Biomedical subjects
J Fareed
Publications and source records attributed to J Fareed.
Pharmacokinetics of argatroban in primates: evidence on endogenous uptake.
BACKGROUND: Antithrombin agent, argatroban, is currently undergoing several clinical trials for cardiovascular indications. Because of its solubility, this drug is usually administered via an intravenous bolus followed by infusion. The purpose of this study was to determine the pharmacokinetics of argatroban after intravenous bolus injection in primates. METHODS: Parallel in vitro studies in primate whole blood were carried out to simulate a one-compartment system. Argatroban (range 1.0-7.5 mg/kg) was administered to four groups of primates and blood samples were drawn at various time periods. Argatroban measurements were made in plasma using functional (aPTT, Heptest, TT) and HPLC methods. RESULTS: In vitro, argatroban primarily distributed in the plasma in proportionate amounts. Relative uptake of argatroban to the blood cells (leukocytes and erythrocytes) was minimum. However, in vivo, argatroban followed a complex pharmacokinetics. Within 5 min after the bolus administration, only <20% of argatroban was recovered. The recovered amount was proportionate to the dosage and followed the expected kinetics with a half-life of <20 min. Simultaneous quantitation of M1-metabolite of argatroban revealed only a fraction of recovered argatroban (approximately 25%) converted into M1 in these experimental settings. Results obtained from the functional and absolute methods correlated well. HPLC profile did not reveal the presence of any other metabolite(s). CONCLUSIONS: These observations suggest that argatroban may be endogenously taken up by the vascular or other sites and may exhibit a complex kinetics. In acute settings, the metabolic transformation of argatroban to M1 is relatively low. To further clarify the pharmacokinetics/pharmacodynamics of this drug, additional studies are warranted.
Heparins in the new millennium: will unfractionated heparin survive? Epilogue.
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Usefulness of intravenous enoxaparin for percutaneous coronary intervention in stable angina pectoris.
This pilot study was designed to determine whether the low molecular weight heparin, enoxaparin, could be used for elective percutaneous coronary intervention (PCI) to provide antithrombotic effects without the full systemic anticoagulation that occurs with the use of unfractionated heparin. Sixty patients were randomized to receive intravenous enoxaparin (1 mg/kg bolus dose) or unfractionated heparin at the time of coronary intervention. Laboratory testing was performed at baseline, 5 minutes, and 4 hours after study drug to test if a single bolus dose of intravenous enoxaparin can consistently achieve therapeutic antithrombotic effect, thus eliminating the need for multiple doses of heparin and closely monitoring levels of anticoagulation during PCI. Thirty percent of patients who received unfractionated heparin required a second bolus of intravenous heparin to achieve the target-activated clotting time of 300 seconds before PCI. Enoxaparin showed antithrombotic properties comparable to that of unfractionated heparin as measured by anti-Xa levels, with less inhibition of thrombin (factor IIa) at the time points measured (p <0.0001). Angioplasty success rates, in-hospital ischemia, bleeding, and vascular complications were similar in both groups. Thus, intravenous enoxaparin has predictable and effective antithrombotic effects during elective PCI. Although the level of anticoagulation attained with enoxaparin is significantly lower than that after unfractionated heparin, no increase in ischemic complications were noted. The use of a single bolus of intravenous enoxaparin, without the need for measuring the activated clotting time or titrating heparin anticoagulation, has the potential for simplifying the performance and perhaps enhancing the safety of PCI.
Laboratory monitoring of pentasaccharide in a dog model of hemodialysis.
Varying dosages of pentasaccharide (400-800 nmol/kg) were compared to a 250-U/kg single bolus dosage of unfractionated heparin (UFH) in a dog model of hemodialysis. Several laboratory assays were used to monitor the effects of pentasaccharide and UFH. The pentasaccharide did not produce any anticoagulant effects as measured by the activated partial thromboplastin time. However, in the anti-Xa chromogenic assay and the Heptest assays, there was a dose-dependent prolongation after pentasaccharide administration. In the group of dogs administered 800 nmol/kg of pentasaccharide, there was a 50% decrease in the thrombin antithrombin (TAT) complex level after 60 minutes on dialysis. In the UFH-treated dogs, wide variations in assays were observed. There was a marked elevation in the activated partial thromboplastin time and Heptest assays up to 6 hours after UFH administration. Both anti-Xa and anti-IIa activity was measured up to 4 hours. In the TAT assay, UFH was found to have a stronger effect in suppressing the formation of TAT in comparison to the pentasaccharide. These results suggest that pentasaccharide can be used as a replacement for UFH in a dog model of hemodialysis to keep the dialysis circuit patent. In addition, the anti-Xa-based assays such as the Heptest and the chromogenic anti-Xa assays can be used to monitor the effects of pentasaccharide in this model.
Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.
A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.
Acquired defects of fibrinolysis associated with thrombosis.
Physiologic regulation of fibrinolysis plays an important role in the control of hypercoagulable states and thrombogenesis. Both the hereditary and acquired conditions leading to fibrinolytic deficit result in thrombotic complications leading to arterial and venous occlusive disorders. Several changes in physiologic states such as pregnancy, old age, stress, obesity, and temperature alterations lead to the modulation of the fibrinolytic system. Various disease states, surgery, radiation, and diet can also trigger mechanisms leading to impaired fibrinolytic states. Several drugs, including anticancer agents, oral contraceptives, cytokines, and blood components can also produce transitory fibrinolytic deficit which can predispose patients to thrombotic complications. The identification of the patient populations with an impaired fibrinolytic state is an important step toward the prevention of thrombotic complications which may lead to such catastrophic events as myocardial infarction and thrombotic strokes. Both functional and immunologic methods have currently become available for the rapid diagnosis of fibrinolytic deficit. Thus, it is important to evaluate patients who are at risk of thrombotic complications due to fibrinolytic deficit. Currently, specific guidelines are developed to identify high risk groups and propose methods to manage these groups of patients.
Inactivation of factor Xa by the synthetic inhibitor DX-9065a causes strong anticoagulant and antiplatelet actions in human blood.
In an in vitro study, anticoagulant and antiplatelet effects of the synthetic, direct factor Xa inhibitor DX-9065a, (+)-2S-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7-a midino-2-naphthyl]propanoic acid hydrochloride pentahydrate, which shows a high affinity and selectivity towards the enzyme, were investigated. Anticoagulant actions of DX-9065a were studied in human plasma using global clotting assays [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and Heptest]. The effect on thrombin generation was measured in whole blood by determining the plasma concentration of prothrombin fragment F1.2. The influence on agonist-induced platelet activation in whole blood was studied using flow cytometric analysis. DX-9065a caused a concentration-dependent prolongation of clotting times in the PT and APTT assay, whereas Heptest was less affected and TT was not influenced. Furthermore, DX-9065a strongly inhibited the generation of thrombin without and after coagulation activation. The factor Xa inhibitor did not affect platelet activation mediated by either thrombin receptor activating peptide, arachidonic acid or y-thrombin, but prevented tissue factor- and factor Xa-induced activation of platelets in a concentration-dependent manner. Inactivation of factor Xa by a highly effective and selective inhibitor, and the resulting inhibition of thrombin generation leads to strong anticoagulant and antiplatelet actions. The interference with the coagulation system at the early level of factor Xa is expected to be an effective approach for a successful anticoagulant/antithrombotic therapy.
Heparin-induced thrombocytopenia: clinical considerations of alternative anticoagulation with various glycosaminoglycans and thrombin inhibitors.
Heparin-induced thrombocytopenia (HIT), the most common complication of heparin therapy, is also the most common form of the drug-induced thrombocytopenias. HIT is classified as type I and type II, the first being benign and the latter severe. HIT type II is attributed to an immune response characterized by complexes of heparin and platelet factor (PF) 4. Enzyme-linked immunosorbent assays allow easy and simple determination of these antibody titers; however, because specificity and sensitivity is not optimal, there is concern that the clinical relevance may be low. In clinical trials many patients were shown to form HIT-IgG in response to heparin without developing manifestations of HIT type II. Therefore, routine screening of clinically asymptomatic patients for antiheparin/PF 4 antibodies is not recommended. HIT type II is a clinico-pathologic syndrome that ideally should be confirmed by laboratory testing. If any clinical suspicion arises, however, heparin and low molecular weight heparin therapy should be discontinued and an alternative anticoagulant therapy started. Alternative drugs have been evaluated in significant numbers of patients including danaparoid and thrombin inhibitors. In the case of danaparoid, it is highly recommended that an in vitro test for cross-reactivity be performed before the onset of therapy. If testing cannot be performed, immediate administration of a thrombin inhibitor is preferred.
