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Recombinant Tissue Plasminogen Activators (rtPA): A Review P Gurman1,3, OR Miranda1, A Nathan1,2, C Washington1, Y Rosen1 and NM Elman1 INTRODUCTION

Acute ischemic stroke (AIS), acute myocardial infarction (AMI), and pulmonary embolism (PE) represent main causes of morbidity and mortality worldwide.1 These clinical conditions result from an imbalance of the hemostatic system, leading to thrombosis. Recombinant tissue plasminogen activators (rtPAs) are used in patients with AIS, AMI, and PE to treat thrombus. This review focuses on the pharmacology and clinical applications of rtPAs, and therapeutic strategies to improve thrombolytic therapy. PHYSIOPATHOLOGY OF HEMOSTASIS: THROMBOSIS AND FIBRINOLYSIS

The hemostatic system is a combination of biochemical and cellular events occurring in the blood of arteries and veins designed to maintain the blood in a fluid state (fibrinolytic system) and prevent blood loss upon the injury of a blood vessel wall (coagulation system).2,3 Primary hemostasis results from small injuries to blood vessels that result in vasoconstriction and platelet activation, aggregation, and adhesion to the subendothelium of the damaged vessel wall, resulting in a platelet clot. Secondary hemostasis refers to the reinforcement of the platelet plug formed during primary hemostasis, through conversion of the soluble protein fibrinogen into an insoluble meshwork of fibrin. This process is carried out by the coagulation system in response to a larger vessel injury. The coagulation system is a complex mechanism involving coagulation factors, a number of plasma proteins, which work in a coordinated fashion to generate fibrin that together with the platelet clot becomes a consolidated thrombus. The interested reader is referred to the literature2–6 for a comprehensive review of the hemostatic system and mechanisms of thrombogenesis. Fibrinolysis is one of the components of the hemostatic system that functions to counteract the coagulation process and dissolve insoluble fibrin clots. The fibrinolytic system is a proteolytic enzymatic process that consists of an inactive proenzyme, plasminogen, which has the ability to be converted to the active enzyme,

plasmin, by tissue plasminogen activator (tPA). Structurally, tPA is a 70 kDa globular protein with serine proteinase activity, consisting of five domains including finger (F domain), epidermal growth factor (E domain), two kringle domains (K1 and K2), and the protease region (P domain). While the finger domains and the second kringle domain are involved in fibrin binding, the F and E domains are involved in tPA clearance by the liver, while the protease region displays plasminogen-specific proteolytic activity.7,8 tPA is synthesized primarily by endothelial cells.9 Plasminogen belongs to a class of proteins known as zymogens. These proteins are present in fibrin and remain in an inactive form until activated via hydrolysis, a kinase coupled reaction, or a change in configuration. Specifically, tPA binds to fibrin in a thrombus and converts the entrapped plasminogen to plasmin, thereby initiating local fibrinolysis. tPA has the property of fibrinenhanced conversion of plasminogen to plasmin. It produces limited conversion of plasminogen in the absence of fibrin. Plasmin is inactivated by alpha-2 antiplasmin, a serine protease inhibitor. tPA can be deactivated by a tissue plasminogen activator inhibitor known as PAI-1. In this manner, the fibrinolytic process is a tightly regulated system, designed to avoid systemic fibrinolysis, and thus excessive bleeding. Figure 1 summarizes the mechanism of action of tPA and fibrinolysis inhibitors present in the plasma.10,11 Under certain conditions, however, the fibrinolytic system can be bypassed by procoagulation states, such as alterations in blood flow or blood constituents, promoting the development of a thrombus, as shown in Figure 2.12 In these situations, external intervention with synthetic tPA agents may be necessary. These synthetic forms of tPA are known as recombinant tissue activators, rtPAs, or thrombolytics. THROMBOLYTIC THERAPY General considerations Pharmacokinetics. All thrombolytic agents are administered intravenously (i.v.). Intraarterial thrombolysis (IAT) has emerged as a potential strategy for thrombolysis in patients who do not match inclusion criteria for i.v. therapy such as time window or

1

Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 2Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel; 3Department of Materials Science and Bioengineering, University of Texas at Dallas, Richardson, Texas, USA. Correspondence: N Elman ([email protected]) Received 25 August 2014; accepted 4 November 2014; advance online publication 00 Month 2015. doi:10.1002/cpt.33

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Figure 1 Schematic representation of the mechanism of action of tPA. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plasminogen activator (tPA). tPA can be inhibited by tissue plasminogen activator inhibitor or PAI-1. Free plasmin in the blood is rapidly inactivated by a2antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with large vessel occlusions. Although a number of clinical studies have been performed to determine whether IAT could offer an alternative to the i.v. thrombolysis, further large, prospective, randomized clinical trials comparing IAT with standard i.v. therapy will be needed to demonstrate a clinical advantage of IAT over i.v. thrombolysis.13

ng/mL, increasing the chances of a hyper fibrinolytic state resulting in hemorrhage.15 Risk factors associated with intracranial hemorrhage during thrombolytic therapy include patients age

Indications for thrombolytic therapy. Thrombolytics have been approved by the US Food and Drug Administration (FDA) for clinical use in the treatment of AIS, AMI, and PE, as shown in Figure 3.14 Contraindications for thrombolytic therapy. Contraindications in the use of thrombolytics include: serious gastrointestinal bleeding during the last 3 months; surgery within 10 days including organ biopsy, puncture of noncompressible vessels, serious trauma, and cardiopulmonary resuscitation; history of hypertension (diastolic pressure >110 mmHg); active bleeding; previous cerebrovascular accident or active intracranial process; aortic dissection and acute pericarditis.15 Side effects of thrombolytic therapy. Bleeding is the major risk of thrombolytic therapy, particularly intracranial hemorrhage. The causes of bleeding result from systemic activation of plasmin outside the thrombus that leads to systemic fibrinolysis. This might be attributed to the fact that under physiological conditions the concentration of tPA around the fibrin clot (5–10 ng/mL) makes the systemic conversion of plasminogen to plasmin unlikely. When external administration of rtPAs becomes necessary, however, the plasma concentration of rtPAs could rise to 300–3,000 2

Figure 2 Schematic depicting the evolution of a thrombus in the vasculature system. The thrombogenic process involves activation, aggregation, and adhesion of platelets to the subendothelium, precipitation of fibrinogen into a fibrin meshwork, and subsequent trapping of red blood cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] VOLUME 00 NUMBER 00 | MONTH 2015 | www.wileyonlinelibrary/cpt

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Figure 3 FDA-approved uses of rtPAs. (A) Acute ischemic stroke. (B) Pulmonary embolism (PE). (C) Acute myocardial infarction (AMI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

>70 years old and those patients who had taken aspirin before starting thrombolytic therapy.16 Thrombolytic agents

Significant advances in thrombolytic therapies have been made since the 1980s. Since 2010 several thrombolytics have been developed. Currently, there are five principal thrombolytic agents approved for clinical use: 1) recombinant tissue plasminogen activators (rtPAs) including alteplase, reteplase, and tenecteplase; 2) streptokinase (SK); and 3) urokinase (UK).17–26

SK is a bacterial product and thus antigenic, resulting in the production of antibodies that preclude repeat doses of SK. In addition, SK is nonfibrin-selective.27 UK has been shown to be more expensive than alteplase and has suffered from manufacturing shortfalls.28 For these reasons, rtPAs are among the most widely adopted thrombolytic drugs in the clinical setting for the management of thrombolytic diseases. Therefore, this review will focus on the pharmacology and clinical applications of rtPAs. Table 1 summarizes key pharmacological and nonpharmacological thrombolytic therapies.

Table 1 Summary of pharmacological and nonpharmacological approaches in thrombolytic therapies Drug name

Advantages

Limitations

Stage of development

Streptokinase

First thrombolytic discovered

Allergenic

FDA-approved (streptase)

Urokinase

Second thrombolytic discovered

 Expensive  Manufacturing issues

Withdrawn from the market in 1999. Reintroduced in 2002 (abbokinase)

Alteplase, Reteplase, Tenecteplase

Current standards for Stroke, AMI, and PE

 Poor selectivity towards fibrin  Long infusion time (alteplase)  Neurotoxicity

FDA-approved (activase, retavase, TNKase)

Desmoteplase

 Potential use after 6 hours stroke onset  Long half-life allowing single bolus administration  High fibrin selectivity  Lack of neurotoxicity

Under clinical development the DIAS-2 clinical trial has demonstrated higher mortality rates with higher doses, without clinical improvement

Clinical trials: DIAS-3, DIAS-4 studies (ongoing)

Mechanical thrombectomy

Can be performed in patients where rtPAs are contraindicated

More clinical trials needed to assess clinical outcome as endpoints

MERCI retriever FDA-approved

Mechanical thrombectomy

Successful recanalization demonstrated after 8 hours of the onset of stroke

 Cost (requires interventional neurologist and angiography team)  Equipment is expensive  Careful selection of patients is needed

Trevo stent retriever FDA-approved

AMI, acute myocardial infarction; PE, pulmonary embolism; FDA, Food and Drug Administration; DIAS, Desmoteplase in Acute Ischemic Stroke Trial. CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 00 NUMBER 00 | MONTH 2015

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REVIEWS Recombinant tissue plasminogen activators (rtPAs)

rtPAs are produced using genetic engineering techniques through mutations in the DNA sequence of native tPA. These new therapeutic agents exhibit longer half-lives than native tPA, allowing convenient bolus dosing, enhanced fibrin specificity, and higher resistance to inactivation by PAI-1. Three tPA analogs are approved in the United States for use as therapeutic agents in thrombotic disorders including: 1) alteplase, 2) tenecteplase, and 3) reteplase. Desmoteplase, a fourth recombinant form of tPA, is currently being tested in clinical trials.29,30 In this section, key features of tPA analogs are summarized. Alteplase (Activase) is synthesized using the complementary natural cDNA sequence of native tPA. Alteplase is administered i.v. in patients experiencing AIS, AMI, or PE. Alteplase is administrated in a single i.v. bolus and then in a 3-hour or 90-minute (accelerated) infusion regime. Circulating fibrinogen levels decrease about 16% to 36% when 100 mg of alteplase is administered. Alteplase has a half-life of 4–8 minutes, requiring long infusion times to achieve recanalization of occluded arteries. The liver mediates clearance of alteplase from the plasma. The most frequent adverse reaction to alteplase in all approved indications is bleeding. Alteplase has been associated with neurotoxic properties.31 This is because alteplase has been shown to activate matrix metalloproteinases (MMP), resulting in breakdown of the blood–brain barrier (BBB) with an increased risk of cerebral hemorrhage and edema. In addition, alteplase has been shown to interact with Nmethyl-D-aspartate (NMDA) receptors and elicit calcium excitotoxicty and cell death. The failure of alteplase to achieve rapid reperfusion, the increased risk of cerebral hemorrhage, and its potential neurotoxicity has led to the development of newer thrombolytic agents, as described below. Reteplase is another recombinant form of tissue plasminogen activator. Reteplase is composed of the second kringle domain and protease domain of native tPA and is normally used for patients who experience AMI. It has a longer half-life than alteplase (13–16 minutes), which makes reteplase easier to administer than alteplase, allowing a double bolus injection (second injection given 30 minutes after the first injection) and thus avoiding the longer infusion times needed for alteplase. Reteplase has been shown to possess similar specificity towards fibrin but with lower binding affinity than alteplase. This property allows reteplase to penetrate the thrombus more efficiently and improve the reperfusion time in occluded arteries compared to alteplase. Clinical trials comparing the efficacy and safety of both thrombolytics in AMI, however, did not find a significant difference in mortality rates between either agent. The liver and kidneys mediate reteplase clearance from plasma, another difference with alteplase, which is cleared mainly by the liver. Similarly, the most common adverse effect of reteplase is excessive bleeding. Tenecteplase was designed by multiple point mutations of the native tPA DNA sequence resulting in a molecule with longer half-life (20–24 minutes compared to 5–10 minutes), enhanced fibrin specificity, and increased resistance to PAI-1 when com4

