R2R01: A long-acting single-chain peptide agonist of RXFP1 for renal and cardiovascular diseases.

BACKGROUND: The therapeutic potential of relaxin for heart failure and renal disease in clinical trials is hampered by the short half-life of serelaxin. Optimization of fatty acid-acetylated single-chain peptide analogues of relaxin culminated in the design and synthesis of R2R01, a potent and selective RXFP1 agonist with subcutaneous bioavailability and extended half-life. EXPERIMENTAL APPROACH: Cellular assays and pharmacological models of RXFP1 activation were used to validate the potency and selectivity of R2R01. Increased renal blood flow was used as a translational marker of R2R01 activity. Human mastocytes (LAD2 cells) were used to study potential pseudo-allergic reactions and CD4+ T-cells to study immunogenicity. The pharmacokinetics of R2R01 were characterized in rats and minipigs. KEY RESULTS: In vitro, R2R01 had comparable potency and efficacy to relaxin as an agonist for human RXFP1. In vivo, subcutaneous administration of R2R01 increased heart rate and renal blood flow in normotensive and hypertensive rat and did not show evidence of tachyphylaxis. R2R01 also increased nipple length in rats, used as a chronic model of RXFP1 engagement. Pharmacokinetic studies showed that R2R01 has a significantly extended terminal half-life. The in vitro assays with LAD2 cells and CD4+ T-cells showed that R2R01 had low potential for pseudo-allergic and immunogenic reactions, respectively. CONCLUSION AND IMPLICATIONS: R2R01 is a potent RXFP1 agonist with an extended half-life that increases renal blood flow in various settings including normotensive and hypertensive conditions. The preclinical efficacy and safety data supported clinical development of R2R01 as a potential new therapy for renal and cardiovascular diseases.

TEAD Inhibitors Sensitize KRASG12C Inhibitors via Dual Cell Cycle Arrest in KRASG12C-Mutant NSCLC.

KRASG12C is one of the most common mutations detected in non-small cell lung cancer (NSCLC) patients, and it is a marker of poor prognosis. The first FDA-approved KRASG12C inhibitors, sotorasib and adagrasib, have been an enormous breakthrough for patients with KRASG12C mutant NSCLC; however, resistance to therapy is emerging. The transcriptional coactivators YAP1/TAZ and the family of transcription factors TEAD1-4 are the downstream effectors of the Hippo pathway and regulate essential cellular processes such as cell proliferation and cell survival. YAP1/TAZ-TEAD activity has further been implicated as a mechanism of resistance to targeted therapies. Here, we investigate the effect of combining TEAD inhibitors with KRASG12C inhibitors in KRASG12C mutant NSCLC tumor models. We show that TEAD inhibitors, while being inactive as single agents in KRASG12C-driven NSCLC cells, enhance KRASG12C inhibitor-mediated anti-tumor efficacy in vitro and in vivo. Mechanistically, the dual inhibition of KRASG12C and TEAD results in the downregulation of MYC and E2F signatures and in the alteration of the G2/M checkpoint, converging in an increase in G1 and a decrease in G2/M cell cycle phases. Our data suggest that the co-inhibition of KRASG12C and TEAD leads to a specific dual cell cycle arrest in KRASG12C NSCLC cells.

Characterization of a new potent and long-lasting single chain peptide agonist of RXFP1 in cells and in vivo translational models.

Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.

Potent Lys Patch-Containing Stapled Peptides Targeting PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), identified as a regulator of low-density lipoprotein receptor (LDLR), plays a major role in cardiovascular diseases (CVD). Recently, Pep2-8, a small peptide with discrete three-dimensional structure, was found to inhibit the PCSK9/LDLR interaction. In this paper, we describe the modification of this peptide using stapled peptide and SIP technologies. Their combination yielded potent compounds such as 18 that potently inhibited the binding of PCSK9 to LDLR (KD = 6 ± 1 nM) and restored in vitro LDL uptake by HepG2 cells in the presence of PCSK9 (EC50 = 175 ± 40 nM). The three-dimensional structures of key peptides were extensively studied by circular dichroism and nuclear magnetic resonance, and molecular dynamics simulations allowed us to compare their binding mode to tentatively rationalize structure-activity relationships (SAR).

Tumor imaging using P866, a high-relaxivity gadolinium chelate designed for folate receptor targeting.

