Selective Antagonist Research Articles

Discovery of first-in-class highly selective TRPV1 antagonists with dual analgesic and hypoglycemic effects

Analgesia and blood sugar control are considered as two main unmet clinical needs for diabetes related neuropathic pain patients. Transient receptor potential vanilloid type-1 (TRPV1) channel is a highly validated target for pain perception, while no TRPV1 antagonists have been approved due to hyperthermia side effects. Herein, two series of new TRPV1 antagonists with flavonoid skeleton were designed by the structure-based drug design (SBDD) strategy. After comprehensive evaluation, compound CX-3 was identified as a promising TRPV1 antagonist. CX-3 exhibited equivalent TRPV1 antagonistic activity with classical TRPV1 antagonist BCTC in vitro, and exerted better analgesic activity in vivo than that of BCTC in the formalin induced inflammatory pain model without hyperthermia risk. Moreover, CX-3 exhibited robust glucose-lowering effects and showed high selectivity over other ion channels. Overall, these findings identified a first-in-class highly selective TRPV1 antagonist CX-3, which is a promising candidate to target the pathogenesis of diabetes related neuropathic pain.

Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

АНТАГОНИСТ РЕЦЕПТОРОВ СИГМА-1, СОЕДИНЕНИЕ BD-1063, ПОДАВЛЯЕТ Са2+-ОТВЕТЫ, ВЫЗЫВАЕМЫЕ МОЛИКСАНОМ В МАКРОФАГАХ

Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Acting as chaperones, sigma-1 receptors modulate a wide range of cellular processes in health and disease, including Ca2+ signaling processes. The pharmacological analogue of oxidized glutathione, drug molixan®, is used as an immunomodulator and cytoprotector in the complex therapy of bacterial, viral and oncological diseases; effective in the prevention and treatment of coronavirus infection COVID-19. To elucidate the involvement of sigma-1 receptors in the effect of molixan on the intracellular Ca2+ concentration in macrophages and in the regulation of Ca2+ signaling processes in macrophages in general, the effect of the sigma-1 receptor selective antagonist, compound BD-1063, on Ca2+ responses induced by molixan in rat peritoneal macrophages was investigated. Using Fura-2AM microfluorimetry we have shown for the first time that compound BD-1063 significantly suppresses both Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by molixan in peritoneal macrophages. The data obtained indicate the involvement of sigma-1 receptors in the complex signaling cascade triggered by molixan and leading to intracellular Ca2+ concentration increase in macrophages. The results also suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

NMDA Receptor Antagonists: Emerging Insights into Molecular Mechanisms and Clinical Applications in Neurological Disorders

Neurodegenerative disorders (NDs) include a range of chronic conditions characterized by progressive neuronal loss, leading to cognitive, motor, and behavioral impairments. Common examples include Alzheimer’s disease (AD) and Parkinson’s disease (PD). The global prevalence of NDs is on the rise, imposing significant economic and social burdens. Despite extensive research, the mechanisms underlying NDs remain incompletely understood, hampering the development of effective treatments. Excitotoxicity, particularly glutamate-mediated excitotoxicity, is a key pathological process implicated in NDs. Targeting the N-methyl-D-aspartate (NMDA) receptor, which plays a central role in excitotoxicity, holds therapeutic promise. However, challenges, such as blood–brain barrier penetration and adverse effects, such as extrapyramidal effects, have hindered the success of many NMDA receptor antagonists in clinical trials. This review explores the molecular mechanisms of NMDA receptor antagonists, emphasizing their structure, function, types, challenges, and future prospects in treating NDs. Despite extensive research on competitive and noncompetitive NMDA receptor antagonists, the quest for effective treatments still faces significant hurdles. This is partly because the same NMDA receptor that necessitates blockage under pathological conditions is also responsible for the normal physiological function of NMDA receptors. Allosteric modulation of NMDA receptors presents a potential alternative, with the GluN2B subunit emerging as a particularly attractive target due to its enrichment in presynaptic and extrasynaptic NMDA receptors, which are major contributors to excitotoxic-induced neuronal cell death. Despite their low side-effect profiles, selective GluN2B antagonists like ifenprodil and radiprodil have encountered obstacles such as poor bioavailability in clinical trials. Moreover, the selectivity of these antagonists is often relative, as they have been shown to bind to other GluN2 subunits, albeit minimally. Recent advancements in developing phenanthroic and naphthoic acid derivatives offer promise for enhanced GluN2B, GluN2A or GluN2C/GluN2D selectivity and improved pharmacodynamic properties. Additional challenges in NMDA receptor antagonist development include conflicting preclinical and clinical results, as well as the complexity of neurodegenerative disorders and poorly defined NMDA receptor subtypes. Although multifunctional agents targeting multiple degenerative processes are also being explored, clinical data are limited. Designing and developing selective GluN2B antagonists/modulators with polycyclic moieties and multitarget properties would be significant in addressing neurodegenerative disorders. However, advancements in understanding NMDA receptor structure and function, coupled with collaborative efforts in drug design, are imperative for realizing the therapeutic potential of these NMDA receptor antagonists/modulators.