pared to alteplase. Tenecteplase is approved for the treatment of AMI. Tenecteplase can be administered in a single i.v. bolus over 5 seconds, which was demonstrated to provide similar efficacy to a 90-minute infusion of alteplase. A recent phase IIb randomized controlled trial comparing tenecteplase vs. alteplase has shown better reperfusion rates as measured by magnetic resonance imaging (MRI), as well as better clinical outcomes after 24 hours of administration of the drugs, without a significant difference in intracranial hemorrhage between the groups. Tenecteplase is cleared from the plasma by the liver. Desmoteplase is a recombinant form of native tPA derived from a chemical found in the saliva of vampire bats with similar structure to native tPA. Desmoteplase has a half-life of 4 hours and higher selectivity for fibrin than alteplase. Due to its high fibrin specificity that avoids systemic activation of plasminogen, and the lack of neurotoxic effects, researchers have sought to replace alteplase by desmoteplase. Desmoteplase alpha I (DSPA a1) exhibits the most favorable profile based on preliminary biochemical and pharmacological analysis and therefore has been chosen for most clinical studies. DSPA a1 shares 70% structural homology with native tPA, but they differ in their proteolytic activities. Currently, a phase III clinical trial, the Desmoteplase in Acute Ischemic Stroke Trial (DIAS-4), is being conducted to study the efficacy and safety of a single i.v. bolus of 90 lg/kg dose of desmoteplase given between 3–9 hours after the onset of AIS. Table 2 summarizes the key pharmacological features of rtPAs CURRENT CLINICAL USES OF rtPAs Acute ischemic stroke (AIS)

Stroke remains as the main cause of disability and the third cause of mortality in industrialized nations. Stroke can be divided into ischemic and hemorrhagic. Ischemic stroke accounts for 85% of cases of stroke while hemorrhagic stroke accounts for 15% of the cases of stroke.32 Ischemic stroke results from a region of the brain that becomes hypoperfused as a consequence of the obstruction of a vessel with a thrombus or embolus. Diagnosis of ischemic stroke requires computed tomography (CT) that must be performed as soon as a stroke is suspected, as shown in Figure 4A. While management of hemorrhagic stroke remains elusive, ischemic stroke can be managed pharmacologically or mechanically.33 One of the drugs that has demonstrated more success in the management of acute ischemic stroke is alteplase. Alteplase has been shown to be effective for the treatment of acute ischemic stroke and was approved for this use in the US in 1996. Clinical trials to assess the optimal therapeutic window for rtPAs in AIS. Traditionally, thrombolytic therapies have been shown to be

effective within the first 3 hours after the onset of stroke symptoms.34,35 Recent studies, however, have suggested that some rtPAs could also be effective up to 4.5 hours after the onset of the symptoms.36,37 Furthermore, ongoing clinical trials are studying the effectiveness of desmoteplase within 3–9 hours of the onset of the symptoms of AIS. Determining the optimal therapeutic window for administration of rtPAs after the onset of AIS results is critical for an VOLUME 00 NUMBER 00 | MONTH 2015 | www.wileyonlinelibrary/cpt

REVIEWS Table 2 Summary of key pharmacological features of rtPAs Drug name

Pharmacokinetics

Fibrin selectivity

Clinical use/FDA status

Study

Alteplase

Single bolus followed by 90 minutes to 3 hours infusion Half-life: 4–8 minutes Clearance mediated by liver

11

Stroke, AMI PE (FDA-approved) (activase)

NIDDS, ECASS-3, IST-3 Wardlaw et al. Goldhaber et al. Yamasawa et al. Dong et al. GUSTO

Reteplase

Double bolus injection (2nd injection given 30 minutes after 1st injection) Half-life: 13–16 minutes Clearance mediated by liver and kidneys

1

AMI (FDA-approved) (retavase)

GUSTO-3

Tenecteplase

Single bolus injection given in 5 seconds Half-life: 20–24 minutes Clearance mediated by liver

111

AMI (FDA-approved) (TNKase)

ASENT-2

Desmoteplase

Single bolus Half-life: 4 hours

111111

Stroke (phase III clinical trial)

DIAS-4

AMI, acute myocardial infarction; FDA, Food and Drug Administration; PE, pulmonary embolism; NIDDS, National Institute of Neurological Disorders and Stroke Trial; ECASS, European Cooperative Acute Stroke Study; IST, International Stroke Trial 3; GUSTO, Global Utilization of Streptokinase and Tissue plasminogen activator for occluded coronary arteries.

effective treatment and will provide an opportunity to broaden the inclusion criteria for rtPAs therapies and thus benefit more patients previously excluded from rtPAs treatments. A brief description of some of the clinical trials designed to study the best therapeutic window for rtPAs therapy is detailed below.

The National Institute for Neurological Disorders and Stroke Study (NIDDS): rtPAs within 3 hours of symptoms onset

One of the key clinical studies assessing the effectiveness of tPA within 3 hours of the onset of stroke was the National Institute for Neurological Disorders and Stroke study (NINDS). This study demonstrated early improvement of neurological symptoms

Figure 4 (A) CT images of the brain depicting a thrombus formation in the brain vasculature system. The hyperdense image shown at the right represents a clot formation in the M1 segment of the middle cerebral artery (MCA), while the loss of gray-white differentiation in the insular cortex represents an infarct secondary to the thrombus (courtesy of Dr Eble, Dept. of Medical Imaging, University of Arizona). (B) Axial image from a contrast-enhanced CT of the chest demonstrating a saddle embolus in the main pulmonary artery with extension into the lobar arterial branches bilaterally (courtesy of Dr Oliva, Dept. of Medical Imaging, University of Arizona). CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 00 NUMBER 00 | MONTH 2015

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REVIEWS after 24 hours of symptoms onset and improved clinical outcomes after 3 months. The study also showed an increased incidence of intracerebral hemorrhage in the tPA group compared to the placebo group. European Cooperative Acute Stroke Study III (ECASS-3 trial): evaluation of alteplase within 3–4.5 hours of the onset of AIS symptoms

In the ECASS-3 study, the safety and efficacy of alteplase administered between 3–4.5 hours after the onset of stroke symptoms was evaluated. ECASS III demonstrated an improvement in clinical outcomes in a 90-day period in the groups treated with tPA compared with the placebo group. Safety analysis showed that, in spite of the fact that intracranial hemorrhage was more frequently found in patients receiving alteplase compared to patients receiving placebo, the overall mortality did not change significantly between the groups. A meta-analysis by Lansberg et al.36 involving the clinical studies ECASS-1, ECASS-2, and ECASS-3 and the Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke (ATLANTIS), concluded that rtPAs therapy between 3–4.5 hours after the onset of AIS was associated with increased chances of favorable outcomes without adversely affecting mortality. The International Stroke Trial (IST-3): alteplase within 6 hours of the onset of AIS symptoms onset

The International Stroke Trial (IST-3) was a multicenter, randomized, open-label study designed to study whether more patients could benefit from thrombolytic therapy by extending the therapeutic window for rtPAs administration beyond 6 hours after the onset of the AIS. Patients were allocated to either rtPA or placebo. The primary endpoint of the study was the proportion of patients alive and independent after 6 months. The study confirmed the need for an early intervention of thrombolytic therapy after the onset of stroke symptoms, although it also provided data that some patients might benefit from rtPAs administration even 6 hours after the onset of the symptoms of stroke. The study also provided data that justify the use of rtPA in patients older than 80 years and did not support any restriction in the use of rtPA according to stroke severity. The study also encouraged that more controlled clinical trials are needed to evaluate the efficacy of rtPAs beyond 4.5 hours of the onset of stroke symptoms. DIAS-4 trial: desmoteplase within 3–9 hours of the onset of AIS symptoms

DIAS-4 is an ongoing randomized, double-blind, parallel group placebo-controlled phase III study to determine the efficacy and safety of desmoteplase vs. placebo within 3–9 hours of the onset of AIS symptoms. The study started in 2009 and is expected to be completed by 2015. SYSTEMATIC REVIEW AND META-ANALYSIS OF rtPAs IN AIS

A recent systematic review and meta-analysis of clinical trials involving i.v. rtPAs vs. placebo was conducted by Wardlaw 6

et al.38 The meta-analysis included 12 randomized controlled trials totaling 7,012 patients with i.v. rtPAs vs. placebo (including NIDDS, ECASS III, and IST-3) administrated up to 6 hours of the development of stroke symptoms. In terms of the optimal therapeutic window for rtPAs therapy, the meta-analysis concluded that rtPAs should be initiated as early as possible, but did not rule out the possibility that some patients might benefit from rtPAs therapy even 6 hours after the onset of ischemic stroke. The study reported that interindividual variability could explain why some individuals might benefit even after 6 hours of the onset of stroke while others do not. In addition, the metaanalysis also concluded that the reason that later treatment (after 6 hours) could not benefit more patients than early treatment (between 3–6 hours) might be attributed to less benefit in tissue to salvage rather than increased odds of intracranial hemorrhage. Acute myocardial infarction

Acute myocardial infraction (AMI) is a life-threatening condition that results from oxygen deprivation to the heart tissue (ischemia) that occurs when a coronary artery becomes occluded. AMI produces the death of myocardial tissue affecting cardiac output with a drop in blood pressure. The drop in blood pressure increases sympathetic reflexes leading to vasoconstriction, which decreases coronary flow hindering even more cardiac contractility, ultimately leading to cardiogenic shock and death. Therefore, AMI must be treated immediately to save as much tissue as possible before cardiogenic shock can occur. Current approaches in the management of AMI aim at restoring the blood flow to myocardial cells in the shortest time possible. These include minimally invasive procedures such as percutaneous coronary interventions (PCI), and pharmacological therapies such as rtPAs.39 Due to the increasing importance of prehospital management of AMI in terms of mortality gain with decreasing delay time to reperfusion after the onset of the symptoms, prehospital thrombolysis became a very important therapeutic tool. Prehospital thrombolysis offers several advantages, such as immediate access (only 25% of US hospitals provide primary angioplasty and the delay time to receive PCI treatment cannot exceed 90 minutes), and the possibility to add adjunctive pharmacotherapy.40 Alteplase, reteplase, and tenecteplase are the thrombolytics approved for the management of AMI in the US. Tenecteplase has found a place in the management of AMI during prehospital management, before PCI is available.41 The ASSENT-2 clinical trial demonstrated that tenecteplase was similar to alteplase in terms of mortality rate, with an additional advantage in terms of major bleeding and reduced rate of congestive failure.41 Additional advantages of tenecteplase over alteplase include its ease of administration (single bolus), its higher affinity to fibrin than alteplase, its 80 times less inhibition by PAI-1 than alteplase, and that it is not affected by nitrates, as seems to occur with alteplase. It is important to note that, although PCI has been demonstrated to be superior to fibrinolysis therapy in reducing mortality in patients with ST elevation, there seems to be a synergistic effect when PCI is performed after thrombolytic therapy. VOLUME 00 NUMBER 00 | MONTH 2015 | www.wileyonlinelibrary/cpt

REVIEWS Goodman and Cantor reviewed the clinical studies comparing early fibrinolytic therapy with PCI in patients entering the emergency room with AMI with ST elevation.42 These studies, which include the “TRANSFER”AMI trial (Trial of Routine Angioplasty and Stenting after Fibrinolysis to Enhance Reperfusion in Acute Myocardial Infarction), have shown a benefit in administering early fibrinolytic therapy followed by PCI.42 Pulmonary embolism

Pulmonary embolism (PE) is one of the manifestations of venous thromboembolism together with thrombophlebitis and deep vein thrombosis. PE can range from asymptomatic findings to lifethreatening clinical entities (massive pulmonary embolism) with mortality rates of up to 65%. PE refers to the migration of emboli to the lung capillaries leading to pulmonary complications including a decrease in gas exchange from the lungs to the systemic circulation and pulmonary edema, leading to a fall in oxygen saturation in the blood, difficulty breathing, and increase in heart rate. PE increases pulmonary artery pressure affecting the right ventricle. In severe cases, PE leads to a failure of the left ventricle with cardiogenic shock. Figure 4B shows a CT image of PE of the main pulmonary artery and lobar arterial branches. Fibrinolytics have an important role in pulmonary embolism by dissolving the thrombus and releasing the pressure on the pulmonary arteries, improving cardiac output. Pioneer studies conducted by Buneameux and Goldhaber and coworkers demonstrated the usefulness of intravenous tPA for the management of this serious condition.43–50 Yamasawa et al.47 compared the effect on oxygen pressure and lung perfusion of tPA vs. heparin alone in 45 patients, demonstrating a clinical benefit of tPA vs. heparin in these parameters. Meneveau49 highlighted the need for thrombolytic therapy as a first-line treatment in patients presenting pulmonary embolism with signs of cardiogenic shock. Wan et al.50 reported a meta-analysis of randomized controlled trials comparing heparin alone vs. thrombolytic therapy. The meta-analysis showed that no clear benefit of using thrombolytic therapy was observed. However, subgroup analysis showed that those patients with massive pulmonary embolism at risk of death have a benefit from thrombolytics compared with heparin alone. NOVEL USES OF rtPAs Frostbite

Frostbite has been defined as the freezing of tissues resulting from exposure of intact skin to temperatures below the freezing point. This traumatic freezing leads to devastating ischemic-based damage to distal extremities, which in severe cases leads to amputation of devitalized tissue.51 The damage caused by frostbite can be divided into two separate categories: mechanical and ischemic. Mechanical damage refers to the injury caused by the formation of ice crystals, whereas ischemic damage refers to injury caused by thrombosis. Intravenous rtPAs administration, together with heparin and aspirin, has been suggested as a pharmacological approach in the CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 00 NUMBER 00 | MONTH 2015

management of frostbite. tPA has been recommended to be administered as fast as possible after exposure to freezing temperature. It is thought that tPA would limit the formation of microvascular thrombus, avoiding reperfusion injuries.52 The Wilderness Medical Society (WMS)53 developed one of the most detailed guidelines on frostbite management. This is because frostbite tends to occur in wild environments and reports of cases in urban settings is very limited. The strongest level of clinical evidence provided to date in the use of tPA for frostbite is detailed below. Twomey et al.54 performed an open label study to assess the safety and efficacy of rtPA for treating severe frostbite. Patients included in the study had suffered severe frostbite, exhibited no improvement with rewarming, lacked Doppler pulses in distal limb or digits, and had no perfusion indicated by the use of a technetium (Tc) 99m three-phase bone scan. Six patients received intra-arterial rtPA, while 13 patients were treated with intravenous tPA. In addition, all patients of the study received i.v. heparin. Patients who did not respond to thrombolytic therapy had more than 24 hours exposure to cold, warm ischemia greater than 6 hours, or multiple freeze-thaw cycles. Twomey et al. saw a reduction in digits requiring amputations from a predicted, at-risk number of 174 to only 33 digits amputated (in 18 patients) after treatment with i.v. rtPA. The authors indicated that i.v. rtPA with heparin after rapid rewarming was safe and reduced significantly the amount of predicted digit amputations. In another open-label study conducted by Cauchy et al.,55 three treatment regimens were randomly assigned to 47 patients with severe frostbite. The regimens assigned randomly included either of the following therapies for 8 days: aspirin plus buflomedil, aspirin plus prostacyclin, or aspirin plus iloprost along with recombinant rtPA for the first day as the additive therapy. Cauchy et al. indicated that adding rtPA should be based on a case-by-case basis, depending on severity level (at least stage 4), presence of trauma such as head trauma, related contraindications, and amount of time since rewarming. Cauchy et al. did not rule out the possible additive effects of rtPA in selected patients. Submacular hemorrhage

