Pichia pastoris as a host for secretion of toxic saporin chimeras.

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino-terminal fragment of human urokinase (ATF) fused to the plant ribosome-inactivating protein saporin could be recovered. ATF-saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti-saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport-competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon-usage optimization greatly increased the expression levels of active saporin but not those of an active-site mutant SAP-KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine-tagged ATF-saporin chimera showing an IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras.

Oligonucleotide microarrays reveal regulated genes related to inward arterial remodeling induced by urokinase plasminogen activator.

Accumulating evidence suggests that urokinase plasminogen activator (uPA) is involved in vascular remodeling and lumen stenosis after angioplasty and stenting. We have shown previously that increased uPA expression greatly promotes neointima formation and inward arterial remodeling after balloon injury. To evaluate the role of inflammation in early mechanisms responsible for inward arterial remodeling induced by uPA and elucidate the mechanisms of remodeling, we characterized changes in the expression profiles of 8,799 genes in injured rat carotid arteries 1 and 4 days after recombinant uPA treatment compared to vehicle. We used a standard model of the balloon catheter injury of the rat carotid followed by periadventitial application to the injured vessel of either uPA dissolved in Pluronic gel, or plain gel. Vessels were harvested and analyzed by immunohistochemistry, morphometry, microarray gene expression profiling and quantitative RT-PCR. Periadventitial application of uPA significantly reduced lumen size and vessel area encompassed by the external elastic lamina at both 1 and 4 days after treatment. Inflammatory cells accumulated in the arterial adventitia at both 1 and 4 days after uPA treatment. On the 4th day, increases in the areas and arterial cell numbers of all arterial layers were found. Among 79 differentially expressed known genes 1 day after uPA application, 12 proinflammatory genes, including TNF-alpha and TACE, and 15 genes related to mitochondrial metabolism and oxidative stress regulation were identified. Four days after injury in uPA-treated arteries, 3 proinflammatory and 2 oxidation-related genes were differentially expressed. We conclude that uPA likely promotes inward arterial remodeling by regulating oxidative stress and inflammation after arterial injury.

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate.

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.

Establishment of a rat model of lumbar facet joint osteoarthritis using intraarticular injection of urinary plasminogen activator.

Lumbar facet joint (LFJ) osteoarthritis (OA) is an important etiology of low back pain. Several animal models of LFJ OA have been established using intraarticular injection of various chemicals. This study aimed to establish a rat model of LFJ OA using urinary plasminogen activator (uPA). Sprague-Dawley rats were treated with intraarticular injection in the L5-L6 facet joints with uPA (OA group, n = 40) or normal saline (vehicle group, n = 40). Mechanical and thermal hyperalgesia in the ipsilateral hind paws were evaluated using von Frey hairs and a thermoalgesia instrument, respectively. Toluidine blue staining, hematoxylin-eosin staining, and immunohistochemical examination of the LFJ was performed. Treatment with uPA induced cartilage damage, synovitis, and proliferation of synovial cells in the fact joints. The OA group showed significantly higher hyperalgesia in the hind paws in comparison with the vehicle group and normal controls (P < 0.05). Expression of IL-1ß, TNF-α, and iNOS in the LFJ cartilage in the OA group was significantly increased (P < 0.05). A rat model of LFJ OA was successfully established using intraarticular injection of uPA. This animal model is convenient and shows good resemblance to human OA pathology.

Effect of urokinase on corneal endothelium.

Urokinase in concentrations ranging from 1,000 to 5,000 units/mL was used to perfuse the corneal endothelium of rabbits for three hours in the specular microscope. No notable corneal swelling was noted, and the corneal endothelium appeared normal when observed with scanning electron microscopy.

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man.

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.

Lysis of intraventricular blood clot with urokinase in a canine model: Part 2. In vivo safety study of intraventricular urokinase.