Thrombogenesis in myocardial infarction and related syndromes: the role of molecular markers in diagnosis and management.
Anticoagulant therapy has undergone some major developments in recent years. Conventional drugs that produce anticoagulant effects such as heparin and oral anticoagulants are no longer considered the only candidates for the anticoagulant/antithrombotic management of patients. Recombinant hirudin, glycoproteins IIb/IIIa targeting antibodies, synthetic peptides such as Hirulog and efegatran are being tested for their efficacy. These drugs produce their effects at different sites. To monitor their overall effects on the hemostatic system, molecular markers offer a practical and reliable approach. Markers of thrombin generation are useful for the monitoring of antithrombin drugs whereas, the efficacy of antiplatelet drugs can be assessed by monitoring the platelet release products. Furthermore, polytherapy using several anticoagulant and antithrombotic drugs in combination has been considered. In these situations, the use of molecular markers may also prove to be invaluable. The introduction of simple technology such as the test strip or particle agglutination methods may be available for the measurement of many of these markers in the near future. This will be useful for ready availability and reduced cost for individual marker testing. Furthermore, this type of technology can be used at bedside, off-site, and in doctor's offices. It is clear that the molecular marker profiling provides useful information on the nature of pathophysiology of a given thrombotic disorder. However, for practical use, a cost-effective and simpler assay-based approach will enhance their use, and these tests will be readily accepted at the laboratory and clinical levels.
Simultaneous monitoring of argatroban and its major metabolite using an HPLC method: potential clinical applications.
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.
Synthetic pentasaccharides do not cause platelet activation by antiheparin-platelet factor 4 antibodies.
A synthetic selective inhibitor of factor Xa, the pentasaccharide SR90107A/Org31540 is in clinical development for the prophylaxis of postsurgical deep vein thrombosis. Another synthetic pentasaccharide with even more sustained inhibition of factor Xa, SanOrg34006, has also been developed. Both of these agents were tested in comparison to unfractionated heparin and a low molecular weight heparin (enoxaparin) for their relative platelet activation potential in heparin-induced thrombocytopenia assays. Sera from patients (n = 30) with heparin-induced thrombocytopenia were pooled and validated for heparin-dependent aggregation responses. Using heparin-platelet factor 4 Sepharose columns, antibodies to heparin-platelet factor 4 were purified from the same pool. The effects of heparin, enoxaparin, SR90107A/Org31540, and San-Org34006 were evaluated in a platelet aggregation assay using platelet donors (n = 10). At comparable concentrations, heparin and enoxaparin consistently produced platelet activation, whereas both pentasaccharides failed to produce a response at a concentration up to 100 micrograms/mL (approximately 50 microM). Similarly, in the 14C-serotonin release and flow cytometric assays, heparin and enoxaparin produced positive responses (n = 30), whereas the two pentasaccharides consistently failed to produce any effect. These observations suggest that the two pentasaccharides with highly selective anti-Xa activity are devoid of generating antiheparin-platelet factor 4 antibody, do not produce heparin-induced thrombocytopenic responses and may inhibit active heparin-induced thrombocytopenia antibody platelet activation.
Functional heterogeneity of antiheparin-platelet factor 4 antibodies: implications in the pathogenesis of the HIT syndrome.
Heparin-induced thrombocytopenia represents one of the most severe drug-induced disorders of platelets. This syndrome is believed to be mediated through antibodies generated against a heparin-platelet factor 4 complex. Complexation of a sulfated mucopolysaccharide chain of heparin with a platelet granular protein (platelet factor 4) produces an allosteric modification of platelet factor 4 resulting in neoepitope formation and the generation of antiheparin-platelet factor 4 antibodies. These antibodies are capable of activating platelets by binding to heparin, platelet factor 4 and the Fc receptor on platelets, resulting in a complex pathophysiology involving ischemic, thrombotic, and inflammatory processes. To characterize this antibody, IgG fractions were obtained from the serum of patients with heparin-induced thrombocytopenia using ammonium sulphate precipitation and heparin-platelet factor 4-sepharose 4B affinity chromatography methods. With the affinity purification, two major components, peaks I and II, with high antiheparin-platelet factor 4 antibody titers were eluted. The purity of all the fractionated immunoglobulins was established by sodium dodecylsulphate-polyacrylamide gel electrophoretic analyses. While peak I did not induce 14C-serotonin release from platelets in the heparin-dependent assay for heparin-induced thrombocytopenia antibodies (14C-serotonin release assay), peak II and the IgGs obtained with the ammonium sulphate precipitation method exhibited a strong and concentration-dependent activation in the presence and absence of heparin and low molecular weight heparin. These immunoglobulins were treated with heparinase, a cationic ion-exchange resin (Heparsorb), or dialyzed to remove traces of heparin, and when tested in the 14C-serotonin release assay, showed the same high degree of activity. These data are suggestive of the generation of heparin-induced thrombocytopenia antibodies capable of activating platelets directly in a nonheparin-dependent manner. These observations underscore the complex pathophysiology of heparin-induced thrombocytopenia syndrome and suggest that the severity of this syndrome in some patients may be due to the generation of "super-active" heparin-induced thrombocytopenia antibodies capable of activating platelets without the requirement of heparin. This could explain why the cessation of heparin in patients does not necessarily correct the symptoms of heparin-induced thrombocytopenia or associated thrombosis.
