Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

Are Color Experiences the Same across the Visual Field?

It seems obvious to laypeople that neurotypical humans experience color equivalently across their entire visual field. To some neuroscientists, psychologists, and philosophers, though, this claim has been met with skepticism, as neurophysiological evidence indicates the mechanisms that support color perception degrade with eccentricity. However, the argument that this entails altered color experience in peripheral vision is not universally accepted. Here, we address whether color experience is essentially equivalent between central and peripheral vision. To assess this, we will obtain similarity relationships between color experiences across the visual field using both online and laboratory-based far-field displays, while removing the confounds of saccades, memory, and expectation about color experiences. Our experiment was designed to provide clear evidence that would favor either unchanged or altered color experience relationships in the periphery. Our results are consistent with lay people's phenomenological reports: Color experiences, as probed by similarity relationships in central vision and the far field (60°), are equivalent when elicited by large stimuli. These findings challenge the widespread view in philosophy and cognitive science that peripheral color experiences are illusory, and are discussed in the context of their related neurophysiological, psychophysical, and philosophical literature.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Renal tubular transporter-mediated interactions between mirogabalin and cimetidine in rats.

Cimetidine at a clinical dosage decreased the renal clearance (CLr) of mirogabalin in humans by inhibition of renal secretion. Mirogabalin is a substrate of human OAT1/3, OCT2, MATE1 and/or MATE2-K. To clarify the mechanism behind the above interaction, it was investigated whether cimetidine inhibits the process of mirogabalin uptake at the basolateral side or the process of its efflux at the apical side in rat kidney in vivo.Cimetidine was administered to rats by a constant infusion to achieve an unbound plasma concentration of 7.0 µM and examine its effect on the renal disposition of [14C]metformin, [3H]p-aminohippuric acid (PAH), and [14C]mirogabalin.Cimetidine significantly induced the intrarenal accumulation of radioactivity (Kp, kidney) and decreased the renal clearance (CLr) of [14C]mirogabalin. These effects resulted in significantly decreased total clearance (CLt). Kp, kidney, and CLr of [14C]metformin, except CLt, were also affected, but no parameters of [3H]PAH were affected by cimetidine.These findings clarified that an unbound plasma concentration of cimetidine of 7.0 µM inhibited the apical efflux not the basolateral uptake of [14C]mirogabalin in rat kidney, suggesting that mirogabalin/cimetidine interaction was caused by inhibiting the apical efflux transporter, human MATE1 and/or MATE2-K, not the basolateral uptake transporter, human OCT2, in the kidney.

Pharmacokinetic and pharmacodynamic assessment of histamine H3 receptor occupancy by enerisant: a human PET study with a novel H3 binding ligand, [11C]TASP457.

PURPOSE: Histamine H3 receptor antagonists and inverse agonists have been extensively developed to treat sleep-wake, neurocognitive, and allied disorders. However, potential adverse effects, including insomnia, hampered the clinical use of these drugs, possibly due to their persistent interaction with the target molecules. The purpose of the present study was to estimate the pharmacokinetics and pharmacodynamics of enerisant, a novel antagonist and inverse agonist for histamine H3 receptors. METHODS: To measure the histamine H3 receptor occupancy by enerisant, positron emission tomography studies using [11C]TASP457, a specific radioligand for histamine H3 receptors, were performed in 12 healthy men at baseline and at 2 h after oral administration of enerisant hydrochloride. For three of these subjects, two additional scans were performed at 6 and 26 h after the administration. Relationships between the receptor occupancy by enerisant and its dose and plasma concentrations were then analyzed. RESULTS: Administration of enerisant hydrochloride decreased the radioligand binding in a dose-dependent manner. The estimated receptor occupancy values at 2 h varied as a function of its dose or plasma concentration. The time course of the occupancy showed persistently high levels (> 85%) in the two subjects with higher doses (25 and 12.5 mg). The occupancy was also initially high at 2 h and 6 h with the lower dose of 5 mg, but it decreased to 69.7% at 26 h. CONCLUSION: The target engagement of enerisant was demonstrated in the brains of living human subjects. The occupancy of histamine H3 receptors by enerisant at 2 h can be predicted by applying the plasma concentration of enerisant to Hill's plot. The preliminary time-course investigation showed persistently high brain occupancy with high doses of enerisant despite the decreasing plasma concentration of the drug. Five milligrams or less dose would be appropriate for the treatment for narcolepsy with initially high occupancy allowing for effective treatment of narcolepsy, and then the occupancy level would be expected to decrease to a level to avoid this drug's unwanted side effect of insomnia at night, although further research is warranted to confirm the statement since the expected decrease is based on the finding in one subject. TRIAL REGISTRATION: This study was retrospectively registered with ClinicalTrials.gov (NCT04631276) on November 17, 2020.

