Translational initiatives in thrombolytic therapy

Abstract

Once thrombi have formed as part of the pathology defining myocardial infarction, ischemic stroke, peripheral arterial disease, deep venous thrombosis or other embolic disorders, the only clinically meaningful thrombolytic agents available for reversing the thrombogenic process are various plasminogen activators. These agents are enzymes that reverse fibrin polymerization underlying the coagulation process by converting endogenous plasminogen to plasmin, which cleaves the fibrin network to form increasingly smaller protein fragments, a process known as fibrinolysis. For the most part, the major clinically used thrombolytics, tissue plasminogen activator, urokinase and streptokinase, as well as the experimentally investigated agent staphylokinase, are the products of recombinant DNA technology, which permits molecular optimization of clinical efficacy. In all cases of molecular optimization and targeting, however, the primary challenge of thrombolytic therapy remains hemorrhagic side effects, which are especially devastating when they occur intracerebrally. Currently, the best strategy to ameliorate this adverse effect is nanoparticulate encapsulation or complexation, and many strategies of this sort are being actively pursued. This review summarizes the variety of targeted and untargeted thrombolytic formulations that have been investigated in preclinical studies.

Keywords

thrombolytics / nanomedicine / plasminogen activators

Introduction

Clinical limitations of thrombolytics and attempts to overcome them

Thrombo-occlusive diseases, such as myocardial infarction (MI), ischemic stroke, peripheral arterial disease (PAD) and pulmonary embolism resulting from deep venous thrombosis (DVT) and surgical sequelae are responsible for great mortality and morbidity throughout the world, accounting for a sizable portion of the deaths due to cardiovascular disease. The introduction of recombinant tissue plasminogen activator (rtPA) for clinical thrombolytic therapy of acute MI and ischemic stroke during the 1980s represented a major advance in cardiovascular acute care practice that has been reflected in the morbidity and mortality outcome data for these conditions. Other thrombolytics, especially urokinase (uPA) and streptokinase (sPA), have also continued to be employed within various contexts of the same or similar applications.

The use of thrombolytic agents for the treatment of acute ischemic and neurologic events is limited, however, by the potential nonspecific activation of plasminogen at sites other than the occluded vessel, resulting in hemorrhagic events that could be catastrophic, especially when they occur in the cerebral vasculature [ 1]. Hemorrhagic complications are also exacerbated by the need for systemic administration of pharmacologic doses of thrombolytics to provide effective concentrations of the agents for plasminogen activation at the desired sites [ 2, 3]. Continual infusion of native thrombolytics is necessary to compensate for their very short half lives in vivo, while bolus administration is possible for later generation agents with longer half-lives [ 4]. Furthermore, thrombi of ischemic stroke, PAD and DVT tend to be refractory to thrombolytic intervention, possibly because of plasminogen depletion in the local environment [ 5– 7].

Thrombolytics are a particularly good example of pharmacologic agents that require reduction of the therapeutic window because of problems associated with systemic administration, especially toxicity issues. In addition, the efficacy of these agents is adversely affected by physiological environmental factors, namely enzyme inhibition by plaminogen activator inhibitors (PAI), such as PAI-1 and PAI-2 [ 8, 9]. From the time of Paul Ehrlich early in the twentieth century, molecular targeting strategies have been recognized as a primary approach to narrowing the therapeutic window of pharmacologic agents. In this case, sequestration methods, such as encapsulation of the agent in controlled release formulations, would provide added benefit.

All thrombi contain components of the thrombogenic process, including platelets, coagulation cascade factors and fibrin. Platelet markers, coagulation factors such as thrombin, and fibrin have been employed as molecular targets for thrombus detection or treatment. Numerous attempts have been made to ameliorate the unfavorable pharmacokinetics and pharmacodynamics of clinical thrombolytic therapy by employing both actively and passively targeted thrombolytic formulations. This review will consider properties of the major thrombolytics that are relevant to the formulations thus far studied and describe untargeted and targeted formulations of these thrombolytics within the context of the delivery vehicles (or lack thereof) employed in them.

Thrombolytics used in experimental formulations

Thrombolytics necessarily are agents that disrupt either major component of thrombi, i.e., the fibrin network or aggregated platelets. All of the fibrinolytic agents are proteases of microbial, as well as of mammalian origin. In the former case, they serve as virulence factors that liquefy fibrin clots, enabling bacterial access to host circulation and tissue [ 10]. While thrombolytics are agents that disrupt thrombi once they have formed, anticoagulants, such as heparin, warfarin, clopidogrel and other clotting factor inhibitors, antagonize the formation of clots. Because they may predispose patients receiving thrombolytics to hemorrhagic effects, anticoagulants are often contraindicated in thrombolytic protocols [ 11].

Streptokinase

Streptokinase (sPA) is a monomeric protein of MW 47 kDa [ 12]. Unlike tPA and uPA, sPA is not a protease. By forming an equimolar complex with endogenous plasminogen or plasmin, it confers plasminogen specificity upon these species, converting them into plasminogen activators that can cleave the Arg₅₆₀-Val₅₆₁ of that protein [ 13]. Although sPA is significantly less costly than tPA and the two biotherapeutic agents exhibit comparable efficacy, as a microbial protein, sPA is highly immunogenic; it has caused severe, even fatal, anaphylactic responses, restricting multiple treatments with the thrombolytic for stability, as well as safety, reasons [ 14, 15].

In addition, sPA is degraded by plasmin, so that its half-life in vivo is about 30 min [ 16]. While this is significantly greater than the 5 min half-life of tPA, which is susceptible to inhibition by PAI-1, it is insufficient for effective clinical thrombolytic therapy. Thus, an acylated plasminogen-sPA complex (APSAC), which exhibits an extended half-life in vivo, has gained wide clinical usage [ 17, 18]. Like tPA, sPA has been genetically engineered to increase its stability. Based on recognition that Lys59 and Lys386 are sPA target residues for plasmin activity, mutant forms of sPA that are relatively resistant to plasmin proteolysis, while retaining thrombolytic activity, have been prepared [ 16, 19]. Glycosylated and PEG-conjugated sPA have also been shown to be more stable than the native molecule because of their relative resistance to proteolysis and their reduced immunogenicity [ 20– 22]. Likewise, molecular variants of reduced immunogenicity have been produced by recombinant DNA technology, based on knowledge of immunogenic sPA domains [ 23].

