Effect of BI 1358894 on Cholecystokinin-Tetrapeptide (CCK-4)-Induced Anxiety, Panic Symptoms, and Stress Biomarkers: A Phase I Randomized Trial in Healthy Males.

INTRODUCTION: Depression, anxiety, and/or panic disorder are often comorbid and have a complex etiology mediated through the same neuronal network. Cholecystokinin-tetrapeptide (CCK-4), a synthetic analog of the endogenous neuropeptide cholecystokinin (CCK), is thought to be implicated in this network. The CCK-4 challenge model is an accepted method of investigating the pathophysiology of panic and has been shown to mediate neuronal activation via the transient receptor potential canonical (TRPC) ion channels. OBJECTIVES: This study aimed to assess the pharmacodynamic effects of BI 1358894, a small-molecule inhibitor of TRPC ion channel members 4 and 5 (TRPC4/5), on CCK-4-induced anxiety/panic-like symptoms and evaluate circuit engagement. METHODS: Twenty healthy male CCK-4-sensitive volunteers entered a Phase I, double blind, randomized, two-way cross-over, single dose, placebo-controlled trial. Randomization was to oral BI 1358894 100 mg in the fed state followed by oral placebo in the fed state, or vice versa. Treatments were administered 5 h prior to intravenous CCK-4 50 µg. The primary endpoint was maximum change from baseline of the Panic Symptom Scale (PSS) sum intensity score after CCK-4 injection. Further endpoints included the emotional faces visual analog score (EVAS), the Spielberger State-Trait Anxiety Inventory (STAI), plasma adrenocorticotropic hormone (ACTH), and serum cortisol values. The safety and tolerability of BI 1358894 was assessed based on a number of parameters including occurrence of adverse events (AEs). All pharmacodynamic, pharmacokinetic, and safety endpoints were analyzed using descriptive statistics. RESULTS: Single oral doses of BI 1358894 were generally well tolerated by the healthy male volunteers included in this study. Adjusted mean maximum change from baseline in PSS sum intensity score was 24.4 % lower in volunteers treated with BI 1358894 versus placebo, while adjusted mean maximum change from baseline of EVAS was reduced by 19.2 % (BI 1358894 vs placebo). The STAI total score before CCK-4 injection was similar in both groups (placebo: 25.1; BI 1358894: 24.3). Relative to placebo, BI 1358894 reduced CCK-4-induced mean maximum plasma ACTH and serum cortisol values by 58.6 % and 27.3 %, respectively. Investigator-assessed drug-related AEs were reported for 13/20 participants (65.0 %). There were no serious or severe AEs, AEs of special interest, AEs leading to discontinuation of trial medication, or deaths. CONCLUSIONS: Overall, BI 1358894 reduced psychological and physiological responses to CCK-4 compared with placebo, as measured by PSS, subjective EVAS and objectively measured stress biomarkers. BI 1358894 had a positive safety profile, and single oral doses were well tolerated by the healthy volunteers. This trial (NCT03904576/1402-0005) was registered on Clinicaltrials.gov on 05.04.19.

Somatostatin Receptor 4 Agonism Normalizes Stress-Related Excessive Amygdala Glutamate Release and Pavlovian Aversion Learning and Memory in Rodents.

Background: Excessive processing of aversive life events is a major pathology in stress-related anxiety and depressive disorders. Current pharmacological treatments have rather nonspecific mechanisms of action. Somatostatin is synthesized and released as an inhibitory co-neurotransmitter by specific GABA (gamma-aminobutyric acid) interneurons, and one of its receptors, SSTR4 (somatostatin receptor 4), is localized in brain regions involved in adaptive aversion processing and implicated in negative valence neuropathology, including the amygdala. Methods: Rat and mouse experiments were conducted to investigate effects of specific SSTR4 agonism on neurobehavioral aversion processing, including any normalization of stress-related hyperresponsiveness. A mouse experiment to investigate stress and SSTR4 agonism effects on reward processing was also conducted. Results: In male rats (n = 5-10/group) fitted with glutamate biosensors in basolateral amygdala, SSTR4 agonism attenuated glutamate release to restraint stress in control rats and particularly in rats previously exposed to chronic corticosterone. In male mice (n = 10-18/group), SSTR4 agonism dose-dependently attenuated Pavlovian tone/footshock learning and memory measured as freezing behavior, in both control mice and mice exposed to chronic social stress, which induces excessive Pavlovian aversion learning and memory. Specificity of SSTR4 agonism effects to aversion learning/memory was demonstrated by absence of effects on discriminative reward (sucrose) learning/memory in both control mice and mice exposed to chronic social stress; SSTR4 agonism did increase reward-to-effort valuation in a dose-dependent manner and in both control mice and mice exposed to chronic social stress, which attenuates reward motivation. Conclusions: These neuropsychopharmacological findings add substantially to the preclinical proof-of-concept evidence for SSTR4 agonism as a treatment in anxiety and depressive disorders.

