Factor XIa (IC50 = 2.8 nM); tryptase (IC50 = 5 nM)

Compared to trypsin, urokinase, plasma kallikrein, plasmin, thrombin (factor IIa), and factor IXa (IC50=0.005, 0.05, 0.542, 0.55)—more than 70 times greater than 1.7, 10.5, and 17.4 μM, respectively—BMS-262084 is more selective for human factor XIa (IC50=2.8 nM) [1]. The activated thromboplastin time in human and rat plasma (concentrations of 0.14 and 2.2 μM) is doubled by BMS-262084 (1-100 μM) [1].

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In vitro selectivity [1]
BMS-262084 was a potent inhibitor of human factor XIa (IC50 = 2.8 nM) and human tryptase (IC50 = 5 nM) as shown in Table 1. It was at least 70-fold more selective for factor XIa compared to other serine proteases involved in coagulation (thrombin, factor Xa, factor IXa, factor XIIa, tissue factor: factor VIIa) and fibrinolysis (tissue plasminogen activator, urokinase, plasmin), but was weakly active against trypsin (IC50 = 50 nM). When compared to isolated enzymes, BMS-262084 was less potent in plasma based clotting times, however its activity was consistent with inhibition of intrinsic (activated partial thromboplastin time) and not extrinsic (prothrombin time) coagulation. In rat plasma, BMS-262084 doubled the activated partial thromboplastin time at a concentration of 2.2 ± 0.5 μM (Fig. 2). It had no effect on the prothrombin time which could be prolonged by activity against factor VIIa, factor Xa or thrombin. BMS-262084 was 15 times more potent in human plasma where the activated partial thromboplastin time was doubled at a concentration of 0.14 ± 0.02 μM (n = 5). The prothrombin time in human plasma was unaffected by BMS-262084 concentrations of 1, 10 and100 μM (maximum change ± 5%, n = 3).

Rats with carotid artery thrombus weight reduction, improved vascular patency, and integrated blood flow are treated with BMS-262084 (2-12 mg/kg + 2-12 mg/kg/h; iv) [1].

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Selective anticoagulation in response to BMS-262084 was also observed ex vivo in rats with dose-dependent prolongation of the activated partial thromboplastin time, but not the prothrombin time (Fig. 2). No significant effects on clotting times were observed with vehicle infusion (not shown). The increase in activated partial thromboplastin time at each BMS-262084 dose level varied at most by 17% over the 5 to 90 min infusion, and therefore clotting time increases averaged over 5 to 90 min were used to construct the dose-response. Significant prolongation of activated partial thromboplastin time was observed at all doses studied, but the anticoagulant effect peaked at 3.0 to 3.2 times control at doses (mg/kg + mg/kg/h) of 6 + 6 ,12 + 12 and 24 + 24. This plateau was not observed in vitro, where the clotting time increased up to 5.9 times control in rat plasma (Fig. 2). In human plasma the activated partial thromboplastin time was increased to 6.9 times control at a 5-μM concentration of BMS-262084.
Aggregation of rat platelets measured by the impedance method in whole blood without coagulation (low calcium) was used to test for in vitro effects on platelet function. We first determined that a collagen concentration of 20 μg/ml elicited a half maximal peak aggregation response of 28 ± 1 Ω (n = 3). This collagen response was unaffected by BMS-262084 concentrations of 0.5 μM (27 ± 3 Ω, n = 3), 2 μM (31 ± 1 Ω, n = 3) and 100 μM (30 ± 2 Ω, n = 3). [1]

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Antithrombotic activity [1]
The 12 mg/kg + 12 mg/kg/h dose of BMS-262084 administered as a pre-treatment reduced carotid artery thrombus weight by 73 ± 5%, and it improved both vessel patency and integrated flow (Fig. 3). Lower dose levels were not effective. In other experiments administration of vehicle or BMS-262084 was delayed 10 min, at which time thrombi had reached 25% of the final weight observed in vehicle-treated rats (Fig. 4). Using this post-treatment regimen, BMS-262084 produced a 80 ± 3% thrombus weight reduction, and prevented occlusion in all drug-treated rats. This effect amounted to prevention of further thrombus growth from the time of BMS-262084 treatment.