Physiologically based pharmacokinetic modelling to predict the clinical effect of CYP3A inhibitors/inducers on esaxerenone pharmacokinetics in healthy subjects and subjects with hepatic impairment.

PURPOSE: Esaxerenone is a novel, oral, nonsteroidal treatment for hypertension. Physiologically based pharmacokinetic (PBPK) modelling was performed to predict the drug-drug interaction (DDI) effect of cytochrome P450 (CYP)3A modulators on esaxerenone pharmacokinetics in healthy subjects and subjects with hepatic impairment. METHODS: In our PBPK model, the fraction of esaxerenone metabolised by CYP3A was estimated from mass-balance data and verified and optimised by clinical DDI study results with strong CYP3A modulators. The model was also verified by the observed pharmacokinetics after multiple oral dosing and by the effect of hepatic impairment on esaxerenone pharmacokinetics. The model was applied to predict the DDI effects on esaxerenone pharmacokinetics with untested CYP3A modulators in healthy subjects and with strong CYP3A modulators in subjects with hepatic impairment. RESULTS: The PBPK model well described esaxerenone pharmacokinetics after multiple oral dosing. The predicted fold changes in esaxerenone plasma exposure after coadministration with strong CYP3A modulators were comparable with the observed data (1.53-fold with itraconazole and 0.31-fold with rifampicin). Predicted DDIs with untested moderate CYP3A modulators were less than the observed DDI with strong CYP3A modulators. The PBPK model also described the effect of hepatic impairment on esaxerenone plasma exposure. The predicted DDI results with strong CYP3A modulators in subjects with hepatic impairment indicate that, for concomitant use of CYP3A modulators, caution is advised for subjects with hepatic impairment, as is for healthy subjects. CONCLUSION: The PBPK model developed predicted esaxerenone pharmacokinetics and DDIs and informed concurrent use of esaxerenone with CYP3A modulators.

Functional robustness of adult spermatogonial stem cells after induction of hyperactive Hras.

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.

Resting-state brain activity can predict target-independent aptitude in fMRI-neurofeedback training.

Neurofeedback (NF) aptitude, which refers to an individual's ability to change brain activity through NF training, has been reported to vary significantly from person to person. The prediction of individual NF aptitudes is critical in clinical applications to screen patients suitable for NF treatment. In the present study, we extracted the resting-state functional brain connectivity (FC) markers of NF aptitude, independent of NF-targeting brain regions. We combined the data from fMRI-NF studies targeting four different brain regions at two independent sites (obtained from 59 healthy adults and six patients with major depressive disorder) to collect resting-state fMRI data associated with aptitude scores in subsequent fMRI-NF training. We then trained the multiple regression models to predict the individual NF aptitude scores from the resting-state fMRI data using a discovery dataset from one site and identified six resting-state FCs that predicted NF aptitude. Subsequently, the reproducibility of the prediction model was validated using independent test data from another site. The identified FC model revealed that the posterior cingulate cortex was the functional hub among the brain regions and formed predictive resting-state FCs, suggesting that NF aptitude may be involved in the attentional mode-orientation modulation system's characteristics in task-free resting-state brain activity.

Dual orexin receptor antagonist (DORA-12) treatment affects the overall levels of Net/maoA mRNA expression in the hippocampus.

