Thrombin

Melagatran is a novel, reversible, and selective inhibitor of thrombin of 429.5 dalton. The Ki value for thrombin, measured with a chromogenic substrate assay, is 2 nM, which is 300 times lower than the inhibition of fibrinolytic enzymes by two-chain tissue plasminogen activator and plasmin. [2]
The synthetic direct thrombin inhibitor Melagatran and its oral form H 376/95 is areversible and selective direct inhibitor of thrombinwith a molecular weight of 429 Da. The inhibitionconstant (K i ) for thrombin as measured with achromogenic substrate assay, is 2 nmol/l, which isapproximately 300 - 400 times lower thancorresponding K i values for fibrinolytic enzymes [1].

For this purpose Melagatran, a direct synthetic thrombin inhibitor with a molecular weight of 429 Da, was employed. Melagatran does not significantly interact with any other enzymes in the coagulation cascade or fibrinolytic enzymes aside from thrombin. Furthermore, melagatran does not require endogenous co-factors such as antithrombin or heparin co-Factor II for its antithrombin effect, which is important, as these inhibitors are often consumed in septic patients. We have shown that melagatran exerts a beneficial effect on renal function, as evaluated by plasma creatinine and urinary output, during experimental septic shock. These effects were most pronounced during the later phase of the experimental period, after the infusion of melagatran had been discontinued. Prevention of intrarenal coagulation may be attributable to this finding. In addition, melagatran had beneficial effects on systemic haemodynamics (left ventricular stroke work index, pulmonary capillary wedge pressure and systemic vascular resistance index) in endotoxaemic pigs. This result may be explained by the ability of melagatran to inhibit thrombin, thereby counteracting thrombin's cellular effects. Thus, it can be seen, using this experimental model of septic shock, that Melagatran may help to alleviate some of the damaging effects of endotoxaemia, although more research is required to test this further.

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Intravenous administration of thrombin inhibitors, such as hirudin, has been shown to decrease the frequency of coronary artery reocclusion after thrombolysis. However, recent findings in large clinical trials in patients with unstable angina and myocardial infarction have failed to demonstrate a sustained antithrombotic effect after cessation of drug treatment. These findings indicate a need for a prolonged antithrombotic regimen, preferably an orally active thrombin inhibitor. To test the hypothesis that a regimen consisting of oral thrombin inhibitor will delay or prevent the formation of occlusive clot, anesthetized dogs were given saline (n = 9) or a single dose of a novel active site low-molecular-weight thrombin inhibitor Melagatran by nasogastric tube (1.5 mg/kg, n = 6; 2.5 mg/kg, n = 6), and 15 min later, a potent thrombogenic stimulus in the form of anodal current (100 microA) was applied to the intimal surface of the narrowed left anterior descending coronary artery (LAD). All saline-treated dogs developed stable thrombus, indicated by zero flow at 34 +/- 7 min after initiation of direct current. On the other hand, one of the six dogs given high-dose melagatran did not develop thrombotic occlusion of the LAD during the entire 4 h of observation. Mean time to occlusive thrombus formation in 11 other dogs was prolonged 4-5 times as compared with that in the saline-treated dogs (p < 0.001). Spontaneous thrombolysis was observed in three of 11 dogs after initial clot formation. Overall, the coronary artery was patent for 68% (low dose) and 75% (high dose) of the observation period in melagatran-treated dogs (vs. 14% of observation period in saline-treated dogs). Peak plasma concentration was 0.87 +/- 0.22 microM in dogs given low-dose and 1.38 +/- 0.30 microM in dogs given high-dose melagatran. The activated partial thromboplastin time (aPTT) increased 1.5-fold at peak plasma concentration of Melagatran. These observations imply (a) thrombin generation plays a critical role in thrombus formation in narrowed coronary arteries, (b) oral melagatran prevents or delays thrombus formation, whereas the aPTT is only modestly prolonged, and (c) the thrombus formed in the presence of melagatran is prone to spontaneous lysis in this canine model of coronary thrombosis. [2]

