Publications by authors named "Philip Janiak"

30 Publications

  • Page 1 of 1

A G-protein-biased S1P1 agonist, SAR247799, improved LVH and diastolic function in a rat model of metabolic syndrome.

PLoS One 2022 14;17(1):e0257929. Epub 2022 Jan 14.

Diabetes and Cardiovascular Research, Sanofi US Services, Bridgewater, NJ, United States of America.

Aim: Heart failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with no approved treatment. Left ventricular hypertrophy (LVH) and diastolic dysfunction represent the structural and functional components of HFpEF, respectively. Endothelial dysfunction is prevalent in HFpEF and predicts cardiovascular events. We investigated if SAR247799, a G-protein-biased sphingosine-1-phosphate receptor 1 (S1P1) agonist with endothelial-protective properties, could improve cardiac and renal functions in a rat model of metabolic syndrome LVH and diastolic function.

Methods: 31- and 65-week-old obese ZSF1 (Ob-ZSF1) rats, representing adult and aged animals with LVH and diastolic dysfunction, were randomized to a chow diet containing 0.025% (w/w) of SAR247799, or control (CTRL) chow for 4 weeks. Age-matched lean ZSF1 (Le-ZSF1) rats were fed control chow. Echocardiography, telemetry, biochemical and histological analysis were performed to evaluate the effect of SAR247799.

Results: Echocardiography revealed that Ob-ZSF1 rats, in contrast to Le-ZSF1 rats, developed progressive diastolic dysfunction and cardiac hypertrophy with age. SAR247799 blunted the progression of diastolic dysfunction in adult and aged animals: in adult animals E/e' was evaluated at 21.8 ± 1.4 for Ob-ZSF1-CTRL, 19.5 ± 1.2 for Ob-ZSF1-SAR247799 p<0.01, and 19.5 ± 2.3 for Le-ZSF1-CTRL (median ± IQR). In aged animals E/e' was evaluated at 23.15 ± 4.45 for Ob-ZSF1-CTRL, 19.5 ± 5 for Ob-ZSF1-SAR247799 p<0.01, and 16.69 ± 1.7 for Le-ZSF1-CTRL, p<0.01 (median ± IQR). In aged animals, SAR247799 reduced cardiac hypertrophy (g/mm mean ± SEM of heart weight/tibia length 0.053 ± 0.001 for Ob-ZSF1-CTRL vs 0.046 ± 0.002 for Ob-ZSF1-SAR247799 p<0.01, Le-ZSF1-CTRL 0.035 ± 0.001) and myocardial perivascular collagen content (p<0.001), independently of any changes in microvascular density. In adult animals, SAR247799 improved endothelial function as assessed by the very low frequency bands of systolic blood pressure variability (mean ± SEM 67.8 ± 3.41 for Ob-ZSF1-CTRL 55.8 ± 4.27 or Ob-ZSF1-SAR247799, p<0.05 and 57.3 ± 1.82 Le-ZSF1-CTRL), independently of any modification of arterial blood pressure. In aged animals, SAR247799 reduced urinary protein/creatinine ratio, an index of glomerular injury, (10.3 ± 0.621 vs 8.17 ± 0.231 for Ob-ZSF1-CTRL vs Ob-ZSF1-SAR247799, respectively, p<0.05 and 0.294 ± 0.029 for Le-ZSF1-CTRL, mean ± SEM) and the fractional excretion of electrolytes. Circulating lymphocytes were not decreased by SAR247799, confirming lack of S1P1 desensitization.

Conclusions: These experimental findings suggest that S1P1 activation with SAR247799 may be considered as a new therapeutic approach for LVH and diastolic dysfunction, major components of HFpEF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0257929PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759645PMC
January 2022

High Glucose Activates YAP Signaling to Promote Vascular Inflammation.

Front Physiol 2021 4;12:665994. Epub 2021 Jun 4.

Cardiovascular Research Unit, Sanofi R&D, Chilly-Mazarin, France.

Background And Aims: The YAP/TAZ signaling is known to regulate endothelial activation and vascular inflammation in response to shear stress. Moreover, YAP/TAZ signaling plays a role in the progression of cancers and renal damage associated with diabetes. However, whether YAP/TAZ signaling is also implicated in diabetes-associated vascular complications is not known.

Methods: The effect of high glucose on YAP/TAZ signaling was firstly evaluated on endothelial cells cultured under static conditions or subjected to shear stress (either laminar or oscillatory flow). The impact of diabetes on YAP/TAZ signaling was additionally assessed in mice.

Results: , we found that YAP was dephosphorylated/activated by high glucose in endothelial cells, thus leading to increased endothelial inflammation and monocyte attachment. Moreover, YAP was further activated when high glucose was combined to laminar flow conditions. YAP was also activated by oscillatory flow conditions but, in contrast, high glucose did not exert any additional effect. Interestingly, inhibition of YAP reduced endothelial inflammation and monocyte attachment. Finally, we found that YAP is also activated in the vascular wall of diabetic mice, where inflammatory markers are also increased.

Conclusion: With the current study we demonstrated that YAP signaling is activated by high glucose in endothelial cells and in the vasculature of diabetic mice, and we pinpointed YAP as a regulator of high glucose-mediated endothelial inflammation and monocyte attachment. YAP inhibition may represent a potential therapeutic opportunity to improve diabetes-associated vascular complications.
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http://dx.doi.org/10.3389/fphys.2021.665994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213390PMC
June 2021

Apelin improves cardiac function mainly through peripheral vasodilation in a mouse model of dilated cardiomyopathy.

Peptides 2021 08 12;142:170568. Epub 2021 May 12.

