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Drug Annotation
Discovery and safety profiling of a potent pre-clinical candidate, (4-[4[[(3R)-3-(hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]-Nmethyl-benzamide) (CM-352), for the prevention and treatment of hemorrhage. Josune Orbe, Jose A Rodriguez, Juan A Sanchez-Arias, Agustina Salicio, Miriam Belzunce, Ana Ugarte, Haisul C. Y. Chang, Obdulia Rabal, Julen Oyarzabal, and Jose A Paramo J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501939z • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Medicinal Chemistry
Discovery and safety profiling of a potent pre-clinical candidate, (4-[4[[(3R)-3-(hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]N-methyl-benzamide) (CM-352), for the prevention and treatment of hemorrhage.
Authors: Josune Orbe,1 José A. Rodríguez,1 Juan A. Sánchez-Arias,2 Agustina Salicio,1 Miriam Belzunce,1 Ana Ugarte,2 Haisul C.Y. Chang,3 Obdulia Rabal,2 Julen Oyarzabal2,∗ and José A. Páramo,1,4,∗ 1
Atherosclerosis Research Laboratory, 2Small Molecule Discovery Platform, Molecular
Therapeutics Program; 3Experimental Hepathology, Center for Applied Medical Research (CIMA); 4Hematology Service, Clínica Universidad de Navarra. University of Navarra. Pamplona, Spain.
∗
To whom correspondence should be addressed. For J.O.: phone, +34 948 194700 (ext
2044); e-mail,
[email protected]. For J.A.P.: +34 948 194700 (ext. 3015), e-mail,
[email protected] 1 ACS Paragon Plus Environment
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Abstract Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics; e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMP) participate in thrombus dissolution; then, we designed a novel series of optimized MMP inhibitors that went through phenotypic screening consisting of thromboelastometry and mouse tail-bleeding. Our optimized lead compound, CM-352 (2), inhibited fibrinolysis in human whole blood functional assays and was more effective than the current standard of care, 1, in the tail-bleeding model using a 30,000 times lower dose. Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect. Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP, defines a new class of antihemorrhagic agents without interfering with normal hemostatic function. Furthermore, 2 represents a pre-clinical candidate for the acute treatment of bleeding.
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Introduction Bleeding represents a dangerous complication that can result in the need for blood transfusion and is associated with substantial morbidity and mortality. Thus, this adverse event has significant health and economic repercussions.1 In trauma patients, severe hemorrhaging is responsible for 50% of deaths within the first 24 h, with approximately 10% of military and 3–5% of civilian trauma patients requiring massive blood transfusions.2-5 Hyperfibrinolysis constitutes an important contributor to major bleeding events. Therefore, antifibrinolytic agents have become indispensable in various clinical settings, including surgery and trauma. In this regard, the synthetic lysine analogues, tranexamic acid (TXA, 1) and ε-aminocaproic acid (EACA, 3) (see Chart 1), delay fibrinolysis by competitively reducing the binding of plasminogen to fibrin, thereby enhancing clot stability.6 Although aprotinin, which is a bovine-derived protease inhibitor (monomeric globular polypeptide derived from bovine lung tissue), was found to effectively reduce fibrinolysis-mediated blood loss during cardiac surgery, major safety issues led to its withdrawal from the market;7 however, very recently the European Medicines Agency (EMA) has recommended that the suspension be lifted for a restricted range of indications.8 Nevertheless, molecules 1 and 3 are safer than aprotinin and appear to be effective for treating inherited and acquired clinical conditions involving excessive fibrinolysis (e.g., cardiac or liver surgery, trauma, and postpartum hemorrhage).9-15 Unfortunately, 1 has been associated with adverse side effects,16 thromboembolic events and mortality in trauma and surgical patients;12,14 while 3 is even less effective.17 The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate to regulate thrombus dissolution and extracellular matrix proteolysis. In fact, the plasminogen/plasmin system is capable of activating specific MMPs (e.g., MMP-1, -3, and -9), which in turn participate in the dissolution of fibrin through direct fibrin/ogen proteolysis or by modifying plasmin 3 ACS Paragon Plus Environment
