Recombinant Hirudin Research Articles

Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate

The effects of the specific growth rate and methanol concentration on the degradation of hirudin produced by recombinant Pichia pastoris were investigated. When a methanol-limited state and the specific growth rate of 0.02 h−1 were maintained during the fermentation, a minimum degradation of hirudin and a maximum specific hirudin production rate were achieved. By this strategy, the production of intact recombinant hirudin Hir65 reached 0.7 g l−1 in fed-batch fermentation. Its proportion was 35% to all forms of hirudin.

Hirudin in renal insufficiency.

Recombinant hirudins (r-hirudins) are potent direct thrombin inhibitors increasingly used for alternative anticoagulation, especially in heparin-induced thrombocytopenia. R-hirudins are almost exclusively eliminated by the kidneys, and a close correlation between r-hirudin clearance and endogenous creatinine clearance has been observed. Accordingly, the pharmacokinetics of r-hirudin are altered in patients with renal insufficiency. A decline of renal r-hirudin clearance is associated with an increase of r-hirudin half-life and the area under the curve (AUC). Therefore, renal impairment necessitates reduction of r-hirudin dose to avoid overdose or inadequate accumulation of the thrombin inhibitor. To this end, close monitoring of r-hirudin anticoagulation is required, which at best is performed by measuring r-hirudin blood levels by ecarin clotting times (ECT) or chromogenic assays, in addition to activated partial thromboplastin time (aPTT). Recent studies showed that r-hirudin anticoagulation is feasible in acute or chronic renal failure treated with continuous or intermittent renal replacement therapy, if appropriate r-hirudin dosing and adequate monitoring are warranted. High-volume hemofiltration with r-hirudin-permeable hemodialyzers constitutes a valuable means to markedly reduce r-hirudin blood concentration and total r-hirudin body content in case of r-hirudin overdose or r-hirudin-associated bleeding. In the future, the hepatically eliminated direct thrombin inhibitor argatroban may facilitate alternative anticoagulation in patients with renal insufficiency.

Pharmacology of recombinant hirudin.

Hirudin is the anticoagulative product of the salivary glands of the medical leech Hirudo medicinalis. It is characterized by a direct, bifunctional inhibition mechanism and a high, exclusive specificity and a strong ability to bind to thrombin (tight binding). Further characteristics are the organic-chemical structure of hirudin (peptide), which allows only parenteral administration; the missing metabolism in the organism; and the exclusive glomerular filtration of hirudin in kidneys as the effective elimination mechanism. Recombinant hirudin (r-hirudin) is a product of genetic engineering that is identical to the hirudin found in nature and has the same biochemical and pharmacological characteristics as natural hirudin.

Biological relevance of anti-recombinant hirudin antibodies--results from in vitro and in vivo studies.

This article provides an overview of the clinically relevant characteristics of antibodies directed toward recombinant (r) hirudin, with emphasis on the different ways in which these antibodies may influence pharmacokinetics and pharmacodynamics of r-hirudin. A high incidence of anti-hirudin antibody (AHAb) formation, mainly of the immunoglobulin G (IgG) subclass, was reported in up to 74% of patients treated with r-hirudin for more than 5 days. Like other drug-induced antibodies, AHAb may be responsible for accumulation or neutralization of the drug. Current clinical data support this assumption with reports on reduced metabolism, enhanced activity, and accumulation and neutralization of r-hirudin in the presence of AHAb. By examining AHAb developed in patients, we were able to demonstrate that AHAbs are capable of neutralizing r-hirudin in vitro. In addition, the anticoagulant activity of r-hirudin administered to Sprague-Dawley rats was almost completely abolished when a monoclonal mouse AHAb with known r-hirudin neutralizing capacity in vitro was injected intravenously. Because r-hirudin is mainly eliminated via the kidneys, formation of r-hirudin-AHAb complexes may, due to their size, result in accumulation of r-hirudin. We investigated filtration of r-hirudin incubated with monoclonal mouse AHAb by using high-flux hemodialyzers in a suitable in vitro model. Whereas sieving coefficients (SC) were high in the absence of AHAb, they were minimal (SC < 0.05) in the presence of AHAb. These findings may also be important when AHAb-positive patients treated with r-hirudin undergo hemofiltration procedures, which have recently been described as a rescue measure in case of r-hirudin overdosage. In vivo, the influence of a non-neutralizing monoclonal mouse AHAb on r-hirudin pharmacokinetics was examined in rats. Pharmacokinetic data compared with those of a control group without AHAb administration revealed that r-hirudin elimination half-life was significantly prolonged (59 +/- 25 minutes versus 142 +/- 25 minutes). This was accompanied by a decrease of total plasma clearance of r-hirudin. The volume of distribution of r-hirudin was significantly decreased (275 +/- 112 mL/kg versus 35 +/- 3 mL/kg), indicating that r-hirudin bound by AHAb is mainly distributed to the intravascular compartment. Taken together, both the half-life prolongation and the decrease of the volume of distribution contribute to r-hirudin accumulation and may explain respective findings in patients. In summary, two different effects of AHAbs seem to be clinically relevant: decreasing anticoagulant activity of r-hirudin by neutralization and accumulation of r-hirudin by reducing renal clearance. Formation of AHAbs has not yet been correlated with enhanced major bleeding complications. However, close monitoring of coagulation parameters is recommended in AHAb-positive patients during r-hirudin treatment.

