Effect of lepirudin on the international normalized ratio.

BACKGROUND: Many patients receiving direct thrombin inhibitor (DTI) therapy require transition to warfarin. This transition may be complicated by DTI-induced elevations in the international normalized ratio (INR). While the effect of argatroban on the INR has been characterized, data assessing the effect of lepirudin on the INR are limited. OBJECTIVE: To evaluate the effect of lepirudin on the INR. METHODS: Patients receiving lepirudin therapy between January 2000 and May 2001 were identified using the pharmacy database, and a retrospective chart review was conducted. Patients were included for analysis if they had paired activated partial thromboplastin time (aPTT) and INR data while receiving lepirudin monotherapy. RESULTS: Fifty-three paired aPTT and INR data points from 8 patients receiving lepirudin monotherapy were collected. The Organon MDA 180 instrument was used for aPTT and prothrombin time (PT) determination. Organon MDA Platelin L reagent was used for the aPTT and Organon Simplastin L reagent was used for the PT. The international sensitivity index (ISI) of the Simplastin L thromboplastin was 2.0. The mean +/- SD lepirudin dose was 0.05 +/- 0.04 mg/kg/h. Linear regression was used to identify the INRs that correspond to a therapeutic aPTT value of 45-75 seconds (1.5-2.5 times mean laboratory normal of 30 sec). The correlation between aPTT and INR was 0.77. An aPTT of 45-75 seconds with lepirudin correlated to an INR of 1.6-3.2. CONCLUSIONS: Based on laboratory results, when using a thromboplastin with an ISI of 2, lepirudin appears to elevate the INR in the absence of warfarin.

Monitoring of hirudin therapy with the Thrombelastograph.

Lepirudin is a specific thrombin inhibitor that is used for anticoagulation in patients with heparin-induced thrombocytopenia who are also undergoing cardiopulmonary bypass (CPB). Monitoring of lepirudin during CPB has been carried out with activated clotting time and ecarin clotting time. Correlation is poor with the former method, whereas ecarin clotting time is not widely available. A patient is described with heparin-induced thrombocytopenia who underwent CPB, where coagulation monitoring was accomplished with the Thrombelastograph. This more widely available method may be useful in such patients.

Bivalirudin: an anticoagulant for acute coronary syndromes and coronary interventions.

Heparin is a commonly used anticoagulant in patients with coronary artery disease but its use does not always result in low rates of ischaemic and bleeding events, so the search for new anticoagulants continues. Thrombin plays a key role in both thrombosis and haemostasis and direct thrombin inhibitors modelled on the hirudin molecule found in the saliva of the medicinal leech, Hirudo medicinalis, have recently been developed. To date, the only direct thrombin inhibitor shown to reduce both the ischaemic and the bleeding complications associated with percutaneous coronary intervention (PCI) is bivalirudin, which is approved for this indication in the US and New Zealand. This agent is currently being studied in patients undergoing PCI with or without glycoprotein IIb/IIIa inhibitors and stenting. Bivalirudin has been shown to significantly reduce the risk of reinfarction in patients with acute myocardial infarction (MI) treated with streptokinase, but its use for this indication is not approved in the US. It may also prove to be beneficial in patients with acute MI treated with other fibrinolytic regimens or with primary or facilitated PCI. Bivalirudin is suitable for use as an alternative to heparin in the majority of patients undergoing PCI and in patients receiving streptokinase for acute MI.

Bivalirudin: a direct thrombin inhibitor.