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activity.18 Indeed, previous studies from our group have revealed a role for MMP-10 (stromelysin-2) in fibrinolysis. We have demonstrated that MMP-10 was among the genes induced by C-reactive protein in endothelium and that thrombin, via protease-activated receptor-1, markedly enhanced endothelial MMP-10 expression in vitro and in vivo.19-22 In addition, we recently revealed an unexpected role for MMP-10 in promoting the dissolution of fibrin thrombi by enhancing tissue plasminogen activator (tPA)-induced fibrinolysis, which we observed both in vitro and in vivo using an experimental model of stroke.23 Importantly, the role of MMPs in hemorrhaging is supported by the fact that increased levels of certain MMPs are associated with prolonged bleeding in human and animal studies, whereas genetic deletion of MMPs prevented bleeding in experimental models.24,25 In this regard, fibrinolytic activity and bleeding time were found to be markedly reduced in Mmp10–/– mice.23 Also, MMP-3, which is a fibrinolytic MMP18 that is 82% homologous to MMP-10, deserves particular attention as it has been consistently associated with intracranial hemorrhage.26 This collective body of evidence led us to hypothesize that inhibition of MMP-10 and other MMPs might constitute a new, effective and safe therapeutic strategy for the prevention of bleeding. Therefore, we initially analyzed the effect of GM6001 (Ilomastat, 4) (Chart 1), a synthetic broad-range MMP inhibitor, on fibrinolysis and bleeding.27 Then, we designed and synthesized a novel series of optimized MMP inhibitors. Following initial biochemical profiling, prioritized molecules underwent a phenotypic screening process consisting of functional thromboelastometry assays in vitro and bleeding tests in vivo.28 Overall, these analyses revealed that our optimized lead compound, 2, was significantly more effective than the current standard of care, the antifrinolytic drug 1. Moreover, this inhibitor 2 exhibited suitable ADME/toxicity and pharmacokinetic profiles, as well as cardiac safety profiling, as it did not result in thrombus formation or other unwanted side effects. Here, we provide a detailed characterization of this pre-clinical candidate for the acute treatment of bleeding. 4 ACS Paragon Plus Environment
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Chart 1. Chemical structure of compounds 1, 3 and 4
1
3
4
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Results Validation of MMPs as therapeutic targets in hemorrhage Due to the fact the MMP-10 plays a role in fibrinolysis,23 we assessed whether antibodymediated inhibition of MMP-10 would reduce fibrinolysis and prevent bleeding. We first determined the concentration of antibody required for effective inhibition of MMP-10 activity, IC50 of 2.5 nM; and, as expected, no inhibition of MMP-3 was detected (Fig. 1A). Anti-MMP-10 prolonged clot lysis time (CLT) in the thromboelastometry assay, delaying fibrinolysis by 50% (EC50) at a concentration of 2 nM, without effecting changes in coagulation activation and clot firmness (Fig. 1B). In addition, we tested the ability of this antibody to control tPA-induced bleeding in a mouse tail transection model. We observed significant reductions in bleeding time (Fig. 1C) and blood loss (Fig. 1D) upon intravenous (i.v.) treatment with 500 ng/kg anti-MMP-10 as compared to isotype control (IgG2a). Administration of a very high dose of compound 1 (300 mg/kg; LD50=1350mg/kg)29 achieved a similar effect on tail-bleeding time but with no reduction in blood loss (Fig. 1C, D). These results are consistent with reduced bleeding observed in Mmp10–/– mice23 and reveal a novel mechanism for controlling hemorrhaging. Since various MMPs within whole blood can enhance fibrin degradation,18 we investigated whether inhibition of MMPs might reverse the fibrinolytic activity in whole blood by performing thromboelastometric assays in the presence of a broad-spectrum MMP inhibitor. Thus, compound 4 was utilized as pharmacological tool to validate, in-vitro proof-of-concept, proposed antifibrinolytic strategy. Interestingly, thromboelastometric assay showed that 4 effectively delayed CLT by 50% (EC50) at a concentration of 6.2 nM (Fig. 2A), without changes in coagulation activity (Fig. 2B,C). Notably, 4 was more effective at lower concentrations than 1 (EC50=3 µM; Fig. 2E-G). Compound 4 also reduced in-vivo tailbleeding time when compared to saline control (16.4 ± 3.9 min vs. 29.3 ± 0.4 min, p