The orexinergic system plays a significant role in regulating proper sleep/wake maintenance. Dual orexin receptor antagonist (DORA) is widely prescribed for insomnia symptoms. The antagonist acts on orexin 1 and 2 receptors located in certain brain areas, including the locus coeruleus and dorsal raphe. Nevertheless, its effects on monoamine-related gene expression remain unclear. Here, we measured the expression levels of monoamine-related genes in DORA-treated mice. DORA treatment significantly affected overall levels of noradrenalin transporter/monoamine oxidases A mRNA expression in the hippocampus. Our findings suggest that DORA contributes to noradrenalin-related gene expression regulation in the central nervous system.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

Drug-Drug Interaction Risk Assessment of Esaxerenone as a Perpetrator by In Vitro Studies and Static and Physiologically Based Pharmacokinetic Models.

Esaxerenone (CS-3150) is a novel, oral, nonsteroidal, selective mineralocorticoid receptor blocker approved for the treatment of hypertension in Japan. Here, the drug-drug interaction (DDI) potential of esaxerenone was evaluated in vitro, and its impact in clinical practice was estimated. Esaxerenone exhibited time-dependent inhibition and induction of CYP3A. When the clinical impacts of esaxerenone on the inhibition and induction of CYP3A were estimated separately by using a mechanistic static model, the predicted area under the curve ratios (AUCRs) of midazolam, a typical CYP3A substrate, were 1.80 and 0.31, respectively, suggesting that the DDI potential of esaxerenone cannot be neglected. Because it was suggested that DDIs mainly occur in the intestine, predictions using concentration-time profiles in each segment of the gastrointestinal tract were performed with GastroPlus, a physiologically based pharmacokinetic (PBPK) modeling software. The predicted AUCR of midazolam was approximately 1.2, which is close to that in a clinical study, despite the difficulty of predicting DDIs for compounds with both inhibition and induction effects. When only inhibition or induction was incorporated into a model, the AUCR of midazolam changed depending on the dosing period and dose level of esaxerenone and the timing of midazolam administration. However, the AUCR calculated by incorporating both effects remained almost constant. This study shows the ability of PBPK models to simulate weak DDIs via intestinal CYP3A and that esaxerenone has low DDI potential as a perpetrator because of the offset of inhibition and induction. SIGNIFICANCE STATEMENT: Weak CYP3A inhibition and/or induction sometimes cause DDIs in the intestine but not the liver. Because strong inhibitors maximally inhibit intestinal CYP3A, the predictability of weak DDIs in the intestine should be evaluated further. Here, we simulate the DDIs of esaxerenone as a perpetrator by using physiologically based pharmacokinetic modeling focusing on the intestine and offset of inhibition and induction.

Critical Impact of Drug-Drug Interactions via Intestinal CYP3A in the Risk Assessment of Weak Perpetrators Using Physiologically Based Pharmacokinetic Models.

A great deal of effort has been being made to improve the accuracy of the prediction of drug-drug interactions (DDIs). In this study, we addressed CYP3A-mediated weak DDIs, in which a relatively high false prediction rate was pointed out. We selected 17 orally administered drugs that have been reported to alter area under the curve (AUC) of midazolam, a typical CYP3A substrate, 0.84-1.47 times. For weak CYP3A perpetrators, the predicted AUC ratio mainly depends on intestinal DDIs rather than hepatic DDIs because the drug concentration in the enterocytes is higher. Thus, DDI prediction using simulated concentration-time profiles in each segment of the digestive tract was made by physiologically based pharmacokinetic (PBPK) modeling software GastroPlus. Although mechanistic static models tend to overestimate the risk to ensure the safety of patients, some underestimation is reported about PBPK modeling. Our in vitro studies revealed that 16 out of 17 tested drugs exhibited time-dependent inhibition (TDI) of CYP3A, and the subsequent DDI simulation that ignored these TDIs provided false-negative results. This is considered to be the cause of past underestimation. Inclusion of the DDI parameters of all the known DDI mechanisms, reversible inhibition, TDI, and induction, which have opposite effects on midazolam AUC, to PBPK model was successful in improving predictability of the DDI without increasing false-negative prediction as trade-off. This comprehensive model-based analysis suggests the importance of the intestine in assessing weak DDIs via CYP3A and the usefulness of PBPK in predicting intestinal DDIs. SIGNIFICANCE STATEMENT: Although drug-drug interaction (DDI) prediction has been extensively performed previously, the accuracy of prediction for weak interactions via CYP3A has not been thoroughly investigated. In this study, we simulate DDIs considering drug concentration-time profile in the enterocytes and discuss the importance and the predictability of intestinal DDIs about weak CYP3A perpetrators.