Urokinase

In 1947, Macfarlane and Pilling reported fibrinolytic activity in urine [ 24]. Four years later, Williams demonstrated that the activity was due to a plasminogen activator that was later termed urokinase (uPA) [ 25, 26]. During the 1970s, latent uPA activity was found in the conditioned media of some human cells and was subsequently identified as single-chain uPA (scuPA), or pro-urokinase [ 26– 28]. Secreted by kidney cells and a wide variety of malignant cells as a relatively inactive, fibrin binding zymogen of 411 residues (53 kDa), scuPA binds to the uPA receptor (uPAR), where it is cleaved at Lys158-Ile159 by neighboring, membrane-bound plasmin or other proteases to form two-chain uPA (tcuPA), which does not bind to fibrin and exhibits full enzymic activity [ 26, 29– 31]. The enzyme catalytic site is located in the tcuPA B-chain. Urokinase expression by malignant cells has been shown to correlate with invasive and metastatic potential [ 32], suggesting that, by degrading fibrin matrices, tumor-expressed uPA plays an analogous role to microbially secreted sPA.

Further cleavage of tcuPA at Lys135-Lys136 releases the uPA N-terminal fragment (ATF), which contains an EGF-like and a Kringle domain. The latter is involved in binding of the receptor [ 30]. The ATF carboxy terminus also contains an a_vb₃ integrin binding site that is involved in cell migration [ 33], consistent with uPA’s role in normal tissue remodeling, such as trophoblast implantation and wound healing [ 30, 34]. Urokinase serves a number of functions not related to its thrombolytic activity, many of them through uPAR binding. Receptor binding causes exposure of a vitronectin interaction site that induces actin cytoskeleton rearrangement, contributing to the spreading of cultured cells on vitronectin-coated surfaces [ 35]. Urokinase‒uPAR interaction also is important in protecting cells from apoptosis following cell detachment (anoikis), mediated by uPAR signal transduction through the MEK/ERK and P13K/Akt pathways, resulting in expression of a Bcl anti-apoptotic protein [ 36]. An NF-kB binding site has been identified in the regulatory region of the uPA gene [ 30], indicating conditional feedback regulation of uPA-induced cell growth, attachment and motility functions that are important in tumor establishment, invasiveness and metastasis through the P13K/Akt pathway.

Clinically, uPA has been used widely as a thrombolytic agent for treatment of such thrombotic pathologies as pulmonary embolism, acute myocardial infarction, ophthalmic clots and peripheral arterial occlusion [ 30]. As a multifunctional effector molecule involved in many aspects of neoplasia, uPA serves as a prognostic marker for human cancers and, thus, is both a diagnostic and therapeutic target for clinical management of neoplastic diseases [ 30, 32].

Tissue plasminogen activator (tPA)

After elucidation of the physiological roles and origin of tPA, the detailed structure-function characteristics of the molecule were elaborated during a period (1979‒1985) that roughly coincided with the cloning and expression of the tPA gene. Prior to production of the recombinant protein, much of the material used for these studies was obtained in purified form from a cultured melanoma cell line [ 37].

Biochemical properties

Tissue plasminogen activator is secreted as a single-chain molecule (sctPA), consisting of 530 residues and having an approximate molecular weight of 68 kDa [ 38]. In this form, the molecule possesses 17 intrachain disulfide bonds [ 31]. Plasmin (generated by sctPA), as well as other serine proteases with trypsin specificity, remove the N-terminal tripeptide, leaving L-serine as the first residue, and cleaving the molecule at Arg275-Ile276 to produce two-chain tPA (tctPA). The A (heavy) chain, which possesses the fibrin binding and regulatory functions of the molecule, has a molecular weight of 36 kDa, while the B (light) chain, in which the tPA catalytic activity resides, exhibits a molecular weight of 32 kDa. The two chains are linked by a single disulfide bond between Cys264 and Cys395 [ 31, 38] (Fig. 1).

Unlike uPA, the catalytic activity of sctPA and tctPA are essentially equal, especially in the presence of fibrin. The catalytic activity of tPA in converting plasminogen to plasmin is enhanced two to three orders of magnitude in the presence of fibrin; this effect apparently is mediated by the lysine-dependent K2 fibrin binding site [ 38– 41]. The lysine-independent fibrin binding site in the finger domain is important for tPA binding at low fibrin concentrations in the absence of plasminogen [ 39], which is consistent with the higher binding affinity found for this site. Studies relying on tPA binding to fibrin clots reported relatively low affinities for both domains [ 42, 43].

Other studies, relying on immunoassay methodology and surface plasmon resonance, however, reported much higher affinities, in the low nanomolar range [ 44, 45]. Affinities (K_D) of 5.81±4.04 nmol/L for the finger domain and 30.4±14.7 nmol/L for the K2 domain, reported in the latter studies were confirmed by immunoassay methods [ 46, 47]. In the course of that study, a novel two-stage fibrin pad ELISA was employed to determine binding affinities and thermodynamics of the tPA fibrin binding sites. The latter analysis indicated strong ionic association characteristics for both sites, particularly the finger domain, which implicated involvement of four charged residues near the N-terminal (Fig. 1) [ 46].

The fibrin sites implicated in lysine-dependent enhanced plasminogen activation by tPA include Aa148‒160, g312‒324 and aC (Aa221‒610) [ 45, 48– 50]. Among the components of the fibrin-plasminogen-tPA ternary complex, the first fibrin site preferentially binds plasminogen under physiological conditions [ 51, 52]. The second site interacts exclusively with tPA [ 53, 54] and the third site binds both plasminogen and tPA [ 45]. The first two sites are exposed during fibrin formation and, therefore, are limited to fibrin-specific degradation [ 52].

The tPA A-chain consists of four distinct domains, each having defined non-catalytic functions. The finger domain, which contains the higher affinity fibrin binding site and is homologous with the fibronectin fibrin binding finger domain, comprises residues 4‒50. The growth factor domain (residues 50‒87) is homologous to EGF and the EGF domain of uPA ATF. A recent study suggests that EGFR binding of this domain initiates tPA catalytic site activation of the PAR and EGFR regulatory pathways [ 55]. The remainder of the A-chain comprises two Kringle domains, designated K1 (residues 87‒176) and K2 (residues 176‒256), which share a high degree of homology with the five Kringles of plasminogen [ 37, 56]. The role of K2 in lysine-dependent fibrin binding has been discussed, while that of K1 seems to be associated mainly with its glycosylation site.