A randomized clinical trial to treat submacular hemorrhage caused by wet macular degeneration, a degenerative disorder of the retina, is currently being performed using rtPAs and perfluoropropane (C3F8). The rationale of using tPA is to help to dissolve the clot formed during the hemorrhage, while C3F8 is used to shift the clot away from the central region of the retina (macula) where high resolution vision is achieved.56 Pediatric empyema

Empyema represents a complication of pneumonia where liquid and pus are accumulated in the pleural cavity. In children, the incidence of this condition has been increasing in the last years, requiring prolonged hospitalization for recovery. In order to shorten the hospitalization times, a new therapeutic modality involving rtPAs is being explored. 7

REVIEWS A double-blind, randomized clinical trial using rtPAs and DNAse is ongoing to investigate the effect of rtPAs and DNAse for the treatment of pediatric empyema. It is thought that tPA will break down the organized pus while DNAse will decrease the viscosity of the pus, thus improving the drainage of the liquid entrapped in the pleural space, leading to a faster clinical recovery.57 Peritonitis

Recent literature supports the potential benefits of tPA in preventing abscess formation after surgical treatment of peritonitis.58 A pilot clinical study is being conducted to investigate the safety of intraperitoneal administration of tPA and DNase including adverse events such as bleeding and pain, and biochemical markers of inflammation. The study is being conducted in patients undergoing peritoneal dialysis who suffer from peritonitis.59 NOVEL THERAPEUTIC STRATEGIES FOR rtPAs

In 1996 the FDA approved the first recombinant form of native tPA (alteplase) for the treatment of AIS. In the following decade, new generations of rtPAs were developed and commercialized. Despite the wide adoption of rtPAs in the management of thrombotic disorders, there are still a number of limitations, such as a short time therapeutic window, a low rate of arterial recanalization, a substantial risk of intracranial hemorrhage, and numerous contraindications that limit their use to selected patients.60 All these factors have contributed in the last decade to the development of novel delivery systems to improve the safety and effectiveness of thrombolytic therapy. A number of approaches have been explored to improve the interaction of tPA with fibrin molecules and activate plasminogen to plasmin locally without activating plasmin systemically, the major cause of bleeding during thrombolytic therapy. In the following, a brief description of new therapeutic modalities for tPA delivery is described. Superparamagnetic nanoparticles

Superparamagnetic nanoparticles are nanoparticles that can be magnetized when exposed to an external magnetic field and become demagnetized when the magnetic field is removed. Most magnetic nanoparticles used for medical applications are iron oxides such as ferrite or magnetite, which allow their surface functionalization with different types of biomolecules.61 Magnetic nanoparticles containing tPA have been developed to improve the localization of tPA molecules in the thrombus while avoiding the interaction of tPA with healthy tissues, decreasing the life-threatening side effects (hemorrhage) associated with tPA therapy. When an external magnetic field is applied at the site of the thrombus, the magnetic particles loaded with tPA are attracted by the magnetic field, improving the penetration of tPA molecules into the thrombus. Furthermore, by localizing tPA molecules inside the thrombus, it is possible to decrease the dosage of tPA, improving the safety of the therapy. In this regard, Chen et al.62 developed a nanocarrier delivery system consisting of a superparamagnetic iron oxide core and an 8

SiO2 shell functionalized with tPA molecules. The nanoparticle platform (SiO2-MNP) was prepared by the sol-gel method; 94% of rtPAs was attached to the carrier with 86% full retention of fibrinolytic activity. A reduction in clot lysis time and penetration of SiO2-MNP into blood clots were observed and confirmed under magnetic guidance from microcomputed tomography analysis. Therefore, by conjugating rtPAs to an SiO2-MNP surface, a new form of thrombolytic drug therapy with improved efficacy was demonstrated, resulting in a promising therapeutic modality for the treatment of thrombotic disorders.62 Gelatin-zinc-tPA complex

A formulation comprising tissue-type plasminogen activator (tPA), basic gelatin, and zinc ions, has been developed.63 The system operates reversibly by inhibiting tPA activity in the presence of Zn ions. Upon application of ultrasound waves in the range of 1 MHz, tPA activity is restored. The system represents a first step towards the realization of tPA switchable drug delivery systems, where the activity of tPA could be switched “on” and “off” in the presence of ultrasound waves. Albumin-rtPA nanoparticles

A nanoparticle system made of rtPA and an anionic peptide electrostatically bonded to a protamine-albumin complex was developed. The system maintains rtPA as inactive due to steric hindrance produced by albumin molecules avoiding the contact of rtPA with proteases in the plasma. rtPA is then transported through the circulation in inactive form and becomes activated upon contact with heparin molecules.64 BioMEMS

MEMS drug delivery devices (BioMEMS) have emerged as a new modality for delivery of drugs. The advantages of BioMEMS include active control over drug release, local delivery of drugs avoiding potential systemic toxicity, multiple pharmacotherapies in a single device, and storage of unstable drugs in powder form, opening the possibility of in vivo reconstitution of drugs. The possibility of storage of lyophilized drugs and local delivery are attractive features for delivery of rtPAs. For example, since rtPAs have a very short half-life, a locally implantable BioMEMS could increase the local concentration of rtPAs molecules at the site of action before they become metabolized systemically. Clinical conditions where rtPAs are being used require immediate intervention to prevent serious sequel. Elman et al. described a rapid MEMS drug delivery device for delivery in ambulatory emergency care.65 The microdevice consists of three layers: 1) a sealing membrane layer, 2) a reservoir layer, and 3) an actuation layer consisting of microresistors. The microresistors heat the liquid contained in the reservoir layer generating bubbles that jet the drug outside of the reservoirs, bursting the sealing membranes, and releasing the drug immediately. Finally, as has been demonstrated with MEMS technology, it can be envisioned that MEMS drug delivery devices with rtPAs will operate by a closed-loop feedback, or by remote activation from the hospital, increasing the chances of survival.66 VOLUME 00 NUMBER 00 | MONTH 2015 | www.wileyonlinelibrary/cpt

REVIEWS molecules entrapped inside the liposomes, with the domains of the tPA molecule that interact with fibrin exposed at the liposome surface. When the liposomes reach the fibrin located in the thrombus, the liposomes release the tPA molecules, avoiding broad biodistribution of tPA, which leads to unwanted side effects. Based on preclinical studies using the aortic rabbit thrombus model, as shown in Figure 5, the researchers provided evidence that echogenic liposomes loaded with rtPA can be a promising theranostic modality that allows direct visualization and target delivery of thrombolytics, improving the clinical safety and efficacy of rtPAs. Table 3 summarizes novel therapeutic strategies based on rtPAs. MONITORING THROMBOLYTIC THERAPY IN STROKE Figure 5 Schematic of the rabbit aortic thrombus model used to study thrombolytic drugs. An ultrasound probe is introduced near the location of the thrombus to induce ultrasound thrombolysis using echogenic liposomes loaded with thrombolytic agents (reproduced with permission from Elsevier, ref. 68). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Ultrasound-based thrombolysis

Ultrasound-based thrombolysis represents an emerging technology that is gaining increasing attention for the management of stroke. It is thought that ultrasound could improve the transport of rtPAs molecules to the thrombus, increasing fibrinolytic activity. In addition, mechanical effects such as acoustic cavitation have been postulated as one of the potential mechanisms involved in improved thrombolysis using ultrasound. Ultrasound-based thrombolysis device. One of the key limitations in ultrasound-based thrombolytic therapies is operator dependency, which limits the wide adoption of the technology, as well as hindering the development of new clinical trials due to the need of highly trained operators. In order to address this issue, an operator-independent ultrasound therapeutic device for the treatment of stroke in patients receiving rtPA has been developed.67 The ultrasound device includes a multiple transducer transcranial head frame comprising broadband transducers placed at the temporal and the suboccipital regions. Each transducer is designed to address a specific arterial segment and only one transducer is operating at a certain time, avoiding exposure levels above FDA-mandated ultrasound exposure limits. A computercontrolled ultrasound generator–receiver system energizes the transducers. A safety trial was conducted with 15 healthy volunteers and the platform is currently being tested in phase II and III clinical trials to assess the overall safety and effectiveness of this device against current operator-dependent systems.67 Echogenic liposomes for ultrasound-based thrombolysis. A novel diagnostic-therapeutic modality (“theranostics”) based on echogenic liposomes has been described by Laing et al.68 The system comprises echogenic liposomes that work as both contrast agents and drug-delivery vectors. The echogenicity of liposomes allows using them as contrast agents for molecular imaging during thrombolytic therapy. The drug-delivery component is based on tPA CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 00 NUMBER 00 | MONTH 2015

Thrombolytic therapy in stroke with rtPAs remains a challenge due to the short therapeutic window and unpredicted outcomes, such as hemorrhagic transformation. MRI and CT represent two current imaging modalities that have been widely adopted to assist in early stroke diagnosis and therapeutic decision-making.69 These techniques provide the first critical distinction between ischemic and hemorrhagic stroke, thus excluding patients with hemorrhagic stroke from thrombolytic therapy. These techniques, however, suffer from several limitations such as limited availability, cost, and image resolution problems to discriminate between stroke and clinical entities with stroke-like symptoms such as migraine, epilepsy, and structural brain lesions (CT). In recent years, there has been an intensive search of techniques other than neuroimaging that could provide a high specificity and high sensitivity diagnosis of stroke. In addition, stroke diagnosis in the prehospital setting, where MRI or CT are not available, has prompted the search for alternative techniques. This search has been focused not only on stroke diagnosis, but also on prognosis and therapeutic monitoring. These techniques would eventually allow the physician to perform critical decisions to improve clinical outcomes to a magnitude not achievable today. In this regard, significant efforts have been dedicated to identify disease biomarkers and biomarkers for patient selection and stratification. Disease biomarkers can provide information about stroke progression and stroke severity scales, providing a powerful tool in the differential diagnosis between healthy and stroke patients, and among stroke patients, between patients with ischemic stroke, and patients with hemorrhagic stroke. Stroke biomarkers will be critical for the selection of patients who would benefit from rtPAs, or those patients who may suffer from hemorrhagic transformation after rtPA therapy has been implemented. Diagnosis via biomarkers will allow a more costeffective, prehospital management of stroke without the need of neuroimaging techniques, further reducing the time between stroke onset and treatment. To date, however, only a few biomarkers have been supported by statistical analysis, including C-reactive protein (CRP), Pselectin, homocysteine, and gliar fibrillary acid protein (GFAP) (diagnostic biomarkers); and glucose, glutamate, D-dimer, and fibrinogen (prognostic biomarkers). Above all, high glucose levels at hospital admission have shown to be a stronger predictor of 9

REVIEWS Table 3 Novel therapeutic strategies for rtPAs therapy Technology

Advantages

Limitations

Development stage

References

Superparamagnetic nanoparticles

 Decrease drug dosage  Decrease side effects due to broad biodistribution

Loss of tPA activity when immobilized in nanoparticle surface  Change on magnetic properties upon particle functionalization

