of the GABAaR modulator mechanism of action.

Test-retest reliability of evoked heat stimulation BOLD fMRI.

To date, the blood oxygenated-level dependent (BOLD) functional magnetic resonance imaging (fMRI) technique has enabled an objective and deeper understanding of pain processing mechanisms embedded within the human central nervous system (CNS). In order to further comprehend the benefits and limitations of BOLD fMRI in the context of pain as well as the corresponding subjective pain ratings, we evaluated the univariate response, test-retest reliability and confidence intervals (CIs) at the 95% level of both data types collected during evoked stimulation of 40°C (non-noxious), 44°C (mildly noxious) and a subject-specific temperature eliciting a 7/10 pain rating. The test-retest reliability between two scanning sessions was determined by calculating group-level interclass correlation coefficients (ICCs) and at the single-subject level. Across the three stimuli, we initially observed a graded response of increasing magnitude for both VAS (visual analog score) pain ratings and fMRI data. Test-retest reliability was observed to be highest for VAS pain ratings obtained during the 7/10 pain stimulation (ICC=0.938), while ICC values of pain fMRI data for a distribution of CNS structures ranged from 0.5 to 0.859 (p<0.05). Importantly, the upper and lower confidence interval CI bounds reported herein could be utilized in subsequent trials involving healthy volunteers to hypothesize the magnitude of effect required to overcome inherent variability of either VAS pain ratings or BOLD responses evoked during innocuous or noxious thermal stimulation.

Evidence for orexinergic mechanisms in migraine.

OBJECTIVE: To examine the effect of the orexinergic blockade with a dual orexin receptor antagonist (DORA) on experimental models of peripheral and central trigeminal as well as cortical activation relevant to migraine and migraine aura. METHODS: In this study we used a precursor of suvorexant, a dual orexin receptor antagonist #12 (DORA-12) in established experimental in vivo models of dural trigeminovascular nociception in rat. Neurogenic dural vasodilation and electrophysiological recordings of second order trigeminocervical neurons were used to study trigeminal nociceptive mechanisms directly. KCl-evoked cortical spreading depression was also used as a surrogate for migraine aura. RESULTS: Neurogenically-induced vasodilation of the middle meningeal artery, caused by nociceptive activation of peripheral afferent projections of the trigeminal nerve, was attenuated by intravenous DORA-12 (1 mgkg(-1)). Second-order trigeminocervical complex neuronal activity was significantly inhibited by intravenous DORA-12 (1 mgkg(-1)). DORA-12 significantly reduced susceptibility to KCl-evoked cortical spreading depression. CONCLUSION: The study provides the first direct evidence, that simultaneous antagonism on both orexin receptors is able to attenuate trigeminal nociceptive activity as well as to induce an elevation of the threshold for the induction of a cortical spreading depression (CSD). In the clinical context, these data imply that targeting the hypothalamic orexinergic system may offer an entirely novel mechanism for the preventive treatment of migraine with and without aura.

Functional imaging of olfaction by CBV fMRI in monkeys: insight into the role of olfactory bulb in habituation.

Cerebral blood volume (CBV) fMRI with superparamagnetic iron oxide nanoparticles (USPIO) as contrast agent was used to investigate the odorant-induced olfaction in anesthetized rhesus monkeys. fMRI data were acquired in 24 axial slices covering the entire brain, with isoamyl-acetate as the odor stimulant. For each experiment, multiple fMRI measurements were made during a 1- or 2-h period, with each measurement consisting of a baseline period, a stimulation period, and a recovery period. Three different stimulation paradigms with a stimulation period of 1 min, 2 min, or 8 min, respectively, were used to study the olfactory responses in the olfactory bulb (OB). Odorant-induced CBV increases were observed in the OB of each individual monkey. The spatial and temporal activation patterns were reproducible within and between animals. The sensitivity of CBV fMRI in OB was comparable with the sensitivities reported in previous animal fMRI studies. The CBV responses during the 1-min, 2-min, or 8-min odor stimulation period were relatively stable, and did not show attenuation. The amplitudes of CBV response to the repeated stimuli during the 1- or 2-h period were also stable. The stable CBV response in the OB to both continuous and repeated odor stimuli suggests that the OB may not play a major role in olfactory habituation. The technical approach described in this report can enable more extensive fMRI studies of olfactory processing in OB of both humans and non-human primates.

Characteristic time courses of cortical and medullary sodium signals measured by noninvasive (23) Na-MRI in rat kidney induced by furosemide.

BACKGROUND: To characterize regional kidney sodium response by MRI following NKCC2 inhibition. METHODS: Regional renal sodium signals were monitored noninvasively using (23) Na-MRI at 9.4T with a temporal resolution of 1.5 min in anesthetized rats (N = 14). A mild NKCC2 inhibition was induced using a slow intravenous furosemide infusion. Time course of sodium signal was modeled as an exponential transient with a single characteristic time constant. RESULTS: Under normal physiological conditions, the renal sodium signals in medullary and cortical regions were stable and found to respond differently to furosemide challenge. Furosemide infusion at 1.2 mg/kg/h (N = 7) increased sodium signal in the cortex by 40 ± 6% (P < 7 × 10(-5) ) whereas decreased in the medulla by 29 ± 2% (P < 3 × 10(-6) ) with different temporal kinetics. The characteristic time constants of the change were determined to be: 8 ± 2 and 70 ± 10 min for medulla and cortex. Also, the medullary change occurred 9(±3) times faster than cortical independent of furosemide infusion rate up to 35 mg/kg/h. CONCLUSION: The pharmacological effects in terms of regional kidney sodium signal changes induced by NKCC2 inhibition are region-specific and highly predictable. Using noninvasive sodium MRI, we obtained regional renal sodium kinetics data sets in response to a low dose furosemide infusion in normal rats.

Addition of an NK1 receptor antagonist to an SSRI did not enhance the antidepressant effects of SSRI monotherapy: results from a randomized clinical trial in patients with major depressive disorder.

OBJECTIVE: Aprepitant is a neurokinin 1 receptor antagonist approved for prevention of chemotherapy-induced and post-operative nausea and vomiting. Early studies demonstrated promising antidepressant activity as monotherapy, although this was unsupported by subsequent phase 3 trials. This phase 2 study evaluated whether aprepitant potentiated the antidepressant effects of paroxetine. METHODS: Outpatients with major depressive disorder were randomized to aprepitant 200 mg + paroxetine 20 mg, paroxetine + placebo, or aprepitant + placebo for 6 weeks. The primary endpoint was change in HAMD-17 total score. Secondary/exploratory endpoints included changes in HAMA, CGI-S, CGI-I, and HAMD Item-1 scores at week 6. RESULTS: A total of 79, 78, and 79 patients received aprepitant + paroxetine, paroxetine + placebo, and aprepitant + placebo, respectively. At week 6, mean changes in HAMD-17 were -11.0 (95% confidence interval [CI]: -12.7, -9.4), -11.7 (95% CI: -13.3, -10.0), and -9.5 (95% CI: -10.9, -8.1), respectively. Pairwise comparisons of HAMD-17 change with combination therapy versus paroxetine alone demonstrated no significant difference (p = 0.567). Changes in CGI-S, CGI-I, and HAMD Item-1 scores were also comparable, although there was a greater reduction in anxiety (HAMA) with paroxetine alone than aprepitant + paroxetine (p = 0.045). Adverse events were generally more common with the combination than either monotherapy. CONCLUSION: Concomitant use of aprepitant + paroxetine for 6 weeks did not provide greater antidepressant benefit compared with paroxetine + placebo in patients with major depression.