Role of Bedside Preparation in Reducing "Door-to-Needle" Tissue-Type Plasminogen Activator (Alteplase) Administration Times and Association with Patient Outcomes.

INTRODUCTION: The purpose of this study is to assess the benefit of bedside alteplase preparation as a component of the acute stroke process. METHODS: A retrospective, single center study, designed to evaluate the impact of a bedside alteplase preparation protocol. Stroke patients receiving intravenous (IV) alteplase prepared at bedside were compared to pre-bedside alteplase preparation patients. The primary outcome was to compare door-to-needle (DTN) times between the groups. The secondary outcomes included comparison of pre-bedside alteplase preparation to post-bedside alteplase preparation on the following variables: imaging-to-drug times, order entry to drug administration times, percentage of patients achieving the 60 minute DTN goal, rate of intracranial hemorrhage (ICH), and patient discharge disposition. RESULTS: Patients in the pre-bedside preparation group included those who received IV alteplase between Jan. 1, 2012 and Jan. 31, 2015 and post-bedside preparation patients between Feb. 1, 2015 and March 31, 2016. Thirty-one patients were enrolled in the study, 16 in the pre-bedside preparation group and 15 in the post-beside preparation group. The mean DTN time in the post-bedside alteplase preparation group was significantly reduced, as compared to the pre-bedside preparation group (66.6 minutes vs. 95.9 minutes, p=0.024). Percent of patients meeting the 60 minute DTN time goal was significantly improved when alteplase was prepared at bedside (53.3 percent vs. 18.8 percent) (p=0.044). Rates of ICH were not significantly different between the two populations. CONCLUSIONS: Bedside alteplase preparation significantly reduced DTN times in an academic hospital emergency department.

Synthesis and Characterization of Tissue Plasminogen Activator-Functionalized Superparamagnetic Iron Oxide Nanoparticles for Targeted Fibrin Clot Dissolution.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention in many biomedical fields and are used in preclinical/experimental drug delivery, hyperthermia and medical imaging. In this study, biocompatible magnetite drug carriers, stabilized by a dextran shell, were developed to carry tissue plasminogen activator (tPA) for targeted thrombolysis under an external magnetic field. Different concentrations of active tPA were immobilized on carboxylated nanoparticles through carbodiimide-mediated amide bond formation. Evidence for successful functionalization of SPIONs with carboxyl groups was shown by Fourier transform infrared spectroscopy. Surface properties after tPA immobilization were altered as demonstrated by dynamic light scattering and ζ potential measurements. The enzyme activity of SPION-bound tPA was determined by digestion of fibrin-containing agarose gels and corresponded to about 74% of free tPA activity. Particles were stored for three weeks before a slight decrease in activity was observed. tPA-loaded SPIONs were navigated into thrombus-mimicking gels by external magnets, proving effective drug targeting without losing the protein. Furthermore, all synthesized types of nanoparticles were well tolerated in cell culture experiments with human umbilical vein endothelial cells, indicating their potential utility for future therapeutic applications in thromboembolic diseases.

Heparin-triggered release of camouflaged tissue plasminogen activator for targeted thrombolysis.

A targetable, heparin-triggered release system for tissue plasminogen activator (tPA) was designed to prevent the excessive 'lytic' state associated with the current tPA therapy for acute thrombotic conditions, such as myocardial infarction (MI). The strategy is, upon target accumulation, to trigger tPA release from a prodrug construct by a usual heparin dose. A relatively inactive form of tPA was constructed by conjugating tPA with low-molecular weight heparin followed by complexation with albumin-protamine conjugate, termed 'camouflage'. The modified tPA was ~97% as active as native tPA. The prodrug construct of tPA significantly masked the enzymatic activity, which was fully recovered upon heparin addition. The camouflaged tPA was stable in human blood for at least 30min and was able to trigger enzyme activation in vitro at heparin level of 0.4U/mL. In vivo studies on jugular vein rat thrombosis model showed that the clot lysis of the heparin-triggered camouflaged tPA group was equivalent to the tPA+heparin group without prolongation of activated partial thromboplastin time (aPTT) before and after the treatment. This proof-of-principle study suggests that the activity of the tPA prodrug construct can be triggered at the thrombus site at therapeutic heparin concentration conjunctively used for MI with reduced bleeding risk.

In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2.

A synthetic deca-peptide corresponding to the amino acid sequence Arg(54)-Trp(63) of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibiting endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition(.) These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.

The tissue-type plasminogen activator story.

Milestones in the development of tissue-type plasminogen activator (t-PA) as a fibrin-specific thrombolytic agent include: purification of human t-PA from the culture fluid of the Bowes melanoma cell line, elucidation of the molecular basis of fibrin-specific plasminogen activation, first experimental animal models of thrombosis, first patient (renal allograft) treated with melanoma t-PA, pilot studies in patients with acute myocardial infarction, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials.

Medical innovation, unmet medical needs, and the drug pipeline.

This paper outlines and illustrates the working of a theoretical approach from the social sciences for analyzing medical innovation, unmet medical need, and the drug pipeline. Using the social history of three drugs made from recombinant DNA (insulin, human growth hormone, and tissue-plasminogen activator) the paper shows how drugs can be both technically and organizationally efficient while the needs they satisfy can be created or identified. The paper posits that drugs that require more organizational efficiency tend to satisfy identified, rather then created needs. Key words: Recombinant DNA, technical efficiency, organizational efficiency, anthropology.

Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.

Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control.

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator.

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.