Hirudin

For centuries, the blood-sucking medicinal leech, Hirudo medicinalis, has been used in European medicine to treat a steadily increasing number of diseases. In the 19th century, leech therapy was the most frequently chosen treatment modality, which almost led to the eradication of this animal. Prolonged bleeding from skin lesions produced by leeches was well-known and regarded as part of the therapeutic success. However, it was not until the late 19th century when Haycraft discovered the pharmacologic principle in the secretion of leeches: it inhibits blood coagulation. Decades of intensive research beginning in the 1950s led to detailed characterization of the active substance being produced by the parapharyngeal glands of Hirudo medicinalis: native hirudin, the most potent, natural direct thrombin inhibitor known. Despite constituting an anticoagulant with remarkable potency, however, its limited available amount hindered introduction of purified hirudin into clinical medicine. When the progress in recombinant technology allowed large-scale production of recombinant hirudins (r-hirudins) without markedly diminishing its antithrombin efficacy as compared to their native counterpart, preclinical and clinical trials with r-hirudins were performed. Clinical trials addressing treatment of heparin-induced thrombocytopenia, deep-vein thrombosis, and acute coronary syndromes gave important insights into efficacy and safety of r-hirudin anticoagulation. In 1997, two r-hirudins were approved by the European Agency for the Evaluation of Medicinal Products (EMEA): lepirudin for the treatment of heparin-induced thrombocytopenia (HIT) and thromboembolic disease, and desirudin for the treatment of deep-vein thrombosis in elective hip and knee surgery. FDA approval of lepirudin for the treatment of HIT and thromboembolic disease followed in 1998.

Hirudin as alternative anticoagulant--a historical review.

Advances in separation techniques and biotechnology have contributed to the development of anticoagulant agents from hematophagous animals. The most potent known natural thrombin inhibitor from blood-sucking leeches ( Hirudo medicinalis), hirudin has served as a standard for designing the natural coagulation inhibitors as an anticoagulant drug. The search for the development of hirudin from leech extract to genetically engineered products as an alternative anticoagulant has been resumed. The pharmacological profiling of the isolated thrombin inhibitor has shown that native hirudin is an antithrombotic agent of high quality. However, its clinical use has remained limited, because the substance has not been available in adequate amounts. The progress in molecular biology has stimulated the interest in the structure and function of hirudin. This development resulted in the production of recombinant hirudins (r-hirudins) through gene technology. The biological properties of hirudin combined with the ready availability of recombinant forms make the specific thrombin inhibitor well-suited for use as an antithrombotic drug. Its use should lead to a decisive progress in the management of thromboembolic diseases of both arterial and venous origin. Clinical trials, especially in diseases in which thrombin plays a crucial role, are in progress.

Methods for the monitoring of direct thrombin inhibitors.