Cardiovascular & Metabolism Therapeutic Area, Sanofi R&D, 1 avenue Pierre Brossolette, 91385, Chilly-Mazarin, France. Electronic address:

There is growing evidence that apelin plays a role in the regulation of the cardiovascular system by increasing myocardial contractility and acting as a vasodilator. However, it remains unclear whether apelin improves cardiac contractility in a load-dependent or independent manner in pathological conditions. For this purpose we investigated the cardiovascular effects of apelin in α-actin transgenic mice (mActin-Tg mice), a model of cardiomyopathy. [Pyr]apelin-13 was administered by continuous infusion at 2 mg/kg/d for 3 weeks. Effects on cardiac function were determined by echocardiography and a Pressure-Volume (PV) analysis. mActin-Tg mice showed a dilated cardiomyopathy (DCM) phenotype similar to that encountered in patients expressing the same mutation. Compared to WT animals, mActin-Tg mice displayed cardiac systolic impairment [significant decrease in ejection fraction (EF), cardiac output (CO), and stroke volume (SV)] associated with cardiac ventricular dilation and diastolic dysfunction, characterized by an impairment in mitral flow velocity (E/A) and in deceleration time (DT). Load-independent myocardial contractility was strongly decreased in mActin-Tg mice while total peripheral vascular resistance (TPR) was significantly increased. As compared to vehicle-treated animals, a 3-week treatment with [Pyr]apelin-13 significantly improved EF%, SV, E/A, DT and corrected TPR, with no significant effect on load-independent indices of myocardial contractility, blood pressure and heart rate. In conclusion [Pyr]apelin-13 displayed no intrinsic contractile effect but improved cardiac function in dilated cardiomyopathy mainly by reducing peripheral vascular resistance, with no change in blood pressure.
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http://dx.doi.org/10.1016/j.peptides.2021.170568DOI Listing
August 2021

Generation of iPSC line from MYH7 R403L mutation carrier with severe hypertrophic cardiomyopathy and isogenic CRISPR/Cas9 corrected control.

Stem Cell Res 2021 04 11;52:102245. Epub 2021 Feb 11.

ICAN - Institute for Cardiometabolism and Nutrition, F-75013 Paris, France; Sorbonne Université, INSERM, UMR_S1166, AP-HP, Hôpital Pitié-Salpêtrière, F-75013 Paris, France. Electronic address:

MYH7 is a major gene responsible for hypertrophic cardiomyopathy (HCM). From patient's skin fibroblasts, we derived an iPSC line (CDGEN1.16) harboring the heterozygous MYH7 R403L mutation, a hot-spot codon in HCM. We subsequently corrected the mutated codon using CRISPR/Cas9 editing and obtained the isogenic control line (CDGEN1.16.40.5) preserving the genomic background of the patient. Both lines were pluripotent and could be efficiently committed to beating cardiomyocytes (CM) suitable for subsequent cell or pseudo-tissue study of HCM pathology.
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http://dx.doi.org/10.1016/j.scr.2021.102245DOI Listing
April 2021

SAR340835, a Novel Selective Na/Ca Exchanger Inhibitor, Improves Cardiac Function and Restores Sympathovagal Balance in Heart Failure.

J Pharmacol Exp Ther 2021 05 18;377(2):293-304. Epub 2021 Feb 18.

Cardiovascular and Metabolism TSU (M.P., F.B., C.D., T.L., S.M., G.T., L.L., Ph.B., M.-P.P.-H., P.J., F.C.-G.) and Integrated Drug Discovery (Pa.B.), Sanofi R&D, Chilly Mazarin, France; Preclinical Safety, Sanofi R&D, Alfortville, France (L.L.-N., V.B., J.-M.G., M.T.); Sanofi R&D, Industriepark Höchst, Frankfurt, Germany (K.S., K.W., H.G., P.A., N.R., J.W.); and Integrated Drug Discovery, Sanofi R&D, Vitry sur Seine, France (G.M.)

In failing hearts, Na/Ca exchanger (NCX) overactivity contributes to Ca depletion, leading to contractile dysfunction. Inhibition of NCX is expected to normalize Ca mishandling, to limit afterdepolarization-related arrhythmias, and to improve cardiac function in heart failure (HF). SAR340835/SAR296968 is a selective NCX inhibitor for all NCX isoforms across species, including human, with no effect on the native voltage-dependent calcium and sodium currents in vitro. Additionally, it showed in vitro and in vivo antiarrhythmic properties in several models of early and delayed afterdepolarization-related arrhythmias. Its effect on cardiac function was studied under intravenous infusion at 250,750 or 1500 µg/kg per hour in dogs, which were either normal or submitted to chronic ventricular pacing at 240 bpm (HF dogs). HF dogs were infused with the reference inotrope dobutamine (10 µg/kg per minute, i.v.). In normal dogs, NCX inhibitor increased cardiac contractility (dP/dt) and stroke volume (SV) and tended to reduce heart rate (HR). In HF dogs, NCX inhibitor significantly and dose-dependently increased SV from the first dose (+28.5%, +48.8%, and +62% at 250, 750, and 1500 µg/kg per hour, respectively) while significantly increasing dP/dt only at 1500 (+33%). Furthermore, NCX inhibitor significantly restored sympathovagal balance and spontaneous baroreflex sensitivity (BRS) from the first dose and reduced HR at the highest dose. In HF dogs, dobutamine significantly increased dP/dt and SV (+68.8%) but did not change HR, sympathovagal balance, or BRS. Overall, SAR340835, a selective potent NCX inhibitor, displayed a unique therapeutic profile, combining antiarrhythmic properties, capacity to restore systolic function, sympathovagal balance, and BRS in HF dogs. NCX inhibitors may offer new therapeutic options for acute HF treatment. SIGNIFICANCE STATEMENT: HF is facing growing health and economic burden. Moreover, patients hospitalized for acute heart failure are at high risk of decompensation recurrence, and no current acute decompensated HF therapy definitively improved outcomes. A new potent, Na/Ca exchanger inhibitor SAR340835 with antiarrhythmic properties improved systolic function of failing hearts without creating hypotension, while reducing heart rate and restoring sympathovagal balance. SAR340835 may offer a unique and attractive pharmacological profile for patients with acute heart failure as compared with current inotrope, such as dobutamine.
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http://dx.doi.org/10.1124/jpet.120.000238DOI Listing
May 2021

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

J Med Chem 2021 02 8;64(4):2139-2150. Epub 2021 Feb 8.