The B-chain catalytic site is characterized by the classic serine protease triad of residues, in this case His 322, Asp 371 and Ser 478. It has a high degree of specificity for cleavage of the Arg-Val peptide bond, which it catalyzes between residues 560 and 561 of glu-plasminogen, converting the zymogen to plasmin [ 26, 42, 57]. Furthermore, the tPA B chain is homologous to the catalytic chains of plasmin, thrombin, chymotrypsin, trypsin, elastase and urokinase [ 56– 58].

Both the A and B chains are involved in PAI-1 binding and complexation with tPA. Initial PAI-1 binding to sctPA is to the finger domain, while binding to tctPA is to the Kringle 2 domain, the PAI-1 binding site of which is exposed during conversion of the single-chain to the two-chain form of the enzyme [ 59]. Secondary binding of PAI-1 to tPA, resulting in full enzymically inactive complex formation, involves an exosite of the B chain, comprising the His 297, Arg 298, Arg 299 and Arg 304 residues [ 60]. There is also evidence for a salt bridge interaction between the Arg 304 of tPA and the Glu 350 of the PAI-1 distal reactive center loop [ 60, 61].

Tissue plasminogen activator isolated from melanoma cells is a glycoprotein; it contains up to 12.8% carbohydrate, consisting of 3 polysaccharide chains N-linked to Asn 117 and Asn 184 in the A-chain and to Asn 448 in the B-chain [ 31, 56]. Mannose (39.8 wt.%) and N-acetylglucosamine (25.0 wt.%) were the major saccharide constituents found [ 62]. A tPA variant containing 7.1% carbohydrate and lacking the Asn 184 chain in the K2 domain has also been characterized [ 62, 63]. Various studies have shown that N-linked carbohydrate is not important for catalytic function, fibrin binding or liver clearance, but may be important for intracellular trafficking and secretion [ 62, 64– 68].

Recombinant tPA (alteplase)

A collaboration between long-term tPA researchers Désiré Collen and H. Roger Lijnen and a team of molecular biologists from Genentech, led by Diane Pennica, that began in 1980 resulted in the cloning and expression of sctPA two years later. Messenger (mRNA) from melanoma cells actively producing and secreting tPA was used to construct a cDNA library, which was hybridized with known tPA DNA sequences. An expression vector containing the complete tPA cDNA sequence coding for a 562-amino acid polypeptide was transfected into E. coli, which produced a non-glycosylated protein retaining the full tPA catalytic and fibrin binding activities [ 37]. The glycosylated protein was then expressed more efficiently in mammalian cells and was shown to be biochemically and pharmacokinetically indistinguishable from tPA isolated from melanoma cells [ 69]. The commercial sctPA product (alteplase) is produced in Chinese hamster ovary (CHO) cells [ 37].

The first definitive demonstration of recombinant tPA (rtPA) clinical efficacy for acute myocardial infarction (AMI) was provided by the NIH Thrombolysis in Acute Myocardial Infarction (TIMI) trial led by Eugene Braunwald in 1984 [ 37, 70]. The GUSTO trial established an alteplase administration protocol for AMI consisting of an initial bolus of 15 mg i.v., followed by 0.75 mg/kg over 30 min (not to exceed 50 mg) and then 0.50 mg/kg over 60 min (not to exceed 35 mg), with coadministration of i.v. heparin during and after rtPA treatment [ 71]. In these trials, coronary arterial patency was restored in 60%‒85% of ST-elevation MI (STEMI) patients [ 70, 72]. Clinical use of rtPA has reduced STEMI mortality at 1 month from 13% to 6.3% [ 73]; as of 2004, rtPA was used worldwide in about 300 000 AMI patients per year [ 37].

Intravenous alteplase is now the only US FDA-approved treatment for acute ischemic stroke (AIS) [ 74]. Improvement of neurological outcomes in this condition by rtPA administration within 3 h of stroke onset was established by the National Institute of Neurological Disorders and Stroke (NINDS) rtPA trial in 1995 [ 75]. Based on various functional neurological outcome assessments at 3 months, AIS patients who received rtPA were 30%‒50% more likely to have a good neurological outcome than were controls, but mortality was unaffected by treatment [ 76]. In January 2013, the American Heart Association/American Stroke Association amended their guidelines for treatment of AIS to extend the window for use of i.v. rtPA to 4.5 h. Prior to this time, only 2%‒5% of all eligible patients were treated in most US centers, although the percentage is higher in centers with better levels of expertise. Major reasons for underutilization of the therapy are low levels of public awareness, poor recognition of symptoms, and delays in emergency transport [ 77– 79].

Perhaps most important in explaining rtPA underutilization in treatment of AIS is a fear among emergency room physicians of adverse side effects, especially potentially fatal intracranial hemorrhage (ICH), and a lack of sufficient efficacy [ 80, 81]. The NINDS study reported a 6% incidence of clinically significant ICH in rtPA-treated AIS patients, compared to 0.6% in placebo controls [ 76], and symptomatic ICH rates as high as 16% have been reported [ 82]. An analysis of the GUSTO-1 trial results revealed that gastrointestinal bleeding was more common than ICH. There was a 1.8% incidence of moderate and severe gastrointestinal hemorrhage, while ICH, which was all severe, was seen in 0.7% of the patients [ 83]. An earlier study found an 8% incidence of gastrointestinal bleeding among 386 tPA-treated AMI patients, with only a 0.5% incidence of ICH [ 84]. There is no effective treatment for ICH, which has a 30-day mortality of 34%‒50% [ 85]. Most treatment modalities fall into the category of palliative care. Anti-inflammatory agents are often administered following ICH to inhibit late-stage pathologic effects of ICH [ 86].

The Interventional Management of Stroke Study part I (IMS-I) reported a recanalization rate of 56% following i.v. rtPA administration in a range of intracranial vessel occlusion locations [ 87], but lower rates of complete recanalization have often been reported [ 88].