BMS-262084 given as a pre-treatment was also efficacious in FeCl2-induced vena cava thrombosis (Fig. 5). Greater potency and efficacy were observed in the venous compared to arterial model. Vena cava thrombus weight reductions ranged from 38 ±9% at 0.2 mg/kg + 0.2 mg/kg/h up to 97 ± 2% at 12 mg/kg +12 mg/kg/h. The 2 mg/kg + 2 mg/kg/h dose, which produced a 78 ± 5% thrombus reduction, was also tested in a post-treatment regimen. In control rats vena cava thrombi removed 20 min after the initial application of FeCl2 weighed 11.0 ± 0.5 mg (n = 5). When the test articles were administered at this time, significant growth of venous thrombi growth was observed in vehicle treated rats (26.5 ±1.0 mg, n = 5, P < 0.05 compared to control) but not in BMS-262084-treated rats (14.4 ± 2.1 mg, n = 7; not significant compared to control).

When vena cava thrombosis was induced by infusion of tissue factor, BMS-262084 was inactive at dose levels up to 24 mg/kg + 24 mg/kg/h (Fig. 5).

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Effect on provoked bleeding times [1]
BMS-262084 dose levels (mg/kg + mg/kg/h) of 6 + 6, 12 + 12 and 24 + 24 did not prolong either cuticle, mesenteric or renal bleeding times compared to vehicle treatment (Fig. 6). Heparin was therefore selected as a positive control, testing a dose which matched the maximum systemic anticoagulant effect of BMS-262084.

Heparin infused at 33 units/kg plus 60 units/kg/h increased the activated partial thromboplastin time to 3.16 ± 0.15 times control (n = 9), which equaled the 3.19-fold increase achieved at the highest dose of BMS-262084 (24 mg/kg + 24 mg/kg/h). This heparin dose produced significant effects (P < 0.05) in all three bleeding models. The relative prolongation of bleeding in response to heparin amounted to less than a doubling in the cuticle model (1.62 ± 0.21-fold increase of 179 ± 11 to 280 ± 29 s, n = 9), the mesenteric model (1.64 ± 0.12-fold increase of 53 ± 6 to 89 ± 14 s, n = 5) and the renal model (1.33 ± 0.09-fold increase of 75 ± 4 to 98 ± 4 s, n = 11).

Selectivity against human serine proteases [1]
Factor XIa activity and all other enzyme assays were measured at room temperature in 96-well microplates using a 3-min incubation of enzyme (0.5 nM for factor XIa) with BMS-262084 in a buffered solution (for factor XIa this was 145 mM NaCl, 5 mM KCl, 1 mg/ml PEG 8000, 30 mM HEPES at pH 7.4). After incubation the appropriate synthetic substrate was added to start the reaction (100 μM S-2366 for factor XIa having Km = 86 μM). Enzyme velocity was measured at 405 nm in a Spectro Max Plus plate reader operated in kinetic mode and analyzed using the associated SOFTmax Pro® software. The BMS-262084 concentration producing 50% inhibition (IC50) was calculated by XLfit using data from at least 3 separate experiments.
Applying the method described above, enzymatic activity of human α-thrombin (0.03 U/ml) was measured in 0.145 M NaCl, 0.005 M KCl, 1 mg/ml polyethylene glycol (PEG-8000), 0.030 M HEPES (pH 7.4) using 10 μM S-2238 (Km = 2.54 μM). Factor Xa (0.033 U/ml), tissue plasminogen activator (3703 U/ml), and urokinase (111 U/ml) were assayed in the same buffer as α-thrombin using 100 μM of S-2222 (Km = 87 μM), spectrozyme tissue plasminogen activator (Km = 90 μM), and S-2444 (Km = 31 μM), respectively. Plasmin activity (0.23 nM) was measured in 50 mM Tris (pH 7.8) using 100 μM S-2251 (Km = 98 μM). The factor XIIa (15 nM) assay included 150 mM NaCl, 50 mM Tris (pH 8.2), 50 mM imidazole and 100 μM spectrozyme-FXIIa (Km = 40.2 μM). Factor IXa (20 nM) activity was measured in 100 mM NaCl, 5 mM CaCl2, 33% ethylene glycol, 50 mM Tris (pH 7.5) with 100 μM spectrozyme-factor IXa (Km > 100 μM). Activity of tissue factor complexed to factor VIIa was measured using factor VIIa (1 nM) with equimolar tissue factor in 20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 1 mM CHAPS and 1 mg/ml PEG 6000 (pH to 7.4) and using 100 μM S-2288 (Km > 500 μM). The kallikrein (5 nM) assay utilized 50 mM Tris (pH 8.2), 50 mM imidazole and 150 mM NaCl with 50 μM Spectrozyme-plasma kallikrein (Km = 15.2 μM). Trypsin (0.33 μg/mL) activity was measured in 50 mM Tris (pH 8.0) and 2 mM CaCl2 with 100 μM Chromozyme-TRY (Km = 26.6 μM). The tryptase (0.67 nM) assay included 100 mM Tris (pH 8.0), 200 mM NaCl, and 100 μg/ml low molecular weight recombinant heparin with 200 μM Z-gly-pro-arg-AMC (Km = 237 μM).