BACKGROUND: Studies of the anticoagulant effects of hirudin, which is derived from the saliva of the leech Hirudo medicinalis, led to the development of compounds that can directly inhibit thrombin activity without the need for additional cofactors. One of these is the direct thrombin inhibitor bivalirudin, which has recently been approved by the US Food and Drug Administration for use as an anticoagulant in patients with unstable angina undergoing percutaneous transluminal coronary angioplasty. OBJECTIVE: This is a review of the pharmacologic properties, efficacy, tolerability, and potential cost-effectiveness of bivalirudin in the treatment of ischemic coronary syndromes. METHODS: Articles were identified by searches of MEDLINE (1966-September 2001), International Pharmaceutical Abstracts (1970-September 2001), and the Iowa Drug Information Service (1966-September 2001) using the terms bivalirudin and Hirulog. The reference lists of retrieved articles were also reviewed for relevant articles. RESULTS: Bivalirudin is a synthetic polypeptide that directly inhibits thrombin by binding simultaneously to its active catalytic site and its substrate recognition site. After intravenous administration, peak plasma concentrations occur in 2 minutes. In patients given a 1.0-mg/kg bolus followed by a 2.5-mg/kg per hour infusion, a median activated clotting time of 346 seconds is achieved with little interpatient or intrapatient variability. Clearance of bivalirudin occurs through a combination of renal elimination and proteolytic cleavage, and doses may need to be decreased in the presence of renal dysfunction. In patients undergoing percutaneous coronary interventions, bivalirudin has been associated with equivalent efficacy but lower bleeding rates (P < 0.001) than unfractionated heparin (UFH). Data from the Hirulog Early Reperfusion/Occlusion-2 study suggest no reduction in mortality with bivalirudin compared with heparin when either is added to aspirin and streptokinase in patients with acute myocardial infarction, despite a lower reinfarction rate (P < 0.001). Experience with bivalirudin in patients with unstable angina and heparin-induced thrombocytopenia (HIT), as well as in patients receiving glycoprotein IIb/IIIla inhibitors, is limited. The differences in bleeding rates between bivalirudin and heparin in published clinical trials probably reflect differences in levels of anticoagulation achieved in comparator groups. CONCLUSIONS: Given its high cost, bivalirudin should be reserved for use as an alternative to UFH, primarily in patients with HIT, until clinical trials have more clearly demonstrated its benefits in terms of efficacy or safety.

Use of ecarin clotting time (ECT) with lepirudin therapy in heparin-induced thrombocytopenia and cardiopulmonary bypass.

Heparin-induced thrombocytopenia (HIT) is described as an allergy-like adverse reaction to heparin. It is a potentially severe complication of heparin therapy that can result in serious or life-threatening venous or arterial thromboembolic events. In the United States, lepirudin (Aventis Pharma AG, Strasbourg, France) is an approved therapy for anticoagulation in patients with HIT requiring anticoagulation. Lepirudin is a recombinant form of hirudin, a leech enzyme that is a highly specific direct inhibitor of thrombin. Lepirudin monitoring during surgery can be managed with ecarin clotting time (ECT) (Cardiovascular Diagnostics, Inc., Raleigh, NC), which has recently been approved as a humanitarian device exemption (HDE) for use in the United States in the management of HIT with cardiopulmonary bypass. This case report describes a patient with HIT who was managed successfully with lepirudin and ECT during coronary artery bypass grafting.

[Removal of lepirudin used as an anticoagulant in mechanical autotransfusion with Cell-Saver 5]. / Auswaschverhalten von Lepirudin als Antikoagulans bei der maschinellen Autotransfusion mit dem cell-saver 5.