Soluble adhesion molecules in the HIT syndrome: pathophysiologic role and therapeutic modulation.
Heparin-induced thrombocytopenia pathophysiology is now known to be a complex process that involves platelets, vascular endothelium, and leukocytes/lymphocytes. The activation products from these sites also contribute to the activation of coagulation and fibrinolytic deficit. While many of the markers of hemostatic activation processes have been found to be increased during the acute phase of heparin-induced thrombocytopenia syndromes, the circulating levels of soluble adhesion molecules such as the P, E, and L selectins, and intracellular and vascular cell adhesion molecules have not been reported. Since the pathophysiology of heparin-induced thrombocytopenia involves the activation of platelets, endothelium, and leukocytes, it is expected that the activation products related to these hemostatic systems including soluble selectins and cellular adhesion molecules will also be increased in circulating blood. These alterations may also provide an index of the pathophysiologic process. With the availability of highly sensitive enzyme-linked immunosorbent assays for soluble P, E, and L selectins, intracellular and vascular cell adhesion molecules, it is now possible to measure these adhesion molecules in biological fluids. This study reports on the circulating levels of various adhesion molecules in patients with heparin-induced thrombocytopenia and their modulation after therapeutic interventions by the use of direct thrombin inhibitors. With the availability of recombinant hirudin, it is now possible to treat these patients with alternate antithrombin agents. However, the immunoactivation of platelets and other cells as shown here indicates the possible need for additional adjunct therapeutic approaches to suppress their participation in the thrombotic process. The reported increase in the circulating levels of the soluble adhesion molecules during the heparin-induced thrombocytopenia and heparin-induced thrombocytopenia with thrombosis syndrome suggests that the antiheparin platelet factor 4 antibody is capable of modulating their regulation. The prognostic role of these mediators in the management of heparin-induced thrombocytopenia syndrome warrants further investigation.
Antithrombin agents: the new class of anticoagulant and antithrombotic drugs.
Antithrombin drugs represent a wide group of natural agents, recombinant agents equivalent to some of the naturally occurring proteins, and synthetic agents. This group of drugs is characterized by marked structural and functional heterogeneity. Several of these drugs are currently in various phases of development. Argatroban represents the first clinically approved antithrombin agent, which was made available in Japan several years ago. Two recombinant hirudin preparations, Revasc (Novartis) and Refludan (Aventis), are available for postsurgical DVT prophylaxis and alternate anticoagulant use in patients with heparin-induced thrombocytopenia. A synthetic antithrombin agent based on the combined structures of hirudin and antithrombin peptides, hirulog (Bivalirudin), is undergoing clinical trials in cardiovascular indications. Additional studies on the hirudins are being carried out to test their efficacy as surgical and interventional anticoagulants as replacements for heparin. However, the need for a proper antagonist is one of the limiting factors for the optimal development of hirudin in this indication. Several of the synthetic thrombin inhibitors are also being developed for oral use for the prophylaxis of DVT in surgical patients. Since the therapeutic index of thrombin inhibitors is narrower than that of heparin, this route may not be an optimal approach for the development of these agents. Despite several unresolved developmental issues, the thrombin inhibitors provide a useful alternative to heparin anticoagulation and may prove to be useful in validated clinical use.
Low molecular weight heparins: differences and similarities in approved preparations in the United States.