It was determined from in vitro experiments that the minimal dose of urokinase required to lyse 10 ml of clotted canine blood within a closed space must exceed 10,000 IU. We empirically doubled this minimum effective dose and tested the in vivo safety of injecting 20,000 IU of urokinase every 12 hours for 4 days into the ventricles of six adult mongrel dogs through an implanted catheter-reservoir system. The animals were monitored carefully for local and systemic bleeding by neurological and clinical examination, hematological tests reflecting systemic fibrinolytic status, serial computed tomography, and postmortem histological examinations of the brain, meninges, and peripheral organs. It was found that this intraventricular dose regimen of urokinase did not cause intracranial hemorrhage even though the dogs had recent brain wounds related to transcerebral ventricular catheterization. Mild activation of systemic fibrinolysis, implying passage of the enzyme from ventricle to blood, occurred 4 to 6 hours after each intraventricular injection, but no systemic hemorrhages were seen. This dose regimen also did not cause acute or chronic inflammatory changes in the brain or meninges and did not disturb cerebrospinal fluid circulation.

Regional thrombolysis with urokinase for central venous catheter-related thrombosis in patients undergoing high-dose chemotherapy with autologous blood stem cell rescue.

Fifty-one of 300 patients undergoing high-dose chemotherapy with (n = 245) or without (n = 55) autologous stem cell rescue developed central venous catheter-related thrombosis diagnosed by Doppler sonography or contrast venography. Eighteen of these individuals underwent regional thrombolysis defined as the infusion of urokinase into a superficial vein of the ipsilateral upper extremity in a dose not sufficient to produce systemic fibrinolysis by laboratory criteria. Urokinase was administered at a dose of 75,000-150,000 U/hour for 24 to 96 hours and contrast venography was performed to assess response. All individuals had a partial or complete resolution of clinical signs and symptoms. Fifty percent of patients also achieved a partial radiographic response defined as clot lysis with irregular canalization of the vein. Therapeutic doses of heparin for 5 to 7 days and warfarin for at least 3 months were commenced at the conclusion of urokinase therapy. Twelve catheters were salvaged and utilized subsequently until no longer required. Six catheters were removed because of poor catheter function or rethrombosis. The median interval from diagnosis of the thrombus until extraction of the 12 salvaged catheters was 3 months (range 1-8 months). Only a single patient who developed gastrointestinal bleeding required discontinuation of urokinase. Regional thrombolysis is safe, easy to administer, effective in many instances, less costly than the doses of antifibrinolytic agents required to induce systemic fibrinolysis, and should be considered in patients receiving high-dose chemotherapy with autologous stem cell rescue who develop central venous catheter-related thrombosis.

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.

Effect of a novel octapeptide urokinase fragment, A6, on experimental choroidal neovascularization in the monkey.

PURPOSE: To evaluate the inhibitory effects of a urokinase-derived octapeptide, A 6, on laser-induced choroidal neovascularization (CNV) in monkeys. METHODS: Twenty female cynomolgus monkeys were randomly grouped into weekly or monthly A 6 treatment groups, each consisting of 10 animals. CNV was induced in both eyes by perimacular laser treatment. In each right eye, a single 22.25-mg A 6 dose (monthly group) or 4 22.25-mg A 6 doses each week (weekly group) were given by intravitreal injections. Each left eye received phosphate buffer on the same schedule. Monkeys were observed for 4 weeks by ophthalmic examinations, color photography, and fluorescein angiography. RESULTS: Weekly treated eyes had a 35% reduction of CNV compared with controls (P = 0.23). In contrast, monthly treated eye had a 71% reduction of CNV compared with controls (P = 0.0009). There was no evidence of toxicity at both clinical and pathologic examinations. CONCLUSIONS: Intravitreal A 6 injections effectively inhibited CNV in cynomolgus monkeys without evidence of toxicity. The overall reduction in CNV was greater for monthly treated eyes than for weekly treated eyes. This study suggests that A 6 has promise as a local antiangiogenic treatment of CNV. Further work is indicated to evaluate the potential role of A 6 in therapy for human CNV associated with age-related macular degeneration.

