Evaluation of the Safety, Tolerability, and Pharmacokinetic Profiles of TP0473292 (TS-161), A Prodrug of a Novel Orthosteric mGlu2/3 Receptor Antagonist TP0178894, in Healthy Subjects and Its Antidepressant-Like Effects in Rodents.

BACKGROUND: TP0473292 (the active ingredient of TS-161) is a prodrug of a novel metabotropic glutamate (mGlu) 2/3 receptor antagonist being developed for the treatment of patients with depression. This study evaluated the safety, tolerability, and pharmacokinetics of orally administered TS-161 in healthy subjects. METHODS: This was a first-in-human, phase 1, randomized, double-blind, placebo-controlled, single-ascending dose (15-400 mg TS-161) and 10-day multiple-ascending dose (50-150 mg TS-161) study in healthy subjects, conducted from June 2019 through February 2020. Plasma and urine concentrations of the prodrug and its metabolites, and cerebrospinal fluid (CSF) concentrations of the active metabolite TP0178894 were measured to evaluate the pharmacokinetic profiles after oral administration of TS-161. RESULTS: Following single and multiple doses, TP0473292 was extensively converted into its active metabolite TP0178894. Plasma concentrations of TP0178894 reached peak (Cmax) within 5 hours post dose and declined with a t1/2 <13 hours. Plasma exposures of TP0178894 increased with increasing dose. TP0178894 penetrated into CSF and reached a Cmax of 9.892 ng/mL at a single dose of 100 mg, which was comparable with IC50 values of antagonist activity at mGlu2/3 receptors. The most frequently observed adverse events that showed exposure-related incidence during the study were nausea, vomiting, and dizziness. CONCLUSIONS: The mGlu2/3 receptor antagonist prodrug TP0473292 is safe and well-tolerated, is orally bioavailable in humans with extensive conversion into the active metabolite TP0178894 with sufficient CSF penetration to exert the anticipated pharmacological effects, and is a promising candidate for further clinical development in treatment of patients with depression.

A Novel Potent and Selective Histamine H3 Receptor Antagonist Enerisant: In Vitro Profiles, In Vivo Receptor Occupancy, and Wake-Promoting and Procognitive Effects in Rodents.

Histamine H3 receptor antagonists/inverse agonists are known to enhance the activity of histaminergic neurons in the brain, thereby promoting arousal and cognition. Here, we report the in vitro and in vivo pharmacological profiles for a newly synthesized histamine H3 receptor antagonist/inverse agonist: [1-(4-{3-[(2R)-2-methylpyrrolidin-1-yl]propoxy}phenyl)-1H-pyrazol-4-yl](morpholin-4-yl)methanone monohydrochloride (enerisant hydrochloride). In vitro assays showed that enerisant was a competitive antagonist/inverse agonist with a high affinity and selectivity for human and rat histamine H3 receptors. Enerisant showed antagonist activity in vivo, as assessed using R-α-methylhistamine (a histamine H3 receptor agonist)-induced dipsogenia, and occupied the histamine H3 receptor in the frontal cortex in a dose-dependent manner. Enerisant also enhanced the extracellular levels of histamine in the posterior hypothalamus and the levels of dopamine and acetylcholine in the medial prefrontal cortex of rats. Enerisant exerted a procognitive effect or reversed scopolamine-induced cognitive impairment in a social recognition test and a novel object recognition test in rats at doses at which less than 50% of the histamine H3 receptor were occupied (0.03-0.3 mg/kg, p.o.). In contrast, higher doses (3-10 mg/kg, p.o.) at which nearly all the histamine H3 receptors were occupied were needed to exert wake-promoting effects in rats. These results indicate that enerisant is a potent and selective histamine H3 receptor antagonist/inverse agonist with the potential to promote arousal and procognition in rats. Moreover, the results also suggest that the histamine H3 receptor occupancy required to exert a pharmacological effect may vary depending on the domain that is being tested. SIGNIFICANCE STATEMENT: Enerisant is a novel histamine H3 receptor antagonist/inverse agonist that exerts wake-promoting and procognitive effects in addition to increasing the release of neurotransmitters related to these pharmacological effects in rodents. Moreover, an in vivo receptor binding study revealed that the in vivo occupancy of the histamine H3 receptor required to exert the pharmacological effects of enerisant varied, and such variations in required occupancy should be taken into account when performing dose selection in clinical studies.

