Hyaluronic acid on the urokinase sustained release with a hydrogel system composed of poloxamer 407: HA/P407 hydrogel system for drug delivery.

Pleural empyema is an inflammatory condition characterized by accumulation of pus inside the pleural cavity, which is usually followed by bacterial pneumonia. During the disease process, the pro-inflammatory and pro-fibrotic cytokines in the purulent pleural effusion cause proliferation of fibroblasts and deposition of extracellular matrix, which lead to fibrin deposition and fibrothorax. Urokinase instillation therapy through a chest drainage tube is frequently used for fibrinolysis in patients with empyema. However, urokinase treatment requires multiple instillation (2-3 times per day, for 4-8 days) and easily flows out from the chest drainage tube due to its high water solubility. In this in vitro study, we developed a thermo-responsive hydrogel based on poloxamer 407 (P407) combined with hyaluronic acid (HA) for optimal loading and release of urokinase. Our results show that the addition of HA to poloxamer gels provides a significantly more compact microstructure, with smaller pore sizes (**p < 0.001). The differential scanning calorimetry (DSC) profile revealed no influence on the micellization intensity of poloxamer gel by HA. The 25% poloxamer-based gel was significantly superior to the 23% poloxamer-based gel, with slower gel erosion when comparing the 16th hour residual gel weight of both gels (*p < 0.05; **p < 0.001). The 25% poloxamer-HA gel also exhibited a superior urokinase release profile and longer release time. A Fourier-transform infrared spectroscopy (FT-IR) study of the P407/HA hydrogel showed no chemical interactions between P407 and HA in the hydrogel system. The thermoresponsive P407/HA hydrogel may have a promising potential in the loading and delivery of hydrophilic drugs. On top of that, in vitro toxicity test of this combination demonstrates a lower toxicity.

Adenovirus Biodistribution is Modified in Sensitive Animals Compared to Naïve Animals.

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-ßGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.

A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation.

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbß3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbß3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

A drug carrier targeting murine uPAR for photodynamic therapy and tumor imaging.

Photodynamic therapy (PDT) has been used as an effective therapeutical modality for tumors. In PDT, a photosensitizer was used to capture the light of specific wavelength, leading to the generation of reactive oxygen species and cytotoxicity surrounding the photosensitizer. Modifications of photosensitizers to enhance tumor specificity are common approaches to increase the efficacy and reduce the side effects of PDT. Previously, we developed a human serum albumin (HSA)-based drug carrier fused with the human amino-terminal fragment (hATF), which binds to a tumor surface marker (urokinase receptor, uPAR). However, hATF-HSA binds to murine uPAR much weaker (79-fold) than to human uPAR, and is not optimal for applications on murine tumor models. In this study, we developed a murine version of the drug carrier (mATF-HSA). A photosensitizer (mono-substituted ß-carboxy phthalocyanine zinc, CPZ) was loaded into this carrier, giving a rather stable macromolecule (mATF-HSA:CPZ) that was shown to bind to murine uPAR in vitro. In addition, we evaluated both the photodynamic therapy efficacy and tumor retention capability of the macromolecule (at a dose of 0.05mg CPZ/kg mouse body weight) on murine hepatoma-22 (H22) tumor bearing mouse model. mATF-HSA:CPZ showed more accumulation in tumors compared to its human counterpart (hATF-HSA:CPZ) measured by quantitative fluorescence molecular tomography (FMT). Besides, mATF-HSA:CPZ exhibited a higher tumor killing efficacy than hATF-HSA:CPZ. Together, the macromolecule mATF-HSA is a promising tumor-specific drug carrier on murine tumor models and is an useful tool to study tumor biology on murine tumor models.

Urokinase-coated chitosan nanoparticles for thrombolytic therapy: preparation and pharmacodynamics in vivo.

Blood reperfusion of affected limbs is the most effective therapy for peripheral vascular thrombotic disease, restoring nutrition and blood flow to threatened tissues. Because it is more cost-effective than other thrombolytics, urokinase (UK) is widely used to treat venous thrombosis in China. However, its use is limited because of the risk of UK-related hemorrhagic complications. UK-coated nanoparticles (NPs) may decrease adverse effects while simultaneously increasing thrombolytic benefits. The aim of this study was to combine the sustained-release properties of NPs with the clinical benefits of catheter-directed thrombolysis (CDT) to create a promising new therapy. NPs were prepared via self-assembled chitosan and tripolyphosphate, introduced into a thrombosis model in New Zealand white rabbits, and the ratio of the residual thrombus cross-sectional area to the vascular cross-sectional area was calculated. The NPs had a drug-bearing efficiency of 14.5 ± 1.3%, an encapsulation efficiency of 94.8 ± 2.1% while the particle size of UK-coated NPs was 236 nm. Transmission electron microscopy results showed that the shape of the NPs were spherical and regular. Whether delivered by intravenation or catheter, UK-coated NPs produced a significant increase in the thrombolytic effect compared with free UK and confirmed the superiority of CDT for improving clot lysis over drug-induced systemic thrombolysis. The intravenous NPs caused an abnormal increase in fibrinogen. In conclusion, a water-soluble UK-WCS-NP suspension with good encapsulation efficiency was easily prepared UK-WCS-NPs were capable of maintaining UK activity, provided sustained-release of UK and exhibited better thrombolytic function than free UK.

DOT corrected fluorescence molecular tomography using targeted contrast agents for small animal tumor imaging.

PURPOSE: To demonstrate diffuse optical tomography (DOT) corrected fluorescence molecular tomography (FMT) for quantitatively imaging tumor-targeted contrast agents in a 4T1 mouse mammary tumor model. PROCEDURES: In the first set of experiments, we validated our DOT corrected FMT method using subcutaneously injected 4T1 cells pre-labeled with a near-infrared (NIR) Cy 5.5 dye labeled recombinant amino-terminal fragment (ATF) of the receptor binding domain of urokinase plasminogen activator (uPA), which binds to uPA receptor (uPAR) that is highly expressed in breast cancer tissues. Next, we apply the DOT corrected FMT method to quantitatively evaluate the ability of sensitive tumor imaging after systemic delivery of new uPAR-targeted optical imaging probes in the mice bearing 4T1 mammary tumors. These uPAR-targeted optical imaging probes are ATF peptides labeled with a newly developed NIR-830 dye being conjugated to magnetic iron oxide nanoparticles (IONPs). RESULTS: Our results have shown that DOT corrected FMT can accurately quantify and localize the injected imaging probe labeled 4T1 cells. Following systemic delivery of the targeted imaging nanoprobes into the mice bearing orthotopic mammary tumors, specific accumulation of the imaging probes in the orthotopic mammary tumors was detected in the mice that received uPAR targeted NIR-830-ATF-IONP probes but not in the mice injected with non-targeted NIR-830-mouse serum albumin (MSA)-IONPs. Additionally, DOT corrected FMT also enables the detection of both locally recurrent tumor and lung metastasis in the mammary tumor model 72 hrs after systemic administration of the uPAR-targeted NIR-830-labeled ATF peptide imaging probes. CONCLUSIONS: DOT corrected FMT and uPAR-targeted optical imaging probes have great potential for detection of breast cancer, recurrent tumor and metastasis in small animals.

Ultrasound-triggered thrombolysis using urokinase-loaded nanogels.

To find a way to modulate the effect of thrombolytic proteins by increasing their specificity, minimizing their adverse effect as well as lengthening their circulation time for the treatment of ischemic vascular disease holds great promise. In this work, urokinase-type plasminogen activator (uPA) was encapsulated into hollow nanogels which are generated by the reaction of glycol chitosan and aldehyde capped poly(ethylene glycol) (OHC-PEG-CHO) through a one-step approach of ultrasonic spray. The uPA-loaded nanogels, with size of 200-300 nm, have longer circulation time than that of the nude urokinase in vivo, besides the protein can be triggered to release in faster rate under diagnostic ultrasonic condition of 2 MHz, which significantly enhanced the thrombolysis of clots. The results are promising for increasing the specificity and positive effects of thrombolytic agents like recombinant tissue plasminogen activator (rt-PA) for the current treatment of ischemic vascular disease.

Pharmacokinetics and safety of a new bioengineered thrombolytic agent, human tissue urokinase type plasminogen activator in Chinese healthy volunteers.

The aim of our study was to investigate the pharmacokinetics and safety of human tissue urokinase type plasminogen activator (HTUPA) in healthy Chinese subjects after intravenous administration. Thirty-two subjects were given intravenous injection doses of 5-35 mg of HTUPA for safety evaluation. Twenty-four subjects were given 10, 20 or 30 mg HTUPA for pharmacokinetic assessment. Safety and tolerance were evaluated by monitoring adverse events, laboratory parameters, electrocardiography and vital signs. HTUPA concentration in human serum samples was determined by an enzyme-linked immunosorbent assay (ELISA) method. The main pharmacokinetic parameters were calculated by DAS software. HTUPA was generally well tolerated and in the whole study course no serious adverse events occurred. The main pharmacokinetic parameters were as follows: geometric mean [95% confidence interval, CI] for t1/2 were 1.5 (1.4, 1.6), 1.3 (1.2, 1.4), and 1.2 (1.2, 1.3) h, AUC0-t were 1.0 (0.7, 1.3), 2.1 (1.5, 2.7), and 5.6 (4.7, 6.6) mg h L(-1), AUC0-∞ were 1.1 (0.8, 1.3), 2.1 (1.5, 2.7), and 5.8 (4.7, 6.7) mg h L(-1) for 10, 20, and 30 mg group, respectively. The main pharmacokinetic parameters were not significantly different between males and females (P>0.05). No serious adverse events were reported by the subjects or revealed by clinical or laboratory examinations, suggesting the given doses were safe and well tolerated.

Numerical analysis of forced injection of enzyme during thrombolysis.

Numerical analysis was performed on the enzyme transport and the flow fields in order to predict the effectiveness of forced injection in thrombolytic therapy. The species and momentum transport equations were numerically solved for the case of uniform perfusion of enzyme into the fibrin clot, and the validity of our methods were verified. In order to predict the lysis efficiency of continuous and forced intermittent injections, enzyme perfusion and clot lysis were simulated for the different injection velocities and frequencies. Intermittent injection showed faster clot lysis compared to continuous perfusion, and lysis efficiency was increased as the injection velocity and period increased.

Immunotoxin pharmacokinetics: a comparison of the anti-glioblastoma bi-specific fusion protein (DTAT13) to DTAT and DTIL13.

DTAT13, a novel recombinant bispecific immunotoxin (IT) consisting of truncated diphtheria toxin, an amino-terminal (AT) fragment of the urokinase-type plasminogen activator (uPa), and a fragment of human IL-13 was assembled in order to target receptors on glioblastoma multiforme (GBM) and its associated neovasculature. Previous in vitro studies confirmed the efficacy of DTAT13 against various GBM cell lines expressing both IL-13 receptor or uPA receptor, and previous in vivo testing demonstrated the efficacy of DTAT13 in significantly inhibiting a range of xenograft tumors and showed that DTAT13 was 160- and 8-fold less toxic to the parental fusion IT, DTAT and DTIL13, respectively. To further understand the properties of DTAT13, pharmacokinetic/biodistribution experiments were performed. Binding analysis revealed that the IL-13 domain functioned independently of the uPA domain and that the K (d) for each binding domain was essentially the same as that of DTIL13 and DTAT. Flow cytometry studies indicated that DTAT13 bound better than DTAT or DTIL13. Analysis of the rate of protein synthesis inhibition in U87 MG cells by DTAT13 compared to DTAT revealed a faster rate of inhibition with DTAT13 compared to DTAT. The rate of protein synthesis inhibition of DTAT13 was identical to that of DTIL13 in U373 MG cells. Intracranial biodistribution studies revealed that DTAT13 was able to cross to the contralateral hemisphere unlike DTIL13 but similar to DTAT. These studies show that DTAT13 has properties encompassing those of both DTIL13 and DTAT and warrants further consideration for clinical development.

A6, a urokinase plasminogen activator (uPA)-derived peptide in patients with advanced gynecologic cancer: a phase I trial.

OBJECTIVES: The aim of this study was to define the toxicity, maximum feasible dose (MFD), and pharmacokinetics (PK) of A6, a peptide derived from human urokinase plasminogen activator (uPA), in patients with advanced gynecologic cancers, and to explore anti-tumor activity and the effects of A6 on biomarkers of the urokinase system. METHODS: A6 was administered subcutaneously daily, and doses were escalated in cohorts of three to six subjects. Serial blood specimens were obtained for pharmacokinetics and levels of urokinase plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1). RESULTS: Sixteen patients were enrolled and eligible for evaluation. No serious drug-related adverse events or dose-limiting toxicity occurred. A6-related toxicities were limited to grades 1 and 2 adverse effects including local injection site reactions. Five patients had stable tumor measurements for at least 4 cycles, one of whom stayed on study for 12 months. One patient had a confirmed cancer antigen (CA)-125 response (decrease in CA-125 of >50%) with stable disease on CT scan after 14 cycles and continues on study. Time to peak plasma level of A6 was 1-2 h. C(max) is proportional to dose. The half-life of A6 was approximately 2 h. Baseline biomarker levels did not predict response and trends over time did not correlate with outcome. CONCLUSIONS: A6 given daily continuously is well tolerated at all dose levels, without any dose-limiting toxicity. Based on the preliminary activity of A6, a phase II trial is underway in ovarian cancer.

In vivo behaviour of vesicular urokinase.

Thromboembolic diseases including deep vein thrombus (DVT) are major causes of morbidity and mortality. Detection of DVT in low extremities is difficult. There are some accepted imaging techniques in clinic but most of them have several disadvantages limiting their effective use. Because of this, researchers are still performed to develop a rapid, specific means of detecting and/or imaging venous thrombi-based on the changing composition of the thrombus. Urokinase, fibrinolytic enzyme isolated form human urine, is a direct activator of plasminogen. In thrombus formation, plasminogen seems to be trapped in or absorbed onto fibrin matrix thus leading to a localised concentration of plasminogen. This suggests that radiolabelled urokinase would be a suitable compound for the detection of thrombi. The most important disadvantage of this enzyme is short plasma half life. To overcome this problem, it was decided to encapsulate the enzyme in drug delivery systems such as liposomes, niosomes or sphingosomes. In this study, we prepared, characterized and monitored the biodistribution of three types of vesicular systems containing urokinase. All types of prepared vesicles show in vitro an acceptable encapsulation, stability and release profile. Thrombus uptake was increased by encapsulation of urokinase into vesicles.

Safety, tolerability and pharmacokinetics of subcutaneous A6, an 8-amino acid peptide with anti-angiogenic properties, in healthy men.

AIMS: To assess the safety, tolerability and pharmacokinetics of subcutaneous A6, an 8-amino acid peptide with anti-angiogenic properties, in healthy men. METHODS: Double-blind, placebo-controlled, parallel-group, dose-rising, phase I study of single and repeated doses. In the single dose phase, successive groups of 5 subjects received A6 15, 35, 75, 150, 300 mg, or placebo, as subcutaneous injections in the upper thigh. In the repeat dose phase, 2 groups of 6 subjects received repeat doses of A6 35 mg and 75 mg, or placebo, and 1 group of 5 subjects received 150 mg, or placebo, 12-hourly for 6 days (11 doses in total). In each group, 4 subjects received active treatment, the remainder placebo. Pharmacokinetics of A6 were assessed up to 24 h after single doses, for 12 h after the first of the repeated doses, and up to 24 h after the last of the repeated doses. MATERIALS: A6 for subcutaneous injection in phosphate buffer, pH 5.6-6.0. Phosphate-buffered saline was used as placebo. RESULTS: All dose regimens of A6 were safe and well-tolerated, both systemically and locally. Time to peak plasma concentration was similar (0.5-2.1 h) in all dosage groups. Cmax and AUC(0-inf) were linearly proportional to dose. Mean Cmax ranged from 454-10,333 ng/ml and mean AUC(0-inf) from 1,690-43,371 ng x h/ml after the 15 and 300 mg single doses, respectively. Terminal t(1/2) was 1.4-1.8 h, and there was no evidence of unexpected drug accumulation. Urinary excretion of unchanged A6 was 94.6% (SD 20.7) after the 300 mg single dose (0-24 h collection), and 78.4% (SD 13.0) after the 150 mg repeated dose (0-12 h collection). A6 did not trigger production of anti-A6 IgG antibodies within 14 days of the first dose. CONCLUSION: Single doses of A6 up to 300 mg, and repeated doses up to 150 mg, were well-tolerated and safe in healthy young men. A6 was rapidly absorbed; it was eliminated, mainly unchanged, in urine. Plasma concentrations were dose-proportional. A6 did not trigger an early immunogenic response.

Amediplase. Menarini.

Amediplase, a chimeric protein containing functional domains of both tissue plasminogen activator and pro-urokinase plasminogen activator, is under development by Menarini for the potential treatment of thrombosis. Phase III trials with amediplase were underway by June 2002.

Intraocular properties of urokinase-derived antiangiogenic A6 peptide in rabbits.

To investigate the intraocular properties of an antiangiogenic peptide, A6, a total of 70 New Zealand rabbit eyes were used. For the toxicity study, 0.05 mL of 0.459 M or 0.148 M A6 was injected intravitreally; right eyes received A6, and left eyes received a vehicle. Serial intraocular pressure measurement, slit lamp, and indirect ophthalmoscopy were performed. The rabbit eyes were evaluated by fluorescein angiography, electroretinography, and histology after the scheduled sacrifice. The pharmacokinetics of an intravitreal A6 (0.05 mL of 0.488 M) and a subtenon A6 (0.5 mL of 0.305 M) injection was studied. There was no toxicity observed following the 0.148 M A6 intravitreal injections. In 2 eyes with a 0.459 M A6 intravitreal injection, focal retinal pigmentary change was observed at the injection site, which was contacted by the hyperosmolar drug bolus. Choroidal A6, following the intravitreal injection, remained therapeutic (>or=10 microM) for 72 hours. The vitreous half-life was 19.4 hours. Choroidal concentrations following the subtenon injection were minimal. The low choroidal concentrations observed may relate to the polar nature of A6. More hydrophobic analogs of A6 are likely to cross the retina more efficiently. However, in diseased eyes, in the area of choroidal neovascularization (CNV), the fluid-filled, damaged, edematous retina may permit the drug to enter the choroid in higher concentrations.

Effects of inhaled plasminogen activator on the balance between coagulation and fibrinolysis in traumatized pigs.

A profibrinolytic state is normal in the alveoli, but this may change as a result of trauma, possibly leading to fibrin deposition, a characteristic of acute lung injury/acute respiratory distress syndrome. Therefore, the present study investigated in a double-blind, placebo-controlled manner the effect of severe trauma on the alveolar fibrinolytic/coagulation balance, and the effect here-upon of inhalation of single-chain urokinase plasminogen activator (scu-PA) in pigs. The study shows an increased concentration of scu-PA in the bronchoalveolar lavage fluid of the treated animals in association with an increased plasmin-dependent fibrinolytic activity without increased systemic fibrinolytic activity, the transient increase in the concentration of scu-PA in the plasma being minimal. In conclusion, the study shows that activatable scu-PA can be nebulized to the lower respiratory tract and can increase the alveolar fibrinolysis without any significant systemic effects.

Pharmacokinetics and hemostatic effects of saruplase in patients with acute myocardial infarction: comparison of infusion, single-bolus, and split-bolus administration.

Saruplase, or unglycosylated, single-chain urokinase-type plasminogen activator (scu-PA) selectively activates fibrin-bound plasminogen, and is subsequently converted to its two-chain derivative tcu-PA (urokinase) by plasmin. The efficacy of a 20 mg IV bolus followed by an infusion of 60 mg over 1 hour (standard regimen) has been demonstrated in acute myocardial infarction (AMI). The Bolus Administration of Saruplase in Europe (BASE) study compared the efficacy of standard therapy, single bolus (80 mg), and split bolus (2 x 40 mg at 30-minute intervals) in AMI. In a substudy of BASE, the pharmacokinetics of total u-PA activity (amidolytic activity after plasmin treatment), high molecular weight (HMW) u-PA antigen, and tcu-PA activity were compared in patients receiving standard therapy (n = 4), single bolus (n = 4), or split bolus (n = 5). Total u-PA activity and HMW u-PA antigen were similar. The maximum concentration (C(max,), mean +/- SD) of total u-PA activity was 2.2 +/- 0.3 microg/mL after standard therapy, 16.3 +/- 3.9 microg/mL after single bolus, and 8.2 +/- 1.6 ug/mL after split bolus. The area under the concentration versus time curve (AUC) values of total u-PA activity were 1.7 +/- 0.1 microg/mL*h (standard therapy), 4.0 +/- 0.9 microg/mL*h (bolus), and 3.0 +/- 0.7 microg/mL*h (split bolus). The dominant initial half-lives (t(1/2) alpha) were 7.1 +/- 1.1 minutes (standard), 8.8 +/- 0.8 minutes (bolus), and 5.1 +/- 2.1 minutes (split bolus). Maximum plasma concentrations of of tcu-PA activity were observed at 5.2 +/- 7 minutes (standard), 21 +/- 10 minutes (bolus), and 42 +/- 2 minutes (split bolus). C(max) was lowest after standard therapy (0.6 +/- 0.3 microg/mL), highest after bolus (4.2 +/- 2.2 microg/mL), and approximately twice as high as standard therapy after split bolus (1. 3 +/- 0.8 microg/mL). After standard therapy the mean fibrinogen concentration decreased gradually from approximately 300 mg/dL to 70 mg/dL at 90 and 120 minutes. After a single bolus the fibrinogen concentration decreased below the limit of quantification within 30 minutes and remained there for at least 120 minutes. Directly after the second 40 mg dose of the split bolus, the fibrinogen levels had an accelerated and more pronounced decrease to approximately 65 mg/dL at 90 and 120 minutes. A single bolus results in very high early total u-PA activity, which accelerates the appearance of tcu-PA activity and fibrinogen consumption. The pharmacokinetics and hemostatic effects of the split-bolus regimen are more comparable with those of standard therapy.

Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices.

Fibrin or a fibrinous exudate can facilitate angiogenesis in many pathological conditions. In vitro, the outgrowth of capillary-like structures in fibrin can be mimicked by exposing human microvascular endothelial cells (hMVECs) to an angiogenic growth factor and tumor necrosis factor (TNF)-alpha. Urokinase-type plasminogen activator (u-PA) and plasmin activities are required for this angiogenic process. This study focuses on the role and localization of the u-PA receptor (u-PAR) in newly formed microvascular structures. The u-PAR-blocking monoclonal antibody (MAb) H-2 completely inhibited the formation of capillary-like tubular structures induced by exposure of hMVECs to basic fibroblast growth factor and TNF-alpha. This was accompanied by a several-fold increase in u-PA accumulation in the conditioned medium. The effect of MAb H-2 was not caused by blocking cellular activation by u-PA/u-PAR interaction, as the amino-terminal fragment (ATF) of u-PA, which also activates u-PAR, prevented tube formation. In addition, the inhibition by MAb H-2 was not due to an effect of the antibody on u-PAR-vitronectin binding. These data show that inhibition of tube formation can be caused not only by inhibition of u-PA or plasmin activities but also by unavailability of the u-PAR for cell-bound proteolysis. Immunohistochemical analysis showed that in in vitro angiogenesis u-PAR and u-PA were localized on the invading, tube-forming hMVECs and not on the endothelial cells that are located on top of the fibrin matrix. u-PAR and u-PA were also prominently expressed on endothelial cells of neovessels present in an atherosclerotic plaque. These data may give more insight into the role of u-PAR in repair-associated angiogenesis.

Identification of megalin/gp330 as a receptor for lipoprotein(a) in vitro.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence-labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation.

[Combined thrombolysis by pharmacokinetically different plasminogen activators]. / Sochetannyi trombolizis farmakokineticheski razlichnymi aktivatorami plazminogena.

Prospects for the search for thrombolytic compositions on the basis of short-term and long-term acting plasminogen activators were shown. These will be useful as potential ambulance remedies for effective prehospital treatment. Combined proteolysis by plasminogen activators with complementary action mechanisms and significantly different pharmacokinetic behavior was suggested for this purpose.