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Journal of Cardiovascular Pharmacology:
doi: 10.1097/FJC.0b013e318180188b
Original Article

Serine Protease Inhibitor Nafamostat Given Before Reperfusion Reduces Inflammatory Myocardial Injury by Complement and Neutrophil Inhibition

Schwertz, Hansjörg MD*; Carter, Justin M MRCP*; Russ, Martin MD*; Schubert, Sebastian*; Schlitt, Axel MD*; Buerke, Ute MD*; Schmidt, Martin PhD*; Hillen, Heinz PhD†; Werdan, Karl MD*; Buerke, Michael MD*

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Author Information

From the *Department of Medicine III, Martin-Luther-University, Halle, Germany; and †Abbott Pharmaceuticals, Ludwigshafen, Germany.

Received for publication August 31, 2007; accepted April 4, 2008

Hansjörg Schwertz and Justin M. Carter contributed equally to the completed work.

This study was supported by Grant Bu 819/3-1 of the Deutsche Forschungsgemeinschaft. Ute Buerke is supported by a state grant of Saxony-Anhalt. Justin M. Carter is supported by the South Cleveland Heart Fund.

Reprints: Michael Buerke, MD, Department of Medicine III, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Str. 40,06120 Halle, Germany (e-mail: michael.buerke@medizin.uni-halle.de).

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Abstract

Animal data strongly support a role for inflammation in myocardial ischemia reperfusion injury. Attempts at cardioprotection by immunomodulation (such as with the specific C5 antibody pexelizumab) in humans have been disappointing. We hypothesized that a broader spectrum antiinflammatory agent might yield successful cardioprotection. The serine protease inhibitor nafamostat (FUT-175), which is already in clinical use, is a potent antiinflammatory synthetic serine protease inhibitor with anticomplement activity that we tested in a well-established rabbit model of 1 hour of myocardial ischemia followed by 3 hours of reperfusion. Compared to vehicle-treated animals, nafamostat (1 mg/kg of body weight) administered 5 minutes before reperfusion significantly reduced myocardial injury assessed by plasma creatine kinase activity (38.1 ± 6.0 versus 57.9 ± 3.7I U/g protein; P < 0.05) and myocardial necrosis (23.6 ± 3.1% versus 35.7 ± 1.0%; P < 0.05) as well as myocardial leukocyte accumulation (P < 0.05). In parallel in vitro studies, Nafamostat was a significantly more potent broad spectrum complement suppressor than C1 inhibitor. Nafamostat appears to have capability as an inhibitor of both complement pathways and as a broad-spectrum antiinflammatory agent by virtue of its serine protease inhibition. Administration of nafamostat before myocardial reperfusion after ischemia produced significant, dose-dependant cardioprotection. Reduced leukocyte accumulation and complement activity seem involved in the mechanism of this cardioprotective effect.

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INTRODUCTION

Many mechanisms of tissue injury are thought to underlie ischemia reperfusion injury. These mechanisms bear a striking resemblance between species and also between tissue types within species groups. Many diseases and therapies produce or treat ischemia reperfusion (in a variety of tissue types); therefore, this pathophysiological situation is common. Among the many potential mediators of ischemia reperfusion injury, it seems clear that this process entrains a sequence of proinflammatory events that generate local and or systemic inflammatory activation that contributes to the eventual injury. With respect to myocardial ischemia (MI) reperfusion injury, there is substantial evidence that inflammatory activation contributes to myocardial injury.1 There are also convincing animal model data to show that suppressing the inflammatory response after MI reperfusion injury leads to a reduction in myocardial damage.2 Even in humans, data suggest that inflammatory modulation therapy after ischemia may improve outcomes,3 but the clinical evidence to date has been largely disappointing.