Cardio-Vascular and metabolism, Sanofi R&D, 1 rue Pierre Brossolette, Chilly Mazarin 91385, France.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (, ). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and efficacy (, ).
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http://dx.doi.org/10.1021/acs.jmedchem.0c01533DOI Listing
February 2021

Endothelial-protective effects of a G-protein-biased sphingosine-1 phosphate receptor-1 agonist, SAR247799, in type-2 diabetes rats and a randomized placebo-controlled patient trial.

Br J Clin Pharmacol 2021 05 26;87(5):2303-2320. Epub 2020 Nov 26.

Sanofi US Services, Bridgewater, NJ, USA.

Aims: SAR247799 is a G-protein-biased sphingosine-1 phosphate receptor-1 (S1P ) agonist designed to activate endothelial S1P and provide endothelial-protective properties, while limiting S1P desensitization and consequent lymphocyte-count reduction associated with higher doses. The aim was to show whether S1P activation can promote endothelial effects in patients and, if so, select SAR247799 doses for further clinical investigation.

Methods: Type-2 diabetes patients, enriched for endothelial dysfunction (flow-mediated dilation, FMD <7%; n = 54), were randomized, in 2 sequential cohorts, to 28-day once-daily treatment with SAR247799 (1 or 5 mg in ascending cohorts), placebo or 50 mg sildenafil (positive control) in a 5:2:2 ratio per cohort. Endothelial function was assessed by brachial artery FMD. Renal function, biomarkers and lymphocytes were measured following 5-week SAR247799 treatment (3 doses) to Zucker diabetic fatty rats and the data used to select the doses for human testing.

Results: The maximum FMD change from baseline vs placebo for all treatments was reached on day 35; mean differences vs placebo were 0.60% (95% confidence interval [CI] -0.34 to 1.53%; P = .203) for 1 mg SAR247799, 1.07% (95% CI 0.13 to 2.01%; P = .026) for 5 mg SAR247799 and 0.88% (95% CI -0.15 to 1.91%; P = .093) for 50 mg sildenafil. Both doses of SAR247799 were well tolerated, did not affect blood pressure, and were associated with minimal-to-no lymphocyte reduction and small-to-moderate heart rate decrease.

Conclusion: These data provide the first human evidence suggesting endothelial-protective properties of S1P activation, with SAR247799 being as effective as the clinical benchmark, sildenafil. Further clinical testing of SAR247799, at sub-lymphocyte-reducing doses (≤5 mg), is warranted in vascular diseases associated with endothelial dysfunction.
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http://dx.doi.org/10.1111/bcp.14632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247405PMC
May 2021

A label-free impedance assay in endothelial cells differentiates the activation and desensitization properties of clinical S1P agonists.

FEBS Open Bio 2020 10 4;10(10):2010-2020. Epub 2020 Sep 4.

Diabetes and Cardiovascular Research, Sanofi US Services, Bridgewater, NJ, USA.

Sphingosine-1 phosphate receptor-1 (S1P ) activation maintains endothelial barrier integrity, whereas S1P desensitization induces peripheral blood lymphopenia. The latter is exploited in the approval and/or late-stage development of receptor-desensitizing agents targeting the S1P receptor in multiple sclerosis, such as siponimod, ozanimod, and ponesimod. SAR247799 is a recently described G protein-biased S1P agonist that activates S1P without desensitization and thus has endothelial-protective properties in patients without reducing lymphocytes. As SAR247799 demonstrated endothelial-protective effects at sub-lymphocyte-reducing doses, the possibility exists that other S1P modulators could also exhibit endothelial-protective properties at lower doses. To explore this possibility, we sought to quantitatively compare the biased properties of SAR247799 with the most advanced clinical molecules targeting S1P . In this study, we define the β-arrestin pathway component of the impedance profile following S1P activation in a human umbilical vein endothelial cell line (HUVEC) and report quantitative indices of the S1P activation-to-desensitization ratio of various clinical molecules. In a label-free impedance assay assessing endothelial barrier integrity and disruption, the mean estimates (95% confidence interval) of the activation-to-desensitization ratios of SAR247799, ponesimod, ozanimod, and siponimod were 114 (91.1-143), 7.66 (3.41-17.2), 6.35 (3.21-12.5), and 0.170 (0.0523-0.555), respectively. Thus, we show that SAR247799 is the most G protein-biased S1P agonist currently characterized. This rank order of bias among the most clinically advanced S1P modulators provides a new perspective on the relative potential of these clinical molecules for improving endothelial function in patients in relation to their lymphocyte-reducing (desensitization) properties.
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http://dx.doi.org/10.1002/2211-5463.12951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530392PMC
October 2020

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

ESC Heart Fail 2020 10 20;7(5):2871-2883. Epub 2020 Jul 20.

Department of Internal Medicine II, University Medical Center Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg, Germany.

Aims: Excessive activation of Ca/calmodulin-dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIɣ-selective, ATP-competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC).

Methods And Results: In human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch-clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n - 1' χ test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n - 1' χ test.