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Effect of Melagatran on the parameters of thrombosis [2]
There were no changes in systemic hemodynamics throughout the entire experimental period (Table 1). Baseline LAD blood flows in the three groups of dogs were similar. Time to thrombus formation and duration of coronary blood flow in each dog treated with Melagatran are shown in Fig. 1. Administration of high-dose melagatran completely prevented thrombus formation in one of six dogs over the 4-h period of observation. Mean time to occlusive thrombus formation in the 11 remaining melagatran-treated dogs was markedly prolonged (156 ± 22 min in dogs given low-dose and 139 ± 26 in dogs given high-dose melagatran, both p < 0.001, vs. the time to thrombus formation in the saline-treated group of dogs). Mean time to thrombus formation was similar in low-dose and high-dose melagatran-treated dogs. LAD blood flow reappeared spontaneously in two of six dogs given low-dose melagatran after 3 and 55 min of occlusion, respectively (dogs 3 and 4). Spontaneous dissolution of the thrombus after 44 min of zero flow also occurred in one of the dogs given high-dose melagatran (dog 5). Thus at the end of the 4-h observation period, blood flow in the LAD was present in four of 12 dogs. [2]
Overall, oral Melagatran prevented thrombus formation in one of 12 dogs and significantly prolonged the time to thrombus formation in the remaining 11, with spontaneous thrombolysis in three dogs. Mean patency time over the 4-h period of observation was 164 ± 25 and 180 ± 21 min after low-dose and high-dose melagatran, respectively (68 and 75% of the total observation time). Although both doses of melagatran were significantly better than placebo, there was no difference between the efficacy of the two doses, and the mean blood flow in dogs given low-dose or high-dose melagatran was similar (Table 1).

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Activated partial thromboplastin time (aPTT) [2]
Baseline aPTT (≈25 s) was similar in the three groups of dogs. During stabilization of the thrombus, this parameter was unchanged in the saline-treated group of dogs. The aPTT were prolonged 1.3-1.5 times at 1 h after the administration of melagatran (1.5- or 2.5-mg/kg dose). Whereas the prolonged aPTT value was maintained throughout the entire observation period in dogs given high-dose Melagatran, it returned to baseline at the end of the observation period in dogs given low-dose Melagatran. These results are summarized in Table 2.

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Plasma fibrinogen and TAT-complex levels [2]
As shown in Table 3, fibrinogen levels before treatment were similar in all dogs and remained unchanged throughout the period of thrombus induction and subsequent observation. Thrombin generation, assessed by plasma TAT-complex levels, also are shown in Table 3. Baseline TAT-complex levels were similar in all dogs (≈10 ng/ml). The TAT-complex values increased three- to fourfold during thrombosis in the saline-treated group of dogs, indicating generation of thrombin during thrombosis. In all Melagatran-treated dogs, however, the TAT levels remained unchanged throughout the entire observation period.

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Scanning electron microscopy of coronary arteries [2]
Figure 3 shows representative examples of scanning electron microscopy of the thrombus in the LADs after treatment with saline or Melagatran (2.5 mg/kg). The ultrastructure of the LADs showed extensive endothelial damage, intense platelet-fibrin deposition, and totally occlusive thrombus in saline-treated dogs. Administration of Melagatran resulted in a loose thrombus, and the residual thrombus showed a minimal amount of fibrin on the intimal surface of the coronary artery.

Determination of Melagatran levels in plasma [2]
Femoral venous blood was collected in 0.13 M sodium citrate (9:1, vol/vol) from the dogs before administration of Melagatran, and 15, 30, 45, 60, 90, 120, 180, and 240 min after Melagatran. Concentration of melagatran in plasma was determined with reversed-phase chromatography coupled to ion-spray mass spectrometry. Melagatran was isolated from 200 μl test plasma by solid-phase extraction after the addition of an internal standard and 50 μl of the extract was used for analysis.

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Measurement of activated partial thromboplastin time (aPTT) [2]
Femoral venous blood was collected in 0.13 M sodium citrate (9:1, vol/vol) from the dogs before administration of Melagatran and 15, 30, 60, 120, 180, and 240 min after bolus injection of Melagatran. The aPTT was determined by using 0.02 M calcium chloride and actin FS activated aPTT reagent. The aPTT assay was performed on Electra 900/900C automated machine. The aPTT was measured immediately after blood collection to avoid time-dependent degradation of fibrinogen and other coagulation proteins.