Ziprasidone, a second-generation antipsychotic, affects core clock gene mRNA expression in mice.

Some psychiatric diseases are associated with disruptions in the circadian clock system. Ziprasidone (ZIP), a second-generation antipsychotic, is widely used for psychiatry-related pharmacotherapy but its mechanism has not been clearly elucidated. We measured clock gene fluctuation patterns in the hippocampus and the amygdala in ZIP-treated mice. ZIP significantly increased Per1, Per2, and Bmal1 mRNA 2 h after the lights were turned off (ZT14) in the hippocampus, but not in the amygdala. These results suggest that ZIP might affect clock gene regulation, which could represent the pathway underlying symptom amelioration.

Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle.

Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.

Pharmacokinetics, Metabolism, and Excretion of [14C]Esaxerenone, a Novel Mineralocorticoid Receptor Blocker in Humans.

Esaxerenone (CS-3150) is a novel, nonsteroidal, selective mineralocorticoid receptor blocker. The absorption, metabolism, distribution, and excretion of esaxerenone were assessed in in vitro studies and in a clinical study, where [14C]esaxerenone (150 µCi/20 mg) was administered orally to six healthy male subjects. The plasma concentrations of esaxerenone and its metabolites (M4, M11, and M1) were measured using liquid chromatography-tandem mass spectrometry. The recovery of radioactivity was 92.5%, with 38.5% and 54.0% excreted in the urine and feces, respectively. The half-life of radioactivity in blood and plasma was approximately 30 hours, similar to that of the unchanged form in plasma. The blood-to-plasma ratio was 0.628, demonstrating low partitioning to blood components. In plasma, esaxerenone was the most abundant moiety (40.8%), followed by O-glucuronide (21.4%; M4), acyl-glucuronide of amide-bond hydrolysate (8.0%; M11), and the deshydroxyethyl form (1.7%; M1). In vitro studies showed that esaxerenone was a substrate of CYP3A and multiple UDP-glucuronosyltransferase isoforms. Oxidation contributed approximately 30% to its clearance, as indicated by the excretion ratio of oxidized metabolites into urine and feces. Caco-2 studies showed that esaxerenone was a substrate of P-glycoprotein and breast cancer resistance protein; however, the excretion ratios of the unchanged form in the feces and urine were 18.7% and 1.6%, respectively, indicating that these transporters were not important for the absorption and elimination of esaxerenone. Low urinary excretion of esaxerenone suggested that the plasma exposure of esaxerenone was not affected by renal dysfunction. Multiple elimination pathways including oxidation, glucuronidation, and hydrolysis, and the low contribution of transporters, indicated limited drug-drug interaction potential.

The influence of light sources on sunitinib measurements with photoisomerization.

Sunitinib is an orally administered tyrosine kinase inhibitor. Therapeutic drug monitoring is an important component of the follow-up of patients because of high interpatient variability in the pharmacokinetics of sunitinib and large variabilities in its efficacy and toxicity. The aim of the present study was to examine the light stability of sunitinib and confirm the effects of light exposure on sunitinib measurements by LC-MS/MS. Sunitinib and its active metabolite, SU12662, convert Z isomers to E isomers with exposure to light. The Z-E photoisomerization ratio reached a plateau at 35% for both E isomers in methanol within 15 min of normal light exposure (700 lx). However, the Z isomer of the sunitinib and SU12662 peak area ratios in plasma decreased by 10% within 15 min. These results suggest that sunitinib samples need to be handled without light exposure in all sample preparation steps. Alternatively, it should be measured sunitinib and SU12662 after the sample has reached photoisomerical equilibrium. These results suggest that the sunitinib therapeutic range changes depending on light conditions during sample handling in sunitinib and SU12662 measurements.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, and Its Water-Soluble Prodrug, DS11960558, against Methicillin-Resistant Staphylococcus aureus.

