Multiple Agonists Research Articles

Abstract 13330: Cardiorespiratory Fitness is Associated With Decreased Platelet Reactivity

Introduction: Platelets are key mediators in atherosclerotic cardiovascular disease (CVD). Low cardiorespiratory fitness (CRF) is a major risk factor for atherosclerosis. However, little is known about the effects of CRF on platelet function. Methods: Platelet assays and cardiopulmonary exercise testing were conducted in the Framingham Heart Study (n= 3001). A linear mixed effects model was used to analyze associations between CRF (assessed by peak oxygen uptake [VO 2 ] in participants achieving a respiratory exchange ratio >= 1), and platelet reactivity traits from multiple platelet function assays: whole blood Multiplate impedance aggregometry (MP), Total-Thrombus Formation Analysis System (T-TAS), and light transmission aggregometry (LTA). Statistical models adjusted for relatedness, age, sex, and anti-platelet medications. Bonferroni correction for multiple statistical testing resulted in a significance threshold of p<7.35E-04. Results: Of 68 platelet traits analyzed, 31 traits passed the significance threshold, all with a direction of effect indicating higher CRF associated with decreased platelet reactivity. Significant traits spanned multiple platelet agonists. Our strongest associations were seen with MP after TRAP-6 (velocity, p=1.57E-26; AUC, p=2.33E-21; aggregation, p=5.30E-16) and ADP stimulation (velocity, p=1.63E-19; AUC, p=7.64E-17; aggregation, p=1.00E-11), and LTA after ristocetin stimulation (max aggregation, p=5.33E-11; AUC, p=1.11E-10). Conclusion: Our research adds to past work that shows CRF to be associated with reduced CVD by suggesting that decreased platelet reactivity may be involved mechanistically. We found significant associations with multiple platelet agonists, indicating higher CRF globally inhibits platelets; however, given the multiple strong associations after TRAP-6 and ADP stimulation, PAR-1 and purinergic signaling, respectively, may be most heavily involved. This is notable since each of these receptor pathways is directly or indirectly tied to anti-coagulant or anti-platelet therapies. Ultimately, our results suggest that CRF may be important for additional consideration in tailoring anti-platelet strategies.

STING Agonists in Head and Neck Squamous Cell Carcinoma.

Despite the development of new treatment paradigms and improved biologic understanding of head and neck squamous cell carcinoma (HNSCC), therapeutic resistance remains a substantial problem, and novel treatment approaches are needed. Stimulator of interferon genes (STING) is a critical regulator of the antitumor response through regulation of both immune-dependent and tumor-intrinsic mechanisms. As such, the STING pathway has emerged as a rational pharmacologic target leading to the development of multiple STING agonists. These compounds have impressive preclinical efficacy as single agents and with PD-1 (programmed death-1) axis agents. However, clinical evaluation in this context has yet to show substantial efficacy. In contrast to monotherapy approaches, activation of STING in combination with DNA-damaging agents has been shown to enhance the effect of these agents in preclinical models and represents a promising approach to improve outcomes in patients with HNSCC. In this review, we will discuss the preclinical and clinical data supporting the use of STING agonists and highlight potential avenues of exploration to unlock the potential of these agents in HNSCC.

Rational Design of T-Cell- and B-Cell-Based Therapeutic Cancer Vaccines.

