Objective Aortic valve stenosis (AS) is characterized by fibrosis and calcification of valves leading to aortic valve (AV) narrowing, resulting in high wall shear stress (WSS) across the valves. We previously demonstrated that high shear stress can activate platelet-derived transforming growth factor-β1 (TGF-β1), a cytokine implicated inducing fibrosis and calcification. The aim of this study was to invest the role of shear-induced platelet release of TGF-β1 and its activation in AS. Approach and Results We studied hypercholesterolemic Ldlr−/−Apob100/100/Mttpfl/fl/Mx1Cre+/+ (Reversa) mice that develop AS on western diet (WD) and a surgical ascending aortic constriction (AAC) mouse model that acutely simulates the hemodynamics of AS to study shear-induced platelet TGF-β1 release and activation. Reversa mice on WD for 6 months had thickening of the AVs, increased WSS and increased plasma TGF-β1 levels. There were weak and moderate correlations between WSS and TGF-β1 levels in the progression and reversed Reversa groups, and a stronger correlation in the AAC model in WT mice, but not in mice with a targeted deletion of megakaryocyte and platelet TGF-β1 (Tgfb1flox). Plasma total TGF-β1 levels correlated with collagen deposition in the stenotic valves in Reversa mice. Although active TGF-β1 levels were too low to be measured directly, we found: 1. canonical TGF-β1 (p-Smad2/3) signaling in the leukocytes and canonical and non-canonical (p-Erk1/2) TGF-β1 signaling in AVs of Reversa mice on a WD, and 2. TGF-β1 signaling of both pathways in the AAC stenotic area in WT, but not Tgfb1flox mice. Conclusions Shear-induced, platelet-derived TGF-β1 activation may contribute to AS.
Objective Treatment of myocardial infarction (MI) within the first 1–2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbβ3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbβ3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly (IM) by autoinjector to facilitate its use in the pre-hospital setting. Here we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models. Approach and Results RUC-4 was ~20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60–80 mg/ml). It shared RUC-2’s specificity for αIIbβ3 vs αVβ3, did not prime the receptor to bind fibrinogen, or induce changes in β3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human, but not mouse platelets. IM injection of RUC-4 in non-human primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5–24 hours. Conclusions RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a pre-hospital therapy of MI, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.
Introduction: We are developing the novel αIIbβ3 antagonist, RUC-4, for subcutaneously (SC)-administered first-point-of-medical-contact treatment for ST Segment Elevated Myocardial Infarction (STEMI). Methods: We studied the: 1. pharmacokinetics (PK) of RUC-4 at 1.0, 1.93, and 3.86 mg/kg IV, IM, and SC in non-human primates (NHPs); 2. impact of aspirin on RUC-4 IC50 in human platelet-rich plasma (PRP); 3. effect of different anticoagulants on the RUC-4 IC50 in human PRP; and 4. relationship between αIIbβ3 receptor blockade by RUC-4 and inhibition of ADP-induced platelet aggregation. Results: 1. All doses of RUC-4 were well tolerated, but animals demonstrated variable temporary bruising. IM and SC RUC-4 reached dose-dependent peak levels within 5–15 min, with T½ s between 0.28 and 0.56 hrs. Platelet aggregation studies in NHPs receiving IM RUC-4 demonstrated >80% inhibition of the initial slope of ADP-induced aggregation with all 3 doses 30 minutes post-dosing, with subsequent dose-dependent loss of inhibition over 4–5 hours. 2. The RUC-4 IC50 for ADP-induced platelet aggregation was unaffected by aspirin treatment (40±9 nM vs. 37±5 nM; p=0.39). 3. The RUC-4 IC50 was significantly higher in PRP prepared from PPACK-anticoagulated blood compared to citrate-anticoagulated blood using either TRAP (122±17 vs. 66±25 nM; p=0.05; n=4) or ADP (102±22 vs. 54±13; p<0.001; n=5). 4. There was a close correspondence between receptor blockade and inhibition of ADP-induced platelet aggregation, with aggregation inhibition beginning with ~40% receptor blockade and becoming nearly complete at >80% receptor blockade. Discussion: Based on these results and others, RUC-4 has now progressed to formal preclinical toxicology studies.
Background Transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-β1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-β1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). Methods Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. Results Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-β1 levels (r=0.38, p=0.05 for PLAs and r=−0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=−0.42, p=0.03 for PLAs and r=−0.37, p=0.06 for single leukocytes). Conclusions p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-β1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-β1.