The effects of transient receptor potential cation channel inhibition by BI 1358894 on cortico-limbic brain reactivity to negative emotional stimuli in major depressive disorder.

Abnormal emotional processing in major depressive disorder (MDD) has been associated with increased activation to negative stimuli in cortico-limbic brain regions. The authors investigated whether treatment with BI 1358894, a small-molecule inhibitor of the transient receptor potential cation channel subfamily C leads to attenuated activity in these areas in MDD patients. 73 MDD patients were randomized to receive a single oral dose of BI 1358894 (100 mg), citalopram (20 mg), or matching placebo. Brain responses to emotional faces and scenes were investigated using functional magnetic resonance imaging. Primary endpoints were BOLD signal changes in response to negative faces in cortico-limbic brain regions, i.e. bilateral amygdala (AMY), dorsolateral prefrontal cortex, anterior insula (AI), and anterior cingulate cortex. Secondary endpoints were BOLD signal changes in response to negative scenes. For each region, separate ANOVA models were computed for the comparison of treatments (BI 1358894 or citalopram) vs. placebo. The adjusted treatment differences in the % BOLD signal changes in the faces task showed that BI 1358894 induced signal reduction in bilateral AMY and left AI. In the scenes task, BI 1358894 demonstrated significant signal reduction in bilateral AMY, AI, anterior cingulate cortex and left dorsolateral prefrontal cortex. Citalopram failed to induce any significant reductions in BOLD signal in both tasks. BI 1358894-mediated inhibition of the transient receptor potential cation channel subfamily resulted in strong signal reduction in cortico-limbic brain regions, thereby supporting development of this mechanism of action for MDD patients.

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.

BACKGROUND: Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects. HC-070 IN VITRO: To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases. HC-070 IN VIVO: Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC50) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.

The somatostatin receptor 4 agonist J-2156 reduces mechanosensitivity of peripheral nerve afferents and spinal neurons in an inflammatory pain model.

Somatostatin (SST) is a peptide hormone that regulates the endocrine system and affects neurotransmission via interaction with G protein-coupled SST receptors and inhibition of the release of different hormones. The aim of this study was to investigate whether the analgesic properties of the selective SSTR4 agonist J-2156 are mediated via peripheral and/or spinal receptors. Effect on mechanical hyperalgesia in the Complete Freund׳s Adjuvant (CFA) model was measured after intraperitoneal application of J-2156. Electrophysiological neuronal recordings were conducted 24 h after injection of CFA or vehicle into the paw of Wistar rats. Mechanosensitivity of peripheral afferents of the saphenous nerve as well as of spinal wide dynamic range (WDR) and nociceptive-specific (NS) neurons were measured after systemic or spinal application of J-2156. In CFA animals J-2156 dose dependently reduced hyperalgesia in behavioral studies. The minimal effective dose was 0.1 mg/kg. Mechanosensitivity of peripheral afferents and spinal neurons was significantly reduced by J-2156. NS neurons were dose dependently inhibited by J-2156 while in WDR neurons only the highest concentration of 100 µM had an effect. In sham controls, J-2156 had no effect on neuronal activity. We demonstrated that J-2156 dose-dependently reduces peripheral and spinal neuronal excitability in the CFA rat model without affecting physiological pain transmission. Given the high concentration of the compound required to inhibit spinal neurons, it is unlikely that the behavioral effect seen in CFA model is mediated centrally. Overall these data demonstrated that the analgesic effect of J-2156 is mediated mainly via peripheral SST4 receptors.

Somatostatin 4 receptor activation modulates TRPV1[correction of TPRV1] currents in dorsal root ganglion neurons.