Candida albicans accelerates atherosclerosis by activating intestinal hypoxia-inducible factor2α signaling

The gut microbiota is closely linked to atherosclerosis. However, the role of intestinal fungi, essential members of the complex microbial community, in atherosclerosis is poorly understood. Herein, we show that gut fungi dysbiosis is implicated in patients with dyslipidemia, characterized by higher levels of Candida albicans (C.albicans), which are positively correlated with plasma total cholesterol and low-density lipoprotein-cholesterol (LDL-C) levels. Furthermore, C.albicans colonization aggravates atherosclerosis progression in a mouse model of the disease. Through gain- and loss-of-function studies, we show that an intestinal hypoxia-inducible factor 2α (HIF-2α)-ceramide pathway mediates the effect of C.albicans. Mechanistically, formyl-methionine, a metabolite of C.albicans, activates intestinal HIF-2α signaling, which drives increased ceramide synthesis to accelerate atherosclerosis. Administration of the HIF-2α selective antagonist PT2385 alleviates atherosclerosis in mice by reducing ceramide levels. Our findings identify a role for intestinal fungi in atherosclerosis progression and highlight the intestinal HIF-2α-ceramide pathway as a target for atherosclerosis treatment.

How selective antagonists and genetic modification have helped characterise the expression and functions of vascular P2Y receptors.

Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y1 receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y2 receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y2 receptor knockdown reduced endothelial signalling and endothelial P2Y2 receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y2 receptor knockout were complex. No P2Y4 receptor antagonists are available and P2Y4 knockout did not affect the vascular actions of UTP and UDP. The P2Y6 receptor antagonist, MRS2578, identified endothelial P2Y6 receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y6 knockout abolished, contractions evoked by UDP. P2Y6 receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y11 receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y12/P2Y13 and P2Y14 receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.

Nonsteroidal mineralocorticoid receptor blockers as a new tool for managing cardiorenal risks in type 2 diabetes mellitus

Today, the importance of targeted cardionephroprotection is increasingly increasing as one of the vectors of a multifactorial therapeutic strategy to reduce the risk of development and progression of complications of type 2 diabetes. Pathological hyperactivation of the renin-angiotensin-aldosterone system (RAAS) and mineralocorticoid receptors (MCRs) is considered as one of the mechanisms for the development of cadiorenal syndrome (RCS) in diabetes. Blocking this pathophysiological pathway in patients with CKD and type 2 diabetes can break the vicious circle of mutually aggravating damage to the kidneys and heart. ACE inhibitors and angiotensin receptor blockers (ARBs) are currently the standard of care in patients with CRS due to diabetes. But despite their effectiveness, the residual risk of CKD progression within 4–5 years remains high in almost half of patients with type 2 diabetes, mainly due to multicomponent processes of MCR hyperactivation. This causes a range of pathological reactions affecting the entire body and may contribute to kidney, heart and CD disease in patients with type 2 diabetes by promoting inflammation and fibrosis. Functional and structural changes in the kidneys and heart develop, which leads to the development of metabolic disorders, arterial hypertension, cardiovascular complications and progressive CKD. Pharmacological blockade of aldosterone binding to MCR appears to be an effective additional line for preventing the progression of the pathological cascade of KRS reactions in type 2 diabetes. The recently developed selective non-steroidal MCR antagonist (nsAMPR) finerenone has convincingly demonstrated improved renal and cardiovascular outcomes in patients with CKD and type 2 diabetes. This review covers in detail the role of MCRs in the development of cardiorenal syndrome in type 2 diabetes and CKD, describes the mechanisms of effectiveness of MCR blockade in preventing the progression of cardiorenal syndrome in type 2 diabetes and the difference between non-steroidal MCRs and steroids, and presents the results of RCTs confirming the cardionephroprotective potential of nsAMCRs in CKD and diabetes. type 2, and the place of finerenone as a multifactorial therapeutic strategy for type 2 diabetes in clinical practice.