Conclusions: RA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.
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http://dx.doi.org/10.1002/ehf2.12895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524064PMC
October 2020

Translational engagement of lysophosphatidic acid receptor 1 in skin fibrosis: from dermal fibroblasts of patients with scleroderma to tight skin 1 mouse.

Br J Pharmacol 2020 09 5;177(18):4296-4309. Epub 2020 Aug 5.

Cardiovascular & Metabolism Unit, Sanofi, Chilly-Mazarin, France.

Background And Purpose: Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA ) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis.

Experimental Approach: Dermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model.

Key Results: SAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA receptor. The LPA functional response (Ca ) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice.

Conclusion And Implications: The effects of SAR100842 on LPA-induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA receptor antagonists in systemic sclerosis.
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http://dx.doi.org/10.1111/bph.15190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443477PMC
September 2020

Lipoprotein(a) Cellular Uptake Ex Vivo and Hepatic Capture In Vivo Is Insensitive to PCSK9 Inhibition With Alirocumab.

JACC Basic Transl Sci 2020 Jun 6;5(6):549-557. Epub 2020 May 6.

Laboratoire Inserm UMR 1188 DéTROI, Université de La Réunion, Sainte Clotilde, France.

Lipoprotein(a) (Lp[a]) is the most common genetically inherited risk factor for cardiovascular disease. Many aspects of Lp(a) metabolism remain unknown. We assessed the uptake of fluorescent Lp(a) in primary human lymphocytes as well as Lp(a) hepatic capture in a mouse model in which endogenous hepatocytes have been ablated and replaced with human ones. Modulation of LDLR expression with the PCSK9 inhibitor alirocumab did not alter the cellular or the hepatic uptake of Lp(a), demonstrating that the LDL receptor is not a major route for Lp(a) plasma clearance. These results have clinical implications because they underpin why statins are not efficient at reducing Lp(a).
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http://dx.doi.org/10.1016/j.jacbts.2020.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315184PMC
June 2020

A G protein-biased S1P agonist, SAR247799, protects endothelial cells without affecting lymphocyte numbers.

Sci Signal 2020 06 2;13(634). Epub 2020 Jun 2.

Diabetes and Cardiovascular Research, Sanofi US Services, 55 Corporate Drive, Bridgewater, NJ 08807, USA.

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P These findings demonstrate that sustained S1P activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.
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http://dx.doi.org/10.1126/scisignal.aax8050DOI Listing
June 2020

Lysophosphatidic Acid Receptor Agonism: Discovery of Potent Nonlipid Benzofuran Ethanolamine Structures.

J Pharmacol Exp Ther 2020 08 14;374(2):283-294. Epub 2020 May 14.

Diabetes and Cardiovascular Unit, Sanofi R&D, Chilly-Mazarin, France (E.G., J.C.L.B., P.B., P.J.); Global Research Portfolio and Project Management, Sanofi R&D, Chilly-Mazarin, France (C.P.); Translational Science Unit, Sanofi R&D, Chilly-Mazarin, France (P.P., V.F.); In-silico design, Chilly-Mazarin, France (B.C.); and Integrated Drug Discovery, Sanofi R&D, Vitry-Sur-Seine, France (M.P.).

Lysophosphatidic acid (LPA) is the natural ligand for two phylogenetically distinct families of receptors (LPA LPA) whose pathways control a variety of physiologic and pathophysiological responses. Identifying the benefit of balanced activation/repression of LPA receptors has always been a challenge because of the high lability of LPA and the limited availability of selective and/or stable agonists. In this study, we document the discovery of small benzofuran ethanolamine derivatives (called CpX and CpY) behaving as LPA agonists. Initially found as rabbit urethra contracting agents, their elusive receptors were identified from [S]GTPS-binding and -arrestin2 recruitment investigations and then confirmed by [H]CpX binding studies (urethra, hLPA membranes). Both compounds induced a calcium response in hLPA cells within a range of 0.4-1.5-log lower potency as compared with LPA. The contractions of rabbit urethra strips induced by these compounds perfectly matched binding affinities with values reaching the two-digit nanomolar level. The antagonist, KI16425, dose-dependently antagonized CpX-induced contractions in agreement with its affinity profile (LPA≥LPA>>LPA). The most potent agonist, CpY, doubled intraurethral pressure in anesthetized female rats at 3 µg/kg i.v. Alternatively, CpX was shown to inhibit human preadipocyte differentiation, a process totally reversed by KI16425. Together with original molecular docking data, these findings clearly established these molecules as potent agonists of LPA and consolidated the pivotal role of LPA in urethra/prostate contraction as well as in fat cell development. The discovery of these unique and less labile LPA agonists would offer new avenues to investigate the roles of LPA receptors. SIGNIFICANCE STATEMENT: We report the identification of benzofuran ethanolamine derivatives behaving as potent selective nonlipid LPA agonists and shown to alter urethra muscle contraction or preadipocyte differentiation. Unique at this level of potency, selectivity, and especially stability, compared with lysophosphatidic acid, they represent more appropriate tools for investigating the physiological roles of lysophosphatidic acid receptors and starting point for optimization of drug candidates for therapeutic applications.
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http://dx.doi.org/10.1124/jpet.120.265454DOI Listing
August 2020

Potential Therapeutic Value of Urotensin II Receptor Antagonist in Chronic Kidney Disease and Associated Comorbidities.

J Pharmacol Exp Ther 2020 07 24;374(1):24-37. Epub 2020 Apr 24.

Cardiovascular and Metabolism Therapeutic Area, Sanofi R&D, Chilly-Mazarin, France (M.L.O., V.B., M.P., F.B., P.S., P.B., B.P., P.J.); Preclinical Safety, Sanofi R&D, Chilly-Mazarin, France (J.M.G.);and Chemistry, Sanofi R&D, Chilly-Mazarin, France (F.P., J.M.A., J.P.B.)