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Plasma fibrinogen and thrombin-antithrombin III (TAT) complex [2]
Femoral venous blood was collected in 0.13 M sodium citrate (9:1, vol/vol) from the dogs before administration of Melagatran, 60, 120, 180, and 240 min after bolus injection of Melagatran, and immediately placed on ice. For measurement of fibrinogen, aprotinin (0.003%) and PPACK (D-Phe-Pro-ArgCH2Cl, 0.005%) were added to the tubes. For measurement of TAT complexes, aprotinin alone was added. Blood samples were centrifuged at 1,500 g for 15 min, and plasma samples were stored at −70°C. Measurement of fibrinogen and TAT-complex levels was completed within 4 weeks of blood collection and performed as described earlier. Fibrinogen in plasma was determined as clottable fibrinogen. A sandwich enzyme immunoassay with microtitration plates coated with rabbit antibodies against human thrombin was used for quantitative determination of TAT complexes.

Animal preparation [2]
Twenty-one mongrel dogs of either sex weighing 21 kg (range, 18-25 kg) were fasted for 8 h, anesthetized with pentobarbital sodium (30 mg/kg), intubated, and placed on positive-pressure ventilation with a respirator. A left thoracotomy was performed in the fifth intercostal space, and the heart was suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) was isolated distal to the first diagonal branch. An ultrasonic Doppler flow probe was placed on the LAD for monitoring coronary blood flow. The signal from the Doppler flow probe was calibrated against an electromagnetic flow probe, and flow was expressed in milliliters per minute, as described earlier. In each dog, a nasogastric tube was inserted into the mid-duodenum. Thereafter, the starved animals were orally given saline (n = 9) or a single dose of Melagatran (1.5 mg/kg, n = 6; or 2.5 mg/kg, n = 6) through the nasogastric tube. All dogs were observed for 4 h after administration of saline or Melagatran for evidence of coronary artery occlusion or reperfusion. Presence or absence of blood flow was observed during the 4-h period.

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Induction of coronary artery thrombus [2]
Fifteen minutes after administration of saline or Melagatran, an intracoronary thrombus was induced by using the technique initially described by Romson et al. and subsequently used by researchers. In brief, the LAD was gently rubbed to disrupt the endothelium distal to the flow probe, and a silver-coated copper wire with a 25-gauge needle tip (∼4 mm) bent 90° was inserted distal to the flow probe into the LAD and pulled back to ensure contact with the intimal surface of the vessel. This electrode was connected in series with a 250,000-Ω variable resistor to the anode (positive terminal) of a 9-V nickel-cadmium battery. The cathode (negative terminal) was secured to subcutaneous tissues. In all animals, the LAD was narrowed by a pneumatic occluder placed distal to the flow probe and the electrode. The degree of occlusion was designed to reduce peak reactive hyperemia after a 10-s period of total occlusion by ≥50%. The thrombus formation was initiated with passage of anodal current (100 μA) through the intracoronary electrode and determined by the presence of zero blood flow with the electric current turned off and the plastic occluder removed. The stability of the thrombus was observed from the presence or absence of coronary blood flow.

Plasma concentrations of Melagatran [2]
Plasma concentrations of Melagatran were monitored frequently, and the mean concentration curve over time is illustrated in Fig. 2. The peak plasma concentration (Cmax) was reached within 30-60 min and was 0.82 ± 0.22 and 1.38 ± 0.30 μM in the low- and high-dose dogs, respectively. The elimination phase of the time-concentration curve was analyzed with linear regression to estimate the plasma concentration of Melagatran at the time the thrombus was formed. The plasma concentration of Melagatran in the dog in the high-dose group that did not develop occlusion (dog 4) was 0.56 μM at the end of the experiment. The mean concentration at time for thrombus formation was determined from individual elimination curves and did not differ significantly between the two dose groups (p = 0.13). Data from the two groups was therefore pooled, and the mean concentration at time for thrombus was found to be 0.43 ± 0.19 μM (n = 11).

[1]. Effects of melagatran, a novel direct thrombin inhibitor, during experimental septic shock. Expert Opin Investig Drugs. 2000 May;9(5):1129-37.

[2]. Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery. J Cardiovasc Pharmacol. 1998 Mar;31(3):345-51.