Discovery of MGS0274, an ester prodrug of a metabotropic glutamate receptor 2/3 agonist with improved oral bioavailability.

We previously reported that MGS0008 is a selective group II metabotropic glutamate receptor (mGlu2/3 receptor) agonist that is effective in animal models of schizophrenia. MGS0008 is a highly hydrophilic glutamate analog and is therefore expected to show low oral bioavailability in humans. To improve the oral bioavailability of MGS0008, ester prodrugs of MGS0008 were synthesized and their usefulness was evaluated. Among the prodrugs, the l-menthol-ester prodrug 4h demonstrated preferable lipophilicity, good chemical stability, and a high conversion rate to MGS0008 in human and monkey liver microsomes. A pharmacokinetic study in monkeys revealed that the oral bioavailability of MGS0008 after oral dosing of compound 4h was approximately 15-fold higher than that after oral dosing of MGS0008. Based on these findings, a diastereomer of compound 4h (compound 4j, or MGS0274), was selected as a candidate for clinical drug development, and its besylate is currently under development for the treatment of schizophrenia (Development code: TS-134).

Antipsychotic profiles of TASP0443294, a novel and orally active positive allosteric modulator of metabotropic glutamate 2 receptor.

Glutamatergic dysfunction has been implicated in psychiatric disorders such as schizophrenia. The stimulation of metabotropic glutamate (mGlu) 2 receptor has been shown to be effective in a number of animal models of schizophrenia. In this study, we investigated the antipsychotic profiles of (2S)-5-methyl-2-{[4-(1,1,1-trifluoro-2-methylpropan-2-yl)phenoxy]methyl}-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide (TASP0443294), a newly synthesized positive allosteric modulator of the mGlu2 receptor. TASP0443294 potentiated the response of human mGlu2 and rat mGlu2 receptors to glutamate with EC50 values of 277 and 149 nM, respectively, without affecting the glutamate response of human mGlu3 receptor. TASP0443294 was distributed in the brain and cerebrospinal fluid after peroral administration in rats. The peroral administration of TASP0443294 inhibited methamphetamine-induced hyperlocomotion in rats, which was attenuated by an mGlu2/3 receptor antagonist, and improved social memory impairment induced by 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) in rats. Furthermore, TASP0443294 reduced the ketamine-induced basal gamma hyperactivity in the prefrontal cortex and suppressed rapid eye movement (REM) sleep in rats. These findings indicate that TASP0443294 is an mGlu2 receptor positive allosteric modulator with antipsychotic activity, and that the suppression of aberrant gamma oscillations and REM sleep could be considered as neurophysiological biomarkers for TASP0443294.

Neurophysiologic and antipsychotic profiles of TASP0433864, a novel positive allosteric modulator of metabotropic glutamate 2 receptor.

