Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure in patients of all ages, resulting in high rates of morbidity and mortality despite decades of clinical research. ALI/ARDS is characterized by diffuse alveolar damage leading to disruption of the alveolar capillary barrier, pulmonary edema and neutrophilic inflammation. Extravascular intra-alveolar thrombin formation and fibrin deposition, often evident as hyaline membranes lining the denuded alveolar surface, have long been recognized as pathological hallmarks of ALI/ARDS. These findings suggest that the coagulation cascade and the fibrinolytic pathway, responsible for fibrin clot clearance, are altered in patients with ALI/ARDS [1–4].

The tissue factor (TF)-dependent extrinsic pathway has been suggested as a central mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS. TF is a transmembrane glycoprotein normally expressed on subendothelial cells in the vascular adventitia layer that is not in contact with the circulating blood [5]. Vessel injury or pathological conditions leading to the exposure TF in the vascular adventitia layer or induction of TF expression on endothelial cells and monocytes permits interactions between TF and coagulation factor VIIa (FVIIa) resulting in the formation of the high affinity TF-FVIIa complex. This complex then binds factor × (FX), converting it to the activated form FXa, which ultimately leads to thrombin formation and fibrin deposition [6]. TF-FVIIa complexes also play a role in cell signaling events mediated by the TF cytoplasmic domain and by activation of the protease activated receptors (PARs) either directing or via downstream TF-dependent coagulation proteases [1, 7, 8]. These signaling events stimulate proinflammatory cytokines, growth factors and chemokines, some of which further upregulate TF expression.

A direct role of TF in promoting ALI/ARDS has been suggested based on elevated levels of TF observed in plasma and pulmonary fluid of ALI/ARDS patients compared to control subjects [9–11]. These higher plasma TF levels correlated with the presence of disseminated intravascular coagulation and sepsis in patients with ALI/ARDS, and were associated with prolonged use for mechanical ventilation and increased mortality. Immunohistochemistry of the lung tissue from patients with ALI/ARDS showed prominent TF expression by alveolar epithelial cells as well as intra-alveolar macrophages and hyaline membranes [9], suggesting an active role of intra-alveolar TF in fibrin deposition within the lungs of these patients.

Consequently, development and evaluation of TF antagonists has been of interest as a therapeutic strategy for treating ALI/ARDS [1–3]. ALT-836 is a recombinant IgG4κ chimeric antibody that binds to human TF or the TF-FVIIa complex preventing the association and activation of FX, thereby inhibiting thrombin generation [12]. The results of a preclinical study in a baboon model of established E. coli-induced sepsis demonstrated that ALT-836, administered after the onset of the disease, was able to reverse the course of sepsis-induced lung and organ injury by reducing abnormalities in gas exchange, pulmonary hypertension, lung compliance and other clinically relevant parameters [13]. Additionally, ALT-836 has been administered to subjects with stable coronary artery disease (CAD) where it exhibited dose-dependent anticoagulant effects [14]. Based on these preclinical and clinical findings, we hypothesized that ALT-836 could serve as a potential therapeutic agent for the treatment of sepsis-induced ALI/ARDS. In this randomized, placebo-controlled, dose-escalation Phase I clinical trial, we evaluated the safety and pharmacokinetics of ALT-836 in critically ill patients with sepsis-induced ALI/ARDS.

