Factor XIa (FXIa; Kis = 0.2~0.4 nM)

In vitro, BMS-654457 demonstrates similar activity (Ki=0.2 nM) against FXIa in humans and rabbits (Ki=0.42 nM), as well as selectivity (more than 500 times) against coagulation-related proteases (thrombin, FXa, and FVIIa) in these species.

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In vitro [1]
Enzyme assays [1]
The Lineweaver–Burk plot of the data (Fig. 2 top panel) for inhibition of human FXIa by BMS-654457 indicates that BMS-654457 is a competitor inhibitor versus the chromogenic peptide substrate S-2366 with a Ki of 0.4 nM (Fig. 2 bottom panel). The association rate of BMS-654457 with human FXIa was approximately 1.6 μM−1 s−1 (Fig. 3 top and middle panels). BMS-654457 binding to FXIa was reversible, which was confirmed by pre-incubation/dilution (Fig. 3 bottom panel). BMS-654457 exhibited a moderately slow off-rate of 0.001 s−1 (Fig. 3), which equates to a half-time of dissociation of BMS-654457 from FXIa of approximately 12 min.

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Clotting assays [1]
The aPTT is a clotting time assay sensitive to inhibition of intrinsic coagulation factors such as FXIa. Thus, addition of BMS-654457 to normal human and rabbit plasma prolonged aPTT, but not PT. BMS-654457 had the highest potency in the prolongation of aPTT in human and rabbit plasma (EC2X of 2.2 and 2.4 µM, respectively), but was very weak in rat and dog plasma (EC2X = ≥ 20 µM).

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Platelet aggregation [1]
In vitro platelet aggregation responses to ADP (10 µM), arachidonic acid (250 µM) and collagen (10 µg/ml) averaged 62 ± 2 %, 70 ± 1 % and 71 ± 2 %, respectively in rabbit PRP (n = 5 per group). These platelet responses were not significantly altered by BMS-654457 at 1 µM (64 ± 2, 71 ± 1 and 71 ± 2 %, respectively) and 10 µM (56 ± 3, 67 ± 3 and 73 ± 2 %, respectively) (n = 5 per group).

BMS-654457 reduces bleeding time but is effective in keeping rabbits from developing arterial thrombosis. With a wide therapeutic window, BMS-654457 is a promising antithrombotic treatment [1].

Enzyme assays [1]
Assays were conducted in 0.05 M HEPES buffer containing 0.145 M NaCl, 0.005 M KCl and 0.1 % PEG 8000 adjusted to pH 7.4. Assays were conducted under conditions of excess substrate and inhibitor over enzyme. To determine the mechanism of inhibition, peptide substrate cleavage by FXIa was assayed against nine concentrations of the chromogenic substrate S-2366 and five concentrations of BMS-654457. Reactions were initiated by adding FXIa. The steady state rates of substrate hydrolysis after the initial curved time course region, due to slow-binding inhibition, were measured by continuous monitoring of the absorbance at 405 nm for 60 min using a SpectraMax 384 Plus plate reader and SoftMax. FXIa activity for each substrate and inhibitor concentration pair was determined in duplicate. The Ki values were calculated by non-linear least-squares fitting of the steady-state substrate hydrolysis rates to the equation for competitive inhibition (Eq. 1) using GraFit, where v equals reactions velocity in OD/min, V max equals maximum reaction velocity, S equals substrate concentration, and I equals inhibitor concentration.

The association rate constant for binding of BMS-654457 to FXIa was determined by stopped-flow spectrophotometry in the presence of S-2366 at 25 °C. The increase in absorbance at 405 nm was followed after rapidly mixing a FXIa solution one-to-one with a premixed solution of 400 µM substrate S-2366 and BMS-654457 using the single-mixing mode of an Applied Photophysics SX.18MV stopped-flow device equipped with a circulating water bath for temperature control. The data were acquired on a linear time base, creating a total of 1000 data points in each curve, using two to three repeated measurements at each inhibitor concentration. Non-linear time course data were fit to the slow binding inhibition equation.
where Absorbance t is the optical density (OD) at 405 nm at time t; Absorbance t=0 is the initial OD; v initial and v final are initial and final velocities, respectively; k obs is the observed rate constant. The association rate (k on ) was calculated from the slope of k obs versus BMS-654457 concentration and the relationship:

For determination of the dissociation rate constant the FXIa: BMS-654457 complex was formed by incubation of 1.25 nM FXIa with 5 nM BMS-654457 for 60 min. Based on the steady-state assay the enzyme was fully inhibited under these conditions. The time-dependent increase in FXIa activity was measured by monitoring the change in OD at 405 nm after the FXIa:BMS-654457 complex was diluted 50-fold into reaction mixtures containing 1 mM S-2366 in the same buffer such that the final concentration of FXIa was 25 pM and of BMS-654457 was 100 pM. Under these conditions the observed rate constant from a fit to the slow-binding equation (Eq. 2) is a close approximation of the dissociation rate constant. The half-time for dissociation can be calculated from the natural logarithm of two divided by the dissociation rate constant.