Somatostatin (sst) is a cyclic neuropeptide known to have inhibitory roles in the central nervous system. It exerts its biological effects via the activation of the 5 sst receptor subtypes, which belong to the family of G-protein coupled receptors (GPCR). This peptide has analgesic properties, specifically via the activation of the sst4 receptor subtype. Although this is established, the precise molecular mechanisms causing this have not yet been fully elucidated. This research aimed to identify a possible anti-nociceptive mechanism, showing functional links to the transient receptor potential vanilloid type 1 (TRPV1) within the pain processing pathway. Calcium imaging and whole cell voltage clamp experiments were conducted on DRG neurons prepared from adult rats, utilizing capsaicin stimulations and the sst4 receptor specific agonist J-2156. The complete Freund's adjuvant (CFA) inflammatory pain model was used to examine if effects are augmented in pain conditions. The sst4 receptor agonist J-2156 was able significantly to inhibit capsaicin induced calcium and sodium influx, where the effect was more potent after CFA treatment. This inhibition identifies a contributory molecular mechanism to the analgesic properties of sst4 receptor activation.

Somatostatin 4 receptor activation modulates G-protein coupled inward rectifying potassium channels and voltage stimulated calcium signals in dorsal root ganglion neurons.

Somatostatin has a wide biological profile resulting from its actions on the five receptor subtypes (sst1-5). Recently somatostatin was shown to exert analgesic effects via activation of the sst4 receptor. Although the analgesia in pain models is established, the precise molecular mechanism has yet to be fully elucidated. This research aimed to identify possible anti-nociceptive mechanisms, showing functional links of the sst4 receptor to G-protein coupled inward rectifying potassium (GIRK) channels and reduction of voltage stimulated calcium influx within the pain processing pathway. Whole cell voltage clamp experiments and calcium imaging experiments were conducted on DRG neurons prepared from adult rats. Application of an sst4 receptor selective agonist, J-2156, on DRG neurons induced a GIRK modulated potassium current, and inhibited voltage sensitive calcium current. Both mechanisms are thought to contribute to the analgesic properties of sst4 receptor agonists.

The CGRP receptor antagonist BIBN4096BS peripherally alleviates inflammatory pain in rats.

Calcitonin gene-related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain. Here we focus on its implication in a rat pain model of inflammation, induced by injection of complete Freund adjuvant (CFA). The nonpeptide CGRP receptor antagonist BIBN4096BS reduces migraine pain and trigeminal neuronal activity. Here we demonstrate that the compound reduces inflammatory pain and spinal neuronal activity. Behavioural experiments reveal a reversal of the CFA-induced mechanical hypersensitivity and monoiodoacetate (MIA)-induced weight-bearing deficit in rats after systemic drug administration. To further investigate the mechanism of action of the CGRP antagonist in inflammatory pain, in vivo electrophysiological studies were performed in CFA-injected rats. Recordings from wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord confirmed a reduction of neuronal activity after systemic drug application. The same amount of reduction occurred after topical administration onto the paw, with resulting systemic plasma concentrations in the low nanomolar range. However, spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model. Peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.

Hyoscine butylbromide potently blocks human nicotinic acetylcholine receptors in SH-SY5Y cells.

Hyoscine butylbromide (HBB; tradenames: Buscopan/Buscapina is an antispasmodic drug for the treatment of abdominal pain associated with gastrointestinal cramping. As a hyoscine derivative, this compound competitively inhibits muscarinic acetylcholine (ACh) receptors on smooth muscle cells in the gastrointestinal tract. Preliminary investigations suggested that it might also inhibit nicotinic ACh receptors. This study investigated the effect of HBB on nicotinic ACh receptor-mediated membrane currents in SH-SY5Y cells. ACh and nicotine application-induced comparable membrane currents with EC(50) values of 25.9+/-0.6 and 40.1+/-0.4microM, respectively. When coapplied with 100microM ACh, HBB concentration-dependently suppressed currents with an IC(50) value of 0.19+/-0.04microM, and was approximately seven-times more potent than the ganglionic blocker, hexamethonium (IC(50)=1.3+/-0.3microM). Increasing the agonist concentration to 5mM did not affect the amount of block by HBB, which suggests a non-competitive mode of action. These functional in vitro data demonstrate for the first time that HBB blocks neuronal nicotinic ACh receptors in the same concentration range as it inhibits muscarinic ACh receptors. If one hypothesizes that HBB might also affect nicotinic receptors in autonomic neurons in vivo (e. g. in the enteric nervous system), this effect could contribute to its spasmolytic activity.

CGRP antagonists: unravelling the role of CGRP in migraine.