Chronic kidney disease (CKD) remains a common disorder, leading to growing health and economic burden without curative treatment. In diabetic patients, CKD may result from a combination of metabolic and nonmetabolic-related factors, with mortality mainly driven by cardiovascular events. The marked overactivity of the urotensinergic system in diabetic patients implicates this vasoactive peptide as a possible contributor to the pathogenesis of renal as well as heart failure. Previous preclinical studies with urotensin II (UII) antagonists in chronic kidney disease were based on simple end points that did not reflect the complex etiology of the disease. Given this, our studies revisited the therapeutic value of UII antagonism in CKD and extensively characterized 1-({[6-{4-chloro-3-[3-(dimethylamino)propoxy]phenyl}-5-(2-methylphenyl)pyridin-2-yl]carbonyl}amino) cyclohexanecarboxylic acid hydrochloride (SAR101099), a potent, selective, and orally long-acting UII receptor competitive antagonist, inhibiting not only UII but also urotensin-related peptide activities. SR101099 treatment more than halved proteinurea and albumin/creatinine ratio in spontaneously hypertensive stroke-prone (SHR-SP) rats fed with salt/fat diet and Dahl-salt-sensitive rats, respectively, and it halved albuminuria in streptozotocin-induced diabetes rats. Importantly, these effects were accompanied by a decrease in mortality of 50% in SHR-SP and of 35% in the Dahl salt-sensitive rats. SAR101099 was also active on CKD-related cardiovascular pathologies and partly preserved contractile reserve in models of heart failure induced by myocardial infarction or ischemia/reperfusion in rats and pigs, respectively. SAR101099 exhibited a good safety/tolerability profile at all tested doses in clinical phase-I studies. Together, these data suggest that CKD patient selection considering comorbidities together with new stratification modalities should unveil the urotensin antagonists' therapeutic potential. SIGNIFICANCE STATEMENT: Chronic kidney disease (CKD) is a pathology with growing health and economic burden, without curative treatment. For years, the impact of urotensin II receptor (UT) antagonism to treat CKD may have been compromised by available tools or models to deeper characterize the urotensinergic system. New potent, selective, orally long-acting cross-species UT antagonist such as SAR101099 exerting reno- and cardioprotective effects could offer novel therapeutic opportunities. Its preclinical and clinical results suggest that UT antagonism remains an attractive target in CKD on top of current standard of care.
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http://dx.doi.org/10.1124/jpet.120.265496DOI Listing
July 2020

Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes.

Circ Res 2020 07 10;127(2):e14-e27. Epub 2020 Apr 10.

From the Department of Physiology, Pennsylvania Muscle Institute (C.Y.C., A.K.S., M.A.C., S.C., N.A.K., A.I.B., K.B.M., B.L.P.), University of Pennsylvania Perelman School of Medicine, Philadelphia.

Rationale: Impaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development.

Objective: We aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF.

Methods And Results: Transcriptional profiling revealed that transcript is >10-fold more abundant than in human hearts. Using short hairpin RNAs (shRNAs) against , , and , we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes.

Conclusions: VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334093PMC
July 2020

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.

Sci Rep 2019 07 25;9(1):10811. Epub 2019 Jul 25.

Stanford Cardiovascular Institute and the Department of Medicine, Stanford University, Stanford, CA, 94305, USA.

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.
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http://dx.doi.org/10.1038/s41598-019-46948-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658660PMC
July 2019

Reversion of cardiac dysfunction by a novel orally available calcium/calmodulin-dependent protein kinase II inhibitor, RA306, in a genetic model of dilated cardiomyopathy.

Cardiovasc Res 2020 02;116(2):329-338

Cardiovascular&Metabolism Therapeutic Area, Sanofi R&D, 1 avenue Pierre Brossolette, 91385 Chilly-Mazarin, France.

Aims: Despite improvements in patient identification and management, heart failure (HF) remains a major public health burden and an important clinical challenge. A variety of animal and human studies have provided evidence suggesting a central role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the development of pathological cardiac remodelling and HF. Here, we describe a new potent, selective, and orally available CaMKII inhibitor.

Methods And Results: Chemical optimization led to the identification of RA306 as a selective CaMKII inhibitor. This compound was found potent on the cardiac CaMKII isoforms delta and gamma (IC50 in the 10 nM range), with pharmacokinetic properties allowing oral administration in animal models of HF. RA306 was administered to diseased mice carrying a mutation in alpha-actin that is responsible for dilated cardiomyopathy (DCM) in humans. In two separate studies, RA306 was orally administered at 30 mg/kg either for 2 weeks (twice a day) or for 2 months (once a day). Echocardiography monitoring showed that RA306 significantly improved cardiac function (ejection fraction and cardiac output) as compared to vehicle. These disease modifying effects of RA306 were associated with inhibition of cardiac phosphorylation of phospholamban (PLN) at threonine-17, indicating reduced cardiac CaMKII activity.

Conclusion: This work supports the feasibility of identifying potent orally available CaMKII inhibitors suitable for clinical use to treat heart disease.
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http://dx.doi.org/10.1093/cvr/cvz097DOI Listing
February 2020

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

MAbs 2019 Feb/Mar;11(2):297-304. Epub 2019 Feb 4.

a Centre d'Etude des Pathologies Respiratoires , UMR 1100 , INSERM , Tours, France.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.
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http://dx.doi.org/10.1080/19420862.2018.1556081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380415PMC
July 2019

PCSK9 inhibition with alirocumab reduces lipoprotein(a) levels in nonhuman primates by lowering apolipoprotein(a) production rate.