Cancer vaccines provide an efficient strategy to enhance tumor-specific immune responses by redeploying immune systems. Despite the approval of the first cancer vaccine (Sipuleucel-T) by the U.S. Food and Drug Administration in 2010, most therapeutic cancer vaccines fail in clinical trials. Basically, tumor-specific immune responses rely on not only T-cell but also B-cell immunity, which indicates that cancer vaccines should leverage both arms of the adaptive immune system. For example, CD8+ T cells activated by antigen-presenting cells (APCs) recognize and directly kill tumor cells via peptide-bound major histocompatibility complex (pMHC). B cells recognize antigen with no need of pMHC and require CD4+ T cells for sufficient activation and antibody generation, enabling antibody-mediated nondirect killing on tumor cells. Considering the different mechanisms of T-cell and B-cell activation, the rational design of therapeutic cancer vaccines should consider several factors, including antigen selection and recognition, immune activation, vaccine delivery, and repeatable vaccination, which can be advanced by chemical strategies.In this Account, we summarize our recent contributions to the development of effective T-cell- and B-cell-based therapeutic cancer vaccines. For T-cell-based vaccines, we focus on adjuvants as the key component for controllable APC activation and T-cell priming. Not only synthetic molecular agonists of pattern recognition receptors (PRRs) but also adjuvant nanomaterials were explored to satisfy diversiform vaccine designs. For example, a type of natural cyclic dinucleotide (CDN) that was chemically modified with fluorination and ipsilateral phosphorothioation to activate the stimulator of interferon gene (STING) was found to mediate antitumor responses. It retains structural similarity to the parent CDN scaffold but possesses increased stability, cellular uptake, and immune activation for antitumor treatment. It also facilitates facile conjugation with other agonists, which not only enhances APC-targeting delivery but also balances cellular and humoral antitumor responses. We also explored the intrinsic properties of nanomaterials that allow them to serve as adjuvants. A black phosphorus nanosheet-based nanovaccine was constructed and found to strongly potentiate antigen-specific T-cell antitumor immune responses through multiple immune-potentiating properties, leading to a highly integrated nanomaterial-based adjuvant design. For B-cell-based vaccines, multicomponent and multivalent strategies were applied to improve the immunogenicity. A multicomponent linear vaccine conjugate coordinates helper T (Th) cells and APCs to proliferate and differentiates B cells for enhanced antitumor immunoglobulin G antibody responses. To further improve antigen recognition, clustered designs on a multivalent epitope were applied by generating various structures, including branched lysine-based peptides, natural multivalent scaffold molecules, and self-assembled nanofibers. We also engineered nano- and microvaccine systems to optimize systemic and localized vaccination. A multilayer-assembled nanovaccine successfully integrated antigens and multiple agonists to modulate APC activation. A DNA hydrogel contributed to the control of APC's immune behaviors, including cell recruitment, activation, and migration, and induced robust antitumor responses as an all-in-one designable platform. In this Account, by summarizing strategies for both T-cell- and B-cell-based vaccine design, we not only compare the differences but also address the intrinsic uniformity between such vaccine designs and further discuss the potential of a combined T-cell- and B-cell-based vaccine, which highlights the applicability and feasibility of chemical strategies.

Profiling Differential Effects of 5 Selective Serotonin Reuptake Inhibitors on TLRs-Dependent and -Independent IL-6 Production in Immune Cells Identifies Fluoxetine as Preferred Anti-Inflammatory Drug Candidate

Excessive proinflammatory cytokine production induced by abnormal activation of Toll-like receptor (TLR) signaling, for example, by SARS-CoV-2 infection, can cause a fatal cytokine storm. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and fluvoxamine, used to treat depression, were recently reported to reduce the risk of severe disease in patients with coronavirus disease 2019 (COVID-19), but the mechanisms of the anti-inflammatory effects of SSRIs, and which SSRI would be most suitable as an anti-inflammatory drug, remain unclear. Here, we examined the inhibitory effects of 5 FDA-approved SSRIs, paroxetine, fluoxetine, fluvoxamine, sertraline and escitalopram, on the production of interleukin-6 (IL-6) induced by stimulation with multiple TLR agonists in murine macrophages and dendritic cells, and on the production of cytokines induced by concanavalin A in murine lymphocytes. In J774.1 murine macrophage cells, pretreatment with SSRIs significantly suppressed IL-6 release induced by TLR3 agonist poly(I:C), TLR4 agonist LPS or TLR9 agonist CpG ODN, but did not affect IL-6 release induced by TLR7 agonists imiquimod or resiquimod. In accordance with the results obtained in J774.1 cells, pretreatment with SSRIs also suppressed IL-6 release induced by a TLR3, TLR4 or TLR9 agonist in bone marrow-derived dendritic cells and peritoneal cells of C57BL/6 mice. On the other hand, interestingly, sertraline alone among the SSRIs amplified IL-6 production induced by TLR7 agonists in murine dendritic cells, though not in macrophages. Concanavalin A-induced production of IL-6 or IL-2 in murine lymphocytes was suppressed by SSRIs, suggesting that SSRIs also inhibit TLRs-independent IL-6 production. Since SSRIs suppressed both IL-6 production induced by multiple TLR agonists in macrophages or dendritic cells and TLR-independent IL-6 production in lymphocytes, they are promising candidates for treatment of patients with cytokine storm, which is mediated by overactivation of multiple TLRs in a complex manner, leading to the so-called IL-6 amplifier, an IL-6 overproduction loop. However, the 5 SSRIs examined here all showed different effects. Overall, our results suggest that fluoxetine may be the most promising candidate as an anti-inflammatory drug. An examination of the structural requirements indicated that the N-methyl group of fluoxetine has a critical role in the inhibition of IL-6 production.

