Efegatran di(hydrochloride)   中文名称: 依非加群盐酸盐

Thrombin

凝血酶抑制剂阻断EPC分化[3] 为确认凝血酶的作用是否直接且需要其蛋白水解活性，我们检测了凝血酶抑制剂对VE-钙黏蛋白阳性细胞生成的影响。细胞分别用指定浓度的水蛭素（hirudin）或埃非加群（Efegatran）（已知的凝血酶抑制剂）处理，随后在凝血酶存在条件下培养7天。通过流式细胞术（FACS）分析培养体系中的VE-钙黏蛋白阳性细胞。水蛭素处理完全消除了凝血酶的作用（图6A）。同样，埃非加群处理阻断了90%以上的凝血酶刺激效应（图6B）。单独使用水蛭素或埃非加群均未引起VE-钙黏蛋白阳性细胞数量的显著变化。这些结果表明凝血酶的作用具有特异性，且需要其蛋白水解活性——可能是为了生成凝血酶受体的锚定配体。

Efegatran显示出剂量依赖性体外抗凝血活性，最高剂量为1.2 mg。公斤(1)。H（-1）导致稳态平均活化部分凝血活酶时间值约为基线的三倍。凝血酶时间也增加。在连续的心电图缺血监测中，两种剂量的Efegatran/伊非加群都不能比肝素更大程度地抑制心肌缺血。Efegatran/伊非加群和肝素组在临床结果和大出血方面没有统计学上的显著差异。轻微出血和血栓性静脉炎在Efegatran/伊非加群治疗的患者中发生的频率更高。 结论：给药Efegatran硫酸盐至少0.63 mg。公斤(1)。H（-1）提供的抗血栓作用至少与激活的部分凝血活素时间调整肝素输注相当。没有过多的大出血。依非加群的凝血酶抑制水平，通过激活部分凝血活素时间来测量，似乎比肝素更稳定。因此，像其他凝血酶抑制剂一样，硫酸Efegatran兰比肝素更容易给药。然而，Efegatran/依非加群与肝素相比没有明显的临床益处。[1]

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弥散性血管内凝血（DIC）是一种全身性血栓出血性疾病，与许多临床情况有关，如败血症、恶性肿瘤、产科并发症和血管内溶血。在我们的模型中，连续两次静脉注射大肠杆菌内毒素（80和40微克/千克）诱导家兔播散性血管内凝血。对照组给予0.9%生理盐水治疗。在有内毒素和没有内毒素的情况下，将硫甙的活性与未分离肝素（UFH）和Efegatran进行比较。给药剂量：肝素50、100 IU/kg/h静脉滴注；Efegatran0.25和0.5 mg/kg/h静脉滴注；GYKI 39521 （RGH-1875）和GYKI 39541 (RGH-1962) 12.5和25 mg/kg / os。与内毒素/载药组相比，巯基糖苷未改变模型中的凝血参数[凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）]。给药后TFPI水平的变化与未给药后相似。在我们的模型中，GYKI 39521和GYKI 39541治疗不影响内毒素诱导的白细胞计数变化。GYKI 39521和GYKI 39541可防止纤维蛋白原水平和血小板计数的降低。GYKI 39521和GYKI 39541显著降低了纤维蛋白降解产物和纤维蛋白溶解。在治疗弥散性血管内凝血时，巯基糖苷类药物可能比肝素具有更低的出血风险。[2]

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内毒素家兔给药后凝血时间的变化[2]
应用肝素和Efegatran后，凝血时间呈剂量依赖性延长（表1）。在生理盐水组和巯基糖苷组之间，凝血参数无显著差异。LPS治疗也倾向于延长APTT和PT，这些效果具有统计学意义（表2）。在DIC模型中，给予肝素和Efegatran后凝血时间延长明显大于未给予内毒素的模型。在Sa/LPS、G1/LPS和G2/LPS组之间，凝血时间无显著差异（表2）。

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内毒素家兔给药后TFPI水平的变化[2]
GYKI 39521诱导的TFPI水平升高（两种测试剂量）与低剂量肝素无内毒素时相似。在给药GYKI 39541后，该参数的变化较小。Efegatran没有引起TFPI水平的显著升高（图2）。Sa/LPS组兔DIC模型血浆TFPI平均浓度高于对照组，且与Efegatran/LPS组相近（图3）。在DIC模型中，给药肝素、GYKI 39521和GYKI 39541后TFPI水平的变化与未给药内毒素组相似（图2、图3）。

通过测量出血时间（Ivy, Simplate， Surgicut和Duke法，局部实验室，剂量范围部分）、活化的部分凝血活酶时间（局部和中心实验室）、凝血酶原时间（局部和中心实验室）和纤维蛋白原水平（中心实验室）来评估硫酸依费加群/Efegatran和肝素对血栓和止血标志物的影响。采用ACL和Clauss法测定纤维蛋白原水平。血小板（β -血小板球蛋白、血小板因子4）、凝血（凝血酶原片段1.2、纤维蛋白肽A、凝血酶-抗凝血酶复合物）和纤维蛋白溶解（纤维蛋白降解产物）的活化标志物水平仅在剂量寻找阶段（中心实验室）进行测量。普通血液学（当地实验室）、化学（中心实验室）和尿液分析（当地实验室）也进行了检查。［1］

凝血酶抑制剂对VE - cadherin阳性细胞生成的影响。在不含水蛭素（20 U mL−1）(A)或Efegatran sulfate (30 nm) (B)的情况下，用凝血酶处理图1中制备的细胞，并使用荧光活化细胞分选（FACS）进行分析。[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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。