Evaluating the efficacy of the selective orexin 1 receptor antagonist nivasorexant in an animal model of binge-eating disorder.

OBJECTIVE: Test the efficacy of the selective orexin 1 receptor (OX1R) antagonist (SO1RA) nivasorexant in an animal model of binge-eating disorder (BED) and study its dose-response relationship considering free brain concentrations and calculated OX1R occupancy. Compare nivasorexant's profile to that of other, structurally diverse SO1RAs. Gain understanding of potential changes in orexin-A (OXA) neuropeptide and deltaFosB (ΔFosB) protein expression possibly underlying the development of the binge-eating phenotype in the rat model used. METHOD: Binge-like eating of highly palatable food (HPF) in rats was induced through priming by intermittent, repeated periods of dieting and access to HPF, followed by an additional challenge with acute stress. Effects of nivasorexant were compared to the SO1RAs ACT-335827 and IDOR-1104-2408. OXA expression in neurons and neuronal fibers as well as ΔFosB and OXA-ΔFosB co-expression was studied in relevant brain regions using immuno- or immunofluorescent histochemistry. RESULTS: All SO1RAs dose-dependently reduced binge-like eating with effect sizes comparable to the positive control topiramate, at unbound drug concentrations selectively blocking brain OX1Rs. Nivasorexant's efficacy was maintained upon chronic dosing and under conditions involving more frequent stress exposure. Priming for binge-like eating or nivasorexant treatment resulted in only minor changes in OXA or ΔFosB expression in few brain areas. DISCUSSION: Selective OX1R blockade reduced binge-like eating in rats. Neither ΔFosB nor OXA expression proved to be a useful classifier for their binge-eating phenotype. The current results formed the basis for a clinical phase II trial in BED, in which nivasorexant was unfortunately not efficacious compared with placebo. PUBLIC SIGNIFICANCE: Nivasorexant is a new investigational drug for the treatment of binge-eating disorder (BED). It underwent clinical testing in a phase II proof of concept trial in humans but was not efficacious compared with placebo. The current manuscript investigated the drug's efficacy in reducing binge-like eating behavior of a highly palatable sweet and fat diet in a rat model of BED, which initially laid the foundation for the clinical trial.

The metabolism of the dual orexin receptor antagonist daridorexant.

Daridorexant is a dual orexin receptor antagonist developed for the treatment of insomnia disorder and has shown improvement in sleep outcomes and daytime functioning. The present work describes its biotransformation pathways in vitro and in vivo and provides a cross-species comparison between the animal species used in preclinical safety assessments and man.Daridorexant clearance is driven by metabolism along seven distinct pathways. Metabolic profiles were characterised by downstream products while primary metabolic products were of minor importance. The metabolic pattern differed between rodent species, with the rat reflecting the human pattern better than the mouse.In rodents, daridorexant is mostly excreted via the bile after extensive metabolism while urinary excretion was negligible in the rat. Only traces of the parent drug were detected in urine, bile, or faeces.Daridorexant has three major metabolites which are well covered in these preclinical safety species. All of them retain some residual affinity towards orexin receptors. However, none of these is considered to contribute to the pharmacological effect of daridorexant as their active concentrations in the human brain are too low.

Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.

T cell homeostasis is essential for the functioning of the vertebrate immune system, but the intracellular signals required for T cell homeostasis are largely unknown. We here report that the WD-repeat protein family member coronin-1, encoded by the gene Coro1a, is essential in the mouse for T cell survival through its promotion of Ca2+ mobilization from intracellular stores. Upon T cell receptor triggering, coronin-1 was essential for the generation of inositol-1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate. The absence of coronin-1, although it did not affect T cell development, resulted in a profound defect in Ca2+ mobilization, interleukin-2 production, T cell proliferation and T cell survival. We conclude that coronin-1, through activation of Ca2+ release from intracellular stores, is an essential regulator of peripheral lymphocyte survival.

The Antifibrotic Activity of Prostacyclin Receptor Agonism Is Mediated through Inhibition of YAP/TAZ.