UNLABELLED: Former studies demonstrated that small amounts of heparin might remain in the prepared retransfusion blood during intraoperative autotransfusion. This could lead to serious complications in patients suffering from heparin-induced-thrombocytopenia type II (HIT II). Lepirudin is an approved anticoagulant in HIT II-patients. We studied to what extent lepirudin is washed out during the preparation of retransfusion blood, when it is used as anticoagulant for the autotransfusion device cell saver 5. METHODS: We investigated four different concentrations of lepirudin solutions, 5 mg, 10 mg, 20 mg and 30 mg per litre normal saline. In order to imitate a clinical situation, each lepirudin solution was mixed with human blood in a 1:5-ratio and put into the reservoir of the cell saver. The device was started in the automatic mode using 1000 ml saline as washing solution. Several runs were carried out (five times using the 5 mg/l solution, ten times the 10 mg/l, eleven times the 20 mg/l and eleven times the 30 mg/l solution). The lepirudin concentration in the prepared retransfusion blood was measured. RESULTS: The median percentage reduction of the lepirudin content from the reservoir blood to the retransfusion blood was 100% for the 5 mg/l, 90.4% for the 10 mg/l, 94.3% for the 20 mg/l and 86.3% for the 30 mg/l solution. The differences of percentage reduction are not significant. But the different lepirudin concentrations in the anticoagulant solution have a significant influence on the lepirudin concentration in the retransfusion blood. The lepirudin concentration (median) in the retransfusion blood was 0.00 microgram/ml for the 5 mg/l, 0.16 microgram/ml for the 10 mg/l, 0.19 microgram/ml for the 20 mg/l and 0.66 microgram/l for the 30 mg/l lepirudin solution. CONCLUSION: Lepirudin as an anticoagulant for intraoperative autotransfusion is effectively eliminated using the cell saver 5 device in the automatic mode with 1000 ml saline as washing solution. A clinically relevant disturbance of coagulation is not to be expected, even if the highest concentration of lepirudin anticoagulant solution investigated in this study is used.

Amelioration of collagen-induced arthritis by thrombin inhibition.

The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mice treated with PEG-hirudin had a significantly prolonged clotting time compared with control mice. PEG-hirudin, administered in a preventive way, led to significantly reduced incidence and severity of CIA during most of the treatment period, as assessed by clinical scoring. Accordingly, histological features showed a significant diminution of synovial hyperplasia in PEG-hirudin-treated mice compared with untreated mice. There was also a significant downmodulation of the synovial proinflammatory IL-1beta and IL-12p35 cytokine mRNAs in treated mice. Intra-articular fibrin, evaluated by immunohistochemistry, was significantly reduced in treated mice compared with control mice and correlated with both clinical and histological scorings. Most importantly, once arthritis was established, PEG-hirudin also showed a curative effect. In conclusion, PEG-hirudin can both prevent the onset of CIA in a dose-dependent manner and ameliorate established arthritis, suggesting that thrombin inhibition may offer a new therapeutic approach in arthritis.

Bivalirudin: a new approach to anticoagulation.

Bivalirudin is one of the first of a new class of anticoagulants known as direct thrombin inhibitors. These drugs are able to overcome many of the shortcomings of traditional heparin anticoagulation by virtue of this unique mechanism of action. Bivalirudin is a semisynthetic derivative of hirudin, a modified component of leech saliva. Hirudin has been plagued by bleeding complications, likely due to its high affinity for thrombin. Bivalirudin has lower thrombin affinity than hirudin and therefore is believed to be a much safer compound. Bivalirudin has been shown to be a very effective anticoagulant in laboratory models, though its clinical efficacy remains to be fully proven. Bivalirudin has been studied in the setting of coronary angioplasty, unstable angina, and acute myocardial infarction and has shown some promise in many of these settings, particularly in preventing complications of percutaneous coronary interventions. Bivalirudin has consistently shown less major bleeding compared with standard heparin, although limitations in study methodologies somewhat hinder an accurate interpretation of this finding. Larger-scale studies are indicated and are currently being performed, the results of which will more definitively define the role of bivalirudin for the treatment of cardiovascular disease.

Heparin-induced thrombocytopenia: laboratory diagnosis and management.

Heparin-induced thrombocytopenia (HIT), a drug-induced immunohaematological adverse reaction, is a rare but potentially very severe condition. The main problem for this complex syndrome is its recognition and management, which should be as early as possible to avoid the development of life-threatening complications. Most studies have reported heterogeneous populations of patients with other diseases that potentially induce thrombocytopenia. There is no gold standard diagnostic criteria, and we have established a score with anamnestic criteria that allows us to evaluate the likelihood of HIT. In clinical practice, the diagnosis is based on the analysis of clinical features and laboratory tests. Platelet aggregation test (PAT) and an ELISA test (heparin platelet-induced antibodies) are generally performed by expert laboratories to confirm the occurrence of HIT. In our experience, both tests are concordant in the majority of patients. PAT seems to correlate better with the clinical features while ELISA appears more specific. Regarding their limits, both are complementary in the determination of HIT diagnosis coupled to the clinical score system. The treatment often requires a multidisciplinary approach. Danaparoid (Orgaran) or lepirudin (Refludan) are the two alternative treatments for HIT patients with marketing approval. To avoid further exposure to heparin, every HIT patient should carry a written document that confirms the immunoallergy.

