Chapter 12. Plasminogen Activators

Chapter 12. Plasmlnogen Actlvators Michael J. Ross, Elliott B. Grossbard, Adair Hotchkiss, Deborah Higgins and Stephen Anderson Genentech, Inc.. S . San Francisco, CA

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h t r o d u c t i o n In addition to its uses in deep vein thrombosis, pulmonary embolism, and peripheral arterial occlusion, thrombolytic therapy is rapidly becoming the treatment of choice for acute myocardial infarction (MI) when diagnosed early. Three thrombolytic agents, streptokinase, urokinase and alteplase (tissue plasminogen activator, recombinant) are approved in the U S . and in addition a fourth (APSAC) in Europe. Many new thrombolytic agents are currently in preclinical and clinical development. The agents are large proteins and all work by activating the plasma zymogen plasminogen to plasmin which cleaves fibrin. Streptokinase is a bacterial protein, urokinase and alteplase are human proteins and APSAC is a chemically modified complex of streptokinase and plasminogen. Specific plasma and platelet borne inhibitors have been identified which are thought to modulate the activity of these drugs i n y h Alteplase binds fibrin directly and streptokinase and APSAC bind to fibrin indirectly through plasminogen. However, only alteplase is dramatically activated by fibrin and alteplase degrades comparatively less fibrinogen in yiva compared to urokinase, streptokinase or APSAC.

CLINICAL AND PRECL INICAL RESULTS

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First Generation Plasminoaen ActviUse in Mvocardial I n f a r W A number of reviews have dealt with the large number of studies of streptokinase and urokinase in preclinical animal models(l,2). Treatment of patients with intravenous streptokinase following acute myocardial infarction has been shown to reduce mortality, (3-5)reduce infarct size (6,7), and improve left ventricular function (4) even though intravenous streptokinase has been shown to be relatively ineffective in inducing prompt coronary artery thrombolysis (8,9). Early presentation has been shown to be critical to achieve a greater mortality benefit (3). Data on long-term effects are conflicting ( 1 0 , l l ) but favor sustained benefit. There are little or no data to date regarding the efficacy of intracoronary or intravenous urokinase beyond coronary thrombolysis ( I 2,13), i.e., no controlled trial showing reduced infarct size, improved ventricular function or reduced mortality. One study showed reduced left ventricular end diastolic volume, an important prognostic variable, following intracoronary urokinase (14).

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Second Generation Plasminogm A c t i v w : P-r - Preclinical data have shown tissue plasminogen activator (alteplase) and anisoylated plasminogen streptokinase activator complex (APSAC) to be effective thrombolytic agents in y&Q, and in animal models of myocardial infarction (15). Single chain urokinase (scu-PA, prourokinase) synthesized in mammalian cells has been shown to be an effective thrombolytic in a rabbit has been shown to reopen model (16,17). Nonglycosylated scu-PA made in .EL occluded coronary arteries in baboons and dogs with little loss of fibrinogen (18,19), whereas urokinase was shown to have similar efficacy but caused almost complete defibrinogenation (18). Polyethylene glycol modification has been shown to extend the half life of urokinase in dogs with an increase in fibrinogen consumption over urokinase

(20). ANNUAL REPORTS IN MEDICINAL CHEMISTRY -23

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Copyright C? 1988 hy Academic PRSS, Inc. All rights of reproduction m any form reserved.

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Second (3mxaUm Plasmmgm Activators: T h e i r 1 Ilse in Mvowdial Infarction Early trials with a tissue plasminogen activator utilized a two-chain molecule produced in tissue culture by a pilot scale roller bottle process (9,21-23). Subsequently, a predominantly single-chain molecule called alteplase, produced by a commercial scale suspension culture process, was introduced into clinical trials (24,25). The latter molecule is cleared from the circulation more quickly (26), and the dose had to be adjusted upward (from 80 to 100 mg) (25). Fibrinogen sparing was noted in all trials (21-28) and was present with both roller bottle (21-23,26-28) and suspension culture molecules (24-26). Studies at 150 mg revealed more rapid coronary thrombolysis (25), but an increased risk of toxicity from intracranial bleeding as well (29,30). Studies at 100 mg have shown that early treatment with alteplase can reduce infarct size (31), improve left ventricular function (31-34), reduce the incidence of congestive heart failure (321, and reduce mortality (31). Studies involving treatment of patients up to six hours after onset of infarction showed cornparabte thrornbolytic benefit when alteplase was used to treat patients with early or late presentation (9). Three additional multicenter trials have shown that following coronary thrombolysis with alteplase emergency coronary angioplasty need not be performed, and indeed may be deleterious (35,36). Trials focusing on coronary thrombolysis have shown APSAC to be comparable to streptokinase with regard to overall ability to lyse thrombi in coronary arteries (37,38). A recently reported controlled study with APSAC showed improved ventricular function (39), but a second study failed to confirm this (40). Clear evidence of reduced mortality has been observed (41,42) in APSAC treated patients. Though predicted to be fibrinogen sparing in preclinical models (43), APSAC has not proven to be fibrinogen sparing in clinical evaluation (44-46). Scu-PA has been studied in only a small number of patients (47,48). There are little data on coronary thrombolysis and none on ventricular function or mortality. Fibrinogen sparing has been observed in most but not all patients (47,48). Some synergy between scu-PA and alteplase has been reported in a very small number of patients (49).