Next-generation recombinant thrombolytics

Despite the routine use of thrombolytic therapy in the management of thrombo-occlusive diseases, major challenges remain. These include (1) rapid plasma clearance (3‒6 min half-life) due to binding of PAI-1 in the case of tPA and uPA and recognition by scavenger cells of the glycosylation sites in the A-chain [ 1, 2, 26], and (2) major bleeding episodes, which can be devastating when they occur in the cerebral vasculature. The latter problem is particularly highlighted by the need for systemic administration of tPA in order to overwhelm circulating PAI-1 levels [ 26].

With the ability to manufacture molecularly manipulated plasminogen activators, it became theoretically possible to optimize thrombolytics for clinical treatment of thrombotic disorders by application of genetic engineering techniques. As is often the case, however, the effort produced some unexpected consequences. An apt case in point is tenecteplase (TNKase). Produced as a third-generation thrombolytic successor to alteplase by Genentech, this compound featured a substitution of asparagine for Thr103, introducing a new glycosylation site, and glutamine for Asn117, eliminating a high mannose glycosylation site. A third site-specific mutation involved replacement of the PAI-1 binding site at 296‒299 with four alanine residues. Both the finger and K2 domain fibrin binding sites were fully retained. These alterations served to prolong plasma half-life by reducing scavenger cell recognition [ 37] and PAI-1 binding, thus increasing tPA circulation time [ 1, 2]. Despite these manipulations, however, opinion regarding the clinical utility of TNKase for thrombolytic therapy of acute MI is mixed [ 1, 89].

On the other hand, reteplase (developed by Boehringer Mannheim) lacks the finger, growth factor and K1 domains, leaving only the lower affinity K2 fibrin binding site and the proteolytic B-chain. Since reteplase is produced in E. coli, it is not glycosylated, reducing scavenger cell recognition. Hence, the plasma half-life of this variant is the same as TNKase, 18 min [ 1, 2]. In contrast, however, the reduced fibrin binding affinity of reteplase appears to have improved its efficacy relative to alteplase and TNKase, since reteplase is able to penetrate the thrombus more efficiently, while alteplase and TNKase become sequestered at the apical surface of the clot, where they exhaust the available plasminogen before lysis can proceed to completion [ 90, 91].

The incidence of adverse bleeding events, including hemorrhagic stroke, is comparable between reteplase and alteplase. In contrast, lanoteplase, which also retains only the K2 fibrin binding domain, but has an appreciably extended plasma half-life of 30‒45 min, was withdrawn from development because it exhibited a significantly higher incidence of hemorrhagic stroke compared to alteplase [ 92, 93].

As noted above, in vitro studies have revealed that another general limitation of plasminogen activator-mediated thrombolysis, especially for agents possessing fibrin binding capability, may be the likely depletion of plasminogen within the clot, preventing completion of the fibrinolytic process [ 91, 94]. If the existence of this phenomenon in vivo can be confirmed, strategies will need to be devised to circumvent plasminogen depletion, especially in thrombotic stroke, peripheral arterial disease and deep venous thrombosis.

It can be concluded, therefore, that, although plasminogen activators can be genetically engineered to potentiate therapeutic efficacy (albeit in unexpected ways) and to prolong circulation time, bleeding events continue to represent a major adverse effect of these agents and, in fact, are exacerbated at more extended circulation times.

Staphylokinase

Like streptokinase, staphylokinase (SAK), a product of staphylococcal species, especially S. aureus strains, represents a bacterial virulence factor that promotes microbial invasion of host tissue. SAK is a nonglycosylated single-chain protein of 135 residues and, like sPA, it binds to plasmin or plasminogen, activating them, but requires that they already be bound to fibrin before the SAK can bind [ 26]. Recombinant SAK has been expressed in E. coli [ 95, 96] and recombinant techniques have been used to reduce the thrombolytic’s immunogenicity through site-dircted mutagenesis [ 97]. The CAPTORS trial indicated that SAK exhibited similar clinical efficacy to tPA for recanalization in acute MI and concluded that the favorable efficacy/safety profile of the agent was encouraging, warranting further investigation [ 98].

Formulations for delivery of thrombolytics

Thrombolytic delivery vehicles and strategies fall into three major categories: untargeted, targeted to thrombus components other than fibrin, and targeted to fibrin. Developments in each category since the clinical introduction of recombinant tPA will be reviewed.

Non-targeted formulations

In this category, there are three major considerations: the thrombolytic, the vehicle, and the improvement(s) offered by the formulation relative to the free thrombolytic.

Liposomal formulations of tPA

Several studies have concentrated on the physical properties of tPA encapsulated in liposomes, determined in vitro. In a study of protein stability after encapsulation in freeze-dried liposomes, using trehalose as an adjuvant, Ntimenou et al. found a maximum 16% encapsulation efficiency for tPA, with a maximum 83% recovery of encapsulated tPA after lyophilization [ 99]. Soeda et al. studied the enzyme kinetics of tPA encapsulated in various liposomal formulations, using D-Ile-Pro-Arg-p-nitroanilide as substrate in the presence of fibrin and glu-plasminogen [ 100]. They found a 60% encapsulation efficiency, based on enzyme activity, for bovine brain sulfatide-containing vesicles, which they found to be best for plasminogen activation in the presence of fibrin.

Heeremans et al. [ 101] achieved 93% tPA encapsulation efficiency in anionic liposomes, comprising phosphatidylcholine (PC), phosphatidylglycerol (PG) and cholesterol, in pH 7.5 HEPES buffer at low ionic strength. None of these studies investigated the ability of the formulations to lyse clots, so there was no opportunity to assess their fibrin-targeting properties. Because tPA itself possesses intrinsic fibrin-targeting capabilities that can be exposed in liposomal formulations of the agent, it may not be possible to produce a truly non-targeted liposomal tPA formulation.

For instance, Heeremans et al. [ 101] found that one-third of the “entrapped” tPA was actually adsorbed to the liposomal surface, causing them to conclude that the tPA molecule interacts with the lipid bilayer of the vesicle. In another study, this group exploited the fibrin-targeting capability of plasminogen by encapsulating tPA in small unilamellar vesicles conjugated to glu-plasminogen via a thioether linkage [ 102]. They demonstrated a 67% enhancement of thrombolytic activity in a rabbit jugular vein thrombosis model by liposome-encapsulated tPA relative to tPA alone, which was not improved by plasminogen-conjugation, implying that the intrinsic fibrin-targeting capability of tPA was fully operative in the formulation. Thus, where exposure of tPA fibrin binding sites on liposomal surfaces is documented, tPA-loaded liposomes should be considered as intrinsically fibrin-targeted formulations. Such cases will be described in the section on fibrin-targeted thrombolytic formulations.