Discovery of the Clinical Candidate IDOR-1117-2520: A Potent and Selective Antagonist of CCR6 for Autoimmune Diseases.

Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.

Prazosin Potentiates Mast Cell-Stabilizing Property of Adrenaline

Background/Aims: Adrenaline quickly inhibits the release of histamine from mast cells. Besides β2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. Methods: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. Results: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a β2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Conclusion: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by β2-adrenergic receptors.

Discovery of a brain penetrant small molecule antagonist targeting LPA1 receptors to reduce neuroinflammation and promote remyelination in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood–brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.

Metabolism, Disposition, Excretion, and Potential Transporter Inhibition of 7-16, an Improving 5-HT2A Receptor Antagonist and Inverse Agonist for Parkinson's Disease.

Compound 7-16 was designed and synthesized in our previous study and was identified as a more potential selective 5-HT2A receptor antagonist and inverse agonist for treating Parkinson's disease psychosis (PDP). Then, the metabolism, disposition, and excretion properties of 7-16 and its potential inhibition on transporters were investigated in this study to highlight advancements in the understanding of its therapeutic mechanisms. The results indicate that a total of 10 metabolites of 7-16/[14C]7-16 were identified and determined in five species of liver microsomes and in rats using UPLC-Q Exactive high-resolution mass spectrometry combined with radioanalysis. Metabolites formed in human liver microsomes could be covered by animal species. 7-16 is mainly metabolized through mono-oxidation (M470-2) and N-demethylation (M440), and the CYP3A4 isozyme was responsible for both metabolic reactions. Based on the excretion data in bile and urine, the absorption rate of 7-16 was at least 74.7%. 7-16 had weak inhibition on P-glycoprotein and no effect on the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 transporters. The comprehensive pharmacokinetic properties indicate that 7-16 deserves further development as a new treatment drug for PDP.

17-β-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-β-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2′,7′-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, β-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE−/− mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.

Novel 5-HT7 receptor antagonists modulate intestinal immune responses and reduce severity of colitis.

Inflammatory bowel disease (IBD) encompasses several debilitating chronic gastrointestinal (GI) inflammatory disorders, including Crohn's disease and ulcerative colitis. In both conditions, mucosal inflammation is a key clinical presentation associated with altered serotonin (5-hydroxytryptamine or 5-HT) signaling. This altered 5-HT signaling is also found across various animal models of colitis. Of the 14 known receptor subtypes, 5-HT receptor type 7 (5-HT7) is one of the most recently discovered. We previously reported that blocking 5-HT signaling with either a selective 5-HT7 receptor antagonist (SB-269970) or genetic ablation alleviated intestinal inflammation in murine experimental models of colitis. Here, we developed novel antagonists, namely, MC-170073 and MC-230078, which target 5-HT7 receptors with high selectivity. We also investigated the in vivo efficacy of these antagonists in experimental colitis by using dextran sulfate sodium (DSS) and the transfer of CD4+CD45RBhigh T cells to induce intestinal inflammation. Inhibition of 5-HT7 receptor signaling with the antagonists, MC-170073 and MC-230078, ameliorated intestinal inflammation in both acute and chronic colitis models, which was accompanied by lower histopathological damage and diminished levels of proinflammatory cytokines compared with vehicle-treated controls. Together, the data reveal that the pharmacological inhibition of 5-HT7 receptors by these selective antagonists ameliorates the severity of colitis across various experimental models and may, in the future, serve as a potential treatment option for patients with IBD. In addition, these findings support that 5-HT7 is a viable therapeutic target for IBD.NEW & NOTEWORTHY This study demonstrates that the novel highly selective 5-HT7 receptor antagonists, MC-170073 and MC-230078, significantly alleviated the severity of colitis across models of experimental colitis. These findings suggest that inhibition of 5-HT7 receptor signaling by these new antagonists may serve as an alternative mode of treatment to diminish symptomology in those with inflammatory bowel disease.