In vitro clotting time and platelet function assays [1]
The activated partial thromboplastin time and prothrombin time were measured in plasma using a BBL fibrometer and the procedure described for Dade Actin FSL and Dade Thromboplastin-C reagents, respectively. The international sensitivity index of the prothrombin time reagent was 2.0. The aggregation response of rat platelets to 20 μg/ml collagen was determined in whole blood using the impedance procedure and reagents described for a Model 560-CA Chrono-Log aggregometer. Plasma and blood samples were from freshly drawn arterial blood collected into 1/10 vol of 3.8% Na-citrate obtained from anesthetized rats as described in Section 2.4, or by venipuncture from consenting human volunteers.

Animal/Disease Models: Male Sprague Dawley rats (310-390 g) were treated with FeCl2[1] to induce venous thrombosis at doses: 2 mg/kg + 2 mg/kg/h, 6 mg/kg + 6 mg/kg/h, 12 mg/kg + 12 mg/kg/h
Route of Administration: intravenous (iv) (iv)injection 10 minutes before administration of FeCl2
Experimental Results: At doses of 12 mg/kg + 12 mg/kg/h, carotid artery thrombus weight was diminished by 73%. Improves vascular patency and overall flow.\n

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\n\nAnimal preparation and dosing for in vivo studies [1]
\nMale Sprague Dawley rats (310 to 390 g) were obtained from Harlan labs and all experimental procedures conducted on these animals were approved by our Institutional Animal Care and Use Committee. Rats were anesthetized with Na-pentobarbital (50 mg/kg i.p.) and the left jugular vein was cannulated with polyethylene-50 tubing for drug administration and the trachea was intubated with polyethylene-205 tubing to ensure airway patency. In some animals a carotid artery was cannulated with polyethylene-50 tubing to obtain blood samples.
\nBMS-262084 was dosed as an intravenous infusion due to its limited duration in rats. Test articles were administered as a 5-min bolus (1 ml/kg) followed by continuous infusion (1 ml/kg/h) that was maintained until the end of the experiment. BMS-262084 doses (mg/kg + mg/kg/h) included 0.2 + 0.2, 0.5 + 0.5, 2 + 2, 6 + 6, 12 + 12 and 24 + 24, with doses adjusted for each model. Each rat was subjected to one of the procedures described in 2.5 , 2.6 Arterial thrombosis, 2.7 Venous thrombosis, 2.8 Bleeding times. The numbers of animals in each dose group are indicated in the results and the figures.\n