There is adequate preclinical data to support the differential biochemical and pharmacological behavior of the currently approved low molecular weight heparins (LMWHs) in the United States. Initial studies on the anti-Xa, anti-IIa, and U.S. Pharmacopoeial (USP) potencies have clearly demonstrated differences among these products. Furthermore, the ratios between the anti-X and anti-IIa activities vary from one product to another. This is primarily due to the composition of each product manufactured by using different patented methods. Studies in pharmacologic animal models, using gravimetric dosages or adjusted anti-Xa dosages of the LMWHs, produce product-specific results. The pharmacokinetics and pharmacodynamics of each product also vary markedly and are not predictable on the basis of any pharmacopoeial potency designation. These agents are capable of releasing tissue factor pathway inhibitor (TFPI), an inhibitor of the coagulation process. Its release is also dependent on the type of LMWH. In the United States enoxaprin, dalteparin, and ardeparin have been approved for DVT prophylaxis. Only enoxaparin and dalteparin have been approved for the acute coronary syndrome. Recently the clinical differentiation among these LMWHs has been demonstrated in the treatment of acute coronary syndrome. Similarly, when these drugs are used at high dosages, they are expected to produce product-specific pharmacodynamic effects. It must be noted that while these drugs may be interchangeable at clinically optimized/approved dosages, these drugs are not interchangeable at equivalent anti-Xa dosages. Even at optimized dosages, the clinical provile of each drug may be different. Thus, each of the LMWHs should be considered a distinct entity and their use in a given clinical situation should be validated in proper clinical trials.
Pharmacologic profile of certoparin.
The low molecular weight heparins (LMWHs) are now not only used for the prophylaxis and treatment of deep vein thrombosis (DVT), but also for the management of acute coronary syndromes. Beside these approved usages, the LMWHs have been developed for indications such as thrombotic and ischaemic stroke, cancer-associated thrombotic and vascular disorders, Alzheimer's disease and a variety of inflammatory disorders. In the United States, there are three approved LMWHs (enoxaparin, dalteparin and ardeparin). In Canada, reviparin and tinzaparin are also approved. The European Union has taken the lead; eight LMWHs are approved for various indications. Certoparin represents one of the earlier LMWHs used for DVT prophylaxis and treatment, with additional indications currently under development. Certoparin represents an isoamyl nitrite depolymerised LMWH with comparable structural characteristics to other nitrous acid depolymerised products such as nadroparin and reviparin. While comparable in structure to dalteparin, this agent differs in function due to a secondary purification process that is employed in the manufacture of dalteparin. The preclinical pharmacology of this drug has been extensively investigated. Although indication specific dosing and the optimisation of use in, for example, acute coronary syndromes and thrombotic stroke, may be require, certoparin represents a typical LMWH with comparable performance characteristics to some other agents. This chapter describes some of the preclinical and clinical pharmacologic characteristics of this drug. This information will be useful in designing clinical trials for newer indications of this drug.
Pharmacology of argatroban.
Argatroban, a synthetic peptidomimetic antithrombin agent, is the first clinical anticoagulant solely to target thrombin. For some time, this drug has been used in Japan for the management of thromboembolic disorders. Recently, it has been approved in Japan for use in thrombotic and ischaemic stroke. Despite a large number of preclinical studies on the pharmacology of this agent, clinical trials in Europe and North America were only initiated in 1996. Argatroban produces anticoagulant effects comparable to therapeutic heparinisation at concentrations of approximately 1 microg/ml. At concentrations of 5-10 microg/ml, this agent produces adequate anticoagulation for inteventional cardiovascular procedures and prolongs the activated clotting time (ACT) to 400-600 s. The predictable anticoagulant effect of this agent is relatively short lasting, and may not warrant pharmacologic neutralisation in the majority of patients. However, patients with hepatic dysfunction may need some means of neutralisation. Unlike heparin, this drug produces its anticoagulant effects by direct inhibition of thrombin and thrombin-mediated processes. This agent is not influenced by endogenous factors such as platelet factor 4 and other proteins which bind heparin. Argatroban's use does not lead to the formation of antiplatelet antibodies. Thus, this drug is useful in the management of heparin induced thrombocytopenic (HIT) patients. Although argatroban was initially developed for the management of deep vein thrombosis (DVT), based on its pharmacologic properties, it can be developed for safer anticoagulation in such indications as acute coronary syndromes, as an adjunct to thrombolytics, thrombotic and ischaemic stroke and inflammatory diseases resulting in thrombotic complications.