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 bleedings [1]
Patients receiving Efegatran often developed a superficial thrombophlebitis that seemed to increase, although not significantly, in severity in the higher dose groups, the incidence ranging from 7·7% to 20%, which was significantly higher than with heparin (P=0·0001). For this reason the infusion rate in the highest dose group during the dose ranging phase was increased from 4 ml . h"1 to 40 ml . h"1 with a subsequent decrease in the concentration of Efegatran administered. This regimen was also used for the subsequent phases of the study, and did reduce the severity of the events. However, it did not reduce the overall occurrence of phlebitis. In the second phase of the study, the number of patients with phlebitis in the 0·63 mg . kg"1 . h"1 dose group was 13%, compared to 25% in the 1·2 mg . kg"1 . h"1 dose group and 2% in the patients treated with heparin. In the great majority of patients, the severity of the phlebitis was only mild. The incidence of minor bleeding events was significantly higher in patients treated with Efegatran (P=0·001) ranging from 17% to 32%, against 11% in patients under heparin (Table 4). There were three major bleedings, two of which occurred in patients treated with heparin. Spontaneous gross haematuria was equally distributed and observed in three patients (0·7%). Most minor bleedings were associated with a previous puncture site, and did not require specific measures. There were no strokes associated with administration of trial medication.

[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 effect of Efegatran, a direct thrombin inhibitor with heparin. Administration of Efegatran sulphate at levels of at least 0·63 mg . kg"1 . h"1 provided a pronounced increase in thrombin time, which is at least comparable to activated partial thromboplastin time adjusted heparin infusion. The level of thrombin inhibition by efegatran, as reflected by the activated partial thromboplastin time, appeared to be more stable than with heparin, especially during the first few hours following initiation of therapy, which may be due to the relatively high initial dose of heparin. This may reflect a more predictable dose response, suggesting that efegatran sulphate administration is probably easier to monitor than heparin. As thrombin plays a key role in the coagulation cascade it was expected that the direct effects of efegatran would result in a more potent antithrombotic effect compared to heparin, which acts indirectly, requiring antithrombin III as a cofactor. However, no clinical benefit of efegatran over heparin was apparent whereas minor bleeding was more frequent. Our findings are in concert with other studies investigating direct thrombin inhibitors [1].

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The diminution in fibrinogen level after administration of endotoxin was prevented significantly by treatment with thioglycosides. Between the Sa/LPS, Efegatran/LPS and heparin/LPS groups, there was no significant difference in fibrinogen level in the DIC model. Thioglycosides prevented significantly the diminution in PLT count in the DIC model. Heparin and efegatran did not inhibit significantly this decrease. Diminution in WBC count could be found in rabbits after injection of LPS. The endotoxin-induced leukopenia is mediated by TNF-α. The thioglycosides as well as heparin and efegatran is likely to have less effect on leukocyte activation caused by DIC. The injection of endotoxin into the rabbits caused significant increases in both fibrinolysis and FDP level, probably due to the release of plasminogen activators from endothelial cells. These findings are consistent with data described previously in the literature. All investigated materials caused a significant decrease in the FDP level in blood compared with the Sa/LPS group, and GYKI 39521 was the most effective. The increase in fibrinolysis after administration of endotoxin was prevented significantly by treatment with thioglycosides or Efegatran. There was no difference between Sa/LPS and heparin/LPS groups. [2]

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Endothelial progenitor cells (EPCs) from the bone marrow play an important role in vascular response to injury and ischemia. The mediators involved in the mobilization, recruitment, proliferation and differentiation of EPCs are not fully understood. In this study, the role of coagulation factor thrombin and protease-activated receptor-1 (PAR-1) on bone marrow-derived cell proliferation and differentiation was investigated. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and plated on fibronectin-coated flasks. Cell characteristics, proliferation and the expression of endothelial cell markers were determined using immunohistochemistry, thymidine uptake and fluorescence activated-cell sorting (FACS), respectively. The results show that thrombin stimulated enrichment of bone marrow cells with endothelial morphology, exhibiting acetylated-low-density lipoprotein (LDL) uptake and isolectin staining. Thrombin or PAR-1-activating peptide produced a 2- to 3-fold increase in the total number of cells as well as an increase in vascular endothelial (VE)-cadherin-positive cells. Thrombin treatment of VE-cadherin-negative cells prepared after cell sorting resulted in the generation of 3- to 4-fold higher VE-cadherin-positive cells than the untreated cultures. Increase in VE-cadherin-positive cells was inhibited by hirudin and efegatran. These results provide first evidence for a novel activity of thrombin and PAR-1 on bone marrow progenitor cell proliferation and EPC differentiation, and suggest their potential role in vascular regeneration and recanalization of thrombus. [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.)