Abstract PO2-20-03: VERITAC-3: A randomized phase 3 study, with a lead-in, of first-line vepdegestrant + palbociclib vs letrozole + palbociclib in estrogen receptor–positive/human epidermal growth factor receptor 2–negative advanced breast cancer

Abstract Background: Vepdegestrant (ARV-471) is a selective, orally administered PROteolysis TArgeting Chimera (PROTAC) estrogen receptor (ER) degrader. In the phase 2 portion (VERITAC) of a first-in-human phase 1/2 study (NCT04072952), vepdegestrant 200 mg once daily (QD) was well tolerated and had clinical activity in heavily pretreated patients with ER+/human epidermal growth factor receptor 2–negative (HER2-) advanced breast cancer and was selected as the recommended phase 3 dose (RP3D) for vepdegestrant monotherapy. A phase 1b cohort of the phase 1/2 study is evaluating the safety and clinical activity of vepdegestrant plus palbociclib in patients with ER+/HER2- breast cancer after prior endocrine-based therapy; prior cyclin-dependent kinase (CDK)4/6 inhibitor therapy was permitted. Preliminary results showed encouraging activity for the combination based on clinical benefit rate (CBR; rate of confirmed complete response, partial response, or stable disease ≥24 weeks); an increase in palbociclib exposure was observed relative to historical palbociclib pharmacokinetic data and was accompanied by a higher incidence of grade 3/4 neutropenia compared with prior palbociclib and endocrine therapy combination studies, which was managed by monitoring and standard palbociclib dose modifications. The global, randomized phase 3 VERITAC-3 study (NCT05909397) will compare the efficacy and safety of vepdegestrant plus palbociclib vs letrozole plus palbociclib as first-line treatment for patients with ER+/HER2- advanced breast cancer. An open-label study lead-in (SLI) will evaluate the safety and tolerability of vepdegestrant 200 mg QD plus 2 doses of palbociclib (100 mg QD and 75 mg QD) in patients with ER+/HER2- advanced breast cancer to select the RP3D of palbociclib for this combination. Trial Design: Approximately 50 and 1130 patients will be enrolled in the SLI and the phase 3 parts of the study, respectively. Eligible patients (aged ≥18 years) must have histologically or cytologically confirmed ER+/HER2- locoregionally recurrent or metastatic breast cancer, with no prior treatment in the advanced setting, and no prior treatment in any setting with CDK4/6 inhibitors, vepdegestrant, fulvestrant, elacestrant, or other investigational agents (including novel endocrine therapy, selective ER degraders, selective ER covalent antagonists, and complete ER antagonists). Patients with measurable disease per RECIST v1.1 or nonmeasurable bone-only disease are eligible. Patients with disease recurrence during or ≤12 months after completion of adjuvant endocrine therapy are excluded. For the SLI, patients are randomized 1:1 to receive vepdegestrant 200 mg QD in combination with palbociclib 100 mg QD or 75 mg QD and are treated in 28-day cycles with vepdegestrant given continuously and palbociclib given for 21 days followed by 7 days off treatment. The objective of the SLI is to identify the RP3D of palbociclib in combination with vepdegestrant, which will be determined through the primary outcome measures of incidence of grade 4 neutropenia, study drug dose reduction, and study drug discontinuation within the first 4 cycles of treatment. Secondary outcome measures include safety, antitumor activity (objective response rate, CBR, and duration of response), and plasma concentrations of study drugs. In the planned phase 3 portion of the trial, patients will be randomized to vepdegestrant plus palbociclib or letrozole plus palbociclib. The primary efficacy endpoint of the phase 3 portion is progression-free survival based on blinded independent central review. Enrollment began June 2023 and is ongoing. Citation Format: Seth Wander, Erika Hamilton, Mario Campone, Michael Danso, Sara Hurvitz, Hiroji Iwata, Colombe Chappey, Derek Yang, Julia Perkins Smith, Yuan Liu, Yuanyuan Zhang, Sibyl Anderson, Michelino de Laurentiis. VERITAC-3: A randomized phase 3 study, with a lead-in, of first-line vepdegestrant + palbociclib vs letrozole + palbociclib in estrogen receptor–positive/human epidermal growth factor receptor 2–negative advanced breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-20-03.

