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Argatroban is a peptidomimetic inhibitor of thrombin that is currently undergoing extensive clinical trials as a heparin substitute for thrombotic complications. Argatroban is readily metabolized into a major derivative, M1, that has pharmacological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumulative anticoagulant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatroban and M1-metabolite. A modified method was developed to simultaneouly quantitate M1-metabolite and argatroban in biological fluids. Initial validation studies for the method included clinical trials of argatroban in patients with heparin-induced thrombocytopenia, (ARG 911 Study) and coronary interventional procedures (ARG 310 Study). Plasma samples were extracted with acetonitrile and reconstituted in a mobile phase. Calibration curves were prepared by running known standards of argatroban and M1-metabolite in normal human plasma. Ultraviolet detection was made at 320 nm. The retention times for argatroban and M1-metabolite peaks were found to be 10.5 +/- 0.3 minutes and 3.9 +/- 0.1 minutes, respectively. The extraction efficiency was > 95% (r2 = 0.99). In heparin-induced thrombocytopenia patients with major bleeding complications (n = 30), the relative increase in M1-metabolite compared to argatroban varied widely (two- to eight-fold). The mean concentration of argatroban during the steady infusion period was found to be 0.7 +/- 0.35 microgram/mL, and for M1-metabolite, it was 5.5 +/- 2.8 micrograms/mL. Proportionate results were not seen when higher dosages of argatroban were administered (coronary angioplasty studies). Argatroban and M1-metabolite levels also compared well with the results in global clotting assays. Owing to the simultaneous quantitation of argatroban and M1-metabolite, this method provides a rapid assessment of the pharmacokinetics and pharmacodynamics of argatroban. The differential quantitation may be useful in the assessment of relative metabolic turnover of argatroban that can be related to the hepatic and renal functions in a given patient.

Argatroban is a peptidomimetic reversible thrombin inhibitor that has been used as an anticoagulant in clinical trials related to heparin-induced thrombocytopenia (HIT). To monitor the absolute concentrations of argatroban and to relate the safety and efficacy of this drug with the circulating levels and corresponding prolongation of the activated clotting time (ACT), a specific method for the absolute measurement of this drug was developed. Initial studies for the validation of method included the quantitation of argatroban in normal volunteers administered with escalating doses of argatroban (15-40 μg/ kg/min) on four consecutive days for 4 h (ARG 102 Study). Pre-and postinfusion samples were analyzed by using the high performance liquid chromatography (HPLC) method. Argatroban levels ranged from 0.5-4.5 μg/mL, postinfusion (0.84 ± 0.23 [day 1], 1.55 ± 0.34 [day 2], 2.92 ± 0.15 [day 3], 3.04 ± 0.49 [day 4]). A proportionate increase in the activated partial thromboplastin time (APTT) and ACT was observed. Similarly, ecarin clotting time (ECT) also provided comparable results. Argatroban levels were also measured in a PTCA trial where this agent was used as an anticoagulant at 350 μg/kg bolus followed by 25 μg/kg/min to regulate the ACT between 400-450 sec (ARG 310 Study). In this angioplasty study, the levels of argatroban correlated well with the ACT (r2 > 0.8). In another clinical study, argatroban was used in conjunction with streptokinase for the management of acute myocardial infarction (AMI), the levels of this agent were quantitated at baseline and 2-8 h postthrombolysis (AMI Study). In the AMI study, mean argatroban levels at 2-8 h were between 1.5-2.0 μg/mL. Upon completion of the infusion, a time dependence in circulating argatroban levels was noted. Since heparinization, hemodilution, hypofibrinogenimia due to thrombolysis influence the ACT levels, absolute quantitation of argatroban in these patients provides a reliable means of monitoring and dosage optimization of this anticoagulant. Based on these observations and additional pharmacokinetics data, it is proposed that the currently used dosage of 350 μg/kg bolus followed by 25 μg/ kg/min infusion is optimal to achieve anticoagulation for interventional cardiovascular procedures. This translates into 3-5 μg/mL circulating concentration. For therapeutic anticoagulation to mimic responses observed at an APTT of 70-100 sec, a bolus of 50 μg/kg followed by 10 μg/kg/min resulting in a circulating levels of approximately 1.0 μg/mL concentration is recommended.

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 IU/mg) and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase I. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540, graded concentrations (1.25-100 microg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was employed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37 degrees C) at 1 mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide. SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.

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