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- Studies in en Activ-s in l-ns Other Than MI: Preseveral models of cerebral thromboembolic occlusion have demonstrated a potential role for thrombolytic agents for this indication. Rabbit (50),baboon (51), and dog (52-54) models have responded to local administration of urokinase. Rabbit (55) and rat (56) models have responded to systemic alteplase administration. Alteplase has been shown to have a positive effect in a non-thrombotic model of myocardial ischemia (57); the effect was ascribed to its lytic effect on non-imageable microthrombi. This was recently contradicted by a second study (58). Models of retinal vein thrombosis in the cat (59) and posterior penetrating eye injuries in the rabbit (60) have also been used to test the efficacy of thrombolysis with urokinase. lntraoccular administration of urokinase was effective in opening occluded retinal veins (60). Alteplase was shown to be effective in reversing eye damage when introduced intraocularly in rabbit models (61-63). The opening of thrombosed arterial grafts has been studied in dogs using alteplase or urokinase alone (64) or in combination Streptokinase was with aspirin (65), and alone in limb reimplantations in rats (66). shown to decrease tissue damage following experimental frostbite in rabbits (67).

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Use of P-n I Activ-s in W n s Other UMI MI: C l i n w l - Much recent activity in thrombolytic clinical research has focused on alteplase. An sngiographicallycontrolled multicenter trial showed excellent efficacy in pulmonary embolism (68,69), which was confirmed by a European study (70) and alteplase was shown to be superior to urokinase in a third trial (71). Favorable results were reported in a recently completed trial with urokinase in massive pulmonary embolism (72). Initial efficacy with APSAC in major pulmonary embolism has also been reported (73). A placebo-controlled pilot

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Plasminogen Activators Ross, Grossbard, Hotchkiss, Higgins, Anderson __ 113

study of alteplase in unstable angina showed a favorable angiographic and clinical effect (74); a second placebo-controlfed study showed a beneficial effect on pacing threshold (75). Preliminary data suggest that alteplase will be effective in peripheral arterial occlusion (76,77) and stroke (78,79). Valvular (80,81) and ventricular thrombi (82) have also been successfully treated with alteplase (81) or urokinase (80,82).

PHARMACOKINFTICS AND PHAFiMACODYNAMGS Clearance Mechanisms - The principal clearance mechanism for alteplase appears to reside in the liver (83,84), possibly in hepatocytes which have been shown to have the ability to take up and degrade alteplase (85). The A chain (amino acids 1-275) and B chain (amino acids 276-527) of alteplase have been separated by limited reduction and the patterns of elimination were compared in rats (86). The half lives of intact alteplase and the A and B chains were 2.3, 1.0 and 5.7 min., respectively, suggesting that the clearance domain might reside in the A chain. The presence of a high mannose oligosaccharide at residue 117 appears to contribute to the rapid clearance of alteplase (87,88). Urokinase has been shown to bind to a number of cell types and enhance plasmin and plasminogen binding to these cells (89,90). The amino terminal region of urokinase has been implicated in this cell binding activity (91,92). Alteplase has been shown to bind tightly to endothelial cells (93-95). The bound alteplase was shown to be protected from inhibition by the fast acting plasminogen activator inhibitor (PAI-1) and was still able to activate plasminogen (94). Though displaced by added urokinase, alteplase bound to endothelial cells was not effectively displaced by fibronectin, epidermal growth factor or plasminogen, all of which have known receptors on endothelial cells (94). Alternative OrUg Delivery - The intramuscular administration of alteplase with the use of absorption enhancers has been shown to result in significant plasma concentrations (96,97). These concentrations also caused thrombolysis in a dog coronary artery model with continuing release into the circulation for six hours (98).