Kim et al. [ 103] found 20% tPA encapsulation efficiency in their PEGylated anionic liposomes. They determined fibrin clot lysis qualitatively by a fibrin-infused agarose diffusion method and assumed that the lysis zones after 24 h were entirely due to released free tPA. This group later injected the formulation subconjunctively in a rabbit subconjunctival hemorrhage (SH) model and showed that it enhanced SH absorption and prolonged tPA activity [ 104].

Other tPA formulations

Absar et al. devised what they called a “camouflaged” tPA “prodrug” construct consisting of tPA conjugated to a polyanion (low molecular weight heparin or poly-L-glutamate) complexed with a human serum albumin (HSA)-protamine conjugate [ 105, 106]. They envisioned targeting the prodrug formulation to activated platelets, followed by administration of heparin to displace the tPA-polyanion conjugate at the thrombus site. In a prototypic study, this group induced thrombi in the jugular veins of rats and administered free tPA or the prodrug intravenously, followed 15 min later by i.v. heparin. The prodrug was nearly as effective as free tPA in reducing the excised clot mass [ 106]. Although they found that tPA activity was 60% masked in the prodrug formulation [ 105], enough fibrin-targeting capacity probably remained to consider the construct to be targeted.

Liposomal formulations of streptokinase (sPA)

Like Absar et al., Holt and Gupta [ 107] envisioned targeting their sPA-loaded liposomes, comprising distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine conjugated to PEG (DSPE-PEG) and cholesterol, to GPIIb/IIIa and P-selectin, but only performed preliminary characterization of the formulation thus far. They were able to achieve an encapsulation efficiency of 46% and recovered 30% sPA activity in the construct. Kim et al. [ 108] encapsulated sPA in PEGylated liposomes and demonstrated superior pharmacokinetics compared to free sPA in rats, extending the half-life of the thrombolytic in blood from barely more than 30 min to 5.4 h.

Erdogan et al. [ 109] conducted a pharmacokinetic study of their liposomal sPA formulation, exhibiting only 13% encapsulation efficiency, in rabbits with induced venous thrombosis. They found that most of the liposomes, which did not contain PEG, were localized in the spleen, liver, lungs and kidneys after 4 h and that no more than 5% were associated with the thrombus. Perkins et al. tested the efficacy of a sPA liposomal formulation in a rabbit thrombosis model and found that it was greater than sPA alone, which was equivalent to empty liposomes [ 110]. Streptokinase encapsulated in large unilamellar vesicles reduced recanalization time by 50% relative to free sPA in a dog coronary arterial thrombus model [ 111].

Other streptokinase formulations

A series of sPA-poly(amido amine) dendrimer conjugates were prepared and evaluated [ 112]. A maximum 80% activity retention was achieved, but there was evidence that the sPA-dendrimer linkage was labile in plasma, releasing free sPA. Overall, dendrimer-conjugated sPA was more stable in buffer than the free enzyme, however. In another in vitro study, sPA was encapsulated in PEG microparticles (MESK) [ 113]. The MESK formulation was more effective in microflow clot dissolution, exhibiting better clot penetration, than free sPA.

Liposomal formulations of urokinase (uPA)

In another study of thrombolytic-loaded liposomes, niosomes and sphingosomes constructed with myristoyl PC, surfactants and sphingomyelin, respectively, Erdogan et al. found a maximum 12% uPA encapsulation efficiency for these preparations [ 114]. In a pharmacokinetic study using rabbits with induced venous thrombosis, they found that most of the liposomes, which did not contain PEG, were localized in the spleen, liver, lungs and kidneys after 4 h. Only the liposomal uPA formulation approached 5% localization in the thrombus.

Ultrasound-potentiated thrombolysis with non-targeted contrast agents

Ultrasonic potentiation of concurrently administered free tPA and microbubbles represents the primary non-targeted thrombolytic delivery strategy at this time. Datta et al. [ 115] demonstrated that Definity^®, a microbubble ultrasound contrast agent, when administered with 120 kHz mechanical ultrasound (within the optimal range of ultrasound-assisted thrombolytic therapy of ischemic stroke) and tPA (96 mg/ml), increased thrombolysis in vitro by 63% over a period of 30 min, relative to tPA and ultrasound alone. These investigators found that the microbubble+ ultrasound-enhanced efficacy was mainly due to stable cavitation effects, which promoted acoustic streaming processes that caused increased penetration of both tPA and plasminogen into the clot, as opposed to inertial cavitation, which is associated with tissue damage.

In an extension of the CLOTBUST trial of ultrasound-enhanced tPA acute ischemic stroke therapy, Alexandrov et al. treated 15 acute ischemic stroke patients with tPA+ transcranial Doppler ultrasound (TCD) or tPA+ TCD+ perflutren-lipid microspheres (µS) [ 116]. Addition of µS to TCD thrombolytic therapy markedly increased the incidence of complete recanalization (50% vs. 18%) and of sustained recanalization after 2 h (42% vs. 13%). Symptomatic intracerebral hemorrhage (sICH) was not seen in any subject, but asymptomatic ICH occurred in three µS-enhanced treatment patients vs. one in the control group.

Although improved arterial recanalization and clinical outcomes with the use of tPA, microbubble contrast agents and ultrasound have been demonstrated clinically [ 117], a significant increase in ICH has been observed under these conditions [ 118]. Thus, attempts to potentiate soluble tPA efficacy again appear to exacerbate the agent’s hemorrhagic effects. Urokinase thrombolytic activity with ultrasound administration and microbubbles has also been investigated, but only in a preliminary in vitro study [ 119]. Francis [ 120] reviewed earlier studies of ultrasound-enhanced thrombolytic therapy.

An ultrasound-enhanced nanoparticulate tPA formulation has been developed. PEG coupled to anionic gelatin was complexed to tPA conjugated to cationic gelatin, resulting in a construct possessing 45% of the original tPA activity. Application of ultrasound (1 MHz, 1.0 W/cm²) immediately restored full tPA activity. Complete recanalization was achieved with the formulation in a rabbit thrombus model and the half-life of the complexed tPA in blood circulation was three times that of the free thrombolytic [ 121]. This group was able to target the formulation to thrombi with von Willibrand factor and achieved greatly enhanced recanalization of coronary arteries in a swine MI model compared to free tPA [ 122].