Excess glutamatergic neurotransmission has been implicated in the pathophysiology of schizophrenia, and the activation of metabotropic glutamate 2 (mGlu2) receptor may exert antipsychotic effects by normalizing glutamate transmission. In the present study, we investigated the neurophysiologic and antipsychotic profiles of TASP0433864 [(2S)-2-[(4-tert-butylphenoxy)methyl]-5-methyl-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide], a newly synthesized positive allosteric modulator (PAM) of mGlu2 receptor. TASP0433864 exhibited PAM activity at human and rat mGlu2 receptors with EC50 values of 199 and 206 nM, respectively, without exerting agonist activity at rat mGlu2 receptor. TASP0433864 produced a leftward and upward shift in the concentration-response curve of glutamate-increased guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to mGlu2 receptor. In contrast, TASP0433864 had negligible activities for other mGlu receptors, including mGlu3 receptor, and did not have any affinity for other receptors or transporters. In hippocampal slices, TASP0433864 potentiated an inhibitory effect of DCG-IV [(2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine], a mGlu2/3 receptor agonist, on the field excitatory postsynaptic potentials in the dentate gyrus, indicating that TASP0433864 potentiates the mGlu2 receptor-mediated presynaptic inhibition of glutamate release. Moreover, TASP0433864 inhibited both MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]- and ketamine-increased cortical γ band oscillation in the rat cortical electroencephalogram, which have been considered to reflect the excess activation of cortical pyramidal neurons. The inhibitory effect of TASP0433864 on cortical activation was also observed in the mouse 2-deoxy-glucose uptake study. In a behavioral study, TASP0433864 significantly inhibited both ketamine- and methamphetamine-increased locomotor activities in mice and rats, respectively. Collectively, these findings indicate that TASP0433864 is a selective mGlu2 receptor PAM with antipsychotic activity, and the attenuation of excess glutamatergic neurotransmission may be involved in the action of TASP0433864.

Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor.

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a (11)C-labeled specific 20-HETE synthase inhibitor, N'(4-dimethylaminohexyloxy)phenyl imidazole ([(11)C]TROA). Autoradiographic study showed that [(11)C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [(11)C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [(11)C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [(11)C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia.

The inhibitor of 20-HETE synthesis, TS-011, improves cerebral microcirculatory autoregulation impaired by middle cerebral artery occlusion in mice.

BACKGROUND AND PURPOSE: 20-Hydroxyeicosatetraenoic acid is a potent vasoconstrictor that contributes to cerebral ischaemia. An inhibitor of 20-Hydroxyeicosatetraenoic acid synthesis, TS-011, reduces infarct volume and improves neurological deficits in animal stroke models. However, little is known about how TS-011 affects the microvessels in ischaemic brain. Here, we investigated the effect of TS-011 on microvessels after cerebral ischaemia. EXPERIMENTAL APPROACH: TS-011 (0.3 mg·kg(-1) ) or a vehicle was infused intravenously for 1 h every 6 h in a mouse model of stroke, induced by transient occlusion of the middle cerebral artery occlusion following photothrombosis. The cerebral blood flow velocity and the vascular perfusion area of the peri-infarct microvessels were measured using in vivo two-photon imaging. KEY RESULTS: The cerebral blood flow velocities in the peri-infarct microvessels decreased at 1 and 7 h after reperfusion, followed by an increase at 24 h after reperfusion in the vehicle-treated mice. We found that TS-011 significantly inhibited both the decrease and the increase in the blood flow velocities in the peri-infarct microvessels seen in the vehicle-treated mice after reperfusion. In addition, TS-011 significantly inhibited the reduction in the microvascular perfusion area after reperfusion, compared with the vehicle-treated group. Moreover, TS-011 significantly reduced the infarct volume by 40% at 72 h after middle cerebral artery occlusion. CONCLUSIONS AND IMPLICATIONS: These findings demonstrated that infusion of TS-011 improved defects in the autoregulation of peri-infarct microcirculation and reduced the infarct volume. Our results could be relevant to the treatment of cerebral ischaemia.

Serum S100B, brain edema, and hematoma formation in a rat model of collagenase-induced hemorrhagic stroke.

S100B, a 21-kD Ca(2+) binding protein expressed in Schwann cells and astroglia, has often been reported as a promising biomarker for ischemic stroke. In addition to ischemic stroke, the peripheral S100B level may also be useful as a biomarker for intracerebral hemorrhage (ICH). However, the kinetics and characterization of peripheral S100B in patients or experimental animal models with ICH have not been carefully examined. The present study investigated the kinetics and characteristics of the serum S100B level in a rat collagenase-induced ICH model. The serum S100B kinetics and the time-course of brain edema and hematoma formation were examined. Then, the correlations between the elevated serum S100B level and brain edema or hematoma formation were investigated. A transient elevation of serum S100B that peaked at 6 h after ICH induction was observed. The single measurement of serum S100B at 6 h after ICH induction was significantly correlated with brain edema formation and the maximal extent of the hematoma volumes. These results suggest the significance of serum S100B as a biomarker of brain damage resulting from ICH.