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\n\nEx vivo clotting time [1]
\nThese animals did not undergo a thrombosis or bleeding time procedure, but were used to determine systemic anticoagulation achieved in those procedures. Treatments included vehicle and BMS-262084 doses (mg/kg + mg/kg/h) of 0.2 + 0.2, 0.5 + 0.5, 2 +2, 6 + 6, 12 + 12 and 24 + 24. Carotid artery blood (0.6 mL drawn into 1/10 volume of 3.8% Na-citrate) was sampled before (0 min control) and at 5, 15, 30, 60 and 90 min after the start of test article infusion, and the activated partial thromboplastin time was determined on freshly prepared plasma. The prothrombin time was also determined at BMS-262084 doses (mg/kg + mg/kg/h) of 6 + 6, 12 + 12 and 24 + 24. Clotting times were performed as described in Section 2.3.\n

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\n\nArterial thrombosis [1]
\nThe left carotid artery was exposed and a piece of parafilm “M” was inserted under the vessel. An electromagnetic flow probe (1.0 mm lumen) was placed on the artery and attached to a model MDL 1401 flowmeter. Following baseline flow measurements, a 2-mm by 5-mm strip of filter paper saturated with a 50% solution of FeCl2 was placed on top of the vessel downstream from the flow probe for a period of 10 min. The carotid artery was removed at 60 min after filter paper application. The vessel was opened lengthwise and the thrombus was removed and weighed immediately on a AE50 balance. Carotid blood flow was monitored continuously on a TA3800 physiologic recorder. Integrated carotid blood flow was determined as an area under the flow curve and normalized as percent of baseline (0 min) flow over 60 min to provide a measure of integrated blood flow during thrombus formation.
\nThere were 2 treatment regimens, one in which treatment was administered prior to thrombus formation, and another where treatment was delayed until a thrombus of ∼ 25% maximum weight had formed. In the pretreatment regimen, FeCl2-induced thrombosis was initiated 10 min after the start of test article infusion. Pretreatment groups included vehicle and BMS-262084 doses (mg/kg + mg/kg/h) of 2 + 2, 6 + 6 and 12 +12. In the delayed treatment regimen the infusion of vehicle and BMS-262084 at 12 mg/kg + 12 mg/kg/h was started upon removal of the FeCl2-saturated filter paper. Thrombus weights at the time of FeCl2 removal were also obtained in control rats.\n

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\n\nVenous thrombosis [1]
\nTwo venous thrombosis models were used. In the tissue factor model, vena cava blood flow was disrupted by a severe but non-occlusive fixed stenosis, and tissue factor was infused into a femoral vein. In the vessel injury model, topical FeCl2 was placed on the vena cava with no stenosis.
\nIn the FeCl2-induced injury, the vena cava was isolated via a midline abdominal incision and the surface cleared by blunt dissection between the renal and iliolumbar veins. A 2-mm by 5-mm strip of filter paper saturated with 15% FeCl2 was placed on the vena cava for 1 min. Sixty min after filter paper application the vena cava was dissected free, the thrombus was removed and weighed immediately on the Mettler balance. In the tissue factor model polyethylene-50 tubing was inserted into the left femoral vein and the vena cava was isolated by midline abdominal incision. To create a non-occlusive stenosis, a 26-gauge needle was placed on top of the vena cava just distal to the renal veins and a piece of 3-O silk was threaded under the vein and secured over the needle. The needle was then slipped free from the ligature leaving a fixed stenosis. Thrombosis was induced by a 1.4 ml/kg infusion of human recombinant tissue factor (1/10 dilution RecombiPlasTin, Ortho Diagnostics) administered over 2 min into the femoral catheter. The thrombus was removed and weighed 25 min after tissue factor injection.
\nA pretreatment dosing protocol was employed in both models. BMS-262084 doses (mg/kg + mg/kg/h) included 0.2 +0.2, 0.5 + 0.5, 2 + 2, 6 + 6 in the FeCl2 model and 6 + 6, 12 + 12 and 24 + 24 in the tissue factor model. Thrombotic provocation (tissue factor infusion or FeCl2 application) was delivered 10 min after the start of vehicle or BMS-262084 infusion.
\nIn the FeCl2 model, infusion of vehicle or BMS-262084 (12 mg/kg + 12 mg/kg/h) was also delayed until 20 min after FeCl2 application, which allowed for thrombus growth to 41% of maximum prior to test article administration.\n

