Development of an Ultrasensitive Competitive Double-Enzyme Cascade Fluorescence Signal Amplification Method for Factor XIa Inhibitor Screening.

Coagulation factor XIa (FXIa) is associated with a low risk of bleeding and has been identified as an effective and safe target for the development of novel anticoagulant drugs. In this study, we established an ultrasensitive competitive dual-enzyme cascade signal amplification method for the quantitative analysis and screening of FXIa inhibitors. Due to the specific recognition of FXIa's active site by the aptamer AptE40, the AptE40-QDs-EK recognition probe modified with enterokinase (EK) and the aptamer AptE40, was attached to the MNPs-FXIa capture probe. When FXIa inhibitor was present, it competed with AptE40 for binding to FXIa, resulting in the detachment of AptE40-QDs-EK from MNPs-FXIa. After magnetic separation, the enterokinase of AptE40-QDs-EK in the supernatant hydrolyzed N-terminal hexapeptide of trypsinogen, leading to the production of a large amount of trypsin as part of the first-stage signal cascade amplification. Next, trypsin could hydrolyze the hexameric arginine peptide (RRRRRR, R6), leading to the dissociation of RQDs from the R6-RQDs signal probe; this resulted in a dramatic increase in the fluorescence intensity of the supernatant as the second-stage signal cascade was amplified. The feasibility of the method was investigated using the FXIa inhibitor aptamer FELIAP as a positive model drug. Furthermore, the method was applied to screen the FXIa inhibitors in Eupolyphaga sinensis Walker. Two fractions with more active anticoagulated ingredients were successfully identified and validated via the conventional method, and the results were consistent. The established method provides a key technique for the sensitive detection, high-throughput analysis, and screening of the FXIa inhibitors.

Pharmacokinetics and pharmacodynamics of the factor XIa-inhibiting antibody osocimab in healthy male East Asian volunteers: Results from two phase 1 studies.

The pharmacokinetics, pharmacodynamics, immunogenicity, and safety of osocimab single doses in healthy Chinese and Japanese volunteers over 149 days were evaluated. Two phase 1 single-blinded, placebo-controlled studies with 27 Japanese and 50 Chinese participants were conducted. Osocimab was investigated with IV doses of 0.3, 1.25, and 2.5 mg/kg (Chinese study) and 0.3, 1.25, and 5.0 mg/kg (Japanese study), as well as SC doses of 3.0 and 6.0 mg/kg (Chinese study) and 6.0 mg/kg (Japanese study). The maximum plasma concentration was reached 1-3 h and 4-6 days after IV and SC administration, respectively. Osocimab exhibited a deviation from dose-proportional pharmacokinetics for AUC but not Cmax; higher doses had higher apparent clearance and disproportionately lower total exposure. A slightly lower exposure was observed in Japanese compared with Chinese volunteers after IV administration; conversely, relatively higher exposure in Japanese volunteers with SC dosing was identified. Osocimab was associated with a dose-dependent increase in activated partial thromboplastin time (aPTT). Maximal aPTT prolongations were observed 1-4 h and 2-6 days after IV and SC administration, respectively. Anti-drug antibodies of low titer were detected in 1/9 (11.1%) Japanese volunteers administered placebo and 26/40 (65.0%) Chinese volunteers administered osocimab. Adverse events were reported in 8/18 (44.4%) Japanese and 28/40 (70.0%) Chinese volunteers who received osocimab, as well as in 1/9 (11.1%) Japanese and 6/10 (60.0%) Chinese volunteers who received placebo. In conclusion, data did not suggest a clear dose-proportionality for osocimab within the investigated dose range. The effect of osocimab on aPTT was expected per its mechanism of action. Osocimab was generally well tolerated.

Milvexian, a novel factor XIa inhibitor for stroke prevention: pharmacokinetic and pharmacodynamic evaluation.

INTRODUCTION: Antiplatelets and oral anticoagulants are commonly used to treat patients with various cardiovascular and cerebrovascular diseases. However, the primary concern for clinicians remains the risk of bleeding, thus necessitating the development of new therapies. Milvexian is a new anticoagulant that inhibits factor XIa, preventing the pathological formation of thrombi without increasing bleeding risk. AREAS COVERED: This drug evaluation examines the pharmacokinetic properties of milvexian and provides information on its pharmacodynamics and clinical efficacy in treating some cerebrovascular conditions. EXPERT OPINION: Milvexian shows a good pharmacokinetic profile with low renal elimination rates, justifying its use in patients with a high degree of renal impairment, and without relevant drug-drug interactions. In patients affected by acute non-cardioembolic ischemic stroke or high-risk transient ischemic stroke, milvexian, in addition to dual antiplatelet therapy, seems to have a positive efficacy profile without any safety concerns, especially in terms of intracranial hemorrhage. Two phase 3 trials are ongoing to investigate the efficacy and safety of milvexian for preventing cardioembolic and non-cardioembolic ischemic stroke.