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of Pl-oaen A c t i v m - PAL1 from endothelial cells and platelets is normally present in plasma at pharmacologically insignificant concentrations. When induced by endotoxin,PAl-1 completely inhibited a Yiya measurements of alteplase; however, in thrombolysis was not affected (98,99). Reactivation of the complex during circulation was postulated to explain the results (99). Though local concentrations of PAL1 in the vicinity of a thrombus may play a role in lysis rate or rethrombosis, in animals with normal plasma concentrations of fast acting inhibitor, there appears to be little complex accumulation during treatment with pharmacologic doses of alteplase (99).

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NEW THROMBOLYTlC AGENTS

- ALTEPLASE AND UROKINASE ANALOGS

D e l e W - Alteplase, 1,is a complex multidomain protein (100,101). At the Nterminus there are two domains: a so-called "finger" region, homologous to the finger domains of fibronectin (102), and a "growth factor" region, homologous to epidermal growth factor (103). A pair of "kringle" structures, homologous to similar motifs in urokinase, plasminogen, prothrombin, and Factor XI1 comprise the central two domains (104), and the C-terminus of the molecule consists of a trypsin-like serine protease (100,105). Altepfase has at least two functionally important (and clinically relevant) biochemical properties, namely, fibrin binding and fibrin-stimulated proteolysis of the Arg561Val562 peptide bond of plasminogen. Several groups have carried out deletion mutagenesis studies, producing and characterizing alteplase analogs lacking one or more domains (106-112). The aim of this work was to assign functional properties to these regions and thereby address the problem of how the five domains of alteplase act in

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concert to produce its unique properties. Experiments employing convenient natural restriction sites to construct deletion analogs showed that kringle two and the finger were the two domains most responsible for fibrin binding. The same studies also implicated kringle two as the domain responsible for fibrin stimulation and lysine binding (106108). Other studies, in which newly-introduced interdomain restriction sites (109) or precise oligonucleotide-directed deletions were used (1 10-1 12), have confirmed these results. Deletion mutagenesis studies have also shown that an alteplase variant lacking the two kringle domains is less reactive with PAL1 (110). Finally, it should be noted that a natural, finger-deleted variant of alteplase exists, produced from the wild type gene via alternative splicing in Detroit 562 cells (1 13). The physiological significance of is unknown. such splicing variants, or whether they even exist h.yjy~,

-1 - Alteplase contains four potential N-linked glycosylation sites at asparagines 117, 184, 218, and 448 (103,114). Asparagine 218 is not glycosylated, presumably because proline 219 blocks utilization of this site. Each of the three remaining sites exhibits a different pattern of glycosylation. At position 117 all molecules are modified with a "simple" high mannose-type sugar (115,l 16). At position 184 only approximately half of the alteplase molecules are glycosylated, but when this has occurred it is with a ''complex"-type of carbohydrate (114-116). The site at asparagine 448 is fully glycosylated with a complex sugar (115,116). Several groups have attempted to modify the properties of alteplase by removing one or more of the glycosylation sites, usually by substituting glutamine for the asparagine. Alternatively, glycosidases such as Endo H (which cleaves high mannose type sugars) or N-glycanase have been used to modify the alteplase glycosylation pattern enzymatically, or biosynthesis in the presence of tunicamycin has been used to suppress N-linked glycosylation completely (1 17,118). These experiments have shown that removal of the sugar moiety at residue 117 or 448 changes the pharmacokinetic properties of alteplase, but does not otherwise affect its biochemical behavior (87,88,119). The absence of glycosylation at 184 is correlated with increased in yitrp clot lysis activity (111,120,121), but the presence or absence of a sugar at this position does not seem to affect pharmacokinetics. AnalqgS - Almost all trypsin-like serine proteases are synthesized as inactive zymogen precursors. These molecules are converted to catalytically active enzymes by an obligatory proteolytic cleavage that causes a conformational change