The objective of this study was to evaluate the potential of a high-relaxivity macromolecular gadolinium (Gd) chelate to target folate receptors (FRs). P866 is a dimeric high-relaxivity Gd chelate coupled to a folate moiety. Binding affinity, in vivo biodistribution studies in KB tumor-bearing mice at 1, 4, and 24 h, and dynamic contrast-enhanced (DCE)-MRI (2.35 T) over 4 h were assessed. Binding and internalization of P866 through the FR was demonstrated. Due to the high molecular volume of P866, the binding affinity compared to free FA was decreased (K(D) = 59.3 +/- 1.8 nM and 5.9 +/- 0.2 nM, respectively). Tumor/muscle (T/M) uptake was 5.4 +/- 1.0, 4 h after injection of 15 micromol/kg. Competition with free FA was less effective when the dose was increased due to a saturation of FR. At a dose of 5 micromol/kg, a 70% difference in signal enhancement was observed between P866 and the nonspecific reference compound, thus demonstrating the specificity of FR targeting. While this high-relaxivity folate-Gd chelate has demonstrated its potential capacity to target in vivo FR on tumors, the sensitivity is probably limited to a certain extent by the saturation of the FR and by the decrease in the apparent relaxivity of the internalized part of P866 in the tumor cells.

Allergy-like reactions to iodinated contrast agents. A critical analysis.

Allergy-like reactions may occur following administration of iodinated contrast media (CM), mostly in at-risk patients (patients with history of previous reaction, history of allergy, co-treated with interleukin-2 or beta-blockers, etc.) but remain generally unpredictable. Severe and fatal reactions are very rare events. All categories of CM may induce such reactions, although first generation (high osmolar CM) have been found to induce a higher rate of adverse events than low osmolar CM. However, no differences were found between the two categories of CM with respect to mortality. Delayed reactions can also occur. There are no differences between the various categories of CM except for non-ionic dimers, which are more likely to induce such effect. Numerous clinical studies have evaluated the prophylactic value of drugs (mostly antihistamines and corticosteroids). Results are unclear and highly variable. Any prevention depends upon the mechanism involved. However, the mechanism of CM-induced allergy-like reaction remains disputed. Relatively recent data revived the hypothesis of a type-I hypersensitivity mechanism. Positive skin tests to CM have been reported. However, the affinity of IgE towards CM has been found to be very low in the only study which actually evaluated it. Other pathophysiological mechanisms (involving direct secretory effects on mast cells or basophils, or activation of the complement system associated or not with the plasma contact system) are also much debated. Anaphylaxis and anaphylactoid reactions are, in the end, clinically undistinguishable.

Macrophage endocytosis of superparamagnetic iron oxide nanoparticles: mechanisms and comparison of ferumoxides and ferumoxtran-10.

RATIONALE AND OBJECTIVES: Superparamagnetic iron oxides (SPIO) used as magnetic resonance (MR) contrast agents undergo specific uptake by macrophages. The purpose of this study was first to determine the mechanism of macrophage uptake for Ferumoxides by using competition experiments with specific ligands of scavenger receptors SR-A (I/II) and second, to evaluate and compare the internalization of 2 different contrast agents, Ferumoxides (SPIO) and Ferumoxtran-10 (USPIO: ultrasmall superparamagnetic iron oxide) using macrophages obtained by chemical activation of human monocytic cells. METHODS: Ferumoxides and Ferumoxtran-10 are 2 MR contrast agents, composed of dextran-coated iron oxide nanoparticles. The endocytosis pathway of Ferumoxides was studied using competition experiments on mouse peritoneal macrophages in the presence of specific ligands of scavenger receptors SR-A (types I and II): polyinosinic acid and fucoidan. In vitro assays using THP-1 (human promonocyte) cells activated into macrophages were performed in the presence of the 2 superparamagnetic nanoparticles. The cellular uptake was determined by measuring the iron content using ICP-AES (inductively coupled plasma-atomic emission spectrometry) and by Prussian blue staining. RESULTS: In the presence of polyinosinic acid or fucoidan, the endocytosis of Ferumoxides by mouse peritoneal macrophages was inhibited. This inhibition was obtained using 10 microg/mL of scavenger receptor ligands at a concentration of 62.5 microg Fe/mL of SPIO, and a dose-dependent relationship was observed. Without competitors, the percentage of uptake of Ferumoxides by mouse peritoneal macrophages ranged between 3 and 8%. On the human activated monocyte THP-1 cell assay, Ferumoxides underwent a higher macrophage uptake (between 1.1 and 3%) compared with Ferumoxtran-10 (between 0.03 and 0.12%). This difference is attributed to the larger size of Ferumoxides nanoparticles. CONCLUSIONS: Competition experiments indicate that the cellular uptake of Ferumoxides involves scavenger receptor SR-A-mediated endocytosis. The comparison between Ferumoxides and Ferumoxtran-10 confirms that macrophage uptake of iron oxide nanoparticles depends mainly on the size of these contrast agents.