Aortic valve stenosis (AS) is a chronic disorder involving fibrosis and calcification of the aortic valve (AV), leading to a decrease in AV orifice area and an increase in wall shear stress (WSS) across the valves. We previously demonstrated that the pro-fibrotic cytokine TGF-β1, which is secreted as a latent complex, can be activated by shear both in vitro and in vivo. Thus, we hypothesize that shear-induced TGF-β1 activation contributes to the pathophysiology of AS. In this study, we used Ldlr -/- Apob 100/100 /Mttp fl / fl /Mx1Cre +/+ mice (“Reversa”, kindly provided by Dr. Heistad), which spontaneously develop AS after 6 months of a high fat diet, to test this hypothesis. Treating Reversa mice with a high fat diet for 6 months was associated with a significant decrease in AV cusp separation distance (median [IQR] from 0.85 [0.64-0.97] to 0.46 [0.37-0.65] mm) and significant increases in plasma cholesterol (mean±SD from 273±27 to 837±98 mg/dl), WSS across the AV (from 189 [152-235] to 308 [236-568] dyn/cm 2 ), and plasma total TGF-β1 levels (from 1.24 [1.08-0.38] to 1.66 [1.34-2.10] ng/ml) (all p<0.01). In contrast, no change in AV cusp separation distance, WSS, or plasma total TGF-β1 levels were observed in mice on a chow diet (n=14, all p>0.05). The increase in plasma total TGF-β1 levels in mice on a high fat diet correlated with the increase in WSS (r=0.32, p=0.04). Immunofluorescence staining showed increased Smad2/3 phosphorylation and α-smooth muscle actin (α-SMA) expression in the stenotic valves. We tested the effect of TGF-β1 (1ng/ml) on cultured porcine AV interstitial cells and found increased Smad2/3 phosphorylation and increased expression of α-SMA after 12 or 24 hours treatment. We conclude that AS progression is associated with both an increase in total plasma TGF-β1 levels and evidence of TGF-β1-induced cellular activation in this hypercholesterolemic murine model of spontaneous AS. These data are consistent with a model in which cellular release of TGF-β1 results in increased plasma TGF-β1, activation of latent TGF-β1 by the increased WSS across the AV, and TGF-β1 induces fibrosis and calcification of the AV. Since WSS increases with AS progression, a feed-forward mechanism may lead to accelerated AS progression.
Shear stress (SS) can activate platelets, leading to exposure of surface P-selectin and development of platelet-leukocyte aggregates (PLAs). Increased numbers of PLAs has been reported in the circulation of patients with aortic valve stenosis (AS), who have high SS across the stenotic valves, and aortic valve replacement decreases the SS and number of PLAs. Activated platelets release their granule contents, which includes transforming growth factor-β1 (TGF-β1). Our lab has reported that SS can activate latent TGF-β1 released from platelets both in vitro and in in vivo mouse models of AS. Since current assays are not sensitive enough to detect activated TGF-β1 in plasma, we searched for evidence of active TGF-β1 in a mouse model of AS by assessing the level of phosphorylated Smad2/3, a downstream mediator of the classical TGF-β1 signaling pathway, in both circulating leukocytes free of platelets and PLAs. We studied Ldlr-/-Apob100/100/Mttpfl/fl/Mx1Cre+/+ mice (Reversa), which spontaneously develop AS when fed a western diet (WD) (n=13), and compared them to the same mice fed a chow diet (n=13), who do not develop AS, and wild type (WT) mice (n=13). We identified leukocytes with an antibody to CD45 (clone 30-F11), platelets with an antibody to CD41 (clone MWReg30), PLAs by double staining, and p-Smad2/3 with an antibody to p-Smad2 (pS465/pS467)/Smad3 (pS423/pS425) (clone O72-670). Leukocytes and PLAs were analyzed by both a flow cytometer and an instrument that combines digital fluorescence microscopy with flow cytometry (ImageStream-X). Reversa mice on WD for 3 months developed AS with increased SS across the valves and increased plasma total TGF-β1 levels in circulation as we previously reported (Wang et al., ATVB, 2014). Reversa mice on WD also had a significantly increased percentage of PLAs compared to the other two groups (both p<0.05; Table), suggesting platelet activation under high SS. The intensity of p-Smad2/3 staining in leukocytes free of platelets of Reversa