Clin Sci (Lond) 2018 05 31;132(10):1075-1083. Epub 2018 May 31.

Sanofi R&D, Chilly-Mazarin, France.

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels Six nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [H]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [H]-leucine was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (-28%), LDL-C (-67%), Lp(a) (-56%), apolipoprotein B100 (apoB100) (-53%), and apo(a) (-53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (-42%), but not significantly that of apoB100, compared with IgG1, respectively.In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.
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http://dx.doi.org/10.1042/CS20180040DOI Listing
May 2018

PCSK9 Modulates the Secretion But Not the Cellular Uptake of Lipoprotein(a) Ex Vivo: An Effect Blunted by Alirocumab.

JACC Basic Transl Sci 2016 Oct;1(6):419-427

Inra UMR 1280, Nantes, France.

To elucidate how the proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor alirocumab modulates lipoprotein(a) [Lp(a)] plasma levels, the authors performed a series of Lp(a) uptake studies in primary human hepatocytes and dermal fibroblasts and measured Lp(a) secretion from human hepatocytes. They found that Lp(a) cellular uptake occurred in a low-density lipoprotein receptor-independent manner. Neither PCSK9 nor alirocumab altered Lp(a) internalization. By contrast, the secretion of apolipoprotein (a) from human hepatocytes was sharply increased by PCSK9, an effect that was reversed by alirocumab. They propose that PCSK9 does not significantly modulate Lp(a) catabolism, but rather enhances the secretion of Lp(a) from liver cells.
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http://dx.doi.org/10.1016/j.jacbts.2016.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753417PMC
October 2016

CaMKII as a pathological mediator of ER stress, oxidative stress, and mitochondrial dysfunction in a murine model of nephronophthisis.

Am J Physiol Renal Physiol 2016 06 13;310(11):F1414-22. Epub 2016 Apr 13.

Rare Diseases, Sanofi-Genzyme R&D Center, Framingham, Massachusetts; and

Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.
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http://dx.doi.org/10.1152/ajprenal.00426.2015DOI Listing
June 2016

Pharmacological characterization of SAR407899, a novel rho-kinase inhibitor.

Hypertension 2009 Sep 13;54(3):676-83. Epub 2009 Jul 13.

TD CV Pharmacology, Sanofi-Aventis, Industriepark Hoechst, Frankfurt am Main, Frankfurt, Germany.

Recent advances in basic and clinical research have identified Rho kinase as an important target potentially implicated in a variety of cardiovascular diseases. Rho kinase is a downstream mediator of RhoA that leads to stress fiber formation, membrane ruffling, smooth muscle contraction, and cell motility. Increased Rho-kinase activity is associated with vasoconstriction and elevated blood pressure. We identified a novel inhibitor of Rho kinase (SAR407899) and characterized its effects in biochemical, cellular, tissue-based, and in vivo assays. SAR407899 is an ATP-competitive Rho-kinase inhibitor, equipotent against human and rat-derived Rho-kinase 2 with inhibition constant values of 36 nM and 41 nM, respectively. It is highly selective in panel of 117 receptor and enzyme targets. SAR407899 is approximately 8-fold more active than fasudil. In vitro, SAR407899 demonstrated concentration-dependent inhibition of Rho-kinase-mediated phosphorylation of myosin phosphatase, thrombin-induced stress fiber formation, platelet-derived growth factor-induced proliferation, and monocyte chemotactic protein-1-stimulated chemotaxis. SAR407899 potently (mean IC(50) values: 122 to 280 nM) and species-independently relaxed precontracted isolated arteries of different species and different vascular beds. In vivo, over the dose range 3 to 30 mg/kg PO, SAR407899 lowered blood pressure in a variety of rodent models of arterial hypertension. The antihypertensive effect of SAR407899 was superior to that of fasudil and Y-27632. In conclusion, SAR407899 is a novel and potent selective Rho-kinase inhibitor with promising antihypertensive activity.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.109.134353DOI Listing
September 2009

Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese Zucker fa/fa rats.

Hepatology 2007 Jul;46(1):122-9

Sanofi-Aventis Recherche & Développement, Discovery Research CNS Department, Montpellier, France.

This study investigated the effects of rimonabant (SR141716), an antagonist of the cannabinoid receptor type 1 (CB1), on obesity-associated hepatic steatosis and related features of metabolic syndrome: inflammation (elevated plasma levels of tumor necrosis factor alpha [TNFalpha]), dyslipidemia, and reduced plasma levels of adiponectin. We report that oral treatment of obese (fa/fa) rats with rimonabant (30 mg/kg) daily for 8 weeks abolished hepatic steatosis. This treatment reduced hepatomegaly, reduced elevation of plasma levels of enzyme markers of hepatic damage (alanine aminotransferase, gamma glutamyltransferase, and alkaline phosphatase) and decreased the high level of local hepatic TNFalpha currently associated with steatohepatitis. In parallel, treatment of obese (fa/fa) rats with rimonabant reduced the high plasma level of the proinflammatory cytokine TNFalpha and increased the reduced plasma level of the anti-inflammatory hormone adiponectin. Finally, rimonabant treatment also improved dyslipidemia by both decreasing plasma levels of triglycerides, free fatty acids, and total cholesterol and increasing the HDLc/LDLc ratio. All the effects of rimonabant found in this study were not or only slightly observed in pair-fed obese animals, highlighting the additional beneficial effects of treatment with rimonabant compared to diet. These results demonstrate that rimonabant plays a hepatoprotective role and suggest that this CB1 receptor antagonist potentially has clinical applications in the treatment of obesity-associated liver diseases and related features of metabolic syndrome.
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http://dx.doi.org/10.1002/hep.21641DOI Listing
July 2007

Long-term blockade of angiotensin AT1 receptors increases survival of obese Zucker rats.

