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1.
Bioorg Med Chem Lett ; 29(19): 126604, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445854

RESUMO

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

2.
J Med Chem ; 62(16): 7400-7416, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31246024

RESUMO

In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.

3.
J Med Chem ; 61(17): 7425-7447, 2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-29775297

RESUMO

With the introduction of thrombin and factor Xa inhibitors to the oral anticoagulant market, significant improvements in both efficacy and safety have been achieved. Early clinical and preclinical data suggest that inhibitors of factor XIa can provide a still safer alternative, with expanded efficacy for arterial indications. This Perspective provides an overview of target rationale and details of the discovery and development of inhibitors of factor XIa as next generation antithrombotic agents.

5.
J Med Chem ; 60(23): 9703-9723, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29077405

RESUMO

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.


Assuntos
Anticoagulantes/química , Anticoagulantes/uso terapêutico , Fator XIa/antagonistas & inibidores , Isoquinolinas/química , Isoquinolinas/uso terapêutico , Trombose/tratamento farmacológico , para-Aminobenzoatos/química , para-Aminobenzoatos/uso terapêutico , Animais , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Cães , Descoberta de Drogas , Fator XIa/química , Fator XIa/metabolismo , Humanos , Isoquinolinas/farmacocinética , Isoquinolinas/farmacologia , Masculino , Simulação de Acoplamento Molecular , Coelhos , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Trombose/sangue , para-Aminobenzoatos/farmacocinética , para-Aminobenzoatos/farmacologia
6.
Bioorg Med Chem Lett ; 27(17): 4056-4060, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28780160

RESUMO

A series of macrocyclic factor XIa (FXIa) inhibitors was designed based on an analysis of the crystal structures of the acyclic phenylimidazole compounds. Further optimization using structure-based design led to inhibitors with pM affinity for FXIa, excellent selectivity against a panel of relevant serine proteases, and good potency in the activated partial thromboplastin time (aPTT) clotting assay.


Assuntos
Fator XIa/antagonistas & inibidores , Imidazóis/farmacologia , Compostos Macrocíclicos/farmacologia , Inibidores de Serino Proteinase/farmacologia , Relação Dose-Resposta a Droga , Fator XIa/metabolismo , Humanos , Imidazóis/síntese química , Imidazóis/química , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Estrutura Molecular , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/química , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 27(16): 3833-3839, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687203

RESUMO

Optimization of macrocyclic inhibitors of FXIa is described which focused on modifications to both the macrocyclic linker and the P1 group. Increases in potency were discovered through interactions with a key hydrophobic region near the S1 prime pocket by substitution of the macrocyclic linker with small alkyl groups. Both the position of substitution and the absolute stereochemistry of the alkyl groups on the macrocyclic linker which led to improved potency varied depending on the ring size of the macrocycle. Replacement of the chlorophenyltetrazole cinnamide P1 in these optimized macrocycles reduced the polar surface area and improved the oral bioavailability for the series, albeit at the cost of a decrease in potency.


Assuntos
Amidas/farmacologia , Descoberta de Drogas , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Inibidores de Serino Proteinase/farmacologia , Amidas/síntese química , Amidas/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Fator XIa/metabolismo , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/química , Relação Estrutura-Atividade
8.
Bioorg Med Chem Lett ; 27(12): 2650-2654, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28460818

RESUMO

Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful due to the requirement of a basic P1 group to interact with Asp189 in the S1 binding pocket, limiting their membrane permeability. We have combined recently reported neutral P1 binding substituents with a highly optimized macrocyclic chemotype to produce FVIIa inhibitors with low nanomolar potency and enhanced permeability.


Assuntos
Fator VIIa/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Inibidores de Serino Proteinase/farmacologia , Relação Dose-Resposta a Droga , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Estrutura Molecular , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/química , Relação Estrutura-Atividade
9.
J Med Chem ; 60(3): 1060-1075, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28085275

RESUMO

A novel series of macrocyclic FXIa inhibitors was designed based on our lead acyclic phenyl imidazole chemotype. Our initial macrocycles, which were double-digit nanomolar FXIa inhibitors, were further optimized with assistance from utilization of structure-based drug design and ligand bound X-ray crystal structures. This effort resulted in the discovery of a macrocyclic amide linker which was found to form a key hydrogen bond with the carbonyl of Leu41 in the FXIa active site, resulting in potent FXIa inhibitors. The macrocyclic FXIa series, exemplified by compound 16, had a FXIa Ki = 0.16 nM with potent anticoagulant activity in an in vitro clotting assay (aPTT EC1.5x = 0.27 µM) and excellent selectivity against the relevant blood coagulation enzymes.


Assuntos
Amidas/química , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Descoberta de Drogas , Ligações de Hidrogênio , Ligantes , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Estrutura Molecular , Inibidores de Serino Proteinase/farmacocinética
10.
J Pharmacol Exp Ther ; 360(3): 466-475, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28035006

RESUMO

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.


Assuntos
Coagulação Sanguínea , Fator XIIa/antagonistas & inibidores , Proteínas de Insetos/farmacologia , Embolia Intracraniana , Trombose Intracraniana , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Fibrinolíticos/farmacologia , Embolia Intracraniana/sangue , Embolia Intracraniana/tratamento farmacológico , Trombose Intracraniana/sangue , Trombose Intracraniana/tratamento farmacológico , Modelos Animais , Coelhos , Albumina Sérica Humana
11.
ACS Med Chem Lett ; 7(12): 1077-1081, 2016 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-27994741

RESUMO

Inhibitors of Factor VIIa (FVIIa), a serine protease in the clotting cascade, have shown strong antithrombotic efficacy in preclinical thrombosis models with minimal bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful because known chemotypes have required a highly basic group in the S1 binding pocket for high affinity. A recently reported fragment screening effort resulted in the discovery of a neutral heterocycle, 7-chloro-3,4-dihydroisoquinolin-1(2H)-one, that binds in the S1 pocket of FVIIa and can be incorporated into a phenylglycine FVIIa inhibitor. Optimization of this P1 binding group led to the first series of neutral, permeable FVIIa inhibitors with low nanomolar potency.