Abstract PO1-05-07: Circulating tumor DNA dynamics in acelERA Breast Cancer: a Phase II study of giredestrant for estrogen receptor-positive, HER2-negative, previously treated advanced breast cancer

Abstract BACKGROUND Giredestrant (GIR) is a highly potent, oral, selective estrogen receptor antagonist and degrader (SERD) that exhibits robust estrogen receptor (ER) occupancy. The Phase II, randomized, open-label acelERA Breast Cancer (BC) study (NCT04576455) assessed GIR vs physician’s choice of endocrine therapy (PCET) in second- or third-line ER-positive, HER2-negative advanced BC (ER+, HER2– aBC). The study did not reach statistical significance for the primary endpoint of investigator-assessed progression-free survival (PFS); however, the benefit of GIR was of larger magnitude among patients (pts) with ESR1-mutated tumors (ESR1m; a common cause of acquired resistance to endocrine therapy [ET]). We present an exploratory biomarker analysis of circulating tumor (ct)DNA dynamics. METHODS Pts (n = 303) were randomized 1:1 to GIR or PCET (75% of pts had fulvestrant [FUL]; 25%, an aromatase inhibitor). Partial response (PR), stable disease (SD), and progressive disease (PD) were categorized by RECIST v1.1. Plasma samples taken at Cycle 1, Day 1 (C1D1; n = 229), while on treatment (tx) at C2D1 (n = 220), and at the end of therapy (EOT; n = 155) were evaluated with the FoundationOne Liquid CDx next-generation sequencing assay. Gene mutations were defined as variants with known or likely impact on protein function. Composite tumor fraction (cTF) was defined as the total estimated tumor ctDNA content in each sample, and was only evaluable for a subset of samples (137 C1D1; 136 C2D1; 99 EOT). cTF or ESR1 mutant allele frequency (MAF) changes were calculated as a percent change from C1D1. ESR1m clonality was estimated as variant tumor fraction/estimated tumor fraction. Statistical significance was evaluated using the Mann–Whitney test. RESULTS The overall mutation landscape remained relatively unchanged with GIR tx at C2D1 or EOT, except for ESR1m prevalence, which declined from 43% at C1D1 to 26% on tx and 27% at EOT. With PCET, ESR1m prevalence decreased from 34% at C1D1 to 29% on tx, but increased to 43% by EOT. There were no concordant changes for other top mutated genes in this study, including PIK3CA, TP53, or DNMT3A. cTF decreased on tx in 38/65 (59%) and 32/59 (54%) of evaluable pts with GIR and PCET, respectively; the median change from C1D1 was –23% with GIR compared with –5% with PCET or –15% with FUL. The median change in cTF levels on tx with GIR was significantly greater in pts with a PR (–88%) vs PD (+6%, p = 0.002) or SD (–19%, p = 0.004). The degree of cTF decline on tx with GIR was significantly higher in pts with baseline ESR1m (median change –58%) vs no ESR1m detected (+5%, p = 0.012). Pts with a clonal ESR1m had higher levels of cTF decline on tx vs pts with subclonal ESR1m, suggesting cTF dynamics are a function of tumor heterogeneity. ESR1 MAF decreased on tx in 93% of pts with baseline ESR1m on GIR, vs 60% with PCET and 70% with FUL. ESR1 MAF decline on tx was significantly higher with GIR vs PCET at both C2D1 and EOT (p < 0.0001). The average ESR1 MAF decline on tx with GIR was greater in pts with a PR (–97%) vs PD (–54%, p = 0.025) or SD (–48%, p = 0.051), and all seven GIR-treated pts with a PR showed a near or total loss of ESR1 MAF by C2D1. The ESR1m variants D538G and Y537X had a significantly greater decline in MAF on tx with GIR vs PCET or FUL (p < 0.0001). CONCLUSIONS Data show that cTF and ESR1m ctDNA dynamics were associated with clinical response to GIR in ER+, HER2– aBC. ESR1 MAF decline was significantly greater with GIR vs PCET or FUL. ESR1 MAF declined to a greater degree with GIR compared with cTF, which is consistent with the larger magnitude of PFS benefit seen with GIR in pts with ESR1m tumors. The specific ESR1 variants D538G and Y537X showed greater sensitivity to GIR compared with PCET or FUL. Citation Format: Ann Collier, Aditya Bardia, Joo Hyuk Sohn, Elgene Lim, Marianna Chavez, Miguel Martín, Jorge Martinalbo, Pablo Perez-Moreno, Heather Moore. Circulating tumor DNA dynamics in acelERA Breast Cancer: a Phase II study of giredestrant for estrogen receptor-positive, HER2-negative, previously treated advanced breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-05-07.