Early increases in serum S100B are associated with cerebral hemorrhage in a rat model of focal cerebral ischemia.

S100B is a 21-kD, Ca2+-binding protein that is mainly expressed in astroglial cells and Schwann cells in the nervous system. The S100B level in peripheral blood samples is reportedly elevated in patients with various central nervous system disorders including ischemic stroke. Since an elevated peripheral S100B level seems to be related closely to cerebral vascular damage involving a blood-brain barrier (BBB) disruption, we hypothesized that the peripheral S100B levels may increase earlier and to a greater extent after stroke onset when the cerebral blood vessels are severely damaged and spontaneous cerebral hemorrhage exists. In the present study, the relationship between an increase in the serum S100B level and cerebral hemorrhage was investigated within 24 h of stroke onset. A rat model for focal cerebral ischemia using an intraluminal filament method was utilized because cerebral hemorrhage is sometimes observed as a result of vascular damage caused by the filament. Significant increases in the serum S100B levels of rats with cerebral hemorrhage were observed from 1 h after stroke onset, compared with the levels in rats without cerebral hemorrhage. The early increases in serum S100B were not correlated with the brain infarct volumes at 3 h after stroke. These findings suggest that the serum S100B level increases earlier, reflecting the existence of cerebral hemorrhage.

Relationship between cerebrospinal and peripheral S100B levels after focal cerebral ischemia in rats.

S100B is a 21-kDa, Ca(2+)-binding protein that is expressed in the central nervous system. Although the peripheral S100B level is significantly correlated with stroke outcome, the mechanisms responsible for increase in the peripheral S100B level have not been precisely investigated in animal ischemic stroke models. To justify the use of peripheral S100B as a common biomarker between stroke patients and animal models, the mechanisms responsible for increases in the peripheral S100B level after focal cerebral ischemia should be clarified. In the present study, we investigated correlations between the cerebrospinal and serum S100B levels to determine whether increase in peripheral S100B properly reflect the conditions inside the central nervous system. From each rat, cerebrospinal fluid and serum samples were collected at 24, 48, 72, or 120 h after the onset of photochemically induced thromboembolic stroke in rats. Our results indicated a difference in the kinetics of cerebrospinal and serum S100B. Among the four sampling points, the serum S100B levels were most strongly correlated with the cerebrospinal S100B levels at 48 h after PIT stroke onset. While the serum S100B level may be a useful biomarker of stroke in experimental or clinical studies, the timing of S100B measurements should be carefully selected to ensure that the serum S100B level properly reflects the conditions in the central nervous system.

Quantitative assessments of cerebral vascular damage with a silicon rubber casting method in photochemically-induced thrombotic stroke rat models.

Previous studies have described microvascular disturbances downstream of occluded large vessels arising during the acute phase (several hours) following cerebral ischemic insult. Prolonged microvascular disturbances may cause delayed neuronal cell death in ischemic penumbral regions, leading to expanded brain infarctions and poor neurological and functional outcomes. The lack of simple and quantitative methods for investigating this microcirculation failure suggests the need to develop a new method for clarifying the precise distribution and persistence of post-ischemic microvascular disturbances. The present study used a silicone rubber casting method in quantitative analyses of microvascular conditions in photochemically-induced thromboembolic (PIT) stroke rat models. After the casting procedure in rats with PIT stroke, a 6 microm-thick coronal section was obtained, and quantitative analyses of microvascular density and measurements of the infarct area in the serial section were performed. The major findings of the present study are as follows: (1) Silicone rubber casting techniques can be applied to precise quantitative analyses of microvessels in the same individual in whom brain infarct volume was measured; (2) the persistence and spatial distribution of microvascular disturbances assessed at the ischemic core, ischemic penumbra, and non-ischemic regions strongly suggest that microvascular disturbances affect brain infarct expansion; (3) the current method demonstrated the protective effects of MK-801 on microvessels, indicating that the technique may be useful in investigating factors that provide vascular protection. The experimental procedure introduced here would facilitate future evaluations of vascular protective agents.