Non-fibrin targeted formulations

These are thrombolytic formulations targeted to thrombus components other than fibrin. Additional considerations besides those listed in the previous section are the target and advantages of the targeted formulation relative to its non-targeted counterpart.

Formulations targeted to GPIIb-IIIa

GPIIb-IIIa is a glycoprotein integrin on the surface of platelets that binds the RGD tripeptide of serum proteins such as fibronectin, fibrinogen and von Willebrand factor, once activated by thrombin. Thus, targeting this integrin is an alternative strategy for site-specific delivery of formulations to the platelet component of thrombi. Most GPIIb-IIIa-targeted formulations employ some variety of the RGD tripeptide as ligand.

Gupta et al. [ 123] constructed an empty liposome conjugated to an 11-mer peptide containing the RGD sequence in order to demonstrate the targeting capabilities of the vehicle. Preferential adherence of the targeted fluorescent liposomes to activated platelets without causing platelet aggregation was demonstrated by fluorescence microscopy and flow cytometry. In a novel approach, an analogous liposomal formulation conjugated to a cyclo-RGD peptide was used to inhibit fibrinogen binding to GPIIb-IIIa on activated platelets [ 124]. Based on an IC₅₀ of 0.5 µmol/L for inhibition of platelet aggregation, this study established that the cyclo-RGD peptide exhibits a 3-log higher affinity for the integrin than the linear peptide, but that the best RGD binding affinities are still in the micromolar range.

Streptokinase was loaded into liposomes conjugated to a functionalized lysyl RGD [ 125]. This formulation demonstrated enhanced thrombolytic release in the presence of activated platelets and was more effective in clot lysis in vitro relative to free streptokinase. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles coated with chitosan or chitosan-GRGD were loaded with tPA [ 126]. An in vitro thrombolysis study demonstrated that this formulation resulted in a more precipitant clot dissolution pattern than that produced by free tPA.

Several investigators studied the targeting capabilities and ultrasound-enhanced thrombolytic efficacy of RGDS/thrombolytic associated microbubble preparations. Mu et al. [ 127] admixed fluorophore-labeled uPA and RGDS with SonoVue^® lipid-coated microbubbles and demonstrated targeting to activated platelets by flow cytometry. Thrombolytic efficacy was demonstrated in vitro by clot mass loss and in vivo clot targeting was confirmed by fluorescence microscopy of residual clots retrieved from rabbit femoral arteries.

Hua et al. [ 128] incorporated tPA and RGDS into the lipid coating of microbubbles, demonstrating thrombolytic activity of the formulation, which was potentiated by ultrasound, with agarose fibrin plates. They also showed echogenicity of the formulation in vivo. In a more recent study, this group demonstrated that their formulation was as effective in recanalizing obstructed femoral arteries in a rabbit PAD model as the individual components administered separately, while only 1/15 the amount of tPA was required to do so [ 129].

A 15-mer peptide was used to target tPA-loaded liposomes to the higher affinity binding site of fibrinogen for GPIIb-IIIa (See “Other tPA formulations” in the previous section) [ 130]. Demonstrating a bimodal release profile, these investigators showed that their formulation retained more than 90% of native thrombolytic activity, but exhibited greater thrombolytic efficacy than soluble tPA. In rats, the circulation time of the liposomal tPA formulation was extended at least 15 times relative to free tPA. These investigators conjugated this targeting peptide to HSA in their camouflaged tPA prodrug formulation (Section “Other tPA formulations”) and confirmed targeting to activated platelets by fluorescence microscopy [ 131].

In summarizing the use of RGD peptides for this application, it is relevant to consider the tendency of peptide targeting to exhibit relatively low binding affinities, usually in the micromolar range. In many cases, endogenous ligand binding, such as the case of ICAM-1 and its natural ligand LFA-1, also happens to be in the micromolar affinity range [ 132]. The local concentration of the ligands, however, is in the same range, since the ligand densities on both the leukocytes (integrin) and the vessel walls (adhesins) are in the order of 10¹⁴/m² [ 133], while the effective volume of interaction is approximately 10 µm³.

It should be noted, however, that leukocytes — both monocytes and neutrophils — patrol vascular surfaces, moving slowly over the endothelium and becoming activated to traverse the intima when encountering adhesins [ 134, 135]. Parenterally administered targeted nanoparticles, on the other hand, encounter rapid flow conditions in the peripheral circulation. Such formulations must rely on slower velocities near vessel walls and constricted flow conditions in local environments, such as atherosclerotic coronary arteries and arterioles, to promote adherence of ligands [ 133].

Even so, ligand affinities should be in the nanomolar range to effect clinically relevant adherence under these conditions. As an example, Demos et al. found that picomolar targeted liposome avidities — the product of nanomolar antibody affinities and ligand densities of>1000 antibody molecules per liposome — were necessary to achieve optimal adherence of liposomes to a fibrin matrix in a flow circuit simulating physiological flow rates and shear stresses [ 136, 137]. This indicates that peptides can be useful targeting agents if high enough conjugation efficiencies (>1 × 10⁴ molecules per nanoparticle) can be attained.

Targeting of thrombi represent an especially favorable case, since flow in their vicinity is impeded. Where there is a significant collateral circulation, however, intravenous administration of targeted thrombolytic formulations may be thwarted by a “vascular steal” phenomenon, in which flow in an occluded vessel can be completely reduced by compensatory flow through the collateral vessels [ 138]. In such a case, local administration of the formulation may be the only viable alternative strategy.

Increased efficacy of a higher affinity formulation was implied by an in vitro study of a GPIIb-IIIa-specific antibody directly coupled to tcuPA [ 139]. The conjugate was 31 times more effective in converting plasminogen to plasmin than free tcuPA and 25 times more potent in a clot lysis assay. The conjugate was 7.5 times more effective than a mixture of the components and 125 times more effective than free tcuPA in inhibiting platelet aggregation.