Direct thrombin inhibitors are available for prophylactic as well as therapeutic purposes. Application of hirudin in therapeutic doses has been shown to require drug monitoring. Currently, most experience is available for recombinant hirudin, but the principle aspects of drug monitoring are the same for all direct thrombin inhibitors. Most frequently, activated partial thromboplastin time (aPTT) and modifications of the activated clotting time (ACT) have been used for the monitoring of hirudin therapy. However, these methods are insensitive at plasma levels higher than 0.6 mg/L of hirudin, so that overdoses may be missed despite monitoring. Correlations between ecarin clotting time (ECT), enzyme immunoassays, and chromogenic substrate assays on one side and global tests on the other side are poor. Fully automated chromogenic substrate-based assays, also available as point-of-care tests (POCT), are more precise and sensitive and are not disturbed by interferents such as heparin and antithrombin. Good correlations can be observed between chromogenic assays and the ECT performed in plasma or whole blood samples. ECT can also be determined with POCT systems. Test characteristics such as imprecision and measuring range are comparable to those of the chromogenic assays. In conclusion, therapy with direct thrombin inhibitors should be monitored with chromogenic assays or ECT.

Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli

A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l −1) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as β-methylnorleucine (βMeNle; (2 S,3 S)-2-amino-3-methylhexanoic acid). The modification pathway of βMeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, βMeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of βMeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.

Comparison of benefits and complications of hirudin versus heparin for patients with acute coronary syndromes undergoing early percutaneous coronary intervention

T Global Use of Strategies To Open occluded coronary arteries (GUSTO-IIb) trial enrolled a cross section of patients with acute coronary syndromes (ACS) with and without ST-segment elevation and randomized them to receive unfractionated heparin or recombinant hirudin (desirudin; Ciba-Geigy [now Novartis], Summit, New Jersey). We analyzed patients from GUSTO-IIb who underwent percutaneous coronary intervention (PCI) during the initial study drug infusion, to evaluate the potential benefits of pretreatment with hirudin compared with heparin among patients with ACS who undergo early PCI. • • • Patients were enrolled in the GUSTO-IIb trial if they had ischemic chest pain for 12 hours associated with transient or persistent ST-segment elevation, STsegment depression, or T-wave inversion. Exclusion criteria included active bleeding, previous stroke, and severe hypertension. Patients were randomized to receive either intravenous unfractionated heparin or recombinant hirudin. Standard dosing nomograms were used for the blinded study drug infusion, to maintain a target activated partial thromboplastin time of between 60 and 85 seconds. The study drug was infused for 3 but 5 days. Patients with persistent ST-segment elevation were treated with either streptokinase or accelerated alteplase and were randomly assigned to heparin or hirudin. A subgroup of patients with persistent ST-segment elevation were enrolled in the Primary Angioplasty Substudy and were randomly assigned to primary angioplasty or accelerated alteplase in addition to randomization to heparin or hirudin. Angiography, PCI, and bypass surgery were discouraged during the study drug infusion unless recurrent ischemia developed or patients underwent protocol-mandated angiography as part of the Primary Angioplasty Substudy. When PCI was performed during

Current status of the anticoagulant hirudin: its biotechnological production and clinical practice.

Hirudin is a potent thrombin inhibitor originally derived from the medicinal leech, Hirudo medicinalis. Owing to its high affinity and specificity for thrombin, hirudin has been intensively investigated for research and therapeutic purposes. The investigation of hirudin has contributed greatly to the understanding of the mode of action of thrombin and the clotting system. Hirudin and several hirudin analogues have also been demonstrated to have several advantages as a highly specific anticoagulant over the most widely used drug, heparin. Due to the great demand for hirudin in physicochemical and clinical studies, various recombinant systems have been developed, using bacteria, yeasts, and higher eukaryotes, to obtain the biologically active hirudin in significant quantities. After 10 years of clinical applications, two recombinant hirudins and a hirudin analogue have gained marketing approval from the United States Food and Drug Administration, for several applications. Clinical trials are currently ongoing for other treatments for thrombotic disease. As a consequence, it is conceivable that hirudin may expand its therapeutic utility over heparin in the near future.

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.

Pharmacologic preservation of the hemostatic system during cardiac surgery

Bleeding after cardiac surgery remains a major potential problem. Numerous pharmacologic approaches to attenuating hemostatic system activation in cardiac surgery patients have been studied to further improve patient management. Therapeutic approaches studied include inhibiting thrombin generation or activation, preserving platelet function, and decreasing the need for transfusion of allogeneic blood products. Pharmacologic approaches to reduce bleeding and transfusion requirements in cardiac surgery patients are based on either preventing or reversing the defects associated with the CPB-induced coagulopathy. The increasing use of platelet inhibitors (clopidogrel and IIb/IIIa receptor antagonists) and new anticoagulants (low–molecular weight heparins, pentasaccharide, recombinant hirudin, bivalirudin, and argatroban) also pose interesting problems in managing cardiac surgery patients. Aprotinin and lysine analogues (ϵ-aminocaproic acid and tranexamic acid) have become mainstay therapeutic agents to prevent bleeding and the potential need for allogeneic transfusion. Newer therapies that are important to consider include the potential of recombinant activated factor VIIa as a therapy for refractory bleeding after cardiac surgery.