Abstract 1391: PBMC humanized mouse model for ImmunoOncology drug evaluation

Abstract Since the first immune checkpoint blocker Ipilimumab was approved by US FDA in 2011, more and more immune therapeutic drugs popped up. Humanized PBMC reconstitution in immune deficient mice is becoming a good model for evaluating therapeutic antibodies, especially the BsAb with one arm of anti-CD3 that mediates immune cells and another arm targeting a tumor antigen. However, limited window of dosing regimen triggered by graft-versus-host-disease (GvHD) after 30-40 days of reconstitution, and insufficient tumor-infiltrated immune cells naturally from reconstituted circulatory system hindered the widely application of the model in immunotherapy in development of multiple immune checkpoint inhibitors or immune agonists. To overcome this, LIDE has developed a unique human PBMC-cancer cells co-inoculated model instead. In our platform, cancer-priming PBMCs were mixed with the fresh cancer cells in MatriGel, co-inoculated into NCG mice to form a huPBMC well-infiltrated tumor tissue for immunotherapy. Our platform has been successfully helped evaluate biological function of PD1, Tigit, PVRIG, CD73, CD47, CD38, CD40, GITR Antibodies, Tgfb1/PDL1 BsAb, DLL3/CD3 BsAb in multiple cancers, such as melanoma, breast cancer and lung cancer. Citation Format: Bin Xie, Hongkui Chen, Xin Hou, Danyi Wen. PBMC humanized mouse model for ImmunoOncology drug evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1391.

Hormones that are involved in metabolic homeostasis: Overview of the past century and future perspectives

The year 2021 marks the 100th anniversary of the discovery of insulin. Although insulin industry has reached ∼30 billion US dollars per year, it is not an ultimate cure for diabetes. This is mainly due to the development and progression of insulin resistance in patients with Type 2 diabetes (T2D). Dozens of other hormones have been discovered that are directly or indirectly involved in metabolic homeostasis. Those discoveries have added to our knowledge on the complexity of energy or metabolic homeostasis and advanced our view that hormones may not necessarily be produced in classical endocrine organs. In addition to insulin, important metabolic hormones include incretins (GLP-1 and GIP) produced in the gut, leptin in adipose tissues, and FGF21 in the liver. Among them, FGF21 has been recognized as a target for dietary polyphenol interventions. Here we have summarized the development of GLP-1 receptor agonists as therapeutic agents for T2D and recent clinical trials with engineered FGF21 analogous. We have also presented our view on the existence of GLP-1-FGF21 axis in improving insulin tolerance and discussed recent literature on the development of dual agonists as future therapeutic agents for T2D and other metabolic disorders. • Insulin resistance is the major obstacle in treating diabetes. • In addition to insulin, GLP-1, Leptin, and FGF21 are also key metabolic hormones. • GLP-1 and FGF21 analogues can attenuate insulin resistance. • FGF21 is among the key mediators of dietary polyphenol intervention. • Dual agonists and multiple agonists are potential future therapeutics.

0307 Is Poor Sleep Associated with Use of Multiple Benzodiazepine Receptor Agonists in Older Veterans?

Abstract Introduction Benzodiazepine receptor agonists (BZAs) are often prescribed for insomnia in older adults. Polypharmacy increases risk of adverse events in this population in general, particularly when medications within the same class are prescribed. During an ongoing hypnotic deprescribing trial, we explored use of more than one BZA (multi-BZA use) and self-reported sleep quality among older adults. Methods Telephone surveys were performed for recruitment to an ongoing BZA deprescribing trial. Participants aged ≥ 55 years who were prescribed at least one BZA (i.e., alprazolam, clonazepam, lorazepam, temazepam, or zolpidem) from a Department of Veterans Affairs pharmacy in Southern California were asked about their use of each BZA over the past 3 months for sleep (yes/no). Multi-BZA use was defined as using > 1 different BZA over the past 3 months. Self-reported sleep items included duration of sleep problems (<3, 3-12, or >12 months), sleep quality (very or fairly good, fairly or very bad), and sleep efficiency (mean total sleep time over time in bed). Analyses compared sleep variables between multi-BZA and non-multi-BZA users (Fisher’s Exact or t-tests). Results Among participants (N=359), 152 (42.3%) reported using zolpidem, 41 (11.4%) lorazepam, 39 (10.9%) alprazolam, 31 (8.6%) clonazepam, and 29 (8.1%) temazepam during the past 3 months. 35 (9.8%) participants reported taking more than one of these drugs. 93.9% reported their sleep problems were present for ≥3 months. 68.3% of participants reported their sleep was fairly/very bad, and mean sleep efficiency was 67.9 (SD 18.5). There were no significant differences between multi-BZA versus non-multi-BZA users in duration of sleep complaints (Fisher’s Exact=1.0; p=.842), sleep quality (Fisher’s Exact=0.70; p=0.56) or sleep efficiency (p=0.91). Conclusion We found 1 in 10 older adults prescribed a BZA for sleep reported multi-BZA use over the past 3 months. Multi-BZA use was unrelated to duration of sleep complaints, sleep quality or sleep efficiency. Whether the use was simultaneous or staggered, these findings are concerning, given the elimination half-life of most of the BZAs and that polypharmacy, especially within medication class, may increase risk of adverse events (e.g., falls). Further research is needed to explore factors contributing to multi-BZA use. Support (If Any) NIA R01AG057929, VA IIR 17-234