In a number of pre-clinical models, blockade of TF is capable of preventing mortality during systemic infection and inflammation, inhibiting endothelial cell injury induced by anoxia-reoxygenation and permeability of endothelial cells during systemic inflammation, reducing lung and renal fibrin deposition, and limiting proliferation, migration and invasion of malignant cells [13, 17, 18]. In humans, TF levels are elevated in plasma and pulmonary fluid of ALI/ARDS patients compared to control subjects [9–11]. Taken together, these findings suggest that TF may represent an important therapeutic target for treatment of systemic inflammatory diseases such as ALI/ARDS [1, 2]. However, bleeding-related complications resulting from therapeutic inhibition of TF activity remain a major concern. This was recently illustrated in a Phase II clinical trial in ALI/ARDS patients where administration of the TF-FVIIa complex inhibitor, active site inactivated FVIIa (FVIIai), resulted in a dose-depended increase in major bleeding events (3.8% for the low dose to 17% for the highest dose) and an increased 28-day mortality rate [15]. In contrast to the FVIIai study, we found that ALT-836, an antibody-based TF antagonist, was well tolerated by ALI/ARDS patients with no drug-related deaths or major bleeding event observed following treatment with dose levels from 0.06 to 0.1 mg/kg. A similar safety profile (i.e. no serious bleeding or drug-related SAEs) for ALT-836 at a single dose levels up to 0.3 mg/kg was observed in 26 patients with CAD who were also receiving daily anti-platelet aspirin therapy [14]. In the trial reported here, there was an ALT-836 dose-dependent increase in mild to moderate hematuria observed in some patients that typically started 6 to 8 hours after study drug administration and spontaneously resolved approximately 24 hours post onset. These minor bleeding episodes did not result in hemodynamic instability and were not associated with alterations in renal function or coagulation parameters. Interestingly, analysis of the pooled data of ALT-836-treated patients did not show a correlation between hematuria and higher ALT-836 serum levels or exposure. Thus, it is possible that other clinical confounders, including baseline/previous history of hematuria or concurrent use of urinary catheters, influenced the development of hematuria following ALT-836 administration.

The dose levels of ALT-836 administered, while relatively low in comparison to those of other therapeutic antibodies, appear to be within the range required to inhibit disease-associated TF activity. In the current study, the results of pharmacokinetic analysis indicate that ALT-836 administered at 0.06 mg/kg to 0.1 mg/kg provided a maximum serum concentration of 9 to 12 nM, respectively. In vitro studies have demonstrated that these ALT-836 concentrations are sufficient to inhibit > 90% of TF cofactor activity at a broad range of TF concentrations (0.005-2.3 nM) [12]. It is noteworthy that disease-associated, elevated TF levels found in undiluted pulmonary edema fluid of patients with ALI/ARDS ranged from 0.5 and 2 nM, approximately 100-fold higher than plasma TF levels of the same patients [9]. Similarly higher levels of TF-dependent procoagulant activity in bronchoalveolar lavage (BAL) fluid of ARDS patients was observed three days after clinical recognition of ARDS and decreased thereafter [11]. Thus one would expect that ALT-836 at dose levels tested in the present study when administered shortly after diagnosis of ALI/ARDS should provide sufficient antibody to block pulmonary and intravascular TF activity for several days. Although ALT-836 levels were not measured in patient's pulmonary fluid of patients, we have previously shown that the antibody could be readily detected at the expected nanomolar level in BAL fluid collected from baboons in a sepsis-induced organ failure model [13], suggesting that ALT-836 is capable of distributing between the vascular and alveolar compartments. The pharmacokinetic analysis also revealed differences in the antibody half-life between patients with ALI/ARDS and CAD. Serum ALT-836 levels typically displayed exponential decay kinetics with a terminal half-life of about 21 hours in ALI/ARDS patients, whereas a biphasic decay with an initial plateau phase and a median 72 hour terminal half life was observed in patients with stable CAD [14]. While patient management may have affected the antibody pharmacokinetics, it is possible that elevated levels of the TF antigen in the vascular and pulmonary compartments of the ALI/ARDS patients contributed to the observed increase rate of serum ALT-836 clearance. Such antigen-antibody interactions have been reported to play a role in the elimination kinetics of a variety of other therapeutic antibodies [19].

The abundant evidence of extensive cross-talk between the coagulation and the inflammatory systems have led to numerous but unsuccessful studies utilizing anti-thrombotic and fibrinolytic agents for treatment of ALI/ARDS and sepsis [15, 16, 20–24]. Although most of these agents are classified as anti-coagulants, they are known to have other physiological functions in addition to their anti-thrombotic activities. For example, activated protein C (APC) not only regulates the coagulation pathway by inactivating factor VIIIa and factor Va which decreases thrombin mediated inflammation, but also binds to the endothelial cell protein C receptor to inhibit endotoxin-induced intracellular calcium fluxes and NF-κB nuclear translocation. These effects influence gene expression profiles, inhibit leukocyte chemotaxis and adhesion to activated endothelium, and inhibit endothelial cell apoptosis [25–29]. By stimulating cell signaling, APC can modulate the cellular response to infectious insults leading to anti-inflammatory, cytoprotective and barrier-protective properties which may contribute significantly to APC effectiveness for severe sepsis treatment [30]. TF, either alone or in complex with FVