[Replacement of the pyrogen-test as batch control test for the biological substances aprotinin and urokinase]. / Ablösung des Pyrogentests als Chargenkontrollprüfung bei den Biologika Aprotinin und Urokinase.

In the Pharmeuropa Vol. 10, No. 4, December 1998, the European Pharmacopoeia Commission published an appeal to replace the pyrogen test by the limulus test for a total of 10 biological substances. These substances included the active ingredients aprotinin and urokinase, contained in our sales products Antagosan and Actosolv. After successful validation of the limulus test, we calculated endotoxin limit concentrations for these and submitted these to the European Department for Quality of Medicines (EDQM). We expect - not least on the basis of the data submitted by ourselves - that the pyrogen test will be replaced in future as a quality control test by the limulus test in testing aprotinin and urokinase compounds for the presence of pyrogenic substances. This leads to a reduction in the number of rabbits used as opposed to the current pyrogen test for testing the stated compounds.

Alteplase and tenecteplase: applications in the peripheral circulation.

Alteplase (t-PA), a recombinant analogue of human tissue plasminogen activator, became the first genetically engineered thrombolytic approved by the Food and Drug Administration in 1987 for acute myocardial infarction (AMI). In addition to AMI, alteplase is currently approved for the treatment of acute ischemic stroke and pulmonary embolism, and we anticipate approval for catheter clearance in late 2001 in a 2-mg vial configuration. With the withdrawal of human neonatal kidney cell-derived urokinase, alteplase has become an alternative agent in peripheral vascular applications. Because few interventionalists had prior experience with the handling and dosage of alteplase, the Advisory Panel to the Society of Cardiovascular and Interventional Radiology established practice guidelines for use in noncoronary applications. Emerging clinical experience with contemporary dosing regimens shows a safety and efficacy profile similar to urokinase but with significantly reduced drug costs. Tenecteplase (TNK) is a genetically modified version of alteplase. TNK is the only plasminogen activator available that has shown a significantly enhanced safety profile versus alteplase in AMI. Approved for a 5-second, single-bolus injection in AMI, TNK possesses a longer half-life, increased resistance to plasminogen activator inhibitor, and improved fibrin specificity compared with alteplase. Because of its enhanced safety profile, TNK may be a desirable agent for peripheral vascular applications. Initial clinical studies with TNK in acute arterial and venous disease are ongoing. This article outlines the Advisory Panel guidelines for using alteplase and highlights features of tenecteplase.

Cell surface-bound urokinase-type plasminogen activator facilitates infiltration of freshly isolated granulocytes into fibrin matrix.

Human cell lines of myelo/monocytic origin express the cellular receptor for urokinase-type plasminogen activator (uPA-R). The receptor localizes urokinase-type plasminogen activator (uPA) to the surface of the cell, where it can convert plasminogen to the active serine proteinase plasmin. Plasmin may subsequently account for proteolysis of pericellular proteins. We demonstrated the expression of the uPA-R by freshly isolated neutrophilic granulocytes by using a specific mAb. In freshly isolated granulocytes we detected only a weak occupation of the uPA-R; further uPA binding by granulocytes was saturable and proceeded in a dose-dependent manner. Receptor-bound uPA retained its enzymatic activity. Saturation of isolated granulocytes with exogenous uPA enhanced cellular infiltration into a fibrin matrix in vitro. uPA-dependent infiltration was inhibited by an anti-catalytic monoclonal anti-uPA antibody. The findings show that circulating neutrophilic granulocytes express the cell surface uPA-R and suggest that surface-binding of uPA may facilitate the infiltration of granulocytes into a fibrin clot, a process that might add to thrombolysis in vivo.

[High Molecular Weight Urokinase Reference Standard (Control 901) of National Institute of Health Sciences].

A candidate for "High Molecular Weight Urokinase Reference Standard (HMW-UK RS, Control 901)" of the National Institute of Health Sciences (NIHS) was examined for evaluating the fibrinolytic activity. The urokinase activity of the candidate HMW-UK RS was determined by "two-stage method" as 800 Unit/Amp. against the present Urokinase Reference Standard (Control 881), through the collaborative study involving five laboratories. According to the gel-permeation chromatography of the candidate for the molecular determination, it was confirmed that more than 97% of the total urokinase activity was attributed to the higher molecular fraction, named HMW-UK. Thus it was authorized as the High Molecular Weight Urokinase Reference Standard of the NIHS.