The process of inflammatory activation after MI is complex and involves multiple cascades undergoing sometimes parallel activation. These cascades involve changes in electrolyte flux (such as calcium overload), coagulation activation, thrombin generation and protease activated receptor (PAR) stimulation, cytokine release, and adhesion molecule expression followed by leukocyte activation and tissue infiltration and then degranulation involving the release of cytotoxic mediators like hydrogen peroxide (H2O2), superoxide anion (O), hydroxyl radical (OH), cathepsin G, and elastase, and finally, the aggressive activation of complement.1

Animal data suggest that the contribution of complement activation to this inflammatory response is important.4 Over the past 10 years, studies have shown that each of the 3 complement pathways has a role in MI reperfusion injury. The classical complement pathway is activated by sensitizing antibodies, subcellular fragments (eg, cardiac mitochondrial particles exposed after cell breakdown) and components of the fibrinolytic system such as activated plasmin. Activation of the alternative pathway is based on contact activation by non-host cells or surfaces without a sialic acid or other polyanionic coating and may predominate in complement activation after cardiopulmonary bypass.5 Activation of classical, alternative, or mannose binding6 complement cascades leads to sequential activation of complement factors (many with a serine protease like enzymatic active center) and finally to tissue injury by formation of the membrane attack complex (MAC). MAC accumulates rapidly in ischemic myocardium during reperfusion7 and binds cellular surfaces, causing pore formation and calcium influx and resulting in cell activation or cell swelling and eventually cell lysis.

On the basis of promising animal and preliminary human data, the inhibition of complement using the specific C5a monoclonal antibody pexelizumab was the focus of the largest series trials involving inflammatory modulation for therapy of MI reperfusion. Results from patients undergoing cardiothoracic surgery involving cardiopulmonary bypass were modestly encouraging8 but results from more than 5000 patients with myocardial infarction were resoundingly negative.9 These data have left a question mark over the role of inflammatory modulation therapy in patients with MI reperfusion injury. However, on the basis that inflammatory activation after MI reperfusion involves activation of multiple pro-inflammatory cascades (not only the complement system), we sought to assess the cardioprotective potential of a compound that not only has potent anticomplement activity (against both major complement pathways) but also has recognized broad spectrum antiinflammatory effects directed at a number of other points in the inflammatory cascade.

Nafamostat is an attractive compound in this context. It is a synthetic, small compound serine protease inhibitor already in clinical use (predominantly in Japan) as an antiinflammatory agent. It has been examined in a variety of proinflammatory conditions such as pancreatitis,10 pro-coagulant states,11 cerebral ischemia,12 and haemorrhagic shock.13 It combines potent inhibition of both major limbs of the complement cascade; in addition, like aprotinin (another protease inhibitor), it has an increasingly recognized broad-spectrum antiinflammatory capacity in reducing leukocyte activation, platelet aggregation, and thrombin / PAR activity.14-17

On this background, we examined the cardioprotective effects of the synthetic serine protease inhibitor nafamostat mesilate (FUT-175, 6-amidino-2-naphthyl-p-guanidinobenzoate dimethanesulfonat; Torii Pharmaceutical, Japan) in a well-established rabbit model of MI and reperfusion. In addition, we compared the complement inhibition characteristics of nafamostat with those of the recognized endogenous classical complement pathway inhibitor, C1 esterase inhibitor (C1-INH, Berinert; Centeon, Germany). Thus we undertook the first study to examine combined complement pathway and broad-spectrum antiinflammatory, serine protease-based cardioprotection in an in vivo model.

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METHODS

Experimental Protocol

Full details are published elsewhere18; briefly, adult male rabbits were anesthetized with sodium pentobarbital and placed on artificial ventilation while receiving complete haemodynamic and electrocardiographic monitoring, including the pressure-rate index (PRI), an approximation of myocardial oxygen demand. A thoracotomy was performed to allow access to the left anterior descending (LAD) coronary artery. MI was induced by tightening the reversible LAD ligature so that the vessel was occluded (time-point 0). Fifty-five minutes after LAD occlusion (5 minutes before reperfusion by release of the ligature), 1 mg of nafamostat per kg of body weight (ie, 2 μM/kg of body weight; Torii Pharmaceutical, Japan), or 100 IU C1-INH per kg of body weight (ie, 0.2 mM/kg of body weight, Berinert; Centeon, Germany) or vehicle were given intravenously as a bolus. After a total of 60 minutes of ischemia, the LAD ligature was untied, and the ischemic myocardium was reperfused for 3 hours.