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\n\nBleeding times [1]
\nBleeding times were performed using incision of the renal cortex or cuticle, and needle puncture of small mesenteric blood vessels. In each model bleeding times were determined from the time of injury until bleeding had stopped for 30 s. Replicate bleeds were averaged to a single value for statistical comparison.
\nIn the renal model both kidneys were exposed by a midline abdominal incision. The renal capsules were removed and the cortex was superfused with Ringer's solution at 37 °C. Incisions were created using a Surgicutt template which produced a 5-mm long by 1-mm deep cut from a spring-loaded surgical blade (No. 25, sharp tip), and bleeding was observed under 3×-binocular magnifier. Three bleeding times were determined on the right kidney before, and on the left kidney 15 min after, the start of test article infusion.
\nIn the cuticle model, toenails were cut with a single edged razor blade at the location where the quick meets the nail. The cuticle was immediately superfused with Ringer's solution at 37 °C and bleeding was observed under a 3×-binocular magnifier. Three replicate bleeding times were determined on the left hind paw prior to administration of test article and on the right hind paw 15 min after start of test article infusion.
\nIn the mesenteric model, the small intestine was exteriorized through a midline abdominal incision. The jejunum was held in place with clamps and superfused with Ringer's solution at 37 °C. Small blood vessels that branch perpendicular to the mesenteric artery and course over the jejunum were observed through an SZH10 stereomicroscope. Vessels were punctured with a 30-gauge hypodermic needle. Bleeding times were determined in 3–5 vessels before administration of test article, and in 3–5 vessels 20 min after the start of test article infusion.
\nBMS-262084 was administered at doses (mg/kg + mg/kg/h) of 6 + 6, 12 + 12 and 24 + 24 in each bleeding model. Heparin was also studied as a positive control in each model. In these experiments vehicle or heparin (33 U/kg + 60 U/kg/h) was tested using the same procedure as described for BMS-262084. The dose of heparin was selected to achieve the maximal anticoagulant effect of BMS-262084 based on increase in ex vivo activated partial thromboplastin time.

[1]. Antithrombotic and hemostatic effects of a small molecule factor XIa inhibitor in rats. Eur J Pharmacol. 2007 Sep 10;570(1-3):167-74.

[2]. A stereoselective synthesis of BMS-262084, an azetidinone-based tryptase inhibitor. J Org Chem. 2002 May 31;67(11):3595-600.

This study investigated the inhibitory effect of activated coagulation factor XIa in a rat model of thrombosis and hemostasis. BMS-262084 is an irreversible and selective small molecule factor XIa inhibitor with an IC50 of 2.8 nM for human factor XIa. At concentrations of 0.14 μM and 2.2 μM, BMS-262084 doubled the activated prothrombin time in human and rat plasma, respectively. Consistent with factor XIa inhibition, prothrombin time was unaffected at concentrations up to 100 μM. Intravenous loading followed by continuous infusion of BMS-262084 effectively inhibited FeCl2-induced thrombosis in the inferior vena cava and carotid artery. At a pretreatment dose of 12 mg/kg + 12 mg/kg/h, thrombus weight was reduced by a maximum of 97% and 73%, respectively (P<0.05), while the in vitro activated prothrombin time was prolonged to 3.0 times that of the control group. This dose level also inhibited the growth of venous and arterial thrombi when administered after partial thrombosis. BMS-262084 was most effective against FeCl₂-induced venous thrombosis, with a 38% reduction in thrombus weight at a threshold dose of 0.2 mg/kg + 0.2 mg/kg/h (P<0.05). In contrast, doses up to 24 mg/kg + 24 mg/kg/h had no effect on tissue factor-induced venous thrombosis or in vitro prothrombin time. Dose up to 24 mg/kg + 24 mg/kg/h also did not significantly prolong bleeding time caused by small bowel mesenteric vascular puncture, renal cortical template incision, or skin incision. These results suggest that pharmacological inhibition of factor XIa can achieve antithrombotic efficacy with minimal effect on induced bleeding. [1]