mice on a WD was greater than the intensity in the other two groups (both p<0.01). In all 3 groups, p-Smad2/3 in PLAs was higher than in leukocytes free of platelets (all p<0.001), but Reversa mice on a WD had the highest p-Smad2/3 levels in their PLAs (p<0.05 for both. Fluorescent microscopy showed that p-Smad2/3 was located primarily in the leukocytes of the PLAs (Figure 1). There was no correlation between p-Smad2/3 staining of PLAs and the number of platelets per leukocyte (p=0.78 for WT group, p=0.52 for Reversa chow diet, and p=0.15 for Reversa WD). In summary: (1) PLAs have more leukocyte p-Smad2/3 than leukocytes that are free of platelets, regardless of genotype/diet. (2) Leukocytes free of platelets in Reversa mice on a WD have more p-Smad2/3 than those in the other two groups. (3) Reversa mice on WD have both more PLAs and more leukocyte p-Smad2/3 in their PLAs. We conclude that the increased leukocyte p-Smad2/3 in Reversa mice on WD reflects increased TGF-β1 signaling through both: 1) direct platelet-leukocyte interactions and 2) release of TGF-β1 from platelets into plasma and its subsequent activation by high SS. TableGroupPLA (% of total leukocytes)p-Smad2/3 Mean Fluorescence Intensity (AU)Leukocytes free of plateletsPLAsWT16.9 ± 1.9173.9 ± 15.49204.7 ± 17.9 #Reversa (chow diet)16.4 ± 2.1172.3 ± 15.24200.8 ± 15.9 #Reversa (WD)26.1 ± 3.1*^237.5 ± 17.42*^268.9 ± 17.1 *^# * p<0.05 Reversa (WD) vs. WT; ^ p<0.05 Reversa (WD) vs. Reversa (chow diet); # p<0.001 PLA vs. leukocytes free of platelets. AU: arbitrary unit; WD: western diet Figure 1: Figure 1:. representative images of PLAs in each group from ImageStream-X. Disclosures No relevant conflicts of interest to declare.
We are developing the novel αIIbβ3 pure antagonist, RUC-4, for first point of contact treatment of ST Segment Elevated Myocardial Infarction (STEMI) in combination with aspirin. We have chosen the subcutaneous (SC) route to facilitate its delivery by ambulance and emergency room personnel, and potentially by self-administration. In preparation for initiating human studies, we have conducted preclinical studies of: 1. the pharmacokinetics (PK) and pharmacodynamics (PD) of RUC-4 succinate administered IV, IM, and SC to non-human primates (NHP); 2. the impact of in vitro aspirin on the RUC-4 IC50 in human platelet-rich plasma (PRP); and 3. the effect of different anticoagulants on the IC50 of RUC-4 in human PRP.Non-human primates (Macaca fascicularis; NHPs) received RUC-4 at 1.0, 1.96, and 3.86 mg/kg IV, IM, and SC. Blood samples for IV PK studies were collected at 0, 1, 5, 15, 30, 120, and 270 min; samples for IM and SC PK studies were collected at 0, 5, 15, 30, 45, 120, and 270 min and ~27 hrs, the latter only for animals that received 3.86 mg/kg IM. There were 2 animals per group and 2 groups per dose, so no more than 4-5 samples were collected from a single animal. Platelet aggregation was performed with 5 µM ADP on citrated PRP from the NHPs that received RUC-4 IM. All doses of RUC-4 administered IM and SC were well tolerated, but animals demonstrated variable temporary bruising. None of the animals developed thrombocytopenia despite receiving up to 4 doses of RUC-4 over 3 months. The PK data are shown in the Table; both IM and SC RUC-4 reached dose-dependent peak levels within 5-15 min, with T1/2 s between 0.28 and 0.56 hrs. The bioavailability of RUC-4 IM and SC ranged between 0.60-0.88 at doses of 1.93 and 3.86 mg/kg; there was greater variability in bioavailability at 1 mg/kg (1.2 IM and 0.55 SC). Platelet aggregation studies demonstrated >80% inhibition of the initial slope of aggregation in response to ADP in all samples in which the whole blood RUC-4 concentration was ≥ ~8 nM, with partial inhibition at lower concentrations.Since aspirin is the standard-of-care for initial treatment of STEMI, we plan to administer RUC-4 + aspirin. To assess aspirin-RUC-4 interactions, we compared the RUC-4 IC50 in untreated human citrated PRP to the RUC-4 IC50 in PRP pre-treated with 0.83 mM aspirin for 10 min at 22°C. This aspirin dose completely inhibited platelet aggregation to 1.5 mM arachidonic acid in each donor, indicating inhibition of thromboxane A2 production. In studies on 5 donors using 5 μM ADP as the agonist, the