Hirudin therapy during thrombolysis for venous thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is the most common drug-related thrombocytopenia. Thromboembolic complications occur in approximately 50% of patients with HIT and result in limb amputation and death in up to 20% and 30% respectively. Because patients with a history of HIT may require future intravenous anticoagulation but have a high-risk of thromboembolism if re-challenged with heparin, alternative therapies are necessary when further anticoagulation is indicated. The use of direct thrombin inhibitors in HIT patients who also require thrombolytic therapy offers unique challenges to anticoagulant monitoring and safety. We present a case of progressive ileofemoral deep venous thrombosis in a patient with a history of HIT in order to review the combined use of hirudin and thrombolysis in this setting.

Anticoagulants in acute coronary syndromes.

Antithrombotic and antiplatelet agents, particularly unfractionated heparin and aspirin, are longstanding therapeutic mainstays for acute coronary syndromes such as unstable angina and non-Q-wave myocardial infarction (MI). Early studies demonstrated that aspirin reduces the risk of mortality or nonfatal MI by 50-70% in patients presenting with unstable angina or non-Q-wave MI. Added to aspirin, heparin regimens further diminish the incidence of these myocardial ischemic events in the acute setting. Three major clinical studies demonstrated that such enhanced risk reductions can be achieved without significant increases in bleeding complications. The low-molecular-weight (LMW) heparin, dalteparin, proved superior to placebo but not unfractionated heparin in diminishing the incidence of (1) death or MI; (2) death, MI, or recurrence of angina; or (3) frequency of revascularization procedures. On the other hand, another LMW heparin, enoxaparin, did reduce these events at 14 and 30 days, as well as 1 year after treatment. The principal biophysical limitation of heparins, however, is that they cannot inactivate clot-bound thrombin, which probably contributes to morbidity and mortality in acute coronary syndromes. The natural leech-derived polypeptide hirudin and its derivatives (e.g., lepirudin) inactivate both fibrin-bound and free thrombin. Lepirudin has been approved in certain countries for the treatment of heparin-induced thrombocytopenia and is now being evaluated in the clinical management of acute myocardial ischemic syndromes. The well-documented pathophysiologic foundation for acute coronary syndromes is partial or intermittent thrombotic occlusion of a coronary artery as the result of atherosclerosis. Although a stable atherosclerotic plaque may not be clinically problematic, plaque rupture, which occurs under a variety of stimuli, touches off a cascade of enzymatic and cellular responses that frequently culminate in thrombotic occlusion. In the coronary circulation, such an occlusion may cause transmural MI, unstable angina, or non-Q-wave MI. Because the pathogenetic mechanisms of atherosclerosis with thrombotic complications have been elucidated, this knowledge can be translated into a rational clinical approach using antithrombotic therapies.

Pharmacological effects of a novel recombinant hirudin, CX-397, in vivo and in vitro: comparison with recombinant hirudin variant-1, heparin, and argatroban.

The novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their antithrombotic actions.

Dissolution of mural thrombus by specific thrombin inhibition with r-hirudin: comparison with heparin and aspirin.