Milvexian vs apixaban for stroke prevention in atrial fibrillation: The LIBREXIA atrial fibrillation trial rationale and design.

BACKGROUND: Direct oral anticoagulants are the standard of care for stroke prevention in eligible patients with atrial fibrillation and atrial flutter; however, bleeding remains a significant concern, limiting their use. Milvexian is an oral Factor XIa inhibitor that may offer similar anticoagulant efficacy with less bleeding risk. METHODS: LIBREXIA AF (NCT05757869) is a global phase III, randomized, double-blind, parallel-group, event-driven trial to compare milvexian with apixaban in participants with atrial fibrillation or atrial flutter. Participants are randomly assigned to milvexian 100 mg or apixaban (5 mg or 2.5 mg per label indication) twice daily. The primary efficacy objective is to evaluate if milvexian is noninferior to apixaban for the prevention of stroke and systemic embolism. The principal safety objective is to evaluate if milvexian is superior to apixaban in reducing the endpoint of International Society of Thrombosis and Hemostasis (ISTH) major bleeding events and the composite endpoint of ISTH major and clinically relevant nonmajor (CRNM) bleeding events. In total, 15,500 participants from approximately 1,000 sites in over 30 countries are planned to be enrolled. They will be followed until both 430 primary efficacy outcome events and 530 principal safety events are observed, which is estimated to take approximately 4 years. CONCLUSION: The LIBREXIA AF study will determine the efficacy and safety of the oral Factor XIa inhibitor milvexian compared with apixaban in participants with either atrial fibrillation or atrial flutter. TRIAL REGISTRATION: ClinicalTrials.gov NCT05757869.

Milvexian: evaluating the factor XIa inhibitor for the treatment of acute coronary syndrome.

INTRODUCTION: Balancing the prevention of thrombosis with bleeding risk when combining anticoagulants and platelet antagonists remains a concern among clinicians, particularly in patients with acute coronary syndrome (ACS) who are treated with potent antiplatelet therapy. This may be because the available antiplatelet and anticoagulants are unable to uncouple physiological hemostasis and pathological thrombosis. Therefore, their use is associated with an unavoidable elevated risk of bleeding. AREAS COVERED: Evidence available from studies evaluating FXIa inhibitors and milvexian was collected from a selective literature search. In this review, the authors describe the potential role of FXI/XIa in experimental thrombosis, evidence for FXIa inhibition in the treatment of clinical thrombotic events, and highlight the current evidence supporting the role of milvexian, a novel FXIa inhibitor, in patients with ACS. EXPERT OPINION: The ongoing LIBREXIA-ACS trial is a large-scale study currently investigating milvexian in patients with ACS. This study may support the proof of concept of differentiating physiological hemostasis and pathological thrombosis and achieving maximum antithrombotic efficacy with minimum bleeding risk when used on top of dual antiplatelet therapy with potent P2Y12 receptor blockers.

Pharmacokinetics, pharmacodynamics, and safety of asundexian in healthy Chinese and Japanese volunteers, and comparison with Caucasian data.

There is an unmet clinical need for effective anticoagulant therapies for the management of thromboembolic diseases that are not associated with a relevant risk of bleeding. Asundexian (BAY 2433334) is an oral, direct, small-molecule inhibitor of activated factor XI (FXIa). Phase I data from healthy Caucasian male participants indicated predictable pharmacokinetic (PK) and pharmacodynamic (PD) profiles and no clinically relevant bleeding-related adverse events (AEs). Reported here are data from two phase I, randomized, placebo-controlled, single- and multiple-dose escalation studies of asundexian conducted in 60 healthy men: 24 Japanese and 36 Chinese. Baseline characteristics were comparable between the treatment groups. All treatment-emergent AEs were mild, with no serious AEs or AEs of special interest reported. Systemic exposure to asundexian increased dose proportionally after single or multiple dosing, with relatively low accumulation following multiple once-daily dosing in both Chinese and Japanese volunteers. Asundexian induced dose-dependent prolongation of activated partial thromboplastin time and inhibition of FXIa activity, with no effects on prothrombin time or FXI concentration in Japanese participants. There were no clinically relevant interethnic differences in PK profile across the Japanese, Chinese, and Caucasian (data from the previous phase I study) participants and no clinically relevant difference in PD response between Japanese and Caucasian participants.