Heparin-Induced Thrombocytopenia and Thrombosis: A Review of Pharmacologic Therapy

Objective: To review pharmacological therapy in heparin-induced thrombocytopenia and thrombosis (HITT). Data Sources: Articles and abstracts in English published from 1966 to January 2000 were identified by MEDLINE and International Pharmaceutical Abstracts searches using the terms HIT, Hill, heparin-associated thrombocytopenia, thrombocytopenia, hirudin, lepirudin, argatroban, ancrod, heparinoids, orgaran, danaparoid, and org 10172. Additional articles were identified from the bibliographies of retrieved literature. Data Synthesis: HITT is a devastating drug-induced immunologic complication that occurs in 2–5% of patients on heparin. Often, HITT results in life- or limb-threatening complications. HITT is frequently diagnosed by clinical presentation, and there is no optimal drug treatment approach. The first step of treatment is to discontinue all forms of heparin. If anticoagulation is indicated, several agents have been evaluated. Lepirudin, argatroban, and danaparoid are agents available for use in HITT. Lepirudin is a recombinant hirudin that directly complexes with thrombin. Danaparoid is a heparinoid moiety that works by inactivation factor Xa. Argatroban, a direct semisynthetic thrombin inhibitor, is the newest agent available. Ancrod is derived from pit viper venom and acts as a fibrinolytic agent; its use is not recommended. No studies have evaluated the comparative efficacy and safety of these agents. Conclusions: Argatroban, danaparoid, and lepirudin have shown efficacy and safety in treatment of HITT. Each agent exerts differences in pharmacologic and pharmacokinetic parameters and has advantages and disadvantages in particular patient populations. Therapy must be guided on an individual basis. Further investigation is needed to ascertain an optimal treatment approach.

Venous thrombosis and anticoagulant therapy.

Venous thrombosis and anticoagulant therapy.

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 n M. This result demonstrates the feasibility of the CE–LIF immunoassay method for the determination of r-hirudin in plasma samples.

Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors

Recombinant adeno-associated virus type 2 (rAAV) is a promising vector for in vivo gene therapy. Transduction by rAAV requires binding to heparan sulfate proteoglycan on the cell surface, and heparin can block this binding. Because heparin is administered to most patients undergoing cardiovascular gene transfer in order to prevent thrombotic events, it is important to identify anticoagulants which do not interfere with rAAV transduction. Therefore, we examined the influence of different anticoagulants on rAAV transduction in vitro. rAAV transduction was inhibited by 40.5 +/- 7.9% at heparin concentrations of 0.1 U/ml, and by 81.7 +/- 3.6% at 1.0 U/ml. The low molecular weight (LMW) heparin tinzaparin inhibited rAAV transduction by 20.2 +/- 3.8% at 0.1 U/ml and 37.1 +/- 1.8% at 1.0 U/ml. The inhibitory effect was significantly weaker compared with heparin at 1.0 U/ml, (P < 0.01). The LMW heparinoid danaparoid inhibited rAAV transduction by 8.8 +/- 3.5% at 0.1 U/ml (P < 0.01 compared with heparin). In contrast, recombinant hirudin did not interfere at all with rAAV transduction. In summary, the results demonstrate that inhibition of rAAV transduction by heparin occurs rapidly and at therapeutically used concentrations. LMW heparinoids and above all recombinant hirudin might be alternatives for heparin when vascular gene transfer with rAAV requires transient anticoagulation.

Development and current applications of thrombin-specific inhibitors.