The rabbits were randomly divided into 4 major groups: a Sham MI+R group (Sham, n = 16), an MI+R with vehicle group (Vehicle, n = 8), an MI+R with nafamostat group (Nafamostat, n = 8), and an MI+R with C1-INH group (C1-INH, n = 8). The Sham MI+R group animals underwent an identical surgical procedure as those in the MI+R groups except that the LAD coronary artery suture was not tied (ie, MI+R was not induced). These sham animals received nafamostat (n = 8) or C1-INH (n = 8) and thereby acted as a non-infarction control group in which the effects of the test agent on haemodynamics and other variables could be assessed. All investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

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Plasma Creatine Kinase Analysis

Arterial blood samples (2 mL) were drawn immediately before LAD occlusion and every 60 min thereafter. Samples were assayed for total protein and creatine kinase (CK) according to previously published methods.18 CK results are expressed as IU/g protein.

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Determination of Myocardial Necrosis

At the end of the 180-minute reperfusion period, the ligature around the LAD was again tightened. As previously published,19 the Evans blue/nitroblue tetrazolium technique was used to identify the zones of MI and infarction. The myocardium was divided into the area not at risk (ANAR) and the area at risk (AAR). The AAR was further subdivided into viable and necrotic portions. Results for the 3 portions of myocardium are expressed once indexed to the total LV or AAR mass.

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Determination of Myocardial Myeloperoxidase Activity

The myocardial activity of myeloperoxidase (MPO), an enzyme occurring virtually exclusively in neutrophils, was determined using the previously described method of Bradley et al.20 Results give an approximation of neutrophil accumulation in the myocardium, and 1 unit of MPO is defined as that quantity of enzyme hydrolyzing 1 mmol/min peroxide at 25°C.

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Immunohistochemical Analysis of MAC, C5b-9 Deposition After MI and Reperfusion In Vivo

Immunohistochemical procedures on frozen sections were performed using methods described previously by Hsu et al21 using the avidin-biotin immunoperoxidase technique (Vectastain ABC Elitekit Reagent; Vector Laboratories, Burlingame, CA). Analyses were performed with an antibody directed against MAC (C5b-9, sheep anti rabbit; Prof. Bhakdi, Department of Microbiology, Johannes Gutenberg-University, Mainz, Germany). Incubation of the primary anti-C5b-9 polyclonal antibody (PAb) was carried out at dilutions of 1:500 and 1:1000 of the C5b-9 MAb. Of these dilutions, the 1:1000 dilution gave the highest degree of immunolocalization together with the least nonspecific background staining. The sections were lightly counterstained with Gill's Hematoxylin 3 (Sigma, Deisenhofen, Germany).

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In Vitro Procedures Comparing Nafamostat and C1-INH Activity
Determination of Nafamostat and C1-INH Inhibitory Activity In Vitro

Complement C1s and C1r proteases were purified from human and rabbit plasma according to Lane et al.22 Enzyme activity was measured spectrophotometrically using the artificial substrate Cbz-Gly-Arg-S-blz (Custom synthesis from Polypeptide Inc, Wolfenbuettel, Germany). Release of Bzl-SH was quantified by adding excess of DTNB (5,5′Dinitrobis-2-nitrobenzoic acid; No.43760, Fluka, CH-9470 Buchs) and measured photometrically.

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Determination of Mannose-Binding Lectin- associated Serine Protease (MASP1) Inhibition

MASP-proteases were purified from human plasma according to Tan et al.23 Enzyme activity was measured using the artificial substrate Cbz-Gly-Arg-S-blz. Release of Bzl-SH was quantified by adding excess of DTNB and spectrophotometric measurement of yellow colour (λ = 405 nm).

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Determination of Thrombin-Inhibition

Thrombin purified from human plasma (Sigma; Nr.T8885) was used to assess potency of inhibiton using a standard chromogenic assay (trifocal substrate S2238; Chromogenix, Mölndal, Sweden) and spectrophotometric measurement of yellow colour at λ = 405nm. IC50-curves of inhibition were determined with nafamostat and C1-INH for 10 and 60 minutes of preincubation, respectively, at concentrations of 100 μM and 0.1nM, respectively. These periods were used because nafamostat is a small molecule agent with rapid onset of inhibitory potency.