DS-2969b is a novel GyrB inhibitor under clinical development. In this study, the in vitro activity of DS-2969b and the in vivo activities of DS-2969b and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. DS-2969b inhibited the supercoiling activity of S. aureus DNA gyrase and the decatenation activity of its topoisomerase IV. DS-2969b showed antibacterial activity against Gram-positive aerobes but not against Gram-negative aerobes, except for Moraxella catarrhalis and Haemophilus influenzae DS-2969b was active against MRSA with an MIC90 of 0.25 µg/ml, which was 8-fold lower than that of linezolid. The presence of a pulmonary surfactant did not affect the MIC of DS-2969b. DS-2969b showed time-dependent slow killing against MRSA. The frequency of spontaneous resistance development was less than 6.2 × 10-10 in all four S. aureus isolates at 4× MIC of DS-2969b. In a neutropenic MRSA-induced murine muscle infection model, DS-2969b was more efficacious than linezolid by both the subcutaneous and oral routes. DS-2969b and DS11960558 showed efficacy in a neutropenic murine MRSA lung infection model. The pharmacokinetics and pharmacodynamics of DS-2969b and DS11960558 against MRSA were characterized in a neutropenic murine thigh infection model; the percentage of time during the dosing period in which the free drug concentration exceeded the MIC (fTMIC) correlated best with in vivo efficacy, and the static percent fTMIC was 43 to 49%. A sufficient fTMIC was observed in a phase 1 multiple-ascending-dose study of DS-2969b given orally at 400 mg once a day. These results suggest that DS11960558 and DS-2969b have potential for use as intravenous-to-oral step-down therapy for treating MRSA infections with a higher efficacy than linezolid.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, against Clostridium difficile.

DS-2969b is a novel GyrB inhibitor that is currently under clinical development for the treatment of Clostridium difficile infection (CDI). In this study, the in vitro and in vivo activities of DS-2969b were evaluated. DS-2969b inhibited the supercoiling activity of C. difficile DNA gyrase. DS-2969b showed potent in vitro activity against C. difficile clinical isolates with a MIC90 of 0.06 µg/ml, which was 2-, 32-, and 16-fold lower than the MIC90s of fidaxomicin, vancomycin, and metronidazole, respectively. DS-2969b did not select spontaneously resistant mutants of various C. difficile strains at 4× MIC, and the frequency of resistance development was less than 4.8 × 10-9 In a hamster CDI model, 5-day oral administration of DS-2969b conferred complete protection from recurrence and mortality at 0.3 mg/kg of body weight once a day, in contrast to a 50% survival rate with fidaxomicin at 3 mg/kg once a day and 0% with vancomycin at a 50-mg/kg/dose twice a day. Even a single oral administration of 1 mg/kg of DS-2969b in the CDI model exhibited 100% animal survival without recurrence. DS-2969b was also efficacious by 5-day subcutaneous administration in the CDI model. DS-2969b showed similar levels of fecal excretion after intravenous and oral administrations in rats. These data support further development of DS-2969b as a drug for oral and intravenous treatment of CDI.

Strategies for Utilizing Neuroimaging Biomarkers in CNS Drug Discovery and Development: CINP/JSNP Working Group Report.

Despite large unmet medical needs in the field for several decades, CNS drug discovery and development has been largely unsuccessful. Biomarkers, particularly those utilizing neuroimaging, have played important roles in aiding CNS drug development, including dosing determination of investigational new drugs (INDs). A recent working group was organized jointly by CINP and Japanese Society of Neuropsychopharmacology (JSNP) to discuss the utility of biomarkers as tools to overcome issues of CNS drug development.The consensus statement from the working group aimed at creating more nuanced criteria for employing biomarkers as tools to overcome issues surrounding CNS drug development. To accomplish this, a reverse engineering approach was adopted, in which criteria for the utilization of biomarkers were created in response to current challenges in the processes of drug discovery and development for CNS disorders. Based on this analysis, we propose a new paradigm containing 5 distinct tiers to further clarify the use of biomarkers and establish new strategies for decision-making in the context of CNS drug development. Specifically, we discuss more rational ways to incorporate biomarker data to determine optimal dosing for INDs with novel mechanisms and targets, and propose additional categorization criteria to further the use of biomarkers in patient stratification and clinical efficacy prediction. Finally, we propose validation and development of new neuroimaging biomarkers through public-private partnerships to further facilitate drug discovery and development for CNS disorders.