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We developed a highly stereoselective synthesis method for a novel trypsin inhibitor, BMS-262084. The key to this synthesis lies in the discovery and development of a highly diastereoselective diester 12 demethoxycarbonylation reaction to generate trans-azacyclobutanone 13. BMS-262084 was prepared from D-ornithine in 10 steps with an overall yield of 30%. [2]

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The in vitro activity profile of BMS-262084 is consistent with that of selectively inhibiting factor XIa. Researchers have confirmed the effects of BMS-262084 by using human serine proteases involved in coagulation and fibrinolysis, and by selectively prolonging the activated partial thromboplastin time (APTT) rather than the prothrombin time (PT) in human and rat plasma. However, BMS-262084 is not only a potent inhibitor of factor XIa, but also an equivalent and irreversible inhibitor of trypsin (the main protease secreted by mast cells and thought to be involved in asthma response). Early studies on allergic bronchoconstriction and airway inflammation have shown that nebulized inhalation of BMS-262084 has a protective effect (Sutton et al., 2002). Therefore, before using BMS-262084 as a tool for thrombosis and hemostasis research, the role of mast cell trypsinoids in coagulation and fibrinolysis needs to be considered. Based on their presence in atherosclerotic plaques (Jeziorska et al., 1997) and deep vein thrombosis (Bankl et al., 1999), mast cells containing trypsinoids may be involved in human vascular diseases. In these cases, mast cells were observed to aggregate primarily within the vessel wall, leading to the hypothesis that mast cells are involved in the development of atherosclerosis, vascular repair, and the resolution of thrombotic events (Valent et al., 2002). Some vasoactive activity can be attributed to mast cell-derived heparin and tissue plasminogen activator, but trypsinoids themselves may regulate vascular responses by cleaving endothelial cell protease-activated receptor 2 (PAR2) (Meyer et al., 2005). The direct effects of mast cells on coagulation (Samoszuk et al., 2003) and platelet function (Gardiner et al., 1999) appear to be more related to the release of heparin than to trypsin-like proteins. Current data best support the view that trypsin-like proteins play a complex role in chronic thrombosis and vascular inflammation. In our model, occlusive thrombi rapidly form after oxidation of the major vessel walls, a process unlikely to be influenced by endogenous trypsin-like proteins. Furthermore, there is a lack of evidence that mast cells are involved in hemostasis of transient blood loss caused by small vessel injury. Therefore, the role of BMS-262084 in acute experimental hemostasis and thrombosis is likely due to factor XIa inhibition rather than trypsin-like protein inhibition. BMS-262084 increased the time to activation of partial thromboplastin (PTP) in vitro by up to 3.2-fold. This in vitro plateau phase of activity was not observed in in vitro experiments. The reason for the limited exposure-response to BMS-262084 in rats is unclear, but this prevented us from assessing the effect of maximum level factor XIa inhibition. Nevertheless, the protective effect of BMS-262084 against FeCl2-induced thrombosis is comparable to previous findings using heparin, low molecular weight heparin, and thrombin inhibitors (Schumacher et al., 1993; Schumacher et al., 1996). In the vena cava and carotid artery, BMS-262084 inhibits the formation and growth of occlusive thrombi, whether initiated before or after partial thrombosis. BMS-262084 has no effect on tissue factor infusion-induced venous thrombosis and does not prolong prothrombin time. In these cases, the coagulation process initiated by high levels of tissue factor is almost unaffected by factor XIa. Conversely, studies have found that factor XI can enhance low-level tissue factor-induced thrombosis (von dem Borne et al., 2006). The antithrombotic activity of BMS-262084 is not due to its direct antiplatelet effect, as the aggregation response measured under coagulation-free conditions is unaffected by this antagonist.
In our experiments, oxidative damage led to platelet aggregation and fibrin formation at the site of full-thickness vascular wall injury (Wang et al., 2007). However, the effectiveness of factor XIa inhibitors has also been demonstrated in rabbit iliac artery repeated balloon injury models (Yamashita et al., 2006) and monkey arteriovenous shunt models (Gruber and Hanson, 2003). Mice lacking factor XII (whose contact-related activation of factor XI is impaired) are also protected from arterial thrombosis induced by oxidative or compressive damage (Renné et al., 2005).
An encouraging property of BMS-262084 is that no significant prolongation of bleeding time was observed in three induced bleeding models using antithrombotic doses. These models were sensitive to anticoagulants, such as heparin, which showed a slight prolongation of bleeding time, up to 1.6 times that of the control group. A factor XIa inhibitor that can significantly prolong the in vitro activated partial thromboplastin time (e.g., by 5-fold) would better define the upper limit of bleeding risk for this target. Hemostasis of small vessel bleeding may require limited factor XI activation, thus limiting the incomplete hemostatic effect of factor XIa inhibition. The safety of this target is further supported by reports that the inhibitor exhibited antithrombotic activity in induced bleeding in factor XI knockout mice (Rosen et al., 2002; Wang et al., 2005), and similar results were observed with factor XIa neutralizing antibodies (Minnema et al., 1998; Gruber and Hanson, 2003; Yamashita et al., 2006). The in vivo properties of BMS-262084 suggest that factor XIa is an attractive antithrombotic target with limited bleeding risk. Continued research into the interaction between platelets and coagulation further supports this target. Factor XIa and factor XIIa contribute to optimal thrombin activation and stable thrombus formation in experimental animals (Colman, 2003). Factor XIa production and activity appear to be closely related to platelet activation (Walsh, 2003), which could explain the benefits of inhibiting or depleting factor XIa in platelet-dominant thrombotic diseases. Regulation of thrombin production and fibrinolysis (von dem Borne et al., 2006) may also contribute to antithrombotic efficacy. The potential advantages of factor XIa over more established anticoagulant targets such as thrombin and factor Xa remain an area of active exploration. [1]