Eribulin Enhances Interferon Production by STING Agonists in Models of Triple‐Negative Breast Cancer

Triple‐negative breast cancer (TNBC) is a highly heterogenous disease that lacks effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment of this disease. Strategies to improve the response of TNBC patients to immune checkpoint inhibitors include the upregulation of the cGAS‐STING innate immune sensing pathway. Multiple STING agonists are in clinical development and have shown promise for the treatment of TNBC as single agents and combined with checkpoint inhibitors. We recently demonstrated that eribulin, a microtubule destabilizer used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm (Mol Pharmacol 100: 309‐318, 2021). We further hypothesized that eribulin could enhance the expression of type I interferons in combination with direct STING agonists. Indeed, our results show that the microtubule destabilizer eribulin significantly enhances interferon expression induced by STING agonists with diverse mechanisms of action in human monocytes and TNBC cells. This phenotype is shared with other microtubule destabilizers, including vinorelbine, but not with the microtubule stabilizing agent paclitaxel, which is also used in the treatment of TNBC. The eribulin‐mediated enhancement of interferon expression induced by multiple pharmacological STING agonists, as well as HT‐DNA and cGAMP ligands, occurs within 6 hours and is mechanistically distinct from the previously reported eribulin‐mediated release of mitochondrial DNA into the cytoplasm. This response is dependent on TBK1 and associated with TBK1 phosphorylation but was not inhibited by the STING antagonist H‐151 or by inhibition of the JNK or JAK1/2 signaling pathways in TNBC cells. Ongoing in vivo studies are determining the effects of eribulin on the immunological response to TNBC tumors alone and in combination with pharmacological STING agonists and whether this improves antitumor efficacy. These studies will determine if eribulin, a chemotherapeutic agent already used in the treatment of TNBC, can increase the immunogenicity of TNBC tumors and inform on combination treatment of eribulin with STING agonists and potentially other immunotherapies.

Long‐term Administration of Resveratrol and MitoQ Stimulates Antioxidant Gene Expression in a Mice Castration Model of Erectile Dysfunction

Erectile Dysfunction (ED) is the most common sexual dysfunction in men and is characterized by the inability to achieve or maintain an erection satisfactory for sexual intercourse. Normal erectile function is highly dependent on testosterone so any dysfunction in testosterone production may accelerate ED pathogenesis. Hypogonadism is a direct cause of ED and is characterized by the diminishing functionality of the testes in producing testosterone. Other pathological conditions such as obesity and diabetes, share similar complications of ED due to decreased testosterone levels. Furthermore, these conditions exhibit increased oxidative stress. Decreased testosterone can weaken antioxidant defenses and increase oxidative stress that then leads to endothelial dysfunction by reducing nitric oxide bioavailability and increasing fibrosis which ultimately leads to ED. Current therapies for ED do not directly target antioxidant defenses to reduce oxidative stress damage. Two prospective drugs, Resveratrol and Mitoquinone (MitoQ), have antioxidant properties that limit oxidative stress and may improve erectile function. They have shown to be successful in decreasing oxidative stress damage and improving endothelial function in cardiovascular disease models, so the objective of this study was to explore the effect resveratrol and MitoQ has on antioxidant defense gene expression and erectile function in a model of hypogonadism. We hypothesized that resveratrol and MitoQ treatment will improve antioxidant defenses and improve erectile function. In this study, we used a surgically castrated mouse model to induce ED. We treated mice with resveratrol or MitoQ for eight weeks. Following treatment, we sacrificed and harvested mice corpus cavernosum (CC). We measured the expression of genes related to antioxidant defenses by performing qRT‐PCR. Additionally, ex vivo vasoreactivity of the internal pudendal artery (IPA) and CC were assessed in response to concentration ranges of multiple agonists with DMT myograph system. One‐way ANOVA and Two‐way ANOVA, respectively, was used to compare differences between groups. Upon castration, vasorelaxation of the IPA and CC significantly declined. Antioxidant defenses were reduced with castration as seen by reduced expression in: Gclc, Gpx1, Prdx3, Prdx5, Nqo1, SOD2, SOD3, and Hmox1. After treatment, vasorelaxation did not improve however there were improvements in antioxidant gene expression. Resveratrol significantly increased expression of Gstm, CAT, SOD1. Additionally, MitoQ and resveratrol trended to increase several antioxidant genes. There was a partially restoration of Gpx1, Prdx3, Nqo1 and SOD2 levels and Hmox1 was restored back to sham levels. To conclude, neither long‐term administration of MitoQ nor resveratrol improved relaxation responses of the IPA or CC, however, they were able to stimulate and improve antioxidant gene expression. Even though these drugs were unable to improve erectile function on their own, they may be paired with other treatments such as PDE‐5 inhibitors, to make them more effective in more severe case of ED. Future research should explore co‐treatment in a castrated model of ED.

