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1.
Nat Protoc ; 16(8): 3981-4003, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34215864

RESUMO

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.


Assuntos
Anticoagulantes/farmacologia , Fator XIa/metabolismo , Trombina/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos
2.
Thromb Res ; 204: 1-8, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089982

RESUMO

BACKGROUND: Cardiac arrest and cardiopulmonary resuscitation (CPR) are associated with activated coagulation and microvascular fibrin deposition with subsequent multiorgan failure and adverse outcome. OBJECTIVES: Activated Factor XI-antithrombin (FXIa-AT) complex, activated Factor IX-antithrombin (FIXa-AT) complex and thrombin-antithrombin (TAT) complex were measured as markers of coagulation activation, and evaluated as independent prognostic indicators in out-of-hospital cardiac arrest (OHCA) patients. METHODS: From February 2007 until December 2010 blood samples were collected in close approximation to CPR from patients with OHCA of assumed cardiac origin. Follow-up samples in survivors were drawn 8-12 h and 24-48 h after hospital admission. All measurements were determined by ELISA. RESULTS: Thirty-seven patients presented with asystole and 77 with ventricular fibrillation as first recorded heart rhythm. At 30-days follow-up, 70 patients (61.4%) had died. All patients had elevated levels of FXIa-AT complex, FIXa-AT complex and TAT. Initial levels were significantly higher in non-survivors compared to 30-days survivors. A significant increase in risk of 30-days all-cause mortality was observed through increasing quartiles of all three biomarkers in univariate Cox regression analysis. Compared to the lowest quartile (Q1), only FXIa-AT complex levels in Q3 (HR 3.17, p = 0.011) and Q2 (HR 3.02, p = 0.016) were independently associated with all-cause mortality in the multivariable analysis. FIXa-AT complex and TAT-complex did not behave as independent predictors. CONCLUSIONS: Complexes of FXIa-AT were independently associated with 30-days survival in OHCA-patients. CLINICAL TRIAL REGISTRATION: ClinicalTrials. gov, NCT02886273.


Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca Extra-Hospitalar , Antitrombinas , Fator XIa , Humanos , Prognóstico
3.
J Chromatogr A ; 1651: 462318, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34161834

RESUMO

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Fator XIa/isolamento & purificação , Preparações Farmacêuticas/isolamento & purificação , Água/química , Acetonitrilas , Amônia/química , Cromatografia Líquida de Alta Pressão , Fator XIa/química , Isoquinolinas/química , Metanol/química , Preparações Farmacêuticas/química , Estereoisomerismo , para-Aminobenzoatos/química
4.
J Thromb Haemost ; 19(10): 2407-2416, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34192419

RESUMO

BACKGROUND: Coagulation factor XI (FXI) contributes to the development of thrombosis but appears to play a minor role in hemostasis and is, therefore, an attractive anticoagulant drug target. OBJECTIVES: To evaluate the safety, pharmacokinetic, and pharmacodynamic properties of BAY 2433334, an orally administered small molecule targeting activated FXI (FXIa), in healthy men. PATIENTS/METHODS: This phase 1 study was conducted in two parts. In part 1, 70 volunteers were randomized 4:1 to receive a single oral dose of BAY 2433334 (5-150 mg as oral solution or immediate-release tablets) or placebo. In part 2, 16 volunteers received a single oral dose of five BAY 2433334 5-mg tablets with or without a high-calorie breakfast in a randomized crossover study design. Adverse events, pharmacokinetic parameters, and pharmacodynamic parameters were assessed up to 72 h after drug administration. Volunteers were followed up after 7 to 14 days. RESULTS: BAY 2433334 demonstrated favorable safety and tolerability with a dose-dependent increase in exposure and a terminal half-life of 14.2 to 17.4 h. A high-calorie breakfast reduced mean maximum plasma concentration and exposure by 31% and 12.4%, respectively. AY 2433334 was associated with a dose-dependent inhibition of FXIa activity and an increase in activated partial thromboplastin time. Bleeding times in volunteers who had received BAY 2433334 were similar to those in volunteers who had received placebo. CONCLUSIONS: These data indicate that BAY 2433334 is a promising development candidate for once-daily oral anticoagulation; it is being evaluated in phase 2 dose-finding studies in patients at risk of thrombosis.