BACKGROUND: The presence of residual mural thrombus may predispose to recurrent thrombotic events in acute coronary syndromes. The purpose of this study was to evaluate the effects of antithrombotic and antiplatelet agents on a preformed, fresh mural thrombus during growth of thrombus. METHODS AND RESULTS: A fresh mural thrombus was formed by perfusing severely injured arterial wall with porcine blood for 5 minutes at a shear rate of 1690 s(-1). Thrombus formation was measured by morphometric analysis (mm2/mm). The average size of a mural thrombus formed in 5 minutes was 0.14+/-0.03 mm2/mm. Progression of thrombus growth within 10 minutes triggered by the preformed thrombus was evaluated in pigs treated with r-hirudin (1 mg/kg per hour i.v.) as a probe for thrombin, high-dose heparin (250 IU/kg per hour i.v.), moderate-dose heparin (100 IU/kg per hour), moderate-dose heparin (100 IU/kg per hour) plus aspirin, aspirin alone (5 mg/kg i.v.), and placebo. Hirudin was associated with a significant decrease (48%) of mural thrombus area and significantly reduced growth of thrombus (0.07+/-0.01), even compared with the highest dose of heparin (0.15+/-0.03), although at lower levels of anticoagulation. Inhibition of growth of thrombus with heparin was dose dependent, showing an inverse correlation of thrombus area with mean plasma heparin concentrations (r=.77, P=.0001). Thrombus size was unchanged by aspirin (0.29+/-0.07) compared with controls (0.28+/-0.07). CONCLUSIONS: Direct inhibition of thrombin activity with r-hirudin completely inhibits growth of thrombus, causes dissolution of a preexisting mural thrombus, and is more effective at lower levels of anticoagulation than the highest dose of heparin at shear rates typical of a moderate coronary stenosis.

Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery.

A canine model of electrolytic injury-induced coronary artery thrombosis and rtPA-induced thrombolysis was used to evaluate the relative antithrombotic efficacy of enoxaparin (a low molecular weight heparin), conventional therapy (heparin or heparin plus aspirin), and hirulog (a direct thrombin inhibitor), when used as adjunctive therapy during thrombolysis. After 60 min of clot aging, adjunctive therapy was begun at doses which elevated APTT approximately 2-fold over baseline. Fifteen minutes after the start of adjunctive therapy, recombinant tissue plasminogen activator (rtPA) was administered (100 microg/kg i.v. bolus + 20 microg/kg/min for 60 min). Adjunctive therapy continued for 1 h after termination of rtPA and blood flow was monitored for two additional hours. Enoxaparin (1 mg/kg i.v. bolus + 30 microg/kg/min, n = 10 for each treatment group) was the only adjunctive treatment that significantly increased the total minutes of flow (143 +/- 25 min out of a possible 240 min, vs 54 +/- 25 min for vehicle, p <0.05) and decreased thrombus mass (6.0 +/- 1.3 mg vs 11.8 +/- 3.2 mg for vehicle). Although hirulog (2 mg/kg i.v. bolus + 40 microg/kg/min) did not significantly increase the minutes of flow (120 +/- 27 min, p <0.06) or decrease thrombus mass (8.7 +/- 1.7 mg) compared to vehicle, these values were not significantly different than those measured in the enoxaparin group. However, the results with hirulog were achieved at the expense of a significantly greater increase in template bleeding time than that measured during enoxaparin treatment. Minutes of flow for heparin (50 U/kg i.v. bolus + 0.6 U/kg/min) and heparin plus aspirin (5 mg/kg i.v. bolus) were 69 +/- 20 and 60 +/- 23 min, respectively; thrombus masses were 8.2 +/- 1.3 and 7.3 +/- 1.0 mg, respectively. In summary, enoxaparin was more effective than conventional therapy in this model in terms of vessel patency and thrombus mass, and was as effective as hirulog, at least at a dose of hirulog that only modestly impaired hemostasis. Therefore, enoxaparin may prove to be a safe and effective alternative agent for adjunctive therapy during thrombolysis with rtPA.

Modulating platelet function with selective thrombin inhibitors.

In addition to its pivotal role in blood coagulation, thrombin is one of the most important agonists for platelet recruitment and aggregation. Thrombin inhibitors impede thrombin-induced platelet aggregation but have no effect on aggregation induced by other agonists. A review is presented of selective thrombin inhibitors now in clinical investigation, some of which are also orally active.

New developments in acute anticoagulation therapy: what improvements over traditional heparin are on the horizon?