Abstract PS17-07: Interim analysis of giredestrant + inavolisib in MORPHEUS Breast Cancer: a Phase Ib/II study of giredestrant treatment combinations in estrogen receptor-positive, HER2-negative, locally advanced/metastatic breast cancer

Abstract BACKGROUND Patients with estrogen receptor-positive metastatic breast cancer (ER+ mBC) almost always progress on first-line endocrine therapy (ET), which is usually combined with a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i). Finding effective ET combinations after progression following CDK4/6is remains a challenge. Giredestrant (GIR) is a highly potent, nonsteroidal, oral, selective ER antagonist and degrader. Inavolisib (INAVO) is a highly potent PI3Kα-selective inhibitor that promotes degradation of mutated p110α. After first-line treatment (tx) with a CDK4/6i, PIK3CA mutations (mut), which are common, persist and remain a sensitive oncogenic target in these patients. We present a 16-week interim analysis of GIR vs GIR + INAVO in MORPHEUS BC (NCT04802759). METHODS Patients whose tumors harbored a PIK3CAmut and who had disease progression on up to two lines of endocrine-based therapy (including at least one prior line of CDK4/6i) for locally advanced (LA)/mBC were randomized 1:6 to receive GIR (30 mg orally daily [PO QD]) or GIR + INAVO (9 mg PO QD) tx until disease progression or unacceptable toxicity. Given the small numbers, the GIR arm is presented for overall safety. Primary endpoints are safety and objective response rate (ORR). Other endpoints include progression-free survival (PFS), disease control rate (DCR), and pharmacokinetics. Genetic alterations were identified centrally in baseline circulating tumor DNA using the FoundationOne Liquid CDx assay. In cases where PIK3CAmut could not be determined centrally, local blood or tissue results are used. RESULTS As of April 18, 2023, seven and 15 patients were enrolled in the GIR and GIR + INAVO arms, respectively; 71% (n = 5) and 67% (n = 10) received one prior line of therapy in the LA/mBC setting; 14% (n = 1) and 33% (n = 5) received two prior lines; and one GIR-arm patient received four prior lines. Prior fulvestrant (FUL) was received by 53% of patients in the GIR + INAVO arm (n = 8). For GIR + INAVO, the ORR was 47% (n = 7); the complete response rate was 7% (n = 1); and the partial response (PR) rate was 40% (n = 6). The DCR at 12 weeks was 80% (12/15 patients). The median PFS was 10.3 months (95% confidence interval = 6.5, not evaluable) with 47% of patients (n = 7) having events. In the GIR + INAVO arm, 5/6 patients with an ESR1mut had a PR (83%) and one (17%) had stable disease. No clinically relevant drug–drug interaction was observed. Safety data are presented in the table. Two patients experienced grade 3 hyperglycemia, one of whom had baseline elevated HbA1c and has been on insulin therapy since the event. The other patient received a single day of insulin treatment. In the GIR + INAVO arm, the incidence of rash and stomatitis (all grade 1) was 13% each. CONCLUSIONS An encouraging efficacy signal was observed with GIR + INAVO when compared cross-trial with BYLieve arm A (PMID: 33794206). ORRs were 47% in MORPHEUS BC vs 21% in BYLieve for patients with measurable disease (although no prior FUL was allowed in BYLieve). Safety of GIR + INAVO was aligned to the individual safety profiles of each of the drugs, with no new safety signals identified and a favorable tolerability profile for a PI3Kα inhibitor-based combination. Table. Safety summary Data are % of patients. AE, adverse event; GIR, giredestrant; INAVO, inavolisib; TRAE, treatment-related adverse event; tx, treatment. Citation Format: Hope Rugo, Maria Gion, Cristina Hernando, Kyung Hae Jung, Mafalda Oliveira, Melinda Telli, Gregory Vidal, Sina Vatandoust, Jing Zhu, Richard Schwab, Huy Ngo, Erika Ferreira, Ann Collier, Vanessa Breton, Einav Gal-Yam. Interim analysis of giredestrant + inavolisib in MORPHEUS Breast Cancer: a Phase Ib/II study of giredestrant treatment combinations in estrogen receptor-positive, HER2-negative, locally advanced/metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-07.