Eur J Pharmacol 2006 Mar 6;534(1-3):271-9. Epub 2006 Mar 6.

Cardiovascular Research Department, Sanofi-Synthelabo Research, 1 Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, Hôpital Georges Pompidou, Paris, France.

Despite the well-documented effect of irbesartan, an angiotensin AT1 receptor antagonist, on diabetic nephropathy, its effect on mortality related to multiple metabolic risk factors is unknown. To address this question, obese fa/fa Zucker rats were submitted to a 13-month treatment by irbesartan (30 mg/kg/day p.o.). Vehicle-treated obese fa/fa Zucker rats exhibited an important mortality (72%), which was markedly reduced by irbesartan (22%, P<0.05). Mortality in control lean fa/+ rats attained 12%. Irbesartan diminished the elevation in urinary protein excretion, plasma creatinine and urea nitrogen levels, and reduced the extent of glomerular and tubulo-interstitial lesions together with a reduction of urinary monocyte chemoattractant protein-1 excretion in fa/fa Zucker rats. Irbesartan treatment prevented the rise in plasma total cholesterol, triglycerides and glucose levels, and partially corrected low-density lipoprotein/high-density lipoprotein (LDL/HDL) cholesterol ratio in fa/fa Zucker rats. Therefore, prolonged irbesartan treatment preserves renal function and metabolic profile, and substantially increases survival in obese fa/fa Zucker rats.
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http://dx.doi.org/10.1016/j.ejphar.2006.01.032DOI Listing
March 2006

Serotonin aggravates exercise-induced cardiac ischemia in the dog: effect of serotonin receptor antagonists.

Eur J Pharmacol 2004 Aug;497(1):55-63

Cardiovascular-Thrombosis Research Department, Sanofi-Synthélabo Research, 1 Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France.

We investigated the effects of serotonin (5-HT), SL65.0472 (7-fluoro-2-oxo-4-[2-[4-thieno[3,2-c]pyridine-4-yl)piperazin-1-yl]ethyl]-1,2-dihydroquinoline-1-acetamide, a 5-HT(1B)/5-HT(2A) receptor antagonist) and ketanserin (a 5-HT(2A) receptor antagonist) during exercise-induced cardiac ischemia in conscious dogs. Dogs were administered a hypercholesterolemic diet and an inhibitor of nitric oxide synthetase to produce chronic endothelial dysfunction. Myocardial ischemia was induced by a treadmill exercise test associated with limitation of left anterior descending coronary blood flow. Infusion of serotonin during exercise produced dose-related cardiovascular changes (after 10 microg/kg/min; heart rate +27+/-6 bpm, systolic blood pressure +18+/-3 mm Hg, left circumflex coronary blood flow +64+/-8 ml/min, myocardial segment length shortening in the ischemic zone -5.9+/-1.9%, P<0.05). SL65.0472 blocked serotonin-induced increases in blood pressure, rate pressure product and circumflex coronary artery flow (100 microg/kg i.v., P<0.05) and reduced serotonin-induced ischemic myocardial segment length shortening (300 microg/kg i.v., P<0.05). Ketanserin (30-300 microg/kg i.v.) had no significant effect on any serotonin-induced changes during exercise. Thus, SL65.0472 opposes serotonin-induced myocardial dysfunction in a dog model of exercise-induced ischemia.
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http://dx.doi.org/10.1016/j.ejphar.2004.06.024DOI Listing
August 2004

Role of peripheral benzodiazepine receptors in mitochondrial, cellular, and cardiac damage induced by oxidative stress and ischemia-reperfusion.

J Pharmacol Exp Ther 2003 Sep;306(3):828-37

Sanofi-Synthélabo Research, Toulouse, France.

Mitochondrial dysfunction has been identified as a possible early event in ischemia-reperfusion damage. The peripheral benzodiazepine receptor, a mitochondrial inner membrane protein, has already been proposed to play a role in mitochondrial regulation, although its exact function remains unclear. The aim of this work was to determine the role of peripheral benzodiazepine receptor in ischemia-reperfusion injury and to test the potential beneficial effect of a novel potent peripheral benzodiazepine receptor ligand, 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575). To characterize and link the mitochondrial, cellular, and cardiac consequences of ischemia-reperfusion, we examined the effects of SSR180575 in several in vitro and in vivo models of oxidative stress. Hydrogen peroxide decreased mitochondrial membrane potential, reduced oxidative phosphorylation capacities, and caused cytochrome c release, caspase 3 activation, and DNA fragmentation. SSR180575 (100 nM-1 microM) prevented all these effects. In perfused rat hearts, SSR180575 administered in vitro (100 nM-1 microM) or by oral pretreatment (3-30 mg/kg) greatly reduced the contractile dysfunction associated with ischemia-reperfusion. Furthermore, in anesthetized rats, SSR180575 (3-30 mg/kg p.o.) produced significant reductions in infarct size after coronary artery occlusion/reperfusion. In conclusion, we have demonstrated that peripheral benzodiazepine receptor play a major role in the regulation of cardiac ischemia-reperfusion injury and that SSR180575, a novel peripheral benzodiazepine receptor ligand, is of potential interest in these indications.
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http://dx.doi.org/10.1124/jpet.103.052068DOI Listing
September 2003

SL65.0472 blocks 5-hydroxytryptamine-induced vasoconstriction in a dog hindlimb ischemia model.

Eur J Pharmacol 2003 Aug;474(1):117-20

Sanofi-Synthélabo Research, Cardiovascular Thrombosis Research Department, 1 Avenue Pierre Brossolette, 91380 Chilly-Mazarin, France.