Chap. 12 Plasminogen Activators Ross, Grossbard, Hotchkiss, Higgins, Anderson

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(involving the new N-terminus) and a resultant ordering of the active site residues (122). Alteplase is unique among this family of serine proteases in that it does nat require a proteolytic cleavage to activate the zymogen form. Rather, alteplase can be converted to the fully active conformation upon binding to fibrin, without any accompanying covalent modification. Alteplase contains an arginine at residue 275, a position analogous to the activation clip site of trypsinogen, and the Arg275-Ile276 peptide bond in alteplase is readily cleaved by serine proteases such as plasmin that have trypsin-like specificity. However, uncleaved ("one-chain") alteplase was found to have full plasminogen activating activity in the presence of fibrin (123,124). An analog of alteplase, Glu275, was made which was intrinsically resistant to cleavage by plasmin. [Glu275]alteplase also exhibited full specific activity in the presence of fibrin (124). When assayed in the absence of fibrin, one-chain alteplase was observed to have approximately 20 times less plasminogen activating activity than two-chain alteplase (124). A similar difference in activity between one- and two-chain urokinase, a fibrin-independent plasminogen activator, has been reported to be due to primarily a difference in turnover number (kcat) (125). Other position 275 alteplase analogs have been reported: Gly275 (126); Asp275 (127); His275 Lys275, Thr275 (128); and all 19 possible substitutions (129). Two groups reported that Lys 275 alteplase was plasmin sensitive (128,129); however there is disagreement as to whether the His275 analog is plasmin resistant (129) or plasmin sensitive (128). All other position 275 analogs exhibited properties virtually identical to the prototypical Glu275 analog except Cys275, which had significantly lower specific activity (129). This reduced activity may have been due to improper protein disulfides (i.e., misfolding) or mixed disulfide formation resulting from the extra protein thiol introduced by the Cys275 mutation. Several other properties of Glu275 alteplase are also noteworthy: increased binding to fibrin, relative to two-chain alteplase (124,130); increased sensitivity to plasmin cleavage at Lys277 (131); decreased plasma clearance rate (132).

- Position 277, a lysine residue of alteplase, has been mutated to isoleucine (133,134). The resultant alteplase analogs do not appear to behave differently from the parent molecule. Glu275 alteplase is more sensitive to plasmin inactivation than is alteplase, but the Glu275, He277 double analog is partially protected from plasmin inactivation, implicating position 277 in the inactivation (131). This may play a role in the regulation of alteplase activity in as has been proposed for the thrombincatalyzed inactivation of scu-PA via a clip at Arg156 (135). Deletion of the three Cterminal amino acid residues Met525 Arg526 Pro527 leads to an alteplase analog with significantly increased specific activity (1 20).

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A limited number of single-chain urokinase, 2,analogs have also been made. Lys158 was replaced with Glu or Gly and it was shown that these single-chain urokinase analogs, which could not be converted to the two chain form by plasmin, had a significantly lower specific activity in a plasminogen activating assay (136). Other analogs, in which the Arg 156 and Lys 158 were replaced by Thr, showed similar properties (137).

se - m s e H v W - Hybrid molecules between proteolytic domains from plasminogen activators and amino terminal domains from proteins which bind to fibrin (e.9. plasminogen, alteplase) or non-binding proteins (e.g. urokinase) have been used to further dissect the role of the various domains. Several distinct apprcaches have been used to produce these hybrid molecules, Limited reduction and affinity chromatography (138) have been used to isolate the amino terminal domains of plasmin and the protease domain of urokinase. These domains were then mixed and reoxidized and a hybrid was isolated containing Lys-78 to Arg-561 of plasmin disulfide-bonded to Ile-159 to Leu41 1 of urokinase. The amidolytic activity of the hybrid was 1/2 of that of the high