12.
Bioorg Med Chem ; 24(10): 2257-72, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27073051

RESUMO

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system.


Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Fator XIa/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacocinética , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Cães , Fator XIa/metabolismo , Humanos , Modelos Moleculares , Fenilcarbamatos/administração & dosagem , Fenilcarbamatos/química , Fenilcarbamatos/farmacocinética , Fenilcarbamatos/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética
13.
Bioorg Med Chem Lett ; 26(2): 472-478, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26704266

RESUMO

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8µM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).


Assuntos
Anilidas/farmacologia , Fator XIa/antagonistas & inibidores , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Anilidas/síntese química , Animais , Cristalografia por Raios X , Cães , Fenilalanina/síntese química , Coelhos , Relação Estrutura-Atividade
14.
J Thromb Thrombolysis ; 40(4): 416-23, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26249722

RESUMO

BMS-654457 ((+) 3'-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5'-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90 min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37 mg/kg + 0.27 mg/kg/h produced almost 90 % preservation of iCBF compared to its vehicle (87 ± 10 and 16 ± 3 %, respectively, n = 6 per group) and increased BT by 1.2 ± 0.04-fold (P < 0.05). At a higher dose (1.1 mg/kg + 0.8 mg/kg/h), BMS-654457 increased BT by 1.33 ± 0.08-fold. This compares favorably to equivalent antithrombotic doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) which produced four- to six-fold BT increases in the same model. In summary, BMS-654457 was effective in the prevention of arterial thrombosis in rabbits with limited effects on BT. This study supports inhibition of FXIa, with a small-molecule, reversible and direct inhibitor as a promising antithrombotic therapy with a wide therapeutic window.


Assuntos
Fator XIa/antagonistas & inibidores , Fibrinolíticos/farmacologia , Trombose/tratamento farmacológico , Animais , Tempo de Sangramento , Cães , Fibrinolíticos/química , Humanos , Tempo de Tromboplastina Parcial , Coelhos , Ratos , Especificidade da Espécie , Trombose/sangue
15.
Blood Coagul Fibrinolysis ; 26(8): 893-902, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26192114

RESUMO

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 µmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.


Assuntos
Endodesoxirribonucleases/genética , Fator XII/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Terapia Trombolítica/métodos , Trombose/terapia , Animais , Derivação Arteriovenosa Cirúrgica , Cloretos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ácido Elágico/farmacologia , Endodesoxirribonucleases/metabolismo , Fator XII/genética , Fator XII/metabolismo , Compostos Férricos/farmacologia , Técnicas de Inativação de Genes , Hemorragia/prevenção & controle , Humanos , Fígado/metabolismo , Masculino , Tempo de Tromboplastina Parcial , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Trombina/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologia , Dedos de Zinco/genética
16.
ACS Med Chem Lett ; 6(5): 590-5, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-26005539

RESUMO

Structure-activity relationship optimization of phenylalanine P1' and P2' regions with a phenylimidazole core resulted in a series of potent FXIa inhibitors. Introducing 4-hydroxyquinolin-2-one as the P2' group enhanced FXIa affinity and metabolic stability. Incorporation of an N-methyl piperazine amide group to replace the phenylalanine improved both FXIa potency and aqueous solubility. Combination of the optimization led to the discovery of FXIa inhibitor 13 with a FXIa K i of 0.04 nM and an aPTT EC2x of 1.0 µM. Dose-dependent efficacy (EC50 of 0.53 µM) was achieved in the rabbit ECAT model with minimal bleeding time prolongation.

17.
J Med Chem ; 58(6): 2799-808, 2015 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-25764119

RESUMO

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.


Assuntos
Desenho de Drogas , Fator VIIa/antagonistas & inibidores , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Fator VIIa/metabolismo , Halogênios/química , Halogênios/farmacologia , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Lactamas/metabolismo , Lactamas/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular
18.
Bioorg Med Chem Lett ; 25(7): 1635-42, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25728130

RESUMO

Compound 2 was previously identified as a potent inhibitor of factor XIa lacking oral bioavailability. A structure-based approach was used to design analogs of 2 with novel P1 moieties with good selectivity profiles and oral bioavailability. Further optimization of the P1 group led to the identification of a 4-chlorophenyltetrazole P1 analog, which when combined with further modifications to the linker and P2' group provided compound 32 with FXIa Ki=6.7 nM and modest oral exposure in dogs.


Assuntos
Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Fator XIa/antagonistas & inibidores , Indazóis/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Fator XIa/efeitos dos fármacos , Humanos , Indazóis/administração & dosagem , Indazóis/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
19.
Bioorg Med Chem Lett ; 25(4): 925-30, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25592713

RESUMO

The structure-activity relationships (SAR) of six-membered ring replacements for the imidazole ring scaffold is described. This work led to the discovery of the potent and selective pyridine (S)-23 and pyridinone (±)-24 factor XIa inhibitors. SAR and X-ray crystal structure data highlight the key differences between imidazole and six-membered ring analogs.


Assuntos
Fator XIa/antagonistas & inibidores , Piridinas/farmacologia , Piridonas/farmacologia , Cristalografia por Raios X , Modelos Moleculares , Relação Estrutura-Atividade
20.
Mol Ther Nucleic Acids ; 4: e224, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25625614

RESUMO

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

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