Assuntos
Anticoagulantes , Fator XIa , Administração Oral , Área Sob a Curva , Estudos Cross-Over , Método Duplo-Cego , Humanos , Masculino , Tempo de Tromboplastina Parcial
5.
J Med Chem ; 64(10): 6802-6813, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33974422

RESUMO

Coagulation factor XI (FXI) has emerged as a promising target for the development of safer anticoagulation drugs that limit the risk of severe and life-threatening bleeding. Herein, we report the first cyclic peptide-based FXI inhibitor that selectively and potently inhibits activated FXI (FXIa) in human and animal blood. The cyclic peptide inhibitor (Ki = 2.8 ± 0.5 nM) achieved anticoagulation effects that are comparable to that of the gold standard heparin applied at a therapeutic dose (0.3-0.7 IU/mL in plasma) but with a substantially broader estimated therapeutic range. We extended the plasma half-life of the peptide via PEGylation and demonstrated effective FXIa inhibition over extended periods in vivo. We validated the anticoagulant effects of the PEGylated inhibitor in an ex vivo hemodialysis model with human blood. Our work shows that FXI can be selectively targeted with peptides and provides a promising candidate for the development of a safe anticoagulation therapy.


Assuntos
Anticoagulantes/química , Fator XIa/antagonistas & inibidores , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator XIa/metabolismo , Meia-Vida , Humanos , Isomerismo , Modelos Biológicos , Tempo de Tromboplastina Parcial , Biblioteca de Peptídeos , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Polietilenoglicóis/química , Coelhos , Diálise Renal
6.
Eur J Med Chem ; 220: 113437, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33894565

RESUMO

Factor XIa, as a blood coagulation enzyme, amplifies the generation of the last enzyme thrombin in the blood coagulation cascade. It was proved that direct inhibition of factor XIa could reduce pathologic thrombus formation without an enhanced risk of bleeding. WSJ-557, a nonpurine imidazole-based xanthine oxidase inhibitor in our previous reports, could delay blood coagulation during its animal experiments, which prompted us to investigate its action mechanism. Subsequently, during the exploration of the action mechanism, it was found that WSJ-557 exhibited weak in vitro factor XIa binding affinity. Under the guide of molecular modeling, we adopted molecular hybridization strategy to develop novel factor XIa inhibitors with WSJ-557 as an initial compound. This led to the identification of the most potent compound 44g with a Ki value of 0.009 µM, which was close to that of BMS-724296 (Ki = 0.0015 µM). Additionally, serine protease selectivity study indicated that compound 44g display a desired selectivity, more 400-fold than those of thrombin, factor VIIa and factor Xa in coagulation cascade. Moreover, enzyme kinetics studies suggested that the representative compound 44g acted as a competitive-type inhibitor for FXIa, and molecular modeling revealed that it could tightly bind to the S1, S1' and S2' pockets of factor XIa. Furthermore, in vivo efficacy in the rabbit arteriovenous shunt model suggested that compound 44g demonstrated dose-dependent antithrombotic efficacy. Therefore, these results supported that compound 44g could be a potential and efficacious agent for the treatment of thrombotic diseases.


Assuntos
Desenho de Fármacos , Fator XIa/antagonistas & inibidores , Inibidores do Fator Xa/farmacologia , Relação Dose-Resposta a Droga , Fator XIa/metabolismo , Inibidores do Fator Xa/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
7.
J Immunol ; 206(8): 1784-1792, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33811105

RESUMO

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.


Assuntos
Plaquetas/metabolismo , Fator H do Complemento/metabolismo , Células Endoteliais/metabolismo , Fator XIa/metabolismo , Inflamação/metabolismo , Animais , Coagulação Sanguínea , Complemento C3b/metabolismo , Via Alternativa do Complemento , Fibrinogênio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Papio , Ligação Proteica , Receptor Cross-Talk
8.
J Biotechnol ; 330: 61-69, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33689867

RESUMO

The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.


Assuntos
Precursor de Proteína beta-Amiloide , Fator XIa , Animais , Fibrinolisina , Camundongos , Inibidores de Proteases , Saccharomycetales , Ativador de Plasminogênio Tecidual/genética
9.
Thromb Res ; 200: 16-22, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33513452

RESUMO

BACKGROUND: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification. METHODS: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems. RESULTS: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma. CONCLUSION: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing.


Assuntos
Fator XIa , Trombose , Fator XIa/metabolismo , Humanos , Cinética
10.
Am J Physiol Cell Physiol ; 320(3): C365-C374, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33471623

RESUMO

Factor XI (FXI) has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, whereas FXI is not essential for hemostasis but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in a purified system and increased coagulation Factor IX (FIX) activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by activated coagulation factor XII (FXIIa) and the activation of FXII by kallikrein (PKa). Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets, the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa [by FXIa-platelet membrane immunoprecipitation] showed that the interaction with platelets is zinc dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib. FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.