Thrombin-specific inhibitors directly diminish thrombin-induced coagulation and cellular activities without the side effects of heparin. Hirudin is the most potent natural thrombin-specific inhibitor. Recombinant hirudins (such as desirudin) have been shown to be effective in the treatment of heparin-induced thrombocytopenia (HIT) and in the prevention of thrombotic complications after hip or knee surgery. The application of recombinant hirudin has been limited mainly by hemorrhagic complications. Synthetic thrombin-specific inhibitors, including oligopeptides, tripeptides and non-peptide low molecular weight (LMW) thrombin inhibitors, have been designed according to their interactions with the active sites of thrombin. Bivalirudin (an anti-thrombin oligopeptide) has been approved for preventing thrombosis in unstable angina patients following angioplasty in adjunct to aspirin. Argotroban (a tripeptide thrombin inhibitor) has been used for the treatment of HIT, peripheral and cerebral thrombotic diseases. The benefit of using thrombin-specific inhibitors alone in acute myocardial infarction or unstable angina remains uncertain. A number of LMW thrombin-specific inhibitors have been developed. Some of them can be administrated orally, and cause less increase in bleeding time than other thrombin inhibitors. The efficacy, safety, stability and oral bioavailability of the thrombin inhibitors may be considerably improved through structural optimization. Most of the LMW thrombin inhibitors are currently being tested in animal models or at early stages of clinical trials. In this review, we will present an overview of recent advances in thrombin-specific inhibitors.

The potential role of new therapies in deep-vein thrombosis prophylaxis.

The value of prophylaxis against venous thromboembolism (VTE) is increasingly accepted in most surgical specialties, although the potential reduction in fatal pulmonary embolism has recently been questioned. The burden of VTE in hospital patients nevertheless remains high, partly attributable to underuse of thromboprophylaxis and partly attributable to occurrence of VTE in high-risk patients because recommended antithrombotic therapies fail to provide full protection. Improved physician education has been shown to increase the application of antithrombotic measures, and research efforts are focused on developing novel antithrombotic agents with greater efficacy and safety in clinical use. Several novel indirect and direct thrombin inhibitors have been investigated. The heparinoid danaparoid has shown superiority over unfractionated heparin in several indications, but only the recombinant hirudin, desirudin, has exhibited greater efficacy than low-molecular-weight heparin (LMWH) in thromboprophylaxis for patients undergoing elective hip-replacement surgery, without increasing the risk of bleeding. Three large-scale clinical trials have shown desirudin to be the most effective thromboprophylactic agent currently available in elective hip-replacement surgery; this agent has now been licensed for use in orthopedic surgery. Research is ongoing into the feasibility of still more effective and convenient therapies, such as oral antithrombins.

The potential role of new therapies in deep-vein thrombosis prophylaxis

The value of prophylaxis against venous thromboembolism (VTE) is increasingly accepted in most surgical specialties, although the potential reduction in fatal pulmonary embolism has recently been questioned. The burden of VTE in hospital patients nevertheless remains high, partly attributable to underuse of thrombopro-phylaxis and partly attributable to occurrence of VTE in high-risk patients because recommended antithrom-botic therapies fail to provide full protection. Improved physician education has been shown to increase the application of antithrombotic measures, and research efforts are focused on developing novel antithrombotic agents with greater efficacy and safety in clinical use. Several novel indirect and direct thrombin inhibitors have been investigated. The heparinoid danaparoid has shown superiority over unfractionated heparin in several indications, but only the recombinant hirudin, desirudin, has exhibited greater efficacy than low-molecular-weight heparin (LMWH) in thromboprophylaxis for patients undergoing elective hip-replacement surgery, without increasing the risk of bleeding. Three large-scale clinical trials have shown desirudin to be the most effective thromboprophylactic agent currently available in elective hip-replacement surgery; this agent has now been licensed for use in orthopedic surgery. Research is ongoing into the feasibility of still more effective and convenient therapies, such as oral antithrombins.

Recombinant hirudin in clinical practice: focus on lepirudin.

Clinical applications for recombinant hirudins have been investigated for the past 10 years. The first indication for which a hirudin-lepirudin-has been approved is treatment of heparin-induced thrombocytopenia (HIT). Also, the recently completed trials for use of lepirudin in unstable angina indicate a potentially new indication. This review describes pharmacology and clinical applications of lepirudin with an emphasis on HIT and unstable angina. An overview of usage of lepirudin in acute coronary syndromes is given, as well as a summary of rare indications for lepirudin, such as extracorporeal circulation, for which comprehensive data are lacking.