Deleterious effect of urokinase used to treat experimental intra-arterial thiopental injection injuries.

UNLABELLED: Inadvertent arterial drug injections continue to be an important source of morbidity. Although the clinical picture of thiopental injection has been well defined over the past 50 years, there is still much controversy concerning pathophysiology and treatment regimen. Recently, a case report showed the efficacy of urokinase in treating this problem. The current study used the reliable ear model to study more closely this phenomenon. Rabbits were divided into four groups. Ears in Group 1 rabbits (n = 10) received an intra-arterial thiopental (15 mg/kg) injection. Group 2 rabbits (n = 10) received thiopental followed by a 1-ml saline injection 15 minutes later. Group 3 rabbits (n = 10) received thiopental followed by 50,000 U of urokinase. Finally, Group 4 rabbits (n = 4) received an intra-arterial injection of saline alone. Necrosis was evaluated 2 weeks later and expressed as a percentage. Student's t tests were used to evaluate data significance. RESULTS: Group 1 (thiopental alone) and Group 2 (thiopental and saline) rabbits had significantly more necrosis than Group 4 (saline alone) rabbits, 21.2% and 17.5% versus 0% (p less than 0.001 for both). Group 3 (thiopental and urokinase) rabbits had significantly more necrosis (46.5%) than Groups 1 and 2 rabbits (p less than 0.001 for both). CONCLUSION: From this study, we found that treatment of intra-arterial thiopental injection injuries with urokinase was of no benefit, but more importantly, it increased tissue necrosis by approximately 100%. Clinical use of this treatment is to be discouraged until underlying mechanisms are better defined.

Toxic ocular effects of two fibrinolytic drugs. An experimental electroretinographic study on albino rabbits.

The toxic effects on rabbit eyes of 2 intravitreally injected fibrinolytic substances at different concentrations were studied with repeated clinical observations and registrations of the DC ERG. The fellow, control eye of each animal was injected with saline. Urokinase (Ukidan, Serono) (13 rabbits) initially produced aqueous flare (64%), iris hyperaemia (36%) vitreous opacities (27%) and small retinal haemorrhages (18%). 2-3 months after the injection cataract (50%), vitreous opacities (25%) and retinal changes (13%) were observed. The highest dose (10 000 Ploug units) caused reduction of the ERG b-wave, as a sign of retinal toxicity. Tissue activator (D-44, Centre d'immunologie et de biologie Pierre Fabre) (10 rabbits) produced marked aqueous flare (initially 100%, after 2 weeks 50%) and pronounced, persistent vitreous opacities (25% after 2-3 months). At the late stage corneal blood vessels (38%) and cataract (38%) were also found, but only in eyes injected with the highest dose (1000 units), which was retinotoxic as judged by the ERG (reduced b- and c-waves).

Urokinase, a constitutive component of the inflamed synovial fluid, induces arthritis.

Urokinase plasminogen activator (uPA) is an important regulator of fibrinolysis in synovial fluid. An increase of uPA activity and expression of its receptor have been reported in joints of patients with rheumatoid arthritis (RA). The aim of the present study was to assess the arthritogenic capacity of uPA and the mechanisms by which this effect is mediated. uPA was injected into the knee joints of healthy mice, and morphological signs of arthritis were assessed 4 days after the injection. The prerequisite of different leukocyte populations for the development of uPA-triggered arthritis was assessed by selective cell depletion. The inflammatory capacity of uPA was assessed in vitro. Finally, levels of uPA were measured in 67 paired blood and synovial fluid samples from RA patients. The synovial fluid from RA patients displayed higher levels of uPA compared with blood samples. Morphological signs of arthritis were found in 72% of uPA-injected joints compared with in only 18% of joints injected with PBS (P < 0.05). Synovitis was characterised by infiltration of CD4-Mac-1+ mononuclear cells, by the formation of pannus and by occasional cartilage destruction. The absence of monocytes and lymphocytes diminished the frequency of synovitis (P < 0.01), indicating an arthritogenic role of both these leukocyte populations. Synthetic uPA inhibitor downregulated the incidence of uPA-triggered arthritis by 50%. uPA induced arthritis, stimulating the release of proinflammatory cytokines IL-6, IL-1beta and tumour necrosis factor alpha. Accumulation of uPA locally in the joint cavity is a typical finding in erosive RA. uPA exerts potent arthritogenic properties and thus may be viewed as one of the essential mediators of joint inflammation.