Dual Inhibition of Factor XIIa and Factor XIa Produces a Synergistic Anticoagulant Effect.

ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.

The Safety, Pharmacokinetics, and Pharmacodynamics of SHR2285, an Oral Small Molecule Factor XIa Inhibitor, in Healthy Chinese Volunteers.

BACKGROUND AND OBJECTIVE: There is an unmet need for a safer anticoagulant since bleeding remains a concern with currently approved anticoagulants. Coagulation factor XI (FXI) is an attractive anticoagulant drug target with limited a role in physiological hemostasis. The objective of this study was to evaluate the safety, pharmacokinetics, and pharmacodynamics of SHR2285, a novel small molecule FXIa inhibitor, in healthy Chinese volunteers. METHODS: The study consisted of single ascending doses part (part 1: 25-600 mg) and multiple ascending doses part (part 2: 100, 200, 300, and 400 mg). In both parts, subjects were randomized in a 3:1 ratio to receive SHR2285 or placebo orally. Blood, urine and feces samples were collected to describe its pharmacokinetic and pharmacodynamic profile. RESULTS: In total, 103 healthy volunteers completed the study. SHR2285 was well tolerated. SHR2285 was absorbed rapidly with median time to maximum plasma concentration (Tmax) of 1.50 to 3.00 h. The geometric median half-life (t1/2) of SHR2285 varied from 8.74 to 12.1 h across 25-600 mg single dose. Total systemic exposure of metabolite SHR164471 was approximately 1.77- to 3.61-fold that of the parent drug. The plasma concentration of SHR2285 and SHR164471 reached steady state by the morning of Day 7, with low accumulation ratio (0.956-1.20 and 1.18-1.56, respectively). The increase in pharmacokinetic exposure of SHR2285 and SHR164471 was less than dose proportional. Food has minimal effect on the pharmacokinetics of SHR2285 and SHR164471. SHR2285 produced an exposure-dependent prolongation of activated partial thromboplastin time (APTT) and a decrease in FXI activity. The maximum FXI activity inhibition rate (geometric mean) at steady state was 73.27%, 85.58%, 87.77% and 86.27% for 100-400 mg, respectively. CONCLUSIONS: SHR2285 was generally safe and well tolerated in healthy subjects across a wide range of doses. SHR2285 exhibited a predictable pharmacokinetic profile and an exposure-related pharmacodynamic profile. CLINICALTRIALS: gov Identifier NCT04472819; registered on July 15, 2020.

Clinical Evaluation of Factor XIa Inhibitor Drugs: JACC Review Topic of the Week.

Factor XI/XIa (FXI/FXIa) represents a potential target for improved precision in anticoagulation because it is involved primarily in thrombus formation and plays a much smaller role in clotting and hemostasis. This suggests that the inhibition of FXI/XIa could prevent pathologic thrombi from forming, but largely preserve a patient's ability to clot in response to bleeding or trauma. This theory is supported by observational data showing that patients with congenital FXI deficiency have lower rates of embolic events without an increase in spontaneous bleeding. Small phase 2 trials of FXI/XIa inhibitors have offered encouraging data with regard to bleeding and safety and evidence of efficacy for the prevention of venous thromboembolism. However, larger clinical trials across multiple patient groups are needed for this emerging class of anticoagulants to understand their possible role in clinical use. Here we review the potential clinical indications for FXI/XIa inhibitors, data available to date, and consider future trials.

Pharmacokinetics, pharmacodynamics and safety of BAY 2433334, a novel activated factor XI inhibitor, in healthy volunteers: A randomized phase 1 multiple-dose study.