TLR2 agonistic lipopeptide enriched PLGA nanoparticles as combinatorial drug delivery vehicle

The use of multiple Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been proved to be beneficial against various diseases. Polymeric nanoparticles (Nps) provide wide range of options to incorporate multiple TLR agonists while ensuring optimal delivery and non-toxicity. To synergistically engage the cross-talk of TLR2 and TLR7, we synthesized pathogen mimicking Poly(lactic-co-glycolic acid) (PLGA) Nps encapsulating T7 (TLR7 agonist) and T2 lipopeptide (TLR2 agonist). The single emulsification-solvent evaporation process was employed for the synthesis where both hydrophobic T7 and TLR2 agonists were incorporated in a single step. Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetric (TGA) and Differential Scanning Calorimetry (DSC) analysis of pure PLGA and both agonists (T2 and T7) were compared with that of synthesized PLGA Nps to better understand the chemical bonding and stability. The physicochemical assessment was performed starting with particle size analysis and was confirmed using Transmission Electron Microscopy (TEM). During in-vitro release studies, about 40% of T7 was released for the first 5 hr followed by a two-day period of sustained release. Kinetics modelling results showed that the release kinetics was unaffected by the addition of T2 lipopeptide into PLGA Nps. These findings confirmed successful co-delivery of both agonists in same cargo that can be utilise to generate a balance immune response against various infectious diseases such as malaria, leishmania, hepatitis, influenza. The higher drug loading and the capability of PLGA suspension to get lyophilised paved the way for long-term storage, effective encapsulation, and co-delivery in vaccination.

Dopaminergic signaling regulates zebrafish larvae's response to electricity.

Electrical stimulation of brain or muscle activities has gained attention for studying the molecular and cellular mechanisms involved in electric-induced responses. We recently showed zebrafish's response to electricity. Here, we hypothesized that this response is affected by the dopaminergic signaling pathways. The effects of multiple dopamine agonists and antagonists on the electric response of 6 days-postfertilization zebrafish larvae were investigated using a microfluidic device with enhanced control of experimentation and throughput. All dopamine antagonists decreased locomotor activities, while dopamine agonists did not induce similar behaviors. The D2-selective dopamine agonist quinpirole enhanced the movement. Exposure to nonselective and D1-selective dopamine agonists apomorphine and SKF-81297 caused no significant change in the electric response. Exposing larvae that were pretreated with nonselective and D2-selective dopamine antagonists butaclamol and haloperidol to apomorphine and quinpirole, respectively, restored the electric locomotion. These results reveal a correlation between electric response and dopamine signaling pathway. Furthermore, they demonstrate that electric-induced zebrafish larvae locomotion can be conditioned by modulating dopamine receptor functions. Our electrofluidic assay has profound application potential for fundamental electric-induced response research and brain disorder studies especially those related to the dopamine imbalance and as a chemical screening method when investigating biological pathways and behaviors.

XJ‐8, a natural compound isolated from Sanguis draxonis, inhibits platelet function and thrombosis by targeting MAP3K3

BackgroundVascular injury initiates rapid platelet activation, which is critical for haemostasis, while it also causes fatal thrombotic diseases, such as myocardial infarction or ischemic stroke. ObjectivesTo study the inhibitory effects and underlying mechanisms of XJ‐8, a natural compound isolated from Sanguis draxonis, on platelet activation and thrombosis. MethodsThe regulatory effects of XJ‐8 on the dense granule release, thromboxane A2 (TxA2) synthesis, α‐granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by multiple agonists were investigated in in vitro experiments. The effects of XJ‐8 on bleeding time and FeCl3‐induced carotid artery thrombosis were also evaluated in in vivo experiments. Furthermore, we investigated the underlying mechanisms by which XJ‐8 exerted its pharmacological effects. ResultsXJ‐8 not only significantly inhibited the dense granule release, TxA2 synthesis, and aggregation of platelets induced by multiple agonists, but also exerted extending effects on bleeding time and therapeutic effects on thrombotic disease. In addition, XJ‐8 selectively and moderately inhibited the activity of mitogen‐activated protein kinase kinase kinase 3 (MAP3K3) and the activation of signalling pathways downstream MAP3K3, which play important roles in platelet activation. ConclusionXJ‐8 can inhibit platelet function and thrombosis by targeting MAP3K3 and has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.