Urokinase in experimental vitreous hemorrhage.

Toxicity of intravitreal urokinase was studied by injection of various doses of urokinase in primate eyes. Doses of 22,500 CTA units or less produced no toxic effects on the eye. Higher doses caused retinal degeneration, transient lens opacities, and cloudy vitreous. Urokinase was ineffective in clearing experimentally induced vitreous hemorrhage if injected as early as 24 hours after the intravitreal blood or as late as six months thereafter.

Prophylaxis with urokinase in pediatric oncology patients with central venous catheters.

This study evaluated the effects of urokinase in the prevention of central venous catheter (CVC)-related complications in children with malignancy. Fifteen patients with 16 CVCs (study group A) received an intraluminal application of urokinase (10,000 IU in each catheter lumen for 4 h) once a week. They were monitored prospectively with quantitative blood cultures and ultrasonography (color Doppler ultrasound of the great veins and echocardiography). The rate of complications was compared with that of 15 children with 19 CVCs without thromboprophylaxis, treated the previous significantly lower incidence of CVC dysfunction year (control group B). The authors found a wer incidence of CVC dysfunction (3/16 versus 13/19), no major thrombosis, fewer CVC-related bacteremias (2/16 versus 8/19), and a higher salvage of CVCs (1/16 versus 5/19 CVC removals due to persistent bacteremia) in the thromboprophylaxis group. Asymptomatic thrombosis rate was also lower (7/16 cases in group A versus 9/11 in group B when sonography was performed). No hemorrhagic complications were noted. Thromboprophylaxis with urokinase seems a safe and effective measure for reducing the rate of CVC-related complications.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

The efficacy of plasminogen-urokinase combination in inducing posterior vitreous detachment.

PURPOSE: To investigate the toxicity of intravitreal plasminogen, urokinase, and their combination, and to evaluate their efficacy in the production of posterior vitreous detachment (PVD) in the rabbit eye. METHODS: Fifty-six albino New Zealand rabbits were examined before and after injection using the indirect ophthalmoscope, slit-lamp biomicroscopy, and electroretinography. Various concentrations of urokinase or recombinant plasminogen or a combination were injected intravitreally into the right eyes of four rabbits for each concentration. The left eyes of the animals served as controls and received 0.1 mL balanced salt solution. Group 1 was injected with pure urokinase (1,000, 5,000, or 10,000 IU); Group 2 with recombinant plasminogen (0.1, 0.4, 1.0, 2.0, 4.0, 8.0, or 16.0 caseinolytic units [CU]); and Group 3 with a combination of 1,000 IU urokinase (highest nontoxic dose) and nontoxic concentrations of plasminogen (0.1, 0.4, 1.0, or 2.0 CU). The animals were killed and the eyes enucleated 15 days after injection. Electron and light microscopy were performed. RESULTS: A concentration of 1,000 IU of urokinase was found to be nontoxic to the retina. Plasminogen concentrations of 2.0 CU or less did not produce retinal toxicity, whereas 4.0, 8.0, and 16.0 CU of plasminogen caused minimal-to-severe inflammatory response in the vitreous without histologic or electroretinographic changes. Neither plasminogen nor urokinase alone was successful in producing PVD. The combination of 1,000 IU of urokinase and 1.0 to 2.0 CU of plasminogen was effective without causing retinal toxicity. CONCLUSION: Posterior vitreous detachment can be produced in the rabbit eye using a combination of plasminogen and urokinase.