AIM: To evaluate BAY 2433334, an oral activated factor XI (FXIa) inhibitor, in volunteers. METHODS: Phase 1 study of healthy men at a German centre. Part A: randomized, single-blind, multiple dose-escalation study of BAY 2433334 (25/50/100 mg once daily [OD]) vs. placebo. Part B: similar design to Part A; evaluated BAY 2433334 25 mg twice daily. Part C: nonrandomized, open-label study; evaluated potential interactions between BAY 2433334 (25/75 mg OD) and midazolam (7.5 mg), a CYP3A4 index substrate. Primary variables: treatment-emergent adverse events (TEAEs; Parts A and B); area under the plasma concentration-time curve (AUC) and maximum plasma concentration of midazolam and α-hydroxymidazolam (Part C). STUDY PERIOD: 18 days plus follow-up visit. RESULTS: Parts A and B: 36 participants randomized to BAY 2433334; 12 to placebo. Part C: 48 participants assigned to BAY 2433334 plus midazolam. BAY 2433334 was well tolerated in all study parts. AUC and maximum plasma concentration of BAY 2433334 in plasma appeared dose proportional over 25-100 mg OD, with low-to-moderate variability in pharmacokinetic parameters. Multiple dosing caused minor-to-moderate accumulation and a mean terminal half-life (15.8-17.8 h) supporting once-daily dosing. Dose-dependent FXIa activity inhibition and activated partial thromboplastin time prolongation were observed. BAY 2433334 appeared to have a minor effect on AUC for midazolam (ratio [90% confidence interval]: 1.1736 [1.0963-1.2564]) and α-hydroxymidazolam (0.9864 [0.9169-1.0612]) only for BAY 2433334 75 mg OD on day 10. CONCLUSION: Multiple dosing of BAY 2433334 in healthy volunteers was well tolerated, with a predictable pharmacokinetic/pharmacodynamic profile and no clinically relevant CYP3A4 induction or inhibition.

Persistence Pays Off: Milvexian Emerges from the Industry's Longstanding Search for Orally Bioavailable Factor XIa Inhibitors.

Over the past two decades, Factor XIa inhibitors have emerged as an exciting new class of antithrombic agents. Identification of orally bioavailable small molecule inhibitors has presented a formidable challenge for medicinal chemists. Herein, those challenges and the problem-solving that resulted in the discovery of milvexian will be presented.

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Milvexian for the Prevention of Venous Thromboembolism.

BACKGROUND: Factor XIa inhibitors for the prevention and treatment of venous and arterial thromboembolism may be more effective and result in less bleeding than conventional anticoagulants. Additional data are needed regarding the efficacy and safety of milvexian, an oral factor XIa inhibitor. METHODS: In this parallel-group, phase 2 trial, we randomly assigned 1242 patients undergoing knee arthroplasty to receive one of seven postoperative regimens of milvexian (25 mg, 50 mg, 100 mg, or 200 mg twice daily or 25 mg, 50 mg, or 200 mg once daily) or enoxaparin (40 mg once daily). The primary efficacy outcome was venous thromboembolism (which was a composite of asymptomatic deep-vein thrombosis, confirmed symptomatic venous thromboembolism, or death from any cause). The principal safety outcome was bleeding. RESULTS: Among the patients receiving milvexian twice daily, venous thromboembolism developed in 27 of 129 (21%) taking 25 mg, in 14 of 124 (11%) taking 50 mg, in 12 of 134 (9%) taking 100 mg, and in 10 of 131 (8%) taking 200 mg. Among those receiving milvexian once daily, venous thromboembolism developed in 7 of 28 (25%) taking 25 mg, in 30 of 127 (24%) taking 50 mg, and in 8 of 123 (7%) taking 200 mg, as compared with 54 of 252 patients (21%) taking enoxaparin. The dose-response relationship with twice-daily milvexian was significant (one-sided P<0.001), and the 12% incidence of venous thromboembolism with twice-daily milvexian was significantly lower than the prespecified benchmark of 30% (one-sided P<0.001). Bleeding of any severity occurred in 38 of 923 patients (4%) taking milvexian and in 12 of 296 patients (4%) taking enoxaparin; major or clinically relevant nonmajor bleeding occurred in 1% and 2%, respectively; and serious adverse events were reported in 2% and 4%, respectively. CONCLUSIONS: Postoperative factor XIa inhibition with oral milvexian in patients undergoing knee arthroplasty was effective for the prevention of venous thromboembolism and was associated with a low risk of bleeding. (Funded by Bristol Myers Squibb and Janssen Research and Development; AXIOMATIC-TKR ClinicalTrials.gov number, NCT03891524.).

Development of Selective FXIa Inhibitors Based on Cyclic Peptides and Their Application for Safe Anticoagulation.

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.

Design, synthesis and biological evaluation of novel FXIa inhibitors with 2-phenyl-1H-imidazole-5-carboxamide moiety as P1 fragment.

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.

Dual inhibition of factor XIIa and factor XIa as a therapeutic approach for safe thromboprotection.

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.

Discovery of Benzyl Tetraphosphonate Derivative as Inhibitor of Human Factor Xia.

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.

Osocimab: A Novel Agent in Preventing Venous Thromboembolism.

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.

Studies on fragment-based design of allosteric inhibitors of human factor XIa.

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.