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Complement Mediated Sheep Red Cell Lysis

To determine the anticomplement activity of nafamostat, we used an erythrocyte hemolytic assay as described previously.11 Sensitized sheep erythrocytes (Nobis, Endingen, Germany) were incubated with 0.5 to 20 μL of rabbit serum. Absorbance in the presence of 20 μL rabbit serum was considered as 100% of hemolytic activity. The complement activity was calculated by dividing the absorbance of each sample by the absorbance of the 20 μL of serum X100 and expressed as percent of red cell hemolysis. To compare the effect of nafamostat or the C1 esterase inhibitor, we incubated sensitized sheep red blood cells with serum (ie, 15 μL ≈ 80% red blood cell hemolysis) in the presence of different concentrations of nafamostat (0.001 to 0.3 mg/mL) or C1-INH (0.1 to 5 U/mL) for 60 minutes and determined hemolytic activity as described above. Further, we used complement-depleted serum in addition to the missing component. Before addition of the factor, we preincubated the component with nafamostat or the C1 esterase inhibitor.

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Determination of Biochemical Factor D and Alternative Pathway Activity

Purified human factor D activity (Fa. Calbiochem, Germany) was measured according to a procedure of Kim et al.24 Enzyme activity was measured by spectrophotometry (λ = 405 nm) using the artificial substrate Z-Lys-SBzl*HCl.

The alternative complement pathway activity was determined using an erythrocyte hemolytic assay. Guinea-pig erythrocytes were incubated with human serum, and alternative complement pathway activity was measured spectrophotometrically. IC50-curves for the inhibitors were determined with preincubation in concentrations of 1 mM to 0.1 nM.

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Statistical Analyses

All values in the text, tables, and figures are presented as mean ± SEM derived from independent experiments. All data on infarct size, cardiac MPO, and hemolytic assay were subjected to ANOVA followed by Fisher PLSD assessment. Values of P < 0.05 were considered to be statistically significant.

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RESULTS

Effect of Nafamostat on Electrocardiographic and Hemodynamic Changes

In the Sham rabbits, we observed that administration of nafamostat or C1-INH had no detectable effect on any of the measured hemodynamic, electrocardiographic, or biochemical variables. In all groups of MI+R rabbits, there were no significant differences in any of the variables observed before coronary occlusion (data not shown). After reperfusion, the ST-segment decreased to control levels, indicating that coronary reperfusion had been effective. In all MI+R groups PRI readings were statistically similar (Table 1), suggesting that neither test agent alters myocardial oxygen demand.

Table 1
Table 1
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Effect of Nafamostat on Myocardial Injury After Reperfusion

To evaluate the effects of nafamostat on the degree of myocardial salvage of ischemic tissue after reperfusion, we measured the amount of necrotic cardiac tissue as a percentage of either the AAR or the total left ventricular mass. There was no significant difference in the wet weights of the AAR expressed as a percentage of total left ventricular mass, indicating that a comparable region of myocardium was rendered ischemic in all groups (Figure 1). About 30% of the AAR myocardium became necrotic in the vehicle group. In contrast, nafamostat compared to vehicle, produced a significant reduction in the size of the necrotic myocardium whether expressed as a percentage of the AAR (23.6 ± 3.1% versus 35.7 ± 1.0%; P < 0.05) or a percentage of total left ventricular mass, (6.7 ± 0.5% versus 10.4 ± 0.7%; P < 0.01) (Figure 1). In this model, nafamostat lead to an approximately 40% relative reduction in reperfusion injury.

Figure 1
Figure 1
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There was a statistically similar infarct size reduction in both nafamostat and C1-INH groups. The size of the necrotic area in animals treated with nafamostat versus C1-INH was 23.6 ± 3.1% versus 23.5 ± 4.0% and 6.7 ± 0.5% versus 6.5 ± 1.5% as a percentage of the AAR and the total left ventricle, respectively.