Possibly the most promising performance of a GPIIb-IIIa-targeted formulation was reported by Xie et al. [ 140]. In a swine model of thrombotic occlusion-induced acute ST-elevation myocardial infarction (STEMI), these investigators used high-MI ultrasound (1.5 MHz, 1.9 MI) insonated proprietary GPIIb-IIIa-targeted microbubbles (MRX-802; ImaRx Therapeutics, Inc., Tucson, AZ, USA) to enhance the thrombolytic efficacy of prourokinase (scuPA) treatment. Efficacy was measured as recanalization rate (53% vs. 7% for scuPA alone; P = 0.01) and ST-segment resolution (82% vs. 21% for scuPA alone; P = 0.006).

Physically targeted formulations

In an example of passive targeting, Korin et al. [ 141] targeted areas of vascular narrowing by fabricating nanoparticle aggregates that disaggregate, releasing thrombolytics, in high shear stress conditions characterizing obstructed vessels. They demonstrated proof of concept by effectively delivering tPA to pulmonary emboli in a mouse model.

Several groups have constructed magnetically targeted nanoparticles for delivery of thrombolytics. Kempe et al. [ 142] developed magnetite nanoparticles for delivery of tPA to stents for treatment of in-stent thrombosis. Both safety and efficacy of the formulation were tested in a pig model. Chen et al. [ 143] directed tPA-loaded silica-coated nanoparticles containing a superparamagnetic iron core to clots under magnetic guidance. They demonstrated biocompatibility and thrombolytic efficacy in vitro. Likewise, Yang et al. [ 144] demonstrated that a similar formulation under magnetic guidance restored blood flow in a rat embolism model without triggering hematologic toxicity. Others showed that such superparamagnetic nanoparticles were capable of encapsulating clinically effective doses of tPA [ 145] and uPA [ 146]. Vaidya et al. have reviewed magnetically targeted thrombolytic formulations [ 147].

Formulations targeted to other markers of vascular injury and thrombosis

Platelet targeting of uPA was also accomplished by conjugation of an antibody specific for thrombospondin, a platelet a-granule glycoprotein expressed on thrombin-activated platelets, directly to the thrombolytic [ 148]. Reasoning that co-administration of liposomes targeted to damaged endothelium with tPA would reduce hemorrhagic side-effects, Asahi et al. [ 149] conjugated an antibody specific for actin, which would be exposed by vascular injury. They demonstrated in a rat embolic stroke model that this approach reduced average intracerebral hemorrhage (ICH) volume by nearly half compared to tPA+ nonspecifically targeted liposomes. In a related approach, Underwood et al. [ 150] conjugated a monoclonal antibody raised to damaged endothelial cells directly to uPA for prevention of thrombotic occlusion in saphenous vein grafts used for coronary artery bypass procedures. They demonstrated in vitro efficacy by clot mass loss assessment.

Maksimenko et al. [ 151] altered thrombin with water-soluble carbodiimide (1-ethyl-3(3′-dimethyl aminopropyl) carbodiimide, EDC) to inactivate its fibrinogen-cleaving activity without affecting its clot binding properties. They then conjugated the modified thrombin directly to uPA to produce a clot-targeted thrombolytic formulation. In an arterial thrombotic dog model, these investigators demonstrated that their construct restored blood flow in 80% (4/5) of affected animals, while uPA alone showed no efficacy. In a truly novel approach, Murciano et al. [ 152] conjugated soluble urokinase receptors (suPAR) to red blood cells and allowed the formulation to bind endogenous scuPA, forming a long-circulating surveillant system for thrombotic prophylaxis, since the thrombolytic-bearing RBC would be incorporated into nascent clots.

A thrombin-targeted thrombolytic was constructed by conjugating hirudin directly to SAK [ 153]. A 12-fold enhancement of SAK lysis of thrombin-rich clots by the construct was observed in vitro. Pulmonary emboli were targeted by coupling a monoclonal antibody specific for angiotensin converting enzyme to uPA, tPA and sPA via a biotin-avidin linkage [ 154]. After i.v. injection in rats, localization of the formulations in lung tissue was 6‒20 times that of nonspecifically targeted controls. Preferential inflammatory lung uptake of anti-ICAM-1directly coupled to tPA was observed in rats [ 155].

Fibrin-targeted thrombolytic formulations

Fibrin-targeted thrombolytic formulations thus far reported are summarized in Table 1. The targeting agents used for this purpose fall into two general categories: antibodies and their derivatives, and endogenous ligands.

Antibody targeted formulations

The most obvious targeting strategy for therapeutic formulations is conjugation of monoclonal antibodies directly to the therapeutic agent or to vehicles carrying the agent. In addition, anti-fibrin Ab fragments (e.g., Fab', scFv) have been employed for non-thrombolytic targeting applications [ 156, 157]. It becomes evident from Table 1 that most antibody-targeted fibrin-specific thrombolytic formulations are direct protein conjugates (including Fv fusion proteins) and that nearly all of them were developed before 2000. Why has this approach not continued since then?

The most likely explanation is advances in recombinant tPA (alteplase) engineering, summarized in the tPA section on “Next-generation recombinant thrombolytics.” Tissue plasminogen activator already possesses two fibrin binding sites, one of which (in the finger domain) has an affinity comparable to clinically useful high-affinity antibodies (See the tPA section on “Biochemical properties”). The targeting advantages of retaining both sites are illustrated by the case of TNKase. Third generation alteplase constructs are well-established in the clinical market and there seems to be little advantage in developing antibody-thrombolytic conjugates.

Since 2000, a number of anti-fibrin Ab-targeted thrombolytic carrier formulations have been developed. Most of these were nanoparticulate formulations [ 158– 160]. In addition, the same group that created the camouflaged tPA prodrug construct (See the non-targeted formulations section on “Other tPA formulations”) produced a fibrin-targeted version that they termed ATTEMPTS (Antibody-Targeted Triggered Electrically Modified Prodrug Type Strategy) [ 169, 170]. In this case, heparin-conjugated anti-fibrin MAb was associated with tPA coupled to a cationic peptide or, in a later refinement, tPA engineered to be cationically charged. After binding of the complex to fibrin, the heparin-Ab was displaced with protamine, restoring full tPA catalytic activity. Proof-of-concept was demonstrated in vitro.