Migraine is a complex, debilitating neurovascular disorder. Although knowledge on the main molecular players is still incomplete, recent preclinical and clinical findings indicate that there is a clear correlation between migraine-associated headache and the release of the neuropeptide calcitonin gene-related peptide (CGRP). BIBN4096 was the first CGRP antagonist to be tested in clinical trials for the treatment of migraine. The proven efficacy of this agent, and also the CGRP antagonist MK-0974, to alleviate acute migraine headache provided significant support for the hypothesis that CGRP has an important role in migraine pathophysiology. Moreover, the recently published results from Phase II trials are encouraging and suggest that this new type of drug might offer advantages over existing therapies for patients suffering from migraine and related headaches.

The role of CGRP and nicotinic receptors in centrally evoked facial blood flow changes.

The release of CGRP in humans is associated with the occurrence of migraine headaches. The vasoactive neuropeptide is released by afferent neurones originating in the peripherally located trigeminal ganglion supplying the dura mater. The role of CGRP in migraine is further supported by recently released data showing that the CGRP-antagonist BIBN4096BS is clinically effective for the treatment of migraine headaches. Yet, the trigger for CGRP release during migraine attacks is not identified. It is suggested that the peripheral CGRP release during a migraine attack might be either triggered by direct activation of afferent dural neurones, or, by indirect activation via the central nervous system. Recently, we were able to show that the CGRP-antagonist BIBN4096BS is able to inhibit vasodilation induced by trigeminal ganglion stimulation. Now, we extend our studies to the investigation of facial blood flow changes induced by electrical stimulation of the brainstem trigeminal nucleus caudalis (TNC). Here, we show that stimulation of the TNC leads to a pronounced increase of facial blood flow. The nicotinic antagonist Hexamethonium reduced the evoked flow by approximately 50% (30 mg/kg), while the muscarinic antagonist Atropin did not influence the stimulation evoked blood flow. Application of BIBN4096BS (0.3 mg/kg, i.v.) diminished the evoked flow almost completely. Therefore, we conclude that CGRP represents the key player in TNC-induced facial vasodilation, while activation of nicotinic receptors modulates centrally induced peripheral neurogenic vasodilation.

Voltage-gated calcium channels may be involved in the regulation of the mechanosensitivity of slowly conducting knee joint afferents in rat.

Voltage-gated Ca(2+) channels play an important role in the central processing of nociceptive information. Recently, it has been shown that L- and N-type voltage-gated Ca(2+) channels are also present on peptidergic, fine afferent nerve fibers in the knee joint capsule. Therefore, the influence of specific blockers for L-type (verapamil) or N-type (omega-conotoxin GVIA) Ca(2+) channels on the mechanosensitivity of slowly conducting afferents was tested in the rat knee joint. Topical application of 100 microM verapamil onto the receptive field reduced the mean response to knee joint rotation to 67+/-8% (SEM, n=12), obtained by outward rotations with a torque of 10 mNm above the mechanical threshold and compared with control movements. In the presence of 50 microM omega-conotoxin GVIA, the mean response decreased to 44+/-5% ( n=12), a reduction that was also observed during rotations of other intensities. Simultaneous application of both substances further reduced the response to 25+/-11% ( n=6). In additional experiments it was shown that L- and N-type voltage-gated Ca(2+) channels do not influence activity-dependent changes of the mechanical excitability. In conclusion, the data of the present study indicate that voltage-gated Ca(2+) channels may also be involved in the regulation of the mechanosensitivity of nociceptive nerve fiber endings.

Determination of clotrimazole in mice plasma by capillary electrophoresis.

In addition to its antifungal activity, clotrimazole attracts interest as an anti-inflammatory drug. In order to correlate this effect with plasma concentrations in mice, a capillary electrophoretic method was developed. Sample preparation was carried out by protein precipitation using methanol. Quantification of clotrimazole was achieved by means of capillary electrophoresis using ketoconazole as an internal standard (IS). The background electrolyte (BGE) composed of a Tris buffer solution (100 mM, pH 3.0, adjusted with acetic acid) and methanol (8:2, v/v). Injection was carried out electrokinetically with 10 kV over a time period of 20 s. A special rinsing procedure utilizing a sequence of a SDS/methanol solution, a sodium hydroxide solution, water and BGE, was applied to enhance the reproducibility. With this procedure, an intermediate precision (day-to-day precision) of the area ratios of clotrimazole and IS of 5.0% for 0.5 microg ml(-1) and 2.6% for 10 microg ml(-1) was obtained. In summary, with the described capillary zone electrophoresis (CZE) method it is possible to handle small sample volumes of 60 microl, to detect clotrimazole concentrations of 0.3 microg ml(-1) (limit of detection), and to quantify clotrimazole down to concentrations of 0.5 microg ml(-1) (limit of quantification).