Thrombin

Thrombin inhibitors block EPC differentiation [3]
To determine whether the effect of thrombin was direct, and required its proteolytic activity, we examined the effect of thrombin inhibitors on the generation of VE‐cadherin‐positive cells. Cells were treated with the indicated concentration of either hirudin or Efegatran, known inhibitors of thrombin, and then incubated in the presence of thrombin for 7 days. VE‐cadherin‐positive cells in the cultures were analyzed by using FACS. Hirudin treatment abrogated the effect of thrombin (Fig. 6A). Similarly, Efegatran treatment blocked more than 90% of thrombin stimulation (Fig. 6B). Hirudin or Efegatran alone did not produce significant change in the number of VE‐cadherin‐positive cells. These results show that the effect of thrombin was specific and required its proteolytic activity, perhaps for the generation of the tethered ligand of thrombin receptor.

Efegatran demonstrated dose dependent ex-vivo anticoagulant activity with the highest dose level of 1.2 mg. kg(-1). h(-1)resulting in steady state mean activated partial thromboplastin time values of approximately three times baseline. Thrombin time was also increased. Neither of the efegatran doses studied were able to suppress myocardial ischaemia during continuous ECG ischaemia monitoring to a greater extent than that seen with heparin. There were no statistically significant differences in clinical outcome or major bleeding between the efegatran and heparin groups. Minor bleeding and thrombophlebitis occurred more frequently in the efegatran treated patients. Conclusion: Administration of Efegatran sulphate at levels of at least 0.63 mg. kg(-1). h(-1)provided an anti-thrombotic effect which is at least comparable to an activated partial thromboplastin time adjusted heparin infusion. There was no excess of major bleeding. The level of thrombin inhibition by efegatran, as measured by activated partial thromboplastin time, appeared to be more stable than with heparin. Thus, like other thrombin inhibitors, efegatran sulphate is easier to administer than heparin. However, no clinical benefits of efegatran over heparin were apparent. [1]

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Disseminated intravascular coagulation (DIC) is a systemic thrombohemorrhagic disorder seen in association with many clinical situations, e.g. sepsis, malignancy, obstetrical complications and intravascular hemolysis. In our model, disseminated intravascular coagulation was induced in rabbits by two consecutive intravenous bolus injections of endotoxin from Escherichia coli, 80 and 40 microg/kg. The control group was treated with 0.9% saline. The activity of thioglycosides was compared to unfractionated heparin (UFH) and Efegatran with and without administration of endotoxin. Drugs were administered in the following doses: heparin 50 and 100 IU/kg/h i.v. infusion; Efegatran 0.25 and 0.5 mg/kg/h i.v. infusion; GYKI 39521 (RGH-1875) as well as GYKI 39541 (RGH-1962) 12.5 and 25 mg/kg per os. Thioglycosides did not modify coagulation parameters in this model [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT)] as compared with endotoxin/vehicle group. The changes in TFPI level after administration of thioglycosides and heparin were similar in the mentioned model to those without endotoxin. Endotoxin-induced changes of leukocyte count were not affected by GYKI 39521 and GYKI 39541 treatment in our model. Diminution of fibrinogen level and platelet count was prevented by GYKI 39521 and GYKI 39541. Fibrin degradation products and fibrinolysis were significantly decreased by GYKI 39521 and GYKI 39541. The thioglycosides may have a lower risk of bleeding in the treatment of disseminated intravascular coagulation than heparin. [2]

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Changes in clotting times after administration of references and thioglycosides to rabbits with and without endotoxin [2]
Dose-dependent prolongations of clotting times appeared after application of heparin and Efegatran (Table 1). Between the saline and the thioglycoside groups, there was no significant difference in clotting parameters. Treatment with LPS also tended to prolong APTT and PT, and these effects were statistically significant (Table 2). The prolongation of clotting times was larger after administration of heparin and Efegatran in the DIC model than without endotoxin administration. Between the Sa/LPS, G1/LPS and G2/LPS groups, there was no significant difference in clotting times (Table 2).

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Changes in TFPI level after administration of tested compounds to rabbits with and without endotoxin [2]
GYKI 39521 induced a similar increase in TFPI level (in both tested doses) to heparin in lower dose without endotoxin. The change in this parameter was smaller after administration of GYKI 39541. Efegatran did not cause a significant increase in the TFPI level (Fig. 2). The mean plasma TFPI concentration in the rabbit DIC model in Sa/LPS group was higher than that in the control group and these values were similar to that of the Efegatran/LPS group (Fig. 3). The changes in TFPI level after administration of heparin, GYKI 39521 and GYKI 39541 were similar in the DIC model to those without endotoxin Fig. 2, Fig. 3.