To further evaluate the degree of ischemia reperfusion injury, we measured plasma creatine kinase activity (CK). In the Sham group, plasma CK activity increased modestly throughout the 4-hour experimental period due to surgical preparation. The final mean CK activity was 30.6 ± 2.6 IU/g protein. In the 3 MI+R groups, plasma CK activity increased slightly during the ischemic period, but there was a substantial washout of myocardial CK into the circulation during the first hour of reperfusion. This increase in circulating CK was most marked in rabbits receiving only vehicle (peaking at 65.4 ± 5.9 IU/g protein). In contrast, MI+R rabbits treated with nafamostat or C1-INH had significantly lower plasma peak CK activities (nafamostat, 33.1 ± 6.5 IU/g; C1-INH, 44.8 ± 4.7 IU/g; both P < 0.05). The effect was sustained over the entire reperfusion period (Nafamostat data shown in Figure 2).

Figure 2
Figure 2
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Effects of Nafamostat on Circulating White Blood Cell Counts

To determine whether nafamostat or C1-INH exerted any systemic leukopenic effects that could contribute to cardioprotection, we counted the number of circulating white blood cells (WBCs) initially and every hour throughout the experimental period. WBC counts did not change significantly in the Sham, Vehicle, Nafamostat, or C1-INH groups (Table 2).

Table 2
Table 2
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Neutrophil Accumulation in the At-Risk Myocardium

Accumulation of PMNs into the ischemic area during reperfusion is an important step in reperfusion injury. We therefore measured MPO activity, a marker of polymorphonuclear neutrophil (PMN) accumulation in the ANAR (ie, non-ischemic myocardium) the AAR myocardium and the necrotic myocardium (Figure 3). The ANAR had low levels of MPO activity indicating no significant neutrophil accumulation into non-ischemic myocardium. All groups were statistically similar in this respect. The AAR (including viable and necrotic myocardium) exhibited a modest increase in MPO activity in MI+R- and vehicle-treated animals. MPO activity in the same portion of myocardium from animals treated with nafamostat and C1-INH showed a trend towards reduction (compared to activity seen in vehicle-treated animals), but this trend did not reach statistical significance. The most striking findings applied to the necrotic portion of myocardium. Among vehicle-treated animals, MPO activity in this portion underwent a substantial and significant increase to 1.26 ± 0.25 IU/100 mg tissue (P < 0.01 compared to the ANAR myocardium from the same group). Nafamostat and C1-INH generated significantly lower MPO levels in the necrotic portion to 0.62 ± 0.05 IU/100 mg tissue and 0.56 ± 0.09 IU/100 mg tissue, respectively (both P < 0.01 versus vehicle-treated animals).

Figure 3
Figure 3
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Immunohistochemical Localization of MAC After MI and Reperfusion

The presence of MAC in the ischemic-reperfused myocardium was detected by an anti-MAC PAb using the avidin-biotin immunoperoxidase procedure. Nonischemic sections of cardiac tissue (from any group) did not demonstrate any immunostaining. In contrast to these controls, MAC deposition was clearly evident in AAR tissue sections of vehicle-treated animals (Figure 4A). Intense immunolocalization of the antibody directed against MAC was prevalent on cardiomyocytes and in the coronary vessels (particularly the endothelium). Nafamostat-treated rabbits demonstrated substantially reduced deposition of MAC in similar tissue sections (nafamostat image shown in Figure 4B). These results indicate that reperfusion of ischemic myocardium results in activation of the complement pathway with deposition of MAC and that this is reduced by administration of nafamostat before reperfusion.

Figure 4
Figure 4
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In Vitro Comparison of Nafamostat and C1-INH on Classical Complement Pathway Serine Protease Activity

Inhibitory activity of nafamostat and C1-INH was analyzed on a panel of related serine proteases (rabbit C1s and C1r, human C1s and MASP1, and thrombin). Nafamostat had higher inhibitory activity on rabbit C1s (compared to the endogenous C1 inhibitor C1-INH), but C1r was only inhibited in the micromolar range (Table 3). Among the human enzymes, nafamostat had a similar potency for C1s and a lower activity against MASP-1 than C1-INH. Thrombin was substantially more potently inhibited by nafamostat than C1-INH.