Serum S100B is a useful surrogate marker for long-term outcomes in photochemically-induced thrombotic stroke rat models.

In recent years, serum S100B has been used as a secondary endpoint in some clinical trials, in which serum S100B has successfully indicated the benefits or harm done by the tested agents. Compared to clinical stroke studies, few experimental stroke studies report using serum S100B as a surrogate marker for estimating the long-term effects of neuroprotectants. This study sought to observe serum S100B kinetics in PIT stroke models and to clarify the association between serum S100B and both final infarct volumes and long-term neurological outcomes. Furthermore, to demonstrate that early elevations in serum S100B reflect successful neuroprotective treatment, a pharmacological study was performed with a non-competitive NMDA glutamate receptor antagonist, MK-801. Serum S100B levels were significantly elevated after PIT stroke, reaching peak values 48 h after the onset and declining thereafter. Single measurements of serum S100B as early as 48 h after PIT stroke correlated significantly with final infarct volumes and long-term neurological outcomes. Elevated serum S100B was significantly attenuated by MK-801, correlating significantly with long-term beneficial effects of MK-801 on infarct volumes and neurological outcomes. Our results showed that single measurements of serum S100B 48 h after PIT stroke would serve as an early and simple surrogate marker for long-term evaluation of histological and neurological outcomes in PIT stroke rat models.

Serum S100B indicates successful combination treatment with recombinant tissue plasminogen activator and MK-801 in a rat model of embolic stroke.

In recent years, serum S100B has been used as a secondary endpoint in some clinical trials in which serum S100B has successfully indicated the benefits or harm done by tested agents. However, few reports describe serum S100B as an indicator of the efficiency of neuroprotective treatment in experimental stroke models, although serum S100B may be as useful for histological and functional evaluations of neuroprotective treatments as in clinical trials. The present study seeks to investigate the possibility that serum S100B reflects successful combined treatment with rt-PA and MK-801 in an embolic stroke rat model. An embolic stroke model of rats was produced via intra-arterial autologous clot injection, after which serum S100B levels were measured 24 h after embolism and the association of serum S100B levels with brain edema volume and infarct volume investigated. Combination treatment with rt-PA and MK-801 significantly attenuated the elevation of serum S100B, which correlated significantly with reductions in brain edema resulting from combination treatment. These findings suggest that serum S100B is a simple and objective indicator for successful neuroprotective therapy and would help seeking partners for combination treatments with rt-PA in an embolic stroke rat model. Assessments of the efficacy of combination treatments with rt-PA and neuroprotectants using serum S100B would facilitate translational research bridging laboratory and bedsides because serum S100B functions as a common marker in both rats and human patients suffering from ischemic stroke.

Serum S100B indicates brain edema formation and predicts long-term neurological outcomes in rat transient middle cerebral artery occlusion model.

To assess the usefulness of serum S100B as a biomarker, the present study proceeded by observing serum S100B kinetics in a rat transient middle cerebral artery occlusion (MCAO) model, then assessed the correlation between serum S100B and both brain edema formation and neurological outcomes. Study results showed increases in serum S100B concentrations, peaking 48 h after MCAO. Brain water content in the ipsilateral hemisphere significantly increased from 24 h after MCAO, and reached peak value 72 h after MCAO. A single measurement of serum S100B 48 h after MCAO showed significant correlations with maximal extent of brain edema 72 h after MCAO. Furthermore, S100B concentrations 48 h after MCAO significantly correlated with infarct volumes. Neurological outcomes were estimated in a long-term study, where a gradual recovery was observed up to 168 h after MCAO. Serum S100B 48 h after MCAO was found to show higher correlation with neurological score 168 h after MCAO than those 48 h after MCAO. These findings suggest that serum S100B is an effective biomarker in predicting both extent of brain edema and long-term neurological outcomes in a rat transient MCAO model.