The effect of Efegatran sulphate and heparin on markers of thrombosis and haemostasis was assessed by measuring bleeding time (Ivy, Simplate, Surgicut and Duke method, local laboratory, dose-ranging part only), the activated partial thromboplastin time (local, and central laboratory), prothrombin time (local, and central laboratory) and fibrinogen levels (central laboratory). Levels of fibrinogen were measured using both the ACL and Clauss methods. Levels of activation markers of platelets (beta-thromboglobulin, platelet factor 4), coagulation (prothrombin fragment 1·2, fibrinopeptide A, thrombin–antithrombin complexes) and fibrinolysis (fibrin degradation products) were measured in the dose finding phase only (central laboratory). General haematology (local laboratory), chemistry (central laboratory) and urinanalysis (local laboratory) were also performed. [1]

Effect of thrombin inhibitors on generation of VE‐cadherin‐positive cells. Cells prepared as in Fig. 1 were treated with thrombin in the absence or presence of hirudin (20 U mL−1) (A) or Efegatran sulfate (30 nm) (B) and analyzed by using fluorescence‐activated cell sorting (FACS). [3]

Four hundred and thirty-two patients with unstable angina were enrolled. Five dose levels of Efegatran were studied sequentially, ranging from 0.105 mg. kg(-1). h(-1)to 1.2 mg. kg(-1). h(-1)over 48 h. Safety was assessed clinically, with reference to bleeding and by measuring clinical laboratory parameters. Efficacy was assessed by the number of patients experiencing any episode of recurrent ischaemia as measured by computer-assisted continuous ECG ischaemia monitoring. Clinical end-points were: episodes of recurrent angina, myocardial infarction, coronary intervention (PTCA or CABG), and death. [1]

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Animal model of DIC [2]
In this model, DIC was induced in rabbits by two intravenous (i.v.) bolus injections of endotoxin [100 μg/ml LPS from Escherichia coli in 0.9% saline]. An initial i.v. bolus of 80 μg/kg was given after M1 (M=measurements), and a second bolus of 40 μg/kg was given after M2 (E group). The control (C) group was treated with 0.9% saline (Sa). Coagulation parameters such as prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen level (FBG), fibrin(ogen) degradation products (FDP), fibrinolysis (diluted whole blood clot lysis: DWBCL), platelet count (PLT), leukocyte count (WBC) and tissue factor pathway inhibitor blood level (TFPI) were measured.
The activity of thioglycosides was compared to heparin and Efegatran with and without administration of endotoxin. Drugs were administered in the following doses: heparin (H) 50 and 100 IU/kg/h i.v. infusion; Efegatran (Ef) 0.25 and 0.5 mg/kg/h i.v. infusion; GYKI 39521 (RGH-1875:G1) as well as GYKI 39541 (RGH-1962: G2) 12.5 and 25 mg/kg per os.

Adverse events and bleeding [1] Superficial thrombophlebitis was common in patients treated with Efegatran, although the severity was increased in the higher dose group (although the difference was not significant), with an incidence of 7.7% to 20%, significantly higher than in the heparin group (P=0.0001). Therefore, during the dose exploration phase, the infusion rate of the highest dose group was increased from 4 ml·h"1 to 40 ml·h"1, and the concentration of Efegatran was reduced accordingly. This regimen was also used in subsequent study phases and did reduce the severity of adverse events. However, it did not reduce the overall incidence of phlebitis. In the second phase of the study, the proportion of patients with phlebitis was 13% in the 0.63 mg·kg"1·h"1 dose group and 25% in the 1.2 mg·kg"1·h"1 dose group. Phlebitis occurred in 2% of patients in the 0.63 mg·kg"1·h"1 dose group and 2% of patients treated with heparin. The vast majority of patients had only mild phlebitis. The incidence of minor bleeding events was significantly higher in patients receiving efagaran than in the heparin group (P=0.001), ranging from 17% to 32% and 11%, respectively (Table 4). Three major bleeding events occurred, two of which occurred in the heparin group. Spontaneous gross hematuria was uniformly distributed, occurring in three patients (0.7%). Most minor bleeding events were related to previous puncture sites and required no special treatment. The investigational drug administration did not induce stroke.

[1]. Anticoagulant properties, clinical efficacy and safety of efegatran, a direct thrombin inhibitor, in patients with unstable angina. Eur Heart J. 1999 Aug;20(15):1101-11.

[2]. Effect of some new thioglycosides on endotoxin-induced disseminated intravascular coagulation in rabbits. Thromb Res . 2002 Sep 15;107(6):357-63.

[3]. Thrombin and PAR-1 stimulate differentiation of bone marrow-derived endothelial progenitor cells. J Thromb Haemost . 2006 Mar;4(3):656-63.