Table 3
Table 3
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Inhibitory Effects of Nafamostat on Classical Complement Pathway-Mediated Red Cell Lysis

Incubation of sensitized sheep erythrocytes with rabbit serum resulted in a concentration-dependent hemolysis of the sheep red blood cells. Fifteen microliters of rabbit serum exerted 80% hemolytic activity. We determined the efficacy of C1 esterase inhibition (0.1 to 5 U/mL) resulting in a dose-dependent inhibition of complement-mediated hemolysis (Figure 5A). Coincubation of 15 μL of rabbit serum with nafamostat (0.001 to 0.3 mg/mL) resulted in a similar concentration-dependent inhibition of the hemolytic activity to almost a complete inhibition at 0.3 mg/mL (Figure 5B). These results demonstrate the efficacy of nafamostat in inhibiting complement activation. When compared on an equimolar basis, nafamostat demonstrated greater potency than C1-INH for hemolysis inhibition (IC50; 1.85 nM and 1.6 μM for nafamostat and C1-INH, respectivley).

Figure 5
Figure 5
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In Vitro Comparison of Nafamostat and C1-INH on Alternative Complement Pathway Activity

Inhibitory activity of nafamostat and C1-INH was analyzed on factor D and the alternative complement pathway. C1-INH showed no inhibitory effects on alternative pathway activation or activation of the isolated factor D. In contrast, nafamostat had inhibitory activity towards factor D with an IC50 of 157 μM. Nafamostat also inhibited global alternative complement pathway activity (Table 4).

Table 4
Table 4
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DISCUSSION

The present data identified an impressive cardioprotective effect of the serine protease inhibitor nafamostat in a well-established model of rabbit ischemia and reperfusion. The agent was effective when applied just before reperfusion, which is analogous to the therapeutic window of opportunity during reperfusion for myocardial infarction or reperfusion after cardiopulmonary bypass. The reduction in myocardial injury was evidenced by a reduction in infarct area and CK release.

Our study identified that nafamostat is capable of inhibition of both principle complement pathways, and one could speculate that its greater activity against the alternative pathway may be an advantage over other inhibitors such as C1 inhibitor, particularly in situations where the alternative pathway is additionally activated such as during cardiopulmonary bypass. This, together with our data showing reduced myocardial leukocyte infiltration, suggests the mechanism of nafamostat action may involve reduced endothelial dysfunction, leukocyte transmigration, and complement activation in the myocardium.

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Complement Inhibition

C1 inhibitor (C1 INH) has been widely studied as an inhibitor of reperfusion injury. As an endogenous C1r and C1s inhibitor, it mainly suppresses the classical and mannose-binding complement pathways (although it also inhibits the alternative pathway at supraphysiologic levels).25 C1 INH has been widely studied and has protective effects after ischemia and reperfusion in animal models of myocardial26-29 and liver30 injury as well as in a clinical setting.31

Given the increasing recognition of the role of the alternative32 and mannose-binding6 complement pathways in MI reperfusion, we reasoned that broader-spectrum complement inhibition (involving activity against all 3 pathways) may prove to have greater protective effects than complement inhibitors such as C1 INH. The present study shows nafamostat is a more powerful broad spectrum complement inhibitor that incorporates activity against the alternative complement pathway. Interestingly the additional activity of nafamostat against the alternative pathway may indicate the drug has a particular role in clinical situations where alternative pathway activation is particularly potent, such as during cardiopulmonary bypass for cardiothoracic surgery.33,34 Indeed, positive data exist for both nafamostat and aprotinin in suppressing the response to bypass,17,35 and these data provided some of the encouraging preclinical data preceding the APEX studies.8 Nevertheless, we believe that complement inhibition alone is unlikely to yield sufficient antiinflammatory activity to become effective therapy for reperfusion injury because the generation of the systemic inflammatory response after such insults causes parallel activation of multiple inflammatory pathways, and successful damping of this response is likely to require a broad-spectrum antiinflammatory agent suppressing several such pathways simultaneously.