Serotonin-Affecting Antidepressant Use in Relation to Platelet Reactivity.

Depression is an independent risk factor of cardiovascular disease morbidity. Serotonin is a key neurotransmitter in depressive pathology, contained within platelets, and is a weak activator of platelets. Our study assessed the link between platelet reactivity traits, depression, and antidepressant (AD) use in a large population sample. Our study was conducted in the Framingham Heart Study (n = 3,140), and AD use (n = 563) and aspirin use (n = 681) were noted. Depression was measured using the Center for Epidemiological Studies‐Depression (CES‐D) survey. Platelet reactivity traits were measured across multiple agonists using five distinct assays. We utilized a linear mixed effects model to test associations between platelet traits and depression, adjusting for age, sex, aspirin use, and AD use. Similarly, we analyzed trait associations with any AD use, serotonin‐affecting ADs, and norepinephrine‐affecting ADs, respectively. There were strong associations with reduced platelet function and AD use, particularly with serotonin‐affecting medications. This included lower Optimul epinephrine maximal aggregation (P = 4.87E‐13), higher U46619 half maximal effective concentration (P = 9.09E‐11), lower light transmission aggregometry (LTA) adenosine diphosphate (ADP) final aggregation (P = 1.03E‐05), and higher LTA ADP disaggregation (P = 2.28E‐05). We found similar associations with serotonin‐affecting ADs in an aspirin‐taking subset of our sample. There were no significant associations between platelet traits and depression. In the largest study yet of AD use and platelet function we show that antidepressants, particularly serotonin‐affecting ADs, inhibit platelets. We did not find evidence that depressive symptomatology in the absence of medication is associated with altered platelet function. Our results are consistent with AD use leading to platelet serotonin depletions, decreased stability of platelet aggregates, and overall decreased aggregation to multiple agonists, which may be a mechanism by which ADs increase risk of bleeding and decrease risk of thrombosis.

Rasagiline does not exacerbate autonomic blood pressure dysregulation in early or mild Parkinson’s disease

IntroductionOrthostatic hypotension (OH) and abnormal blood pressure (BP) fluctuations occur mainly due to noradrenergic dysfunction and are clinically important in patients with Parkinson’s disease (PD). They lead to impairments of cognition function, daily activities, and quality of life. Some monoamine oxidase (MAO)-B inhibitors have a sympathomimetic amine, which can be attributed to OH. Therefore, we determined whether rasagiline, a common MAO-B inhibitor used in PD treatment, can contribute to cardiovascular autonomic BP dysregulation in patients with early or mild PD. MethodsNineteen patients with early or mild PD were recruited, and tilt test and 24-h ambulatory BP monitoring (ABPM) were performed before and after rasagiline administration. Early or mild PD was defined as patients with de novo (n = 4), levodopa (n = 10), dopamine agonist (n = 1), levodopa and one dopamine agonist (n = 2), levodopa and droxidopa (n = 1), and levodopa and istradefylline (n = 1). Furthermore, patients with motor fluctuation and multiple dopamine agonists were excluded from our study. ResultsOH and BP frequency were not significantly exacerbated before or after rasagiline administration. No significant differences of type in BP fluctuation on ABPM and the degree of nocturnal BP falls were found before and after rasagiline administration. The Unified Parkinson’s Disease Rating Scale motor score in patients (post-rasagiline administration) was significantly improved compared with before. ConclusionRasagiline seems to be a suitable medication for Parkinsonian symptoms in patients with early and mild PD. It does not exacerbate cardiovascular autonomic responses, circadian rhythm of BP, or both.

Combined delivery of TLR2 and TLR7 agonists by Nanostructured lipid carriers induces potent vaccine adjuvant activity in mice