Idiopathic pulmonary fibrosis is a life-threatening progressive disease characterized by loss of alveolar epithelial cells, inflammation, and aberrant fibroblast activation. The two currently approved therapies do not halt or reverse tissue remodeling, and therefore novel disease-modifying mechanisms are needed. Our results describe YAP/TAZ inhibition through prostacyclin (IP) receptor activation as a novel mechanism that suppresses profibrotic (myo)fibroblast activity. We investigated the antifibrotic properties of the selective IP receptor agonist ACT-333679 using primary human lung fibroblasts. ACT-333679 prevented transforming growth factor ß1-induced fibroblast-to-myofibroblast transition, proliferation, extracellular matrix synthesis, and IL-6 and PAI-1 secretion, and exerted relaxant effects in cell contraction assays. ACT-333679 treatment also reverted an established myofibroblast phenotype. Unbiased analysis of ACT-333679-induced whole-genome expression changes in transforming growth factor ß1-treated fibroblasts identified significant attenuation of genes regulated by YAP/TAZ, two transcriptional cofactors that are essential for fibrosis. We then demonstrated that ACT-333679, via elevation of cAMP, caused YAP/TAZ nuclear exclusion and subsequent suppression of YAP/TAZ-dependent profibrotic gene transcription. In summary, we offer a rationale for further exploring the potential of IP receptor agonists for the treatment of idiopathic pulmonary fibrosis.

The Use of Physiology-Based Pharmacokinetic and Pharmacodynamic Modeling in the Discovery of the Dual Orexin Receptor Antagonist ACT-541468.

The identification of new sleep drugs poses particular challenges in drug discovery owing to disease-specific requirements such as rapid onset of action, sleep maintenance throughout major parts of the night, and absence of residual next-day effects. Robust tools to estimate drug levels in human brain are therefore key for a successful discovery program. Animal models constitute an appropriate choice for drugs without species differences in receptor pharmacology or pharmacokinetics. Translation to man becomes more challenging when interspecies differences are prominent. This report describes the discovery of the dual orexin receptor 1 and 2 (OX1 and OX2) antagonist ACT-541468 out of a class of structurally related compounds, by use of physiology-based pharmacokinetic and pharmacodynamic (PBPK-PD) modeling applied early in drug discovery. Although all drug candidates exhibited similar target receptor potencies and efficacy in a rat sleep model, they exhibited large interspecies differences in key factors determining their pharmacokinetic profile. Human PK models were built on the basis of in vitro metabolism and physicochemical data and were then used to predict the time course of OX2 receptor occupancy in brain. An active ACT-541468 dose of 25 mg was estimated on the basis of OX2 receptor occupancy thresholds of about 65% derived from clinical data for two other orexin antagonists, almorexant and suvorexant. Modeling predictions for ACT-541468 in man were largely confirmed in a single-ascending dose trial in healthy subjects. PBPK-PD modeling applied early in drug discovery, therefore, has great potential to assist in the identification of drug molecules when specific pharmacokinetic and pharmacodynamic requirements need to be met.

Selexipag Active Metabolite ACT-333679 Displays Strong Anticontractile and Antiremodeling Effects but Low ß-Arrestin Recruitment and Desensitization Potential.

Prostacyclin (PGI2) receptor (IP receptor) agonists, which are indicated for the treatment of pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation, and extracellular matrix synthesis. Selexipag (Uptravi, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide) is the first nonprostanoid IP receptor agonist, it is available orally and was recently approved for the treatment of PAH. In this study we show that the active metabolite of selexipag and the main contributor to clinical efficacy ACT-333679 (previously known as MRE-269) behaved as a full agonist in multiple PAH-relevant receptor-distal-or downstream-cellular assays with a maximal efficacy (Emax) comparable to that of the prototypic PGI2 analog iloprost. In PASMC, ACT-333679 potently induced cellular relaxation (EC50 4.3 nM) and inhibited cell proliferation (IC50 4.0 nM) as well as extracellular matrix synthesis (IC50 8.3 nM). In contrast, ACT-333679 displayed partial agonism in receptor-proximal-or upstream-cAMP accumulation assays (Emax 56%) when compared with iloprost and the PGI2 analogs beraprost and treprostinil (Emax ∼100%). Partial agonism of ACT-333679 also resulted in limited ß-arrestin recruitment (Emax 40%) and lack of sustained IP receptor internalization, whereas all tested PGI2 analogs behaved as full agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related receptor-proximal readouts.

Milestones to the Discovery of T-type Calcium Channel Blockers for the Treatment of Generalized Epilepsies.

We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.

From bosentan (Tracleer®) to macitentan (Opsumit®): The medicinal chemistry perspective.