We have studied the ability of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c]pyridin-4-yl)piperazin-1-yl]ethyl]-1,2-dihydroquinoline-1-acetamide), a 5-hydroxytryptamine (5-HT) 5-HT1B/5-HT2A receptor antagonist, to antagonise the vasoconstrictor effects of 5-HT and sumatriptan in a canine model of hindlimb ischemia. Dogs underwent right external iliac artery ligation and right superficial femoral artery excision, resulting in decreased perfusion (-31%, P<0.05) in the right hindlimb. Following pretreatment with L-NAME, phentolamine and propranolol, intra-aortic injection of 5-HT markedly reduced blood flow to the right ischemic hindlimb (-50 +/- 2%, P<0,05). 5-HT induced vasoconstriction was significantly inhibited (-66%, P<0.05) by SL65.0472 (300 microg/kg i.v.), but unaffected by ketanserin (300 microg/kg i.v.), a 5-HT2A receptor antagonist. SL65.0472 also blocked sumatriptan-induced vasoconstriction in ischemic and normally perfused hindlimbs. Thus, SL65.0472 is an effective antagonist of 5-HT-receptor mediated hindlimb vasoconstriction.
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http://dx.doi.org/10.1016/s0014-2999(03)02032-6DOI Listing
August 2003

SSR69071, an elastase inhibitor, reduces myocardial infarct size following ischemia-reperfusion injury.

Eur J Pharmacol 2003 Feb;461(1):49-52

Cardiovascular-Thrombosis Department, Sanofi-Synthelabo Research, 1 Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France.

Neutrophil elastase contributes to the severity of cardiac damage following coronary ischemia and reperfusion. We evaluated the effects of 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyridol[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methyethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide hemihydrate (SSR69071), a novel, potent and selective inhibitor of neutrophil elastase, on infarct size in anaesthetized rabbits subjected to coronary artery occlusion for 30 min followed by reperfusion for 120 min. SSR69071 (3 mg/kg i.v.) reduced cardiac infarct size when administered before ischemia (-39%, P<0.05) or just prior to reperfusion (-37%, P<0.05). Subsequent experiments using the latter administration protocol confirmed the ability of SSR69071 (1 and 3 mg/kg i.v.) to reduce infarct size. This cardioprotective activity was associated with inhibition of cardiac elastase.
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http://dx.doi.org/10.1016/s0014-2999(03)01298-6DOI Listing
February 2003

Serotonin receptor blockade improves distal perfusion after lower limb ischemia in the fatty Zucker rat.

Cardiovasc Res 2002 Nov;56(2):293-302

Cardiovascular-Thrombosis Department, Sanofi-Synthélabo Research, 1 Avenue Pierre Brossolette, 91385 Cedex, Chilly-Mazarin, France.

Objective: Lower limb perfusion following arterial occlusion depends on the recruitment of collateral vessels. Blood flow through these collateral vessels may be jeopardized by hypersensitivity to vasoconstriction by serotonin (5-HT), as has been reported during hyperlipidemia and diabetes. Therefore the aim of this study was to determine the benefits of chronic treatment with SL65.0472, a mixed 5-HT(1B)/5-HT(2A) receptor antagonist, on lower limb ischemia in fatty fa/fa Zucker rats, a strain characterized by obesity, diabetes and hyperlipidemia. Comparison was made with lean control fa/+ Zucker rats.

Methods: SL65.0472 (3 mg/kg/day, n=16) or its vehicle (n=20) were administered orally for 13 days to male fatty fa/fa Zucker rats submitted to lower limb ischemia. Hindlimb ischemia was induced unilaterally by resection of the left femoral and external iliac arteries and embolization of the left internal iliac artery with microspheres. Distal perfusion was measured under mild anesthesia by laser Doppler imaging of both feet. The perfusion deficit (Delta%) was calculated before and 3, 7 and 14 days after induction of hindlimb ischemia. Twenty-four hours after the last administration of SL65.0472, muscular partial oxygen pressure, iliac blood flows, maximal vasodilatory reserve and the vasoreactivity to 5-HT were measured in both limbs.

Results: Chronic administration of SL65.0472 improved the distal perfusion from day 3. At day 14, the deficit of perfusion was limited to -36+/-7% in SL65.0472-treated animals vs. -70+/-6% in the vehicle-treated group (P<0.001) and was accompanied by a significant improvement of partial oxygen pressure in the ischemic limb (SL65.0472: 10.4+/-3.9 mmHg vs. vehicle: 3.5+/-1.1 mmHg, P<0.05). Maximal vasodilatory reserve tended to increase from 2.2+/-0.4 ml/min in the vehicle-treated group to 3.8+/-0.6 ml/min after SL65.0472. SL65.0472 markedly reduced 5-HT-mediated vasoconstriction, which was enhanced in the hypoperfused limb, without altering arterial pressure. Induction of hindlimb ischemia led to the overexpression of both 5-HT(1B) and 5-HT(2A) receptors only in the hypoperfused skeletal muscle as assessed by semi-quantitative RT-PCR.

Conclusion: These results suggest that the recruitment of collateral vessels after the induction of hindlimb ischemia is significantly impaired in obese fa/fa Zucker rats due to a persistent vasoconstriction mediated by 5-HT and involving stimulation of 5-HT(1B) and/or 5-HT(2A) receptors.
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http://dx.doi.org/10.1016/s0008-6363(02)00538-2DOI Listing
November 2002

Unexpected sensitivity of nonobese diabetic mice with a disrupted poly(ADP-Ribose) polymerase-1 gene to streptozotocin-induced and spontaneous diabetes.

Diabetes 2002 May;51(5):1470-6

Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, Paris, France.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic beta-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced beta-cell death.
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http://dx.doi.org/10.2337/diabetes.51.5.1470DOI Listing
May 2002
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