Ahmad Ahsan

Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands

Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.

Simultaneous Monitoring of Argatroban and Its Major Metabolite Using an HPLC Method: Potential Clinical Applications

Argatroban is a peptidomimetic inhibitor of throm bin 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 phar macological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumula tive anticoagulant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatro ban and M1-metabolite. A modified method was developed to simultaneously quantitate M1-metabolite and argatroban in biological fluids. Initial validation studies for the method in cluded clinical trials of argatroban in patients with heparin- induced thrombocytopenia, (ARG 911 Study) and coronary in terventional 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 reten tion 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 com plications (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 μg/mL, and for M1- metabolite, it was 5.5 ± 2.8 μg/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.

Pharmacokinetics and Pharmacodynamics of Argatroban as Studied by HPLC and Functional Methods: Implications in the Monitoring and Dosage-Optimizations in Cardiovascular Patients

Argatroban is a peptidomimetic reversible throm bin 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. Argatro ban 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. Simi larly, ecarin clotting time (ECT) also provided comparable re sults. 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 infarc tion (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 circu lating argatroban levels was noted. Since heparinization, he modilution, 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 inter ventional cardiovascular procedures. This translates into 3-5 μg/mL circulating concentration. For therapeutic anticoagula tion 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.

Molecular weight and biochemical profile of a chemically modified heparin derivative, Suleparoide®

Recently, a new chemically modified derivative of heparin (Suleparoide, Syntex Laboratories Buenos Aires, Argentina) was introduced for the prophylaxis of thrombosis and treatment of vascular disorders. This agent is claimed to contain a depolymerized, chemically modified, heparin derivative with similar biologic actions as heparan sulfate. To study the pharmacologic profile of this agent, we have defined its molecular weight distribution profile, utilizing a computerized gel permeation chromatographic system equipped with ultraviolet and refractive index detectors. Suleparoide exhibited a normal molecular distribution profile with no contaminants. It exhibited a weight average of 9.3 K DA and an apparent peak MW of 8.0 K DA. Approximately 50% of the molecular components were < 5.0 K DA and 40% > 5.0 K DA. The results from these studies on the mechanisms show that Suleparoide has anticoagulant activity primarily mediated through Heparin Cofactor-II (HC-II) and because of its novel mechanism of action, further investigations on the biochemical profile of Suleparoide are carried out. Global clotting tests such as Activated Partial Thromboplastin Time (APTT), Heptest and Thrombin Time (TT) revealed a concentration dependent effect in all assays. Plasma samples supplemented with Suleparoide exhibited no significant anti-Xa and anti-IIa activities. However, in the HC-II mediated inhibitory assay for IIa, Suleparoide exhibited significant activity. In contrast, the Antithrombin-III (AT-III) mediated inhibition of IIa was much weaker.