The endothelin peptides bind to two receptors found on cells of vasculature and in tissues. While the endothelin-A (ETA)-receptor is predominantly expressed in vascular smooth muscle cells, the endothelin-B (ETB)-receptor is also found in endothelial cells, fibroblasts, and neuronal cells. Activation of the endothelin system plays a driving role in several chronic cardiovascular diseases and several endothelin receptor antagonists (ERAs) (bosentan (6), ambrisentan (83) and macitentan (43)) have successfully been introduced as oral treatments for the life threatening condition of pulmonary arterial hypertension (PAH). This digest highlights the medicinal chemistry of the pyrimidine based ERAs 6 and 43 and describes the story that started with bosentan and culminated in macitentan (43). A condensed overview of the competitive landscape in the field of ERAs puts the different strategies and tactics applied by the medicinal chemists involved in this endeavor into perspective.

FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction.

FTY720 phosphate (FTY720-P; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, monodihydrogen phosphate ester) is a nonselective sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P2 receptor subtype. However, we have recently shown that FTY720-P displays significant S1P2 receptor agonist activity in recombinant cells and fibroblasts expressing endogenous S1P2 receptors. To elucidate the S1P2-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and small interfering RNA-based pathway inhibition in recombinant Chinese hamster ovary (CHO)-S1P2 cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P2 cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα12/13 knockdown and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα12/13/Rho/ROCK signaling. S1P similarly activated Gα12/13/Rho/ROCK signaling via S1P2 receptors, whereas the two selective S1P1 receptor agonists (Z,Z)-5-(3-chloro-4-[(2R)-2,3-dihydroxy-propoxy]-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (ponesimond) and 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871) were inactive. In lung myofibroblasts, which mainly expressed the S1P2 receptor subtype, we showed that FTY720-P selectively activated the Gα12/13/Rho/ROCK pathway via the S1P2 receptor. Moreover, the activation of the Gα12/13/Rho/ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or phenotypic assays in native expression systems can uncover activation of additional pathways, such as Gα12/13/Rho/ROCK signaling.

Discovery of novel bridged tetrahydronaphthalene derivatives as potent T/L-type calcium channel blockers.

Chemical evolution of mibefradil resulted in the identification of novel bridged tetrahydronaphthalene derivatives as potent T/L-type calcium channel blockers. A SAR study, in vitro and in vivo DMPK properties as well as the in vivo antihypertensive effect in rats are presented.

Substituted pyrrolidin-2-ones: Centrally acting orexin receptor antagonists promoting sleep. Part 2.

Starting from advanced pyrrolidin-2-one lead compounds, this novel series of small-molecule orexin receptor antagonists was further optimized by fine-tuning of the C-3 substitution at the γ-lactam ring. We discuss our design to align in vitro potency with metabolic stability and improved physicochemical/pharmacokinetic properties while avoiding P-glycoprotein-mediated efflux. These investigations led to the identification of the orally active 3-hydroxypyrrolidin-2-one 46, a potent and selective orexin-2 receptor antagonist, that achieved good brain exposure and promoted physiological sleep in rats.

Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P3 receptors and Smad-independent signaling.

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-ß1. In contrast to TGF-ß1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-ß1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-ß1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-ß1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways.

Discovery of substituted lactams as novel dual orexin receptor antagonists. Synthesis, preliminary structure-activity relationship studies and efforts towards improved metabolic stability and pharmacokinetic properties. Part 1.

Starting from a thiazolidin-4-one HTS hit, a novel series of substituted lactams was identified and developed as dual orexin receptor antagonists. In this Letter, we describe our initial efforts towards the improvement of potency and metabolic stability. These investigations delivered optimized lead compounds with CNS drug-like properties suitable for further optimization.

Pyrazole derivatives as selective orexin-2 receptor antagonists (2-SORA): synthesis, structure-activity-relationship, and sleep-promoting properties in rats.