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Serine Protease Inhibition

Broad-spectrum antiinflammatory activity could arise from serine protease inhibition. The serine proteases are a widespread group of enzymes that mediate many of the cytotoxic effects of the immune response. They are among the key mediators of PMN-induced injury.36 Elastase and Cathepsin G are the two major neutral serine proteases in neutrophils.36 They have direct effects such as elastase's degradation of molecules like elastin, collagen, immunoglobulins, complement, clotting factors, proteoglycans, fibronectin, and even intact cells. There are also indirect effects; both are chemoattractant and exhibit mutual positive feedback by further activation leukocytes. These proteases are also intimately involved in apoptosis.37,38 Thrombin is another serine protease with widespread downstream proinflammatory effects thought also to mediate reperfusion injury. The enzyme is versatile and has multiple functions, including the conversion of fibrinogen to fibrin, platelet activation, and aggregation and in eliciting responses from multiple cell types, including vascular smooth muscle cells, endothelium, monocytes, neurons, nephrons, and osteoclasts.39 Thrombin is widely activated during CABG, myocardial infarction, and other inflammatory states. Thrombin effects are mediated by the protease activated receptors (PAR), of which types 1 through 4 have been identified thus far.40 To date, PAR inhibition (directly or with the use of serine protease inhibition) has been shown to reduce inflammation,15 to improve stroke volume and cardiac output after ischemia and reperfusion,41 and to reduce apoptosis in nephrons42 and neutrophils.43

Thus serine proteases are frequently encountered, potent enzymes with a central role in the mediation of reperfusion injury. They are key to coagulation control, platelet aggregation, leukocyte activation, infiltration, degranulation, and subsequent inflammatory tissue injury. Our data support the notion that the inhibition of serine proteases by inhibitors such as nafamostat adds antiinflammatory potential over and above those agents (such as pexelizumab), whose action is specific to the complement system.

The synthetic serine protease inhibitor nafamostat is in clinical use in Japan and is a small-molecule (MW 539.85) non-peptide inhibitor that is specific for arginyl-lysyl-residuals-cleaving serine proteases, like C1r, C1s, Bb, D, C3bBb, thrombin, plasmin, kallikrein, and trypsin. Nafamostat is a recognized potent inhibitor of serine proteases.14 Antiinflammatory capability has been found with the use of nafamostat in a number of models, including airway inflammation in a murine model of asthma44 and colitis.45 Nafamostat has been successfully used to suppress rejection through inflammatory reaction after xenotransplantation.46 Whole organ protection by nafamostat (or other broad spectrum serine proteases such as aprotinin) after insults such as ischemia/reperfusion has been identified in a number of tissue types such as the liver47 and kidney42 and myocardium.20

One theoretical advantage of nafamostat over aprotinin in the management of ischemia/reperfusion pathology is that nafamostat has been shown to inhibit platelet and microaggregation16,48 (particularly useful in maintaining graft and stent patency), whereas aprotinin has been reported to increase thrombotic graft occlusion risk post CABG.49 Interestingly, given their similar enzymatic profiles, aprotinin and nafamostat have been shown to reduce blood loss during CABG,50 while nafamostat decreased IL-6 and IL-8 levels33 in such patients. The small molecule nature of nafamostat allows for the theoretical advantage of optimal tissue penetration and bioavailability. This may prove an advantage over larger molecules such as aprotinin and pexelizumab, whose transit into the myocardium may be less pronounced.

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Broad Spectrum Inflammatory Suppression to Reduce Cardiac Injury

Many clinical scenarios lead to the onset of systemic inflammation. These include sepsis, dialysis, cardiopulmonary bypass, and ischemia reperfusion of any organ.1 In the case of MI/reperfusion, part of the inflammatory response arises due to local, myocardial inflammation. This is complemented by systemic inflammation generated by hypoperfusion of multiple vascular beds during cardiogenic shock. Gut hypoperfusion degrades mucosal integrity leading to bacterial and toxin translocation, and these are a potent cause of systemic inflammatory response.51 The degree of inflammatory response is related to outcome in such patients. Given the above, the concept of immune modulation to suppress inflammation as an aid to recovery from ischemia reperfusion has been attractive.