Toll-like receptor (TLR) agonists are promising adjuvants and the combination of TLR agonists enhance immune responses by providing synergistic immune activity via triggering different signalling pathways. However, systematic cytotoxicity due to the immediate release of such immune potentiators from the site of injection hampers its clinical performance. Nanostructured lipid carriers (NLCs) offer a possibility to incorporate multiple TLR agonists with high encapsulation efficiency and slow drug release. Herein, we synthesized NLCs from didodecyldimethylammonium bromide (D12DAB) and oleic acid and used these to co-encapsulate a Pam2CS derivative (T-2, TLR2 agonist) with an imidazoquinoline derivative (T-7, TLR7 agonist) as a combination vaccine adjuvant. Hydrodynamic diameter and zeta potential of the prepared NLCs were found to be in the range of 200–500 nm and 23–27 mV, respectively. Spherical shape and size of prepared NLCs were also assessed through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) analysis. In-vitro release studies of T-7 demonstrated sustained release and the addition of lipopeptide T-2 augmented encapsulation efficiency (from 84 to 92.9%) with a slight trigger in the release percentage. All NLC formulations were screened in TLR2/1, TLR2/6, TLR7 and TLR8 reporter cell lines and loaded NLC formulation showed high TLR2 and TLR7 agonistic activity. Adjuvant potency was evaluated through intramuscular immunization of female C57BL/6 mice with recombinant hepatitis B surface antigen and influenza hemagglutinin protein. T-2 and T-7 loaded NLCs induced good protective efficacy in mice challenged with a lethal dose of influenza virus.

Abstract 11580: Platelet Activity and Cardiovascular Events in Patients with Diabetes and Peripheral Artery Disease: Findings from the American Heart Association Cardiometabolic Health Strategically Focused Research Network

Introduction: Peripheral artery disease (PAD) and diabetes mellitus (DM) are significant risk factors for atherothrombosis. Platelets mediate atherothrombosis and are therapeutic targets in PAD and DM. We investigated the relationships between PAD, DM, platelet activity, and cardiovascular risk. Methods: The Platelet Activity and Cardiovascular Events (PACE) study enrolled 289 PAD patients undergoing lower extremity revascularization (LER). Prior to LER, platelet activity was measured via light transmission aggregometry in response to submaximal ADP, collagen (coll), serotonin (ser), epinephrine (epi), and arachidonic acid (AA). Patients were followed for a median of 18 months for the composite of major adverse cardiac or limb events (MACLE; MI, stroke, death, major amputation, and limb revascularization). Results: There were 93 (32%) patients with insulin-dependent DM (IDDM), 60 (21%) with non-insulin dependent DM (NIDDM), and 136 (47%) without-DM. Patients with IDDM were more likely than those without DM to be female, non-white, and Hispanic, and to have history of coronary artery disease, heart failure, and critical limb ischemia (P<0.05 for all). Use of antiplatelet therapy was similar across groups. Maximum platelet aggregation was highest in the IDDM group in response to multiple agonists ( Figure) . MACLE occurred in 51%, 57%, and 69% of the without-DM, NIDDM, and IDDM groups, respectively (P=0.02). Compared to subjects without DM, only those with IDDM were at increased risk of MACLE after multivariable adjustment (aHR 1.6 [95% CI 1.02-2.4]; P=0.04) ( Figure ). After further adjustment for platelet aggregation, the association between IDDM and MACLE was no longer statistically significant (1.4 [0.9-2.3]; P=0.17); platelet aggregation mediated 24% of the excess MACLE risk in individuals with IDDM. Conclusions: In patients with PAD, IDDM is associated with increased platelet activity that mediates, in part, the risk for incident cardiac and limb events.

Development of Potent and Selective Agonists for Complement C5a Receptor 1 with In Vivo Activity.

The anaphylatoxin C5a is a complement peptide associated with immune-related disorders. C5a binds with equal potency to two GPCRs, C5aR1 and C5aR2. Multiple C5a peptide agonists have been developed to interrogate the C5a receptor function but none show selectivity for C5aR1. To address these limitations, we developed potent and stable peptide C5aR1 agonists that display no C5aR2 activity and over 1000-fold selectivity for C5aR1 over C3aR. This includes BM213, which induces C5aR1-mediated calcium mobilization and pERK1/2 signaling but not β-arrestin recruitment, and BM221, which exhibits no signaling bias. Both ligands are functionally similar to C5a in human macrophage cytokine release assays and in a murine in vivo neutrophil mobilization assay. BM213 showed antitumor activity in a mouse model of mammary carcinoma. We anticipate that these C5aR1-selective agonists will be useful research tools to investigate C5aR1 function.