Preclinical

61,62

Zn-gelatin complex

Switchable system. tPA activity controlled by ultrasound

 Early stage of development  Need clinical validation of switchable capacity

Preclinical

63

Albumin-tPA nanoparticles

Prodrug. tPA remains inactive until reach the target

 Early stage of development  Potential interference of plasma proteins

In vitro

64

BioMEMS

 Actively controlled  Local release  Batch fabrication  Integration with electronics  In vitro reconstitution possible

 Small payload  Surgical procedure for implantation might be needed

First in human testing

Ultrasound-based thrombolysis device

 Improve thrombolysis by mechanical cavitation and improved transport of tPA molecules to fibrin  Operator independent  Advanced stage of development (phase II, phase III clinical trials)

 Optimal acoustic exposure parameters not yet determined  Variability between subjects

Phase II, III clinical trials

67

Echogenic liposomes

 Combined diagnostics and therapeutics  Lower dose of tPA required

Early stage of development

Preclinical

68

poor prognosis and hemorrhage transformation after rtPA therapy, and is perhaps the biomarker with the highest chance of clinical translation.70 For example, a patient who is admitted to the emergency room with high glucose levels might not benefit from rtPAs therapy. Instead, another thrombolytic modality, such as mechanical thrombectomy, can provide better clinical outcomes without detrimental side effects. Biomarkers could not only provide benefits for clinical management of stroke, but also assist during the drug discovery, preclinical and clinical testing of novel thrombolytic drugs.71 Biomarkers used during the drug development process could provide the information required to determine if a potential molecular target plays a role in stroke physiopathology and to determine the physical interaction of such targets with the drug being investigated. During preclinical development, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers would define the pharmacokinetic and pharmacodynamic implications of the interaction between the candidate drug and its molecular target. During clinical research, biomarkers for patient selection and stratification would avoid several types of bias related to poor patient selection. New imaging modalities could also provide effective monitoring systems during the pharmacological treatment in the clinical setting. For example, ultrasound imaging combined with ultrasound-induced thrombolysis is a promising therapeutic modality that allows real-time monitoring of thrombolytic ther10

65,66

apy.72 Furthermore, the use of ultrasound imaging combined with echogenic delivery systems such as echogenic liposomes loaded with new drug candidates could provide valuable information to validate novel thrombolytic agents suitable for clinical translation during the drug development process. Finally, genetic biomarkers such as single nucleotide polymorphisms (SNPs) could open a new era in thrombolytic therapy by means of personalized therapy. The development of an SNP test in patients with stroke could provide a powerful tool to select those patients who would benefit from rtPA (patient selection and stratification biomarkers). In this regard, in a prospective clinical study conducted in 497 patients treated with rtPAs, Fernandez-Cadenas et al. identified three SNPs associated with recanalization rates after rtPA therapy including the interleukin1 gene (IL1B) and von Willlebrand factor gene (VWF). According to the authors, these SNPs could influence rtPA efficacy through modulation of coagulation factors activity.73

PERSPECTIVE

Recombinant tissue plasminogen activators (rtPAs) have emerged as a powerful pharmacotherapy for the management of AIS, AMI, and PE. Moreover, an increasing number of clinical studies are reporting novel potential therapeutic applications for these drugs. Thrombolytic therapies, however, suffer from a short therapeutic window, serious side effects, and several contraindications. VOLUME 00 NUMBER 00 | MONTH 2015 | www.wileyonlinelibrary/cpt

REVIEWS Therefore, there is a critical need to develop new therapeutic strategies to improve thrombolytic therapies. New therapeutic modalities such as ultrasound-based delivery, magnetic nanoparticles, and BioMEMS are promising technologies, although they are at a very early stage of development and will require further clinical trials to demonstrate their utility. Meanwhile, other interventions based on education of patients, relatives, and the public in early recognition of symptoms of AIS, AMI, and PE, rapid transport to the hospital, and proper triage assessment in the hospital will contribute to improve the clinical outcomes for these devastating conditions.

6. 7. 8.

9.

10. 11. 12.

CONCLUSION

Thrombolytic diseases are the leading cause of death in the world. Tissue plasminogen activator (tPA), a protein present in the blood and a component of the fibrinolytic system, is responsible for degradation of thrombus formed during the coagulation process, which normally protects against bleeding when a blood vessel is damaged. tPA has now been developed synthetically using genetic engineering techniques for the management of pathological conditions including acute ischemic stroke, acute myocardial infarction, and pulmonary embolism. In these clinical disorders the coagulation processes bypass the fibrinolytic system capacity to maintain the blood in a liquid state, resulting in thrombus formation, vessel obstruction, and, ultimately, ischemia and tissue infarct. These synthetic forms of tPA known as recombinant tissue plasminogen activators or rtPAs have improved some of the features of native tPA, including increase in the half-life and resistance to plasma inhibitors, leading to improved therapeutic regimes. Novel clinical applications of rtPAs are being explored including frostbite, submacular hemorrhage, and pediatric empyema, while novel therapeutic technologies such as ultrasound, nanoparticles. and BioMEMS are being investigated. Therefore, it is foreseen that in the near future rtPAs will positively affect a number of clinical conditions, if their safety issues can be overcome and their benefits fully exploited.

13. 14. 15.

16.

17. 18. 19. 20.

21.

22.

23.

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ACKNOWLEDGMENTS The authors thank T. Hunter, J. Eble, and I. Oliva for contributions with the CT images. This research work was supported by the US Army Research Office via the Institute for Soldier Nanotechnologies (ISN) at MIT (contract: W911NF-07-D-0004).

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CONFLICT OF INTEREST The authors declared no conflict of interest. 28. C 2015 American Society for Clinical Pharmacology and Therapeutics V

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