A collaborative study to establish a standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA).

An international collaborative study involving eleven laboratories located in eight countries was undertaken to establish an International Standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA). The current International Reference Preparation (IRP code numbered 66/46) for urinary-type plasminogen activator (u-PA) or urokinase (see Nomenclature footnote) is a 66/34 molar ratio mixture of low molecular weight (LMW)--and high molecular weight (HMW)--u-PA's and is considered unsuitable as a standard for homogeneous preparations of HMW/u-PA. The putative standard for HMW/u-PA (code number, 87/594) was compared for potency in a clot lysis assay with the current IRP for u-PA (code number, 66/46) and a lyophilised preparation of single chain urinary-type plasminogen activator (SCuPA), the latter being used in the assay without prior activation by plasmin to its active two chain form (TCuPA). Both the proposed standard for HMW/u-PA (87/594) and the SCuPA compared in a statistically satisfactory manner in parallel line bioassays with the current IRP for u-PA (66/46), thus allowing potency estimates to be obtained for these two materials in relation to defined international units. Data from the eleven laboratories indicated that each ampoule of the proposed standard for HMW/u-PA contained 4,300 i.u. of activity and was stable for over 1 year at 4 degrees C. Most participants indicated that SCuPA expressed only a small amount of its activity without a prior activator step and this suggests that SCuPA assays need to be preceded by a plasmin activation step.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinase administration in pediatric patients with occluded central venous catheters.

The effectiveness of urokinase in declotting central venous catheters was retrospectively assessed in a pediatric population of 63 patients ranging in age from 4 days to 22 years (mean, 6.4 years). Results of treatment from 103 episodes of catheter occlusion were evaluated. Urokinase was instilled into occluded single-lumen catheters and into each lumen of double-lumen catheters, as well as into occluded implanted ports. Patency was restored to 101 of 103 occluded catheters, with no side effects observed subsequent to clearance of the catheters with the urokinase infusion. Significant cost savings were seen using urokinase to restore patency as compared to the cost of replacing the catheter. These data clearly support the use of urokinase in a pediatric population as a safe and cost-effective alternative to catheter replacement in cases of thrombotic occlusion.

[Urokinase Reference Standard of National Institute of Hygienic Sciences (Control 881)].

The National Institute of Hygienic Sciences Standard for Urokinase (Control 881) was established in collaboration with five laboratories. This standard contains 1100 international units of urokinase and 1.17 mg of human serum albumin in each ampoule. Urokinase used for the standard is constituted of 1 part of high molecular weight species (M. W. 54000) and 4 parts of low molecular weight species (M. W. 33000).

Urokinase quality: analysis of different preparations.

Five commercially available German Urokinase (UK) preparations were examined for purity, fibrinolytic activity, and molecular weight composition. With the exception of one preparation (B) all samples showed comparable fibrinolytic activity (91-107% of the activity declared). Preparations A, D, and E contained almost exclusively high-molecular-weight UK (HMW-UK), i.e., 82-86%. However, preparation C (from kidney tissue cultures) contained 95% of low-molecular-weight-UK (LMW-UK), and sample B consisted of nearly equal amounts of both HMW-UK and LMW-UK (47:53). Purity criteria (coagulative and lysozyme activity, endotoxin content, protease activity, HBs antigen) were fulfilled by preparation E, and, with certain restrictions, also by preparations A and C.

Possible use of monoclonal antibodies to standardize the production and use of human urokinase.

Urokinase, a plasminogen activator found in urine is used in the treatment of pulmonary embolism, and is supplied commercially by several companies in Europe, U.S.A. and Japan. It exists in two forms, of molecular weights 50-55 000 and 33 000 daltons, which may differ in their therapeutic efficiency. A variety of assay systems exist to measure plasminogen activator activity--clot lysis, casein hydrolysis, amidolytic cleavage of synthetic peptide substrates. These assays are time-consuming, relatively insensitive, and cannot accurately assess the amount of the two molecular species in any preparation of urokinase. Standardization therefore presents a problem to manufacturers, control agencies, and clinicians. We have prepared a variety of monoclonal antibodies to human urokinase and have assessed their value as standardizing different commercial preparations of urokinase and measurement of urokinase for therapeutic monitoring.