We compared the efficacy of the direct thrombin inhibitor efagaran with heparin. Administration of efagaran sulfate at a dose of at least 0.63 mg·kg⁻¹·h⁻¹ significantly prolonged thrombin time, with an effect at least comparable to that of heparin infusion corrected for activated partial thromboplastin time (APT). Thrombin inhibition levels of efagaran (reflected by APT) appeared to be more stable than with heparin, especially in the first few hours after treatment initiation, likely due to the relatively high initial dose of heparin. This may reflect a more predictable dose-response relationship with efagaran, suggesting that efagaran sulfate administration may be easier to monitor than heparin. Given the crucial role of thrombin in the coagulation cascade, the direct action of efagaran was expected to produce a stronger antithrombotic effect than that of heparin (whose action is indirect and requires antithrombin III as a cofactor). However, efagaran did not show a significant clinical benefit compared to heparin, and instead had a higher incidence of minor bleeding. Our findings are consistent with those of other studies on direct thrombin inhibitors [1]. Thioglycoside treatment significantly prevented the decrease in fibrinogen levels following endotoxin administration. In the DIC model, there were no significant differences in fibrinogen levels among the Sa/LPS, efogacran/LPS, and heparin/LPS groups. Thioglycosides significantly prevented the decrease in platelet count in the DIC model. Neither heparin nor efogacran significantly inhibited this decrease. A decrease in white blood cell count was observed after LPS injection in rabbits. Endotoxin-induced leukopenia is mediated by TNF-α. Thioglycosides, as well as heparin and efogacran esters, may have a smaller effect on DIC-induced leukocyte activation. Injection of endotoxin into rabbits significantly increased fibrinolysis and FDP levels, likely due to the release of plasminogen activator from endothelial cells. These results are consistent with previously reported data in the literature. Compared with the Sa/LPS group, all tested drugs significantly reduced blood FDP levels, with GYKI 39521 showing the best effect. Thioglycosides or efogatane significantly inhibited the increase in fibrinolysis after endotoxin administration. There was no difference between the Sa/LPS group and the heparin/LPS group. [2] Bone marrow endothelial progenitor cells (EPCs) play an important role in the vascular response to injury and ischemia. The mediators involved in EPC mobilization, recruitment, proliferation and differentiation have not been fully elucidated. This study investigated the effects of thrombin and protease-activated receptor-1 (PAR-1) on the proliferation and differentiation of bone marrow-derived cells. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and seeded in fibronectin-coated culture flasks. Cell characteristics, proliferation and expression of endothelial cell markers were detected by immunohistochemistry, thymidine uptake and flow cytometry (FACS). The results showed that thrombin stimulated the enrichment of bone marrow cells, which had endothelial cell morphology and exhibited acetylated low-density lipoprotein (LDL) uptake and heterolectin staining. Thrombin or PAR-1 activating peptide increased the total number of cells by 2 to 3 times and increased the number of vascular endothelial (VE) cadherin-positive cells. After thrombin treatment, the number of VE cadherin-negative cells prepared after cell sorting was 3 to 4 times higher than that of untreated cultures. Hirudin and efegaran inhibited the increase of VE cadherin-positive cells. These results confirmed for the first time that thrombin and PAR-1 have novel activities on bone marrow progenitor cell proliferation and endothelial progenitor cell (EPC) differentiation, and suggested that they may play a role in angiogenesis and thrombosis recanalization. [3]

C21H34CL2N6O3

489.44

416.2536

C, 51.53; H, 7.00; Cl, 14.49; N, 17.17; O, 9.81

173006-83-2

Typically exists as solids at room temperature

O=C([C@H]1N(C([C@@H](CC2=CC=CC=C2)NC)=O)CCC1)N[C@@H](CCCNC(N)=N)C=O.[H]Cl.[H]Cl

NRMUVRCVXCYWNB-VWRRVXQQSA-N

InChI=1S/C21H32N6O3.2ClH/c1-24-17(13-15-7-3-2-4-8-15)20(30)27-12-6-10-18(27)19(29)26-16(14-28)9-5-11-25-21(22)23;;/h2-4,7-8,14,16-18,24H,5-6,9-13H2,1H3,(H,26,29)(H4,22,23,25);2*1H/t16-,17+,18-;;/m0../s1

(S)-N-((S)-5-guanidino-1-oxopentan-2-yl)-1-(methyl-D-phenylalanyl)pyrrolidine-2-carboxamide dihydrochloride

LY 294468 dihydrochloride; Efegatran dihydrochloride;RGH 2958; RGH2958; GYKI-14166; GYKI14166; GYKI 14166; RGH-2958; LY 294468; LY-294468; LY294468;

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.)