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Clotting assays [1]
Clotting times were measured in citrated platelet poor plasma (PPP) with an automated coagulation analyzer as described previously. The concentrations of BMS-654457 required to prolong clotting time by twofold (EC2x) in citrated plasma obtained from healthy dogs, rats, and rabbits were determined as described previously.

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Platelet aggregation [1]
Platelet aggregation was measured in hirudin-treated rabbit platelet-rich plasma (PRP) in vitro with a platelet aggregometer according to the manufacturer’s instructions. PRP was obtained from hirudin-treated blood (lepirudin, 25 μg/ml) after centrifuging at 180 g for 10 min. PRP (250 µl) was mixed with 2.5 µl of vehicle or BMS-654457 at 1 and 10 µM and incubated for 2 min at 37 °C. Peak platelet aggregation was determined after the addition of 2.5 µl of the agonist (ADP at 10 µM, arachidonic acid at 250 µM and collagen at 10 µg/ml, final concentration).

In vivo [1]
Surgical preparation [1]
The rabbits were anesthetized and surgically prepared as described previously. Each rabbit was utilized in either an arterial thrombosis or cuticle bleeding time (BT) protocol. BMS-654457 or vehicle (10 % N-N-dimethylacetamide:90 % of 5 % dextrose) was administered as a bolus plus sustaining IV infusion begun 30 min prior to each protocol. The dosing regimen for BMS-654457 was designed based on results from pilot pharmacokinetic studies in rabbits, which determined the IV bolus and infusion doses needed to produce constant plasma levels over the duration of each experiment. Terminal plasma samples were obtained for drug exposure and ex vivo clotting times. Plasma levels of BMS-654457 were measured by a specific and sensitive liquid chromatographic mass spectrometry method.

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Arterial thrombosis model [1]
The rabbit electrolytic-mediated carotid arterial thrombosis (ECAT) model, described by Wong et al, was used in this study. Thrombosis was induced by electrical stimulation of the carotid artery. Carotid blood flow was measured by an electromagnetic flow probe over a 90-min period to monitor thrombotic occlusion. Integrated carotid blood flow (iCBF) over 90 min was measured by the area under the flow-time curve. Treatment groups included BMS-654457 (mg/kg + mg/kg/h) at 0.011 + 0.008, 0.037 + 0.027, 0.11 + 0.08, 0.37 +0.27 and 1.1 + 0.8 or vehicle (n = 6 per group). The plasma concentration that increased iCBF to 50 % of the control (EC50) was estimated for BMS-654457 as described below.

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Cuticle bleeding model [1]
The rabbit cuticle BT model, described by Himber et al., was modified and used in this study. Treatment groups included BMS-654457 (mg/kg + mg/kg/h) at 0.11 + 0.08, 0.37 + 0.27 and 1.1 + 0.8 or vehicle (n = 6 per group). BT was measured before and after treatment in opposite hind-limbs, and was expressed as a ratio of treated over the control value.

[1]. In vitro, antithrombotic and bleeding time studies of BMS-654457, a small-molecule, reversible and direct inhibitor of factor XIa. J Thromb Thrombolysis. 2015 Nov;40(4):416-23.

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

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In summary, BMS-654457 is a small-molecule, reversible and direct inhibitor of FXIa. BMS-654457 pretreatment was able to prevent the rapid formation of thrombotic occlusion in the rabbit ECAT model at doses that had a minimal impact on primary hemostasis. The ex vivo aPTT appeared to be a reasonable biomarker for monitoring the antithrombotic activity in rabbits. Overall this study supports FXIa as an attractive target in the development of anticoagulants possessing a wide therapeutic window. [1]

C36H37N5O4

603.71

603.284

C, 71.62; H, 6.18; N, 11.60; O, 10.60

1004551-41-0

60154989

Typically exists as solid at room temperature

5.3

6

6

9

45

1090

2

CC(C)CC(=O)NC1=CC(=CC(=C1)[C@@H]2C[C@](C3=C(N2)C=CC(=C3)C(=N)N)(C)C4=CC=CC=C4)C5=C(C=C(C=C5)C(=O)N)C(=O)O

PDUMJXCNOKHQKH-SVXHESJVSA-N

InChI=1S/C36H37N5O4/c1-20(2)13-32(42)40-26-15-23(27-11-9-22(34(39)43)17-28(27)35(44)45)14-24(16-26)31-19-36(3,25-7-5-4-6-8-25)29-18-21(33(37)38)10-12-30(29)41-31/h4-12,14-18,20,31,41H,13,19H2,1-3H3,(H3,37,38)(H2,39,43)(H,40,42)(H,44,45)/t31-,36+/m0/s1

2-[3-[(2S,4R)-6-carbamimidoyl-4-methyl-4-phenyl-2,3-dihydro-1H-quinolin-2-yl]-5-(3-methylbutanoylamino)phenyl]-5-carbamoylbenzoic acid

BMS-654457; CHEMBL3127491; 1004551-41-0; 3'-[(2s,4r)-6-Carbamimidoyl-4-Methyl-4-Phenyl-1,2,3,4-Tetrahydroquinolin-2-Yl]-4-Carbamoyl-5'-[(3-Methylbutanoyl)amino]biphenyl-2-Carboxylic Acid; SCHEMBL4936907; BDBM50448583;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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