C18H31N7O5

425.48

425.239

C, 50.81; H, 7.34; N, 23.04; O, 18.80

253174-92-4

253172-74-6 (HCl);253174-92-4;

9802488

White to off-white solid powder

0.616

4

6

6

30

721

2

CC(C)(C)NC(=O)N1CCN(CC1)C(=O)N2[C@@H]([C@H](C2=O)CCCN=C(N)N)C(=O)O

MFTQITSPGQORDA-NEPJUHHUSA-N

InChI=1S/C18H31N7O5/c1-18(2,3)22-16(29)23-7-9-24(10-8-23)17(30)25-12(14(27)28)11(13(25)26)5-4-6-21-15(19)20/h11-12H,4-10H2,1-3H3,(H,22,29)(H,27,28)(H4,19,20,21)/t11-,12+/m1/s1

(2S,3R)-1-[4-(tert-butylcarbamoyl)piperazine-1-carbonyl]-3-[3-(diaminomethylideneamino)propyl]-4-oxoazetidine-2-carboxylic acid

BMS-262084; BMS262084; BMS-262084; 253174-92-4; UNII-I0IR71971G; I0IR71971G; (-)-BMS-262084; 2-Azetidinecarboxylic acid, 3-(3-((aminoiminomethyl)amino)propyl)-1-((4-(((1,1-dimethylethyl)amino)carbonyl)-1-piperazinyl)carbonyl)-4-oxo-, (2S,3R)-; CHEMBL71037; (2S,3R)-1-[4-(tert-butylcarbamoyl)piperazine-1-carbonyl]-3-[3-(diaminomethylideneamino)propyl]-4-oxoazetidine-2-carboxylic acid; BMS 262084

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~117.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)