Melagatran is a member of the class of azetidines that is (2S)-azetidine 2-carboxylic acid in which the carboxylic acid has been converted to the amide corresponding to formal condensation with 4-(aminomethyl)benzenecarboximidamide and in which the hydrogen attached to the azetidine nitrogen is replaced by a (2R)-2-cyclohexyl-2-[(carboxymethyl)amino]acetyl group. It has a role as an anticoagulant, an EC 3.4.21.5 (thrombin) inhibitor and a serine protease inhibitor. It is a carboxamidine, a dicarboxylic acid monoamide, a non-proteinogenic alpha-amino acid, a secondary amino compound and a member of azetidines.

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Endotoxaemia causes activation of prothrombin tothrombin. In both haemostatic and inflammatoryreactions, which are closely related, thrombin is ofcrucial importance. Thus, treatment with an inhibitorof thrombin seems a logical attempt to counteractsome of the deleterious effects of endotoxic shock.These findings indicate a beneficial effect of melaga-tran on renal function during experimental septicshock. Retained plasma creatinine levels wereobserved during endotoxaemia, even after the Melagatran infusion was terminated. The total urinaryoutput during the experimental period (6 h) wassignificantly reduced in the control group ascompared with the Melagatran-treated group [31].Since the kidneys are frequently damaged in septicconditions, these findings may be of importance. Theprotective effect of melagatran on renal functionduring endotoxaemia may be explained by melaga-tran’s ability to reduce thrombin-induced intrarenaldepositions. The endotoxin-induced dysfunction ofthe left side of the heart and systemic haemodynamicresistance, LVSWI, PCWP and SVRI, were less affectedby endotoxaemia during the 3 h period when melaga-tran was infused and the plasma concentrationremained at a steady-state around 0.8 µmol/l [31,32].Thus, Melagatran may have opposed the action ofthrombin from exerting its biological effects on theheart as well as on endothelial and smooth musclecells [30]. Another field of interest is the effects ofseptic shock on the fibrinolytic system. Fibrinolyticdisturbances are associated with poor prognosis inseptic shock [40] and treatment with fibrinolyticagents may be of value [41]. The naturally occurringanticoagulant protein C may be beneficial in certaincases [42]. Bleeding problems may arise when fibrino-lytic drugs are used in septic shock [43,44]. This is anatural consequence of the fact that drugs affectingcoagulation and/or fibrinolysis should be used withcaution, in conditions with severely disturbedhaemostasis. Although only preliminary, the resultspresented in this review suggest that direct thrombininhibitors, such as melagatran, may be appropriateagents for the treatment of septic shock.[1]

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This study addressed the effect of Melagatran on the prevention of arterial thrombosis and on spontaneous thrombolysis in dogs. The study demonstrates that Melagatran was easily and rapidly absorbed from the gastrointestinal tract and that it delayed or prevented or both the formation of platelet-fibrin-rich thrombus formation in dog coronary arteries via inhibition of thrombin. The antithrombotic effect was achieved at a dose that gave only a modest increase in aPTT (≈1.5 times the baseline value), suggesting that long-term oral treatment with melagatran may be an attractive way to prevent arterial thrombosis. However, a precise relation between change in aPTT and inhibition of thrombosis cannot be discerned from this study. [2]

C22H31N5O4

429.51264

429.238

159776-70-2

159776-70-2; 179418-09-8 (HCl);318245-80-6 (hydrate);

183797

HOCOCH2-D-Chg-Aze-NHBn(4-amidino)

White to off-white solid powder

1.41g/cm3

1.67

2.33

5

6

9

31

671

2

C1CCC(CC1)[C@H](C(=O)N2CC[C@H]2C(=O)NCC3=CC=C(C=C3)C(=N)N)NCC(=O)O

DKWNMCUOEDMMIN-PKOBYXMFSA-N

InChI=1S/C22H31N5O4/c23-20(24)16-8-6-14(7-9-16)12-26-21(30)17-10-11-27(17)22(31)19(25-13-18(28)29)15-4-2-1-3-5-15/h6-9,15,17,19,25H,1-5,10-13H2,(H3,23,24)(H,26,30)(H,28,29)/t17-,19+/m0/s1

2-[[(1R)-2-[(2S)-2-[(4-carbamimidoylphenyl)methylcarbamoyl]azetidin-1-yl]-1-cyclohexyl-2-oxoethyl]amino]acetic acid

Melagatran; 159776-70-2; Melagatran [INN]; UNII-2A9QP32MD4; Melagatran (INN); 2A9QP32MD4; CHEBI:43966; MELAGATRAN [MI];

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.)