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KRINGLE

-2 molecular weight form of urokinase on a IU/mg basis. The hybrid did bind to fibrin and also exhibited a 5-fold enhancement of plasminogen activating activity in the presence of soluble fibrin. A Similar approach was taken to prepare a hybrid containing the plasminogen amino terminal and the alteplase protease domain (139). This hybrid had about 40% of the amidolytic activity of alteplase. The plasminogen activating activity was difficult to quantify due to non-parallel dose-response curves, but seemed to be slightly less than that for intact alteplase. The hybrid bound fibrin, but not as well as alteplase. Chimeric proteins have also been made by recombinant DNA techniques. The similarity in the domains of alteplase and urokinase has led several groups to insert domains from one plasrninogen activator into the other protein with or without corresponding deletions. A chimeric protein containing Ser-1 to Thr-263 from alteplase attached to Leu-144 to Leu-411 of urokinase was constructed by fusing the appropriate coding sequences from the cDNAs for these proteins (140,141). The single chain form of the molecule was expressed and characterized. Similar to scu-PA, this molecule had little amidolytic activity until conversion to a two-chain form by plasmin. Both the singlechain and two-chain forms of the protein bound to fibrin to a limited degree and their plasminogen activating activity was enhanced by fibrin peptides. Both forms were inferior to alteplase in both fibrin binding and fibrin stimulation, suggesting the importance of interdomain communication in these functions. The hybrid had decreased inhibition by PAI-1 when compared to alteplase or urokinase (141). A series of alteplase/urokinase chimeras, all of which contain the urokinase protease domain, have been produced (137). All recombinant activators which contained the 'activation' site at Lys-158 in urokinase and could be converted to the two-chain form were active when expressed in mammalian cells. Further analysis (142) of the protein containing residues Ser-1 to Tyr-67 of alteplase attached to Lys-135 to Leu-411 of scu-PA demonstrated that the protein was similar to scu-PA in that it did not bind fibrin. In a plasminogen activating assay the catalytic constants were quite similar although scuPA exhibited a Michaelis constant 3-fold lower than that of the hybrd. However, the presence of a cysteine residue at position 366 (which is not present in the natural protein) as well as an "unpaired" cysteine at position 50 could lead to non-native disulfide bond formation and misfolding of some of the hybrid molecules. Further studies will be necessary to resolve this possibility.

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When the growth factor and kringle domains of urokinase were inserted either before kringle one or after kringle two of full-length alteplase, or used to replace the finger and growth factor domains of alteplase (143), it was found that all proteins had similar amidolytic activity. All three hybrid proteins had somewhat reduced fibrin binding activity, and the plasminogen activating activity was not stimulated to the same degree as with alteplase. A comparison of the three hybrids suggested that the finger/growth factor regions were important in fibrin binding and in fibrin stimulation. The insertion of the urokinase domains after kringle two of alteplase also interfered with fibrin binding and stimulation, but to a lesser extent than removal of the alteplase finger and growth factor domains and to a greater extent than insertion of the urokinase domains before kringle one of alteplase. n--P l alternative manner in which to alter the odv -A characteristics of a plasminogen activator is to covalently couple the activator to antibodies of known specificity. Both anti-fibrinogen antibodies (144) and monoclonal antibodies raised against the amino-terminal residues of the beta chain of fibrin (145) have been chemically cross-linked to urokinase. The conjugates retained amidolytic activity. When the antibody conjugates were compared to urokinase at an equivalent amidolytic activity, a significantly lower amount of antibody conjugate was required to produce an equivalent amount of plasmin as measured in an 1251-fibrin release assay. The same result was obtained with a Fab' fragment - urokinase conjugate. The beta chain amino-terminal peptide completely abolished the enhancement of activity (146). In analogous experiments using alteplase, the conjugate was 2-10 fold more effective than alteplase alone if equal amidolytic activities were infused in an in vivq rabbit thrombolysis model (147). Covalent linkage of alteplase to the beta chain fibrin specific antibody has also been accomplished via recombinant DNA technology. (148) The cDNA for the heavy chain from the beta specific monoclonal antibody was cloned and linked to the coding sequences for the serine protease domain of alteplase. The chimeric gene was expressed in a myeloma cell line which had lost the ability to produce the heavy chain, but still retained expression of the light chain of the antibody. The recombinant protein was purified by affinity chromatography, and presumably contained the correct association of two light chains and two recombinant heavy chains as judged by its molecular weight. The recombinant alteplase-antibody hybrid bound fibrin, although less well than the antibody alone, and retained amidolytic and plasminogen activating activity although to a lesser extent than in free alteplase.

SUMMARY Far broader use of thrombolytic therapy in myocardial infarction as well as other indications should be expected in the near future as the recently published clinical data becomes widely appreciated. The probable increase in the use of this class of agents will inevitably lead to a number of new agents under investigation in the clinic. The next generation of thrombolytic agents may have greater efficacy and potentially a wider therapeutic ratio than the recently marketed second generation agents. References

1. 2. 3. 4.

5.

6. 7. 8.

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Section I1 - Cardiopulmonary and Vascular Agents

Bristol, Ed.

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