RUC-4 IC50 was 40 ± 9 nM in untreated PRP and 37 ± 5 nM in aspirin-treated PRP (p=0.50).Previous studies demonstrated that citrate anticoagulation can variably enhance the antiplatelet effect of some αIIbβ3 antagonists (Phillips et al., Circulation 96:1488, 1997: Kereiakes et al., J Thromb Thrombolysis 12:123, 2001). We therefore compared the IC50 of RUC-4 in human blood anticoagulated with citrate (0.38%), heparin (15.8 U ml), and PPACK (300 nM) when stimulated with a thrombin receptor activating peptide (T6; TRAP) at 20 µM. The resulting IC50 values were 66 ± 25, 111 ± 7, and 122 ± 17, respectively (n=4; p=0.05 for citrate vs PPACK and p=0.04 for heparin vs citrate). With 20 µM ADP activation, the IC50 values for blood anticoagulated with citrate and PPACK (100 µM) were 54 ± 13 and 102 ± 12 nM (p=0.001). In summary: 1. In accord with our studies with RUC-4 free base (Li et al., ATVB 34: 2321, 2014), IM RUC-4 succinate achieves peak blood levels in NHP within 5 min, resulting in >80% inhibition of platelet aggregation that persists for several hrs at a dose of 3.86 mg/kg. 2. SC RUC-4 achieves peak blood levels within 5-15 min; while the peak concentrations are lower than those with IM RUC-4 at the same doses, the blood levels are more sustained, with levels equivalent to the IM values producing ≥80% inhibition of initial slope persisting for several hrs. 3. Aspirin at a concentration that eliminates arachidonic acid-induced platelet aggregation does not affect the IC50 for RUC-4; 4. Assaying RUC-4 IC50 in human PRP from blood anticoagulated with citrate yields a value ~50% of that obtained with PRP prepared from blood anticoagulated with agents that do not affect divalent cation levels. Thus, the values in the presence of citrate may overestimate the in vivo antiplatelet effect. We conclude that the PK and PD of SC RUC-4 are favorable for short-term, high-potency inhibition of platelet aggregation as part of first point of care treatment of STEMI. Table Table. Disclosures Nedelman: Biomere: Employment, Equity Ownership. Coller:Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.
The bromodomain and extraterminal domain (BET) family of proteins play a vital role in gene transcription, making it an attractive therapeutic target for cancer. BET inhibitors can also combine with many anticancer agents to enhance activity. The bromodomains of the BET protein, BD1 and BD2, have unique functions and inhibiting either domain can result in differential responses. To date, non-selective BET inhibitors have failed during early clinical development due to significant on-target toxicity and limited benefit; however, selectively targeting specific bromodomains may result in a more favorable benefit/risk profile. NUV-868 is a novel and highly selective BD2 inhibitor of the BET protein family with ~1500-fold selectivity for BRD4-BD2 relative to BRD4-BD1. Herein, we describe NUV-868 and its activity in multiple in vitro and in vivo solid tumor models. Target engagement, selectivity of bromodomain inhibition, and regulation of BET-mediated gene expression were examined. Additionally, the antitumor activity of NUV-868 in combination with enzalutamide or olaparib was studied in tumor xenograft models of prostate, breast, and pancreatic cancer. NUV-868 demonstrated high selectivity for BD2 and regulated expression of several BET target genes. NUV-868 in combination with enzalutamide inhibited growth of several prostate cancer cell line- and patient-derived xenografts. NUV-868 in combination with olaparib inhibited tumor growth in models of breast, ovarian and pancreatic cancer. Our preclinical data demonstrate that NUV-868, a BD2-selective BET inhibitor, inhibits growth of tumor xenografts in combination with enzalutamide or olaparib and provides rationale for examination of these combinations in the clinic. An ongoing, phase 1 clinical study (NCT05252390) is evaluating NUV-868 as a monotherapy and in combination with olaparib or enzalutamide in patients with advanced solid tumors. Citation Format: Hitisha Patel, Jennifer Hertzog, Laura Heller, Spandana Vootukuri, Yan Zhang, Chris Miller, Gary Hattersley. NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6264.
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