The APEX group of studies (using the specific C5 monoclonal antibody pexelizumab) represent the largest test of immune modulation to reduce MI/reperfusion injury yet undertaken. Interestingly, amongst patients undergoing prolonged cardiothoracic surgical procedures, pexelizumab did yield an improvement in outcomes.8 This benefit may have arisen due to the potent effect of the cardiopulmonary bypass circuit in complement activation and systemic inflammation, a situation in which complement inhibition may be particularly effective. Conversely, the APEX AMI study recruited more than 5000 patients with ST segment elevation myocardial infarction (STEMI) in whom pexelizumab given during reperfusion therapy by PCI did not significantly improve outcomes in any respect.52 The failure of this promising preclinical concept to translate into clinical benefit in such a large study is disappointing,53 but we believe that this negative study does not indicate that immune modulation therapy after ischemia reperfusion is fruitless; but that the right agent has not yet been applied to the right groups of patients. We speculate that the APEX AMI study did not enroll patients with sufficient systemic inflammatory activation for immune-modulating therapy to produce a clear benefit. Although the study sought to enroll particularly high-risk patients with STEMI, mean 30-day mortality was less than 4%. Previous data suggest that the highest risk (and hence the most inflammatory activated) patients have a mortality rate of more than 20%.54 Had such patients been enrolled, we speculate that the degree of inflammatory activation may have been sufficient for antiinflammatory therapy to have yielded a measurable benefit. Other features of the APEX AMI study may limit the observed utility of inflammatory suppression in these patients. Randomization (and administration of pexelizumab) before angiography means that some patients with spontaneous restoration of flow may have received the compound. Patients with large, anterior territory, shock-inducing, myocardial infarction were not preferentially selected, and there was no exclusion of patients with known coronary artery disease (who may have undergone physiological preconditioning and the development of collateral vessels). In all such patients, we would anticipate the effect of antiinflammatory therapy to be lessened. Finally, the APEX AMI data may suggest that complex multimediator cascades causing inflammation are not adequately controlled by specific complement inhibition using agents such as pexelizumab. There may be a role for the broader-spectrum antiinflammatory properties of compounds such as the serine protease inhibitors like nafamostat. Any such potential role for protease inhibition as immune-modulating cardioprotection would require further evaluation in both preclinical and clinical settings. The present study is a preliminary one using a well-described animal model of MI for 60 minutes and reperfusion for 180 minutes. Although these are recognized intervals within the context of an animal model, they do not reflect the reality of the clinical setting, where patients present with substantial variation in ischemia time. Furthermore the changes in CK and necrotic in the present study are only surrogate markers for potential clinical benefit, where important endpoints would be a change in long-term morbidity and mortality. Nevertheless, despite recent disappointing clinical data, we believe the principle of immune modulation-based cardioprotection is attractive and may translate with positive results into the clinical setting.

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CONCLUSION

We have demonstrated that the serine protease inhibitor nafamostat has significant activity against classical and alternative complement pathway activation. Nafamostat was at least as effective as the endogenous inhibitor C1-INH, but it has superior inhibitory activity on the alternative pathway. In vivo administration of nafamostat significantly attenuated myocardial injury after ischemia and reperfusion. This cardioprotective effect was associated with reduced PMN infiltration into the reperfused myocardium. The superior potency of nafamostat against the alternative complement pathway may be an advantage when aggressive activation of this pathway is anticipated, such as during extracorporeal circulation use. Furthermore the non-complement-mediated antiinflammatory effects of nafamostat and other broad spectrum serine protease inhibitors may be of therapeutic potential in conditions where significant systemic inflammatory activation is encountered, such as myocardial infarction complicated by cardiogenic shock or surgical procedures involving long cardiopulmonary bypass times.

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ACKNOWLEDGMENTS

Some results of the present study are included in the thesis of Hansjörg Schwertz. The authors gratefully acknowledge Professor Bhakdi (Department of Microbiology, Johannes Gutenberg University, Mainz, Germany) for his critical comments.

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REFERENCES

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2. Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res. 2002;53:31-47.

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Keywords:

myocardial reperfusion injury; serine protease inhibitors; nafamostat; complement activation; leukocytes

© 2008 Lippincott Williams & Wilkins, Inc.

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