Iron Repletion Increases Platelet Aggregation and Reduces Platelet Count in Iron Deficient Premenopausal Women

Background: Iron deficiency, heavy menstrual bleeding, and anemia are interrelated conditions with high prevalence in women's health. Together, they impact an estimated 310 million premenopausal women worldwide. Iron deficiency has been associated with development of anemia as well as thrombocytosis. However, the effects of iron deficiency on platelet function remain unknown.Objective: We set out to investigate the impact of IV iron repletion on platelet function, platelet count, and other blood cell indices in iron deficient premenopausal women.Methods: We conducted a prospective single-center study of iron deficient premenopausal women who underwent iron repletion with a single dose of 1000 mg of low molecular weight iron. Pre-infusion and post-infusion blood samples were obtained for laboratory analysis. Standard of care monitoring of iron indices and complete blood counts were also included in the analysis. Pre-infusion rates of anemia and thrombocytosis were calculated using minimum hemoglobin and peak platelet count in the 6 months preceding iron infusion. Platelet function was quantified by Fluorescence-Activated Cell Sorting (FACS) quantification of platelet activation marker antibodies for GPIIb/IIIa (PAC1) and P-selectin (CD62P) after exposure to multiple platelet agonists. Platelet aggregation was assessed by flow of anticoagulated whole blood at a shear rate of 300 s -1 for 5 minutes through chambers coated with type I collagen, imaged with differential interference contrast optics, and quantitatively represented through computation of percent total surface coverage. Pre-infusion and post-infusion cell indices and aggregation data were compared by paired samples t-test. All statistical analyses were performed in GraphPad Prism (Version 8.0.0).Results: Thirteen patients were included in the analysis. Heavy menstrual bleeding was the suspected cause of iron deficiency in 83% of patients. Thrombocytosis was present in 15% of patients at pre-infusion and no patients at post-infusion. Average ferritin was 14 µg/L at pre-infusion and 126 µg/L at post-infusion. The peak platelet count within 6 months pre-infusion was 309 K/mm 3 (±89) vs. 274 (±64) K/mm 3 at post-infusion (p<0.05). The average change to 6-month pre-infusion peak and post-infusion platelet count was -35.2 K/mm 3 (95%CI: -66.2, -5.23). The mean 6-month minimum hemoglobin was 11.9 (±1.9) g/dL vs. 13.3 (±1.1) g/dL at post-infusion (p<0.005). Acquisition of FACS platelet reactivity data are ongoing, with preliminary results for the first 7 consecutively enrolled study patients displayed in Figure 1. Platelet aggregation measured at pre-infusion showed 14% surface coverage, with a significant increase to 29% at post-infusion (p < 0.05). None of the 13 women experienced a thrombotic event during the study period.Conclusion: Correction of iron deficiency results in decreased platelet count and improves platelet aggregation over collagen. Iron repletion may also improve platelet reactivity in response to physiologic agonists. These findings may suggest that iron deficiency impairs hemostasis and that correction of low iron may be even more critical for women with heavy menstrual bleeding. [Display omitted] DisclosuresShatzel: Aronora Inc,: Consultancy.

\u03b22-adrenergic receptor regulates ER-mitochondria contacts

Interactions between the endoplasmic reticulum (ER) and mitochondria (Mito) are crucial for many cellular functions, and their interaction levels change dynamically depending on the cellular environment. Little is known about how the interactions between these organelles are regulated within the cell. Here we screened a compound library to identify chemical modulators for ER-Mito contacts in HEK293T cells. Multiple agonists of G-protein coupled receptors (GPCRs), beta-adrenergic receptors (β-ARs) in particular, scored in this screen. Analyses in multiple orthogonal assays validated that β2-AR activation promotes physical and functional interactions between the two organelles. Furthermore, we have elucidated potential downstream effectors mediating β2-AR-induced ER-Mito contacts. Together our study identifies β2-AR signaling as an important regulatory pathway for ER-Mito coupling and highlights the role of these contacts in responding to physiological demands or stresses.

The incretin/glucagon system as a target for pharmacotherapy of obesity.

SummaryObesity is a chronic, multifactorial, relapsing disease. Despite multicomponent lifestyle interventions, including pharmacotherapy, maintaining bodyweight loss is challenging for many people. The pathophysiology of obesity is complex, and currently approved pharmacotherapies only target a few of the many pathways involved; thus, single‐targeting agents have limited efficacy. Proglucagon‐derived peptides, glucagon, and the incretin hormones glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP), represent attractive targets for managing obesity and metabolic disorders because they may have direct roles in multiple mechanisms including satiety, energy homeostasis, and lipolytic activity. Unimolecular dual and triple agonists targeting glucagon and incretin hormone receptors have been shown to promote bodyweight loss, lower glucose levels, and reduce food intake in animal models of obesity. Multiple dual receptor agonists are in clinical development for the treatment of obesity, including GLP‐1/GIP and GLP‐1/glucagon receptor agonists. The extent to which glucagon contributes to treatment effects remains to be understood, but it may promote bodyweight loss by reducing food intake, while concomitant GLP‐1 receptor agonism ensures normal glucose control. Further research is required to fully understand the molecular mechanisms of action and metabolic effects of both dual and triple receptor agonists.