Selective orexin 2 receptor antagonists (2-SORA) such as seltorexant (15) are in clinical development for the treatment of insomnia and other conditions such as depression. Herein, we report our structure-activity-relationship (SAR) optimization efforts starting from an HTS hit (1) (N-(1-((5-acetylfuran-2-yl)methyl)-1H-pyrazol-4-yl)-5-(m-tolyl)oxazole-4-carboxamide) that was derived from an unrelated in-house GPCR-agonist program. Medicinal chemistry efforts focused on the optimization of orexin 2 receptor (OX2R) antagonistic activity, stability in liver microsomes, time dependent CYP3A4 inhibition, and aqueous solubility. Compounds were assessed for their brain-penetrating potential in in vivo experiments to select the most promising compounds for our in vivo sleep model. Our lead optimization efforts led to the discovery of the potent, brain penetrating and orally active, 2-SORA (N-(1-(2-(5-methoxy-1H-pyrrolo[3,2-b]pyridin-3-yl)ethyl)-1H-pyrazol-4-yl)-5-(m-tolyl)oxazole-4-carboxamide) 43 with efficacy in a sleep model in rats comparable to 15.

Discovery of Nivasorexant (ACT-539313): The First Selective Orexin-1 Receptor Antagonist (SO1RA) Investigated in Clinical Trials.

The orexin system consists of two neuropeptides (orexins A and B) and two receptors (OX1 and OX2). Selective OX1 receptor antagonists (SO1RA) are gaining interest for their potential use in the treatment of CNS disorders, including substance abuse, eating, obsessive compulsive, or anxiety disorders. While blocking OX2 reduces wakefulness, the expected advantage of selectively antagonizing OX1 is the ability to achieve clinical efficacy without the promotion of sleep. Herein we report our discovery efforts starting from a dual orexin receptor antagonist and describe a serendipitous finding that triggered a medicinal chemistry program that culminated in the identification of the potent SO1RA ACT-539313. Efficacy in a rat model of schedule-induced polydipsia supported the decision to select the compound as a preclinical candidate. Nivasorexant (20) represents the first SO1RA to enter clinical development and completed a first proof of concept phase II clinical trial in binge eating disorder in 2022.

A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

Synthesis, structure-activity relationship studies, and identification of novel 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine derivatives as dual orexin receptor antagonists. Part 1.

A novel series of non-peptidic OX1R/OX2R orexin receptor antagonists was prepared by heterocyclic replacement of the dimethoxyphenyl moiety contained in the tetrahydroisoquinoline core skeleton of almorexant. Introduction of substituted imidazole moieties delivered potent dual orexin receptor antagonists with nanomolar potency for hOX1R and hOX2R suitable for further fine-tuning. The preparation of these novel orexin receptor antagonists and the outcome of preliminary structure-activity relationship studies are described in this communication.

Structure-activity relationship studies and sleep-promoting activity of novel 1-chloro-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine derivatives as dual orexin receptor antagonists. Part 2.

Replacement of the dimethoxyphenyl moiety in the core skeleton of almorexant by appropriately substituted imidazoles afforded novel 1-chloro-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine derivatives as potent dual orexin receptor antagonists. We describe in this Letter our efforts to further optimize the potency and brain penetration of these derivatives by fine-tuning of the pivotal phenethyl motif, and we comment on the sleep-promoting activity of selected compounds in a rat electroencephalographic (EEG) model.

Novel pyrazolo-tetrahydropyridines as potent orexin receptor antagonists.

A novel series of dual orexin receptor antagonists was prepared by heteroaromatic five-membered ring system replacement of the dimethoxyphenyl moiety contained in the tetrahydroisoquinoline core skeleton of almorexant. Thus, replacement of the dimethoxyphenyl by a substituted pyrazole and additional optimization of the substitution pattern of the phenethyl motif allowed the identification of potent antagonists with low nanomolar affinity for hOX(1)R and hOX(2)R. The synthesis and structure-activity relationship of these novel antagonists will be discussed in this communication. These investigations furnished several suitable candidates for further evaluation in in vivo studies in rats.

The Quest for the Best Dual Orexin Receptor Antagonist (Daridorexant) for the Treatment of Insomnia Disorders.

Since its discovery in 1998, the orexin system has been of interest to the research community as a potential therapeutic target for the treatment of sleep/wake disorders, stress and anxiety disorders, addiction or eating disorders. It consists of two G protein-coupled receptors, the orexin 1 and orexin 2 receptors, and two neuropeptides with agonistic effects, the orexin A and orexin B peptides. Herein we describe our efforts leading to the identification of a promising set of dual orexin receptor antagonists (DORAs) which subsequently went through physiology-based pharmacokinetic and pharmacodynamic modelling>[1] and finally led to the selection of daridorexant, currently in phase 3 clinical trials for the treatment of insomnia disorders.