Assuntos
Plaquetas/metabolismo , Fator XIa/metabolismo , Adolescente , Precursor de Proteína beta-Amiloide/metabolismo , Sítios de Ligação/fisiologia , Coagulação Sanguínea/fisiologia , Feminino , Hemostasia/fisiologia , Humanos , Masculino , Trombina/metabolismo , Trombose/metabolismo
11.
Vox Sang ; 116(6): 656-664, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33277936

RESUMO

BACKGROUND AND OBJECTIVES: In 2010, an intravenous immunoglobulin (IVIG) product was removed from the market due to an association with serious thromboembolic events. Investigations revealed that factor XIa (FXIa) was present as a process-related impurity. This study investigated the ability of two commercial FXIa assays to measure FXIa in immunoglobulin preparations and conducted a survey of FXIa activity in marketed immunoglobulin products. MATERIALS AND METHODS: Factor XIa assays were modified to include spiking of samples with FXIa before testing. An immunoglobulin product and its excipient were used to assess the ability of the assays to recover the spiked FXIa levels. RESULTS: The Biophen FXIa assay required a high pre-dilution of the sample to obtain statistically valid results and complete FXIa recovery. The ROX FXIa assay was more sensitive, giving statistically valid results at a lower sample pre-dilution and FXIa spike level. This modified ROX FXIa assay was used to assay 17 lots of immunoglobulin products for FXIa. Two product lots had measurable FXIa levels without the need for spiking. A further 3 lots produced detectable but not statistically valid FXIa results when left unspiked. Spiking produced statistically valid assays and recoveries above 100%, demonstrating inherent FXIa. CONCLUSION: This study shows marketed immunoglobulin products can contain detectable levels of FXIa. Spiking brings the FXIa levels into the quantifiable range of the assay, allowing measurement of inherent FXIa. Accurate measurement is important to inform on 'safe' levels of FXIa in these products and allow future safety guidelines to be set.


Assuntos
Fator XIa , Imunoglobulinas Intravenosas , Tromboembolia , Testes de Coagulação Sanguínea , Fator XIa/análise , Humanos
12.
J Thromb Haemost ; 19(2): 323-329, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33047454

RESUMO

Clinical practice shows that a critical unmet need in the field of medical device-associated thrombosis prevention is the availability of an anticoagulant therapy without hemorrhagic risk. In the quest for new drugs that are at least as effective as those currently available, while avoiding bleeding complications, molecules that target nearly every step of the coagulation pathway have been developed. Among these molecules, inhibitors of factor XII (FXII) or factor XI (FXI) are promising alternatives as deficiencies in these factors protect against thrombosis without causing spontaneous hemorrhage, as revealed by epidemiological and preclinical data. Ixodes ricinus-contact phase inhibitor (Ir-CPI), a new anticoagulant candidate with an innovative mechanism of action could be this ideal anticoagulant agent for safe prevention from clotting on medical devices. This protein, which selectively binds to FXIIa, FXIa, and plasma kallikrein and inhibits the reciprocal activation of FXII, prekallikrein, and FXI in human plasma, was shown to prevent thrombosis in an ovine cardiopulmonary bypass system associated with cardiac surgeries. Furthermore, as opposed to unfractionated heparin, Ir-CPI appears to be devoid of bleeding risk. This review outlines the rationale for targeting upstream coagulation factors in order to prevent medical device-associated thrombosis; examines the novel approaches under development; and focuses on Ir-CPI, which shows promising properties in the field of thrombosis prevention.


Assuntos
Fator XIIa , Fator XIa , Trombose/prevenção & controle , Animais , Coagulação Sanguínea , Fator XI , Fator XII , Fator XIIa/antagonistas & inibidores , Fator XIa/antagonistas & inibidores , Heparina , Humanos , Ovinos
13.
ChemistryOpen ; 9(11): 1161-1172, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33204588