The quest for an orally active anticoagulant to replace warfarin sodium (Coumadin, Panwarfin, Sofarin) in long-term use has been disappointing. Most advances in oral anticoagulant therapy have involved more judicious and efficacious use of warfarin or one of its analogues. The area of heparin substitutes has experienced some exciting discoveries, with most current interest centered on low-molecular-weight heparins. Their efficacy, safety, and perhaps most important, clinical utility as a once- or twice-daily unmonitored medication have given them a meaningful role in current anticoagulation therapy. Third-generation anticoagulants, such as the direct thrombin inhibitors, are being investigated but are not ready for general clinical use. The role of ancrod (Arvin) from snake venom in patients with heparin-induced thrombocytopenic thrombosis has been clearly established. A practical issue that remains under discussion is the most suitable interaction between fiscal and clinical applications of these medications.

Hirulog in the treatment of unstable angina. Results of the Thrombin Inhibition in Myocardial Ischemia (TIMI) 7 trial.

BACKGROUND: Direct thrombin inhibitors are a new class of drugs that may offer a more effective and potentially simpler alternative to heparin. Hirulog is a synthetic peptide based on the leech-derived compound hirudin and, like hirudin, is a highly specific, direct inhibitor of free and clot-bound thrombin. METHODS AND RESULTS: TIMI 7 was a randomized, double-blind study of Hirulog, given with 325 mg/d aspirin to 410 patients with unstable angina. Patients received a constant infusion of Hirulog for 72 hours at one of four doses: 0.02 (n = 160), 0.25 (n = 81), 0.5 (n = 88), and 1.0 (n = 81) mg.kg-1.h-1. The primary efficacy end point was "unsatisfactory outcome," defined as death, nonfatal myocardial infarction (MI), rapid clinical deterioration, or recurrent ischemic pain at rest with ECG changes by 72 hours. Unsatisfactory outcome was not different among the four dose groups: 8.1%, 6.2%, 11.4%, and 6.2% (P = NS). However, the secondary end point of death or nonfatal MI through hospital discharge occurred in 10.0% of patients treated with 0.02 mg.kg-1.h-1 compared with 3.2% of patients treated with the three higher doses of Hirulog (0.25, 0.5, and 1.0 mg.kg-1.h-1, P = .008). Only 2 of 410 patients (0.5%) experienced a major hemorrhage attributed to Hirulog. CONCLUSIONS: The direct thrombin inhibitor Hirulog is a promising new antithrombotic agent that deserves further study. The results of TIMI 7 lend support to the use of an antithrombin agent with aspirin in patients with unstable angina.

Comparative studies on the anticoagulant and protease generation inhibitory actions of newly developed site-directed thrombin inhibitory drugs. Efegatran, argatroban, hirulog, and hirudin.

Site-directed thrombin inhibitors are being currently assessed clinically for their antithrombotic efficacy. Although these agents are claimed to be specific and direct thrombin inhibitors, their mechanism of inhibition varies. The objective of these studies was to compare four such agents in in vitro systems and to assess their relative anticoagulant efficacy. The four agents utilized in these studies were argatroban, Efegatran, hirulog, and hirudin. While hirulog and hirudin are specific irreversible inhibitors of thrombin, argatroban and Efegatran are reversible. All four agents were found to have a concentration-dependent anticoagulant effect when supplemented into normal human plasma, as assessed in the global clotting tests (PT, APTT, and Heptest). The most potent anticoagulant on a molar basis was hirudin in all three tests. The other three agents had similar anticoagulant actions. All four agents were also capable of inhibiting the generation of thrombin and factor Xa as determined by an amidolytic method after intrinsic or extrinsic activation of fibrinogen-deficient human plasma. Except for hirulog, all agents inhibited the extrinsic generation of thrombin, with hirudin being the most potent agent. The intrinsic generation of thrombin was blocked by the reversible thrombin inhibitors (argatroban and Efegatran) but not by the irreversible inhibitors (hirulog and hirudin). While all agents were capable of inhibiting the intrinsic generation of factor Xa at very low concentrations, only Efegatran was capable of blocking the extrinsic generation of the same factor.(ABSTRACT TRUNCATED AT 250 WORDS)