Endogenous effector targeted formulations

A novel fibrin-targeted thrombolytic formulation was provided by the discovery that the fibrin binding sites of tPA loaded into echogenic liposomes, remained exposed at the liposomal surface, rendering the formulation, termed TELIP, intrinsically fibrin targeted [ 47], which was also confirmed in vivo (Fig. 2). Subsequently, these investigators determined that about one-third of ELIP-loaded tPA was loosely associated on the liposomal surface, while another third was tightly associated, presumably traversing the lipid bilayer, with fibrin binding and catalytic sites exposed [ 185]. Transmission electron microscopic images of the TELIP formulation were consistent with these data, showing aggregates of multilamellar echogenic liposomes averaging approximately 600 nm in diameter (Fig. 3).

Using an in vitro clot mass loss model, Tiukinhoy-Laing et al. [ 178] demonstrated that 2 min of mechanical ultrasound (1 MHz, 2 W/cm²) enhanced TELIP thrombolytic activity, resulting in greater thrombolysis than free tPA or insonification of free tPA in the presence of an ultrasound contrast agent (Optison^®). Subsequently, it was shown that clinically relevant Doppler ultrasound was as effective as mechanical ultrasound in enhancing thrombolysis, presumably through stable cavitation effects [ 186]. This was confirmed in another study relying on colorimetric assay of tPA enzyme activity [ 187]. A preliminary in vivo study using a rabbit aorta clot model demonstrated that Doppler ultrasound enhanced TELIP thrombolysis significantly in 15 min, achieving complete recanalization in all animals treated [ 179]. A follow-up study using this model showed that TELIP had the thrombolytic efficacy of locally delivered rtPA [ 188].

Attempts were made to produce endogenously fibrin-targeted thrombolytic formulations by directly conjugating plasminogen, which possesses two Kringle fibrin binding domains analogous to the low-affinity binding site of tPA, to thrombolytic catalytic domains. Robbins et al. conjugated the fibrin binding domains of plasminogen to the catalytic domain of uPA [ 181] and to the catalytic B-chain of tPA [ 182] via a disulfide linkage, providing constructs with 1–2× fibrinolytic activity of the unconjugated thrombolytic. An anisoylated plasminogen-streptokinase activator complex (APSAC) was tested clinically on 1004 STEMI patients in the AIMS trial [ 180]. Thirty-day survival in the treated group was reduced from 12.2% in placebo controls to 6.4%. These results were nearly identical to those found for alteplase (See the tPA section on “Recombinant tPA (alteplase)”). Apparently, the ensuing dominance achieved by alteplase for this application precluded further clinical use of the APSAC formulation.

As previously mentioned (Non-targeted formulations section on “Liposomal formulations of tPA”), Heeremans et al. conjugated glu-plasminogen to tPA-loaded liposomes via a thioether linkage and found an improvement in thrombolytic efficacyvivo relative to free tPA [ 101, 102, 189]. The fibrin targeting, however, may have been partly or entirely due to exposed tPA fibrin binding sites, rather than the plasminogen fibrin binding sites.

In an approach analogous to Murciano’s use of an erythrocyte-associated thrombolytic to inhibit formation of developing thrombi, described in the non-fibrin targeted formulations section on “Formulations targeted to other markers of vascular injury and thrombosis,” Maksimenko et al. conjugated tcuPA to fibrinogen, reasoning that the fibrinogen would be converted to fibrin as part of clot formation [ 183, 184]. Incorporation of the formulation into the clot yielded an overall 2.5-fold enhancement of thrombolytic efficiency relative to the unconjugated thrombolytic. In a dog femoral arterial thrombus model, the fgn-tcuPA preparation produced complete recanalization in 4 of 6 animals, and extensive recanalization in the rest, while no thrombolysis was observed with tcuPA alone. Administration of the formulation to rabbits resulted in less systemic depletion of fibrinogen than unconjugated tcuPA.

Conclusions

Two major drawbacks have severely limited the clinical usefulness of plasminogen activator thrombolytics for treatment of thrombotic disorders, especially myocardial infarction (MI), ischemic stroke, peripheral artery disease and deep venous thrombosis: devastating, often fatal intracranial hemorrhage (ICH) and refractory clots that resist dissolution. Percutaneous intervention (PCI) has eclipsed thrombolytic therapy for treatment of MI and analogous methods are being developed for treatment of ischemic stroke. Stent restenosis and neoatherosclerosis, requiring lifelong antithrombotic therapy, are important complications of PCI [ 190– 194], however, indicating a continuing need for thrombolytic agents in the treatment of these disorders.

Novel formulations for delivery of thrombolytics should therefore focus on ameliorating the drawbacks of ICH and refractory thrombi. Next-generation recombinant alteplase does not offer a solution to ICH and in fact may exacerbate it by increasing the circulation time and, in the case of TNK, increasing the affinity for fibrin, since the cross-reactivity with fibrinogen causes systemic fibrinogen depletion that impairs coagulation and fosters bleeding [ 195]. Counterintuitively, reteplase has shown improved thrombolytic efficacy relative to TNK because of its lower affinity for fibrin, enabling it to dissociate and re-attach to the clot as it dissolves [ 90, 91].

Novel thrombolytic formulations most likely to solve these problems should be encapsulated in a carrier or otherwise should be shielded from the circulation to prevent systemic thrombolytic effects and they should be targeted to thrombi to expand the pharmacologic window. An added advantage of the latter strategy is the ability to deliver a relatively low effective dose of the thrombolytic, well below the hemorrhagic level. With this in mind, formulations targeted to GPIIb-IIIa with RGD peptides are likely to exhibit relatively low affinities, limiting the clinical efficacy and are likely to cross-react with other integrins such as a_vb₃. Of the non-fibrin clot-targeted formulations, the most promising may be those targeted to damaged endothelium, but there seems to have been no recent developments in this direction.

Of the fibrin-targeted carrier-encapsulated thrombolytic formulations, the most promising recent developments appear to involve intrinsically-targeted tPA in ultrasound-enhanced liposomal formulations, offering added efficacy toward refractory thrombi provided by ultrasound insonation. All encapsulated formulations currently under development are still experimental, with most data thus far resulting from in vitro studies. For the most part, these studies have mainly established retention of native thrombolytic efficacy, with potentiation by delivery modalities, especially ultrasound. Largely unaddressed are in vivo efficacy under clinically pertinent conditions, pharmocokinetics, adverse effects and safety issues, all of which would be the subject of pre-clinical studies and exploratory clinical trials. Hopefully, these kinds of studies are forthcoming for the more promising formulations.

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