RESUMO

The inhibition of factor XIa (FXIa) is a trending paradigm for the development of new generations of anticoagulants without a substantial risk of bleeding. In this report, we present the discovery of a benzyl tetra-phosphonate derivative as a potent and selective inhibitor of human FXIa. Biochemical screening of four phosphonate/phosphate derivatives has led to the identification of the molecule that inhibited human FXIa with an IC50 value of ∼7.4 µM and a submaximal efficacy of ∼68 %. The inhibitor was at least 14-fold more selective to FXIa over thrombin, factor IXa, factor Xa, and factor XIIIa. It also inhibited FXIa-mediated activation of factor IX and prolonged the activated partial thromboplastin time of human plasma. In Michaelis-Menten kinetics experiment, inhibitor 1 reduced the VMAX of FXIa hydrolysis of a chromogenic substrate without significantly affecting its KM suggesting an allosteric mechanism of inhibition. The inhibitor also disrupted the formation of FXIa - antithrombin complex and inhibited thrombin-mediated and factor XIIa-mediated formation of FXIa from its zymogen factor XI. Inhibitor 1 has been proposed to bind to or near the heparin/polyphosphate-binding site in the catalytic domain of FXIa. Overall, inhibitor 1 is the first benzyl tetraphosphonate small molecule that allosterically inhibits human FXIa, blocks its physiological function, and prevents its zymogen activation by other clotting factors under in vitro conditions. Thus, we put forward benzyl tetra-phosphonate 1 as a novel lead inhibitor of human FXIa to guide future efforts in the development of allosteric anticoagulants.


Assuntos
Anticoagulantes/farmacologia , Fator XIa/antagonistas & inibidores , Organofosfonatos/farmacologia , Sítio Alostérico , Anticoagulantes/metabolismo , Ensaios Enzimáticos , Fator XIa/química , Fator XIa/metabolismo , Humanos , Simulação de Acoplamento Molecular , Organofosfonatos/metabolismo , Tempo de Tromboplastina Parcial , Ligação Proteica , Tempo de Trombina
14.
J Cardiovasc Pharmacol ; 76(6): 645-649, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33105325

RESUMO

The nature of orthopedic surgery, and specifically total knee arthroplasty, lends itself to the development of venous thromboembolism given endothelial injury from the surgical procedure, promotion of an acute hypercoagulable state, and the prolonged period of immobilization after surgery promoting stasis; all factors of Virchow's triad. Current guidelines recommend the direct acting oral anticoagulants, enoxaparin, fondaparinux, and warfarin as options for venous thromboembolism prevention. However, these agents may still be prone to unacceptable bleeding risk, given they mostly target the extrinsic pathway of the clotting cascade, and have other characteristics which can be problematic for use. Investigators have determined patients with factor XI deficiency seem to be protected for thrombotic risk and seem to be devoid of bleeding sequelae. This has led to the development of osocimab, a fully humanized monoclonal G1 antibody designed specifically to functionally neutralize factor XIa. Phase 1 clinical trials have demonstrated an agent with a long half-life (∼30 days) with minimal requirement of renal elimination and hepatic metabolism. Phase 2 trials have identified that an optimal dose range, 0.6-1.2 mg/kg, as a 1-time dose preoperatively or postoperatively is effective in preventing thrombotic complications with minimal bleeding risk compared with standard of care for elective total knee arthroplasty patients. Future clinical development will be able to clearly outline the role this agent will play in the future.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticoagulantes/uso terapêutico , Artroplastia do Joelho/efeitos adversos , Fator XIa/antagonistas & inibidores , Tromboembolia Venosa/prevenção & controle , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Anticoagulantes/efeitos adversos , Anticoagulantes/farmacocinética , Hemorragia/induzido quimicamente , Humanos , Medição de Risco , Fatores de Risco , Resultado do Tratamento , Tromboembolia Venosa/etiologia
15.
Front Immunol ; 11: 2072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922409

RESUMO

A dysregulated immune response with hyperinflammation is observed in patients with severe coronavirus disease 2019 (COVID-19). The aim of the present study was to assess the safety and potential benefits of human recombinant C1 esterase inhibitor (conestat alfa), a complement, contact activation and kallikrein-kinin system regulator, in severe COVID-19. Patients with evidence of progressive disease after 24 h including an oxygen saturation <93% at rest in ambient air were included at the University Hospital Basel, Switzerland in April 2020. Conestat alfa was administered by intravenous injections of 8400 IU followed by 3 additional doses of 4200 IU in 12-h intervals. Five patients (age range, 53-85 years; one woman) with severe COVID-19 pneumonia (11-39% lung involvement on computed tomography scan of the chest) were treated a median of 1 day (range 1-7 days) after admission. Treatment was well-tolerated. Immediate defervescence occurred, and inflammatory markers and oxygen supplementation decreased or stabilized in 4 patients (e.g., median C-reactive protein 203 (range 31-235) mg/L before vs. 32 (12-72) mg/L on day 5). Only one patient required mechanical ventilation. All patients recovered. C1INH concentrations were elevated before conestat alfa treatment. Levels of complement activation products declined after treatment. Viral loads in nasopharyngeal swabs declined in 4 patients. In this uncontrolled case series, targeting multiple inflammatory cascades by conestat alfa was safe and associated with clinical improvements in the majority of severe COVID-19 patients. Controlled clinical trials are needed to assess its safety and efficacy in preventing disease progression.