Abstract PO4-04-11: Exposure–response analyses of GO39932: a Phase Ia/b study of giredestrant in estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer

Abstract BACKGROUND Giredestrant (GIR) is a highly potent, nonsteroidal, oral, selective estrogen receptor antagonist and degrader (SERD). The Phase Ia/b GO39932 study (NCT03332797) investigated GIR ± palbociclib (palbo) and ± a luteinizing hormone-releasing hormone agonist for patients with ER-positive, HER2-negative, locally advanced or metastatic breast cancer who had disease progression on prior endocrine therapies. In the single-agent dose-escalation stage, patients received 10, 30, 90, or 250 mg of once-daily (QD) GIR. In the dose-expansion stage, patients received 30, 100, or 250 mg of QD GIR. Safety and tolerability of 100 mg GIR + 125 mg palbo was also explored in the dose-escalation and -expansion stages. Results showed that GIR was well tolerated and potentially clinically active as a single agent and in combination with palbo, including in patients with ESR1-mutated tumors. To inform the selection of the clinical dose for GIR late-stage development based on risk–benefit considerations, exposure–response (E–R) analyses for efficacy and safety endpoints were conducted using single-agent GIR data. METHODS Exposure metrics included maximum concentration at steady state and area under the concentration–time curve at steady state. The association between the exposure metrics and efficacy endpoints (objective response rate [ORR] and clinical benefit rate [CBR]) or safety endpoints (all adverse events [AEs], Grade ≥ 3 AEs, selected AEs of interest [sAEIs], including all-grade hepatotoxicity, and bradycardia) were evaluated. RESULTS Data cutoff was September 17, 2021. A total of 111 patients were enrolled in the single-agent GIR cohort. Of these 111 patients, 107 were pharmacokinetic-evaluable and included in the analysis. ORR in patients with measurable disease at baseline was 20% (16/81 patients) and CBR was 49% (54/111). No significant associations were observed between exposure and ORR or CBR, including in patients with ESR1-mutated tumors and those with no mutation detected (p > 0.05). There were 92/107 patients (86%) with AEs of any grade, overall. Seventy-two (67%) had sAEIs, 19 (18%) experienced hepatotoxicity (all events were reported as liver function test abnormalities), 11 (10%) had bradycardia, and 20 (19%) had Grade ≥ 3 AEs. Increasing exposure did not significantly increase the incidence of all AEs, Grade ≥ 3 AEs, sAEIs, or bradycardia at the dose range of 10 to 250 mg (p > 0.05). For hepatotoxicity, the analysis suggested that higher exposure may lead to a higher probability of events (p = 0.0065); however, at the clinically relevant exposure range for the 30 mg dose, the incidence of hepatotoxicity was low and the predicted exposures were at the shallower slope of the relationship. CONCLUSIONS Single-agent GIR did not exhibit a significant association between exposure and efficacy endpoints at a dose range of 10 to 250 mg, regardless of ESR1 mutation status, indicating that the efficacy of GIR might have reached a plateau at low dose levels. At the clinically relevant exposure range of the 30 mg dose, GIR exposure was not associated with an appreciable increase in the incidence of AEs. The Phase Ia/b study design and subsequent E–R results enabled dose selection to be based on risk–benefit considerations, rather than the traditional maximum tolerated dose paradigm, in line with the United States Food and Drug Administration’s oncology dose optimization recommendations. The E–R results also indicate that GIR may have a relevant wide therapeutic window. Overall, these data support the 30 mg clinical dose of GIR that was selected, with a favorable risk–benefit profile, for further clinical studies. Citation Format: Komal Jhaveri, Yi Ting (Kayla) Lien, Nicholas Turner, Elgene Lim, Maureen Cannon, Richard Anziano, Jurgen Langenhorst, Kenta Yoshida, Vikram Malhi, Mary Gates, Jennifer Eng-Wong, Chunze Li, Mona Shah, Pablo Perez-Moreno, Jiajie Yu, Aditya Bardia. Exposure–response analyses of GO39932: a Phase Ia/b study of giredestrant in estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-11.