Assuntos
Betacoronavirus/efeitos dos fármacos , Proteína Inibidora do Complemento C1/uso terapêutico , Complemento C1/antagonistas & inibidores , Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Sistema Calicreína-Cinina/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Proteína Inibidora do Complemento C1/análise , Fator XIa/antagonistas & inibidores , Feminino , Humanos , Calicreínas/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Pandemias , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2 , Carga Viral/efeitos dos fármacos
16.
Bioorg Med Chem ; 28(23): 115762, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32992249

RESUMO

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa's active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.


Assuntos
Desenho de Fármacos , Fator XIa/antagonistas & inibidores , Inibidores de Serino Proteinase/química , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Dimerização , Fator XIa/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Quinazolinonas/química , Quinazolinonas/metabolismo , Quinazolinonas/farmacologia , Inibidores de Serino Proteinase/metabolismo , Relação Estrutura-Atividade , Sulfatos/química
17.
J Med Chem ; 63(15): 8088-8113, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32551603

RESUMO

The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.


Assuntos
Fator XIa/antagonistas & inibidores , Fator XIa/genética , Inibidores do Fator Xa/administração & dosagem , Inibidores do Fator Xa/química , Administração Oral , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
18.
Thromb Haemost ; 120(6): 883-993, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32375196

RESUMO

Cardiovascular disease, including stroke, myocardial infarction, and venous thromboembolism, is one of the leading causes of morbidity and mortality worldwide. Excessive coagulation may cause vascular occlusion in arteries and veins eventually leading to thrombotic diseases. Studies in recent years suggest that coagulation factors are involved in these pathological mechanisms. Factors XIa (FXIa), XIIa (FXIIa), and plasma kallikrein (PKa) of the contact system of coagulation appear to contribute to thrombosis while playing a limited role in hemostasis. Contact activation is initiated upon autoactivation of FXII on negatively charged surfaces. FXIIa activates plasma prekallikrein (PK) to PKa, which in turn activates FXII and initiates the kallikrein-kinin pathway. FXI is also activated by FXIIa, leading to activation of FIX and finally to thrombin formation, which in turn activates FXI in an amplification loop. Animal studies have shown that arterial and venous thrombosis can be reduced by the inhibition of FXI(a) or PKa. Furthermore, data from human studies suggest that these enzymes may be valuable targets to reduce thrombosis risk. In this review, we discuss the structure and function of FXI(a) and PK(a), their involvement in the development of venous and arterial thrombosis in animal models and human studies, and current therapeutic strategies.


Assuntos
Arteriopatias Oclusivas/sangue , Fator XIa/fisiologia , Calicreína Plasmática/fisiologia , Trombose/sangue , Animais , Coagulação Sanguínea/fisiologia , Transtornos da Coagulação Sanguínea/sangue , Fatores de Coagulação Sanguínea/fisiologia , Modelos Animais de Doenças , Ativação Enzimática , Deficiência do Fator XI/sangue , Fator XIa/química , Fator XIa/imunologia , Inibidores do Fator Xa/uso terapêutico , Humanos , Camundongos , Camundongos Knockout , Pré-Calicreína/deficiência , Pré-Calicreína/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade da Espécie , Trombofilia/tratamento farmacológico , Trombose Venosa/sangue
19.
Blood ; 136(6): 740-748, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32369559

RESUMO

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator IX/administração & dosagem , Fator X/administração & dosagem , Hemofilia A/tratamento farmacológico , Hemorragia/tratamento farmacológico , Modelos Animais , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/imunologia , Quimioterapia Combinada , Fator IX/análise , Fator IX/imunologia , Fator VIII/administração & dosagem , Fator VIII/análise , Fator VIII/uso terapêutico , Fator X/análise , Fator X/imunologia , Fator XIa/farmacologia , Feminino , Hemofilia A/sangue , Hemofilia A/complicações , Hemofilia A/imunologia , Hemorragia/etiologia , Infusões Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tempo de Tromboplastina Parcial , Cauda/lesões , Trombina/biossíntese
20.
J Med Chem ; 63(13): 7226-7242, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32456431

RESUMO

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.


Assuntos
Fator XIa/antagonistas & inibidores , Fibrinolíticos/administração & dosagem , Fibrinolíticos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Fator XIa/química , Fator XIa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Compostos Macrocíclicos/administração & dosagem , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Coelhos , Relação Estrutura-Atividade
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