Abstract PS17-01: Key drivers of therapeutic response and resistance to giredestrant from GO39932: a Phase Ia/b study in patients with estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer

Abstract BACKGROUND Several investigational oral selective estrogen receptor antagonists and degraders (SERDs) are under investigation. These were partly developed to benefit patients (pts) with breast cancer (BC) who acquired resistance to standard-of-care (SoC) endocrine therapies (ET); e.g., through gain of ESR1 mutations that enable estrogen-independent ER activity. One such SERD, giredestrant (G), demonstrates activity in pts with ESR1-wild type (WT) or -mutant (m) tumors, and in pts who progressed on other ETs. Results from Phase I/III trials in ER-positive, HER2-negative, locally advanced/metastatic BC (ER+, HER2– LA/mBC) showed that next-generation SERDs had increased activity in ESR1m tumors vs SoC ETs. However, enthusiasm was hindered by heterogeneous responses, driven in part by study design differences. Across acelERA BC (NCT04576455), EMERALD (NCT03778931), SERENA-2 (NCT04214288), and AMEERA-3 (NCT04059484), 30–50% of pts progressed rapidly (< 2 months) when treated with SoC ETs or next-generation SERDs, while others benefited for ≥ 1 year. We present an exploratory biomarker analysis aimed at understanding the mechanistic basis for heterogeneous responses to SERDs in ER+, HER2– LA/mBC, by assessing a Phase Ia/b G cohort (NCT03332797) with paired baseline and on-treatment (tx) biopsies. METHODS Pts had ≤ 2 prior therapies for LA/mBC; disease recurrence/progression while on adjuvant ET for ≥ 24 months and/or ET for ER+, HER2– LA/mBC; and tumor response/stable disease for ≥ 6 months. Single-agent dose-escalation stage: 10, 30, 90, or 250 mg G once daily (QD) on Days (D) 1–28 of 28-D cycles (C). Dose-expansion stage: 30, 100, or 250 mg G QD. 100 mg G + 125 mg palbociclib on a 21-D on/7-D off schedule was also explored. Pre-/perimenopausal pts received LHRH agonists. Paired pre- and on-tx (C2D8) tumor biopsies (n = 29) were immunolabeled for ER, progesterone receptor, and Ki67, and assessed via bulk RNA-sequencing. Pre-tx liquid biopsies (n = 85) were evaluated by FoundationOne Liquid CDx assay. To validate our clinical observations, we generated a SERD-resistant MCF7 cell line and tested its sensitivity to other therapies. RESULTS We compared fast-progressing pts (FP; progression-free survival [PFS] < 2 months) with those experiencing long-term benefit (LTB; PFS > 12 months). LTB was significantly associated with high tumor baseline ER pathway activity. Although the benefit of G was of larger magnitude among pts with ESR1m tumors compared with SoC ET in acelERA BC, here pts with predominantly ESR1WT tumors received LTB on G tx. At the molecular level, G acted on LTB tumors by suppressing cell cycle- and ER-associated genes. In contrast, G had no effect on the transcriptome of FP tumors, which had lower baseline ER activity than LTB tumors. FP tumors were instead enriched for multiple cancer-associated pathways, e.g., RAS/MAPK and PI3K, which may drive resistance to G and thus enable fast progression. By liquid biopsy, most FP pts (> 90%) did not harbor a mutation in the associated pathway (e.g., NF1, KRAS), and thus could not be identified by genomic profiling alone. FP tumors were instead enriched for multiple cancer-associated pathways by gene expression. We explored these mechanisms further in the SERD-resistant cell line, which was similarly enriched for EGFR and MAPK activation vs standard MCF7 cells. Although cells became resistant to ER-targeted drugs, they acquired sensitivity to EGFR/MAPK inhibitors e.g., gefitinib, cobimetinib. CONCLUSIONS We identified molecular features associated with LTB to G and revealed a set of oncogenic pathways associated with FP pts on tx; demonstrating that these pathways represent acquired dependencies and potential therapeutic targets for SERD-resistant tumors and FP pts. Citation Format: Jackson Liang, Christy Ong, Jennifer Giltnane, Junko Aimi, Ching-Wei Chang, Mary Gates, Jennifer Eng-Wong, Pablo Perez-Moreno, Komal Jhaveri, Elgene Lim, Nicholas Turner, Heather Moore. Key drivers of therapeutic response and resistance to giredestrant from GO39932: a Phase Ia/b study in patients with estrogen receptor-positive, HER2-negative, locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-01.

Terpenes from Cannabis sativa induce antinociception in a mouse model of chronic neuropathic pain via activation of adenosine A 2A receptors.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa . A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, β-pinene, α-humulene, and β-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the potential antinociception and mechanism of these Cannabis terpenes in a mouse model of chronic pain. We first tested for antinociception by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with mouse models of chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal antinociception to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs either alone. We then used the adenosine A 2A receptor (A 2A R) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A 2A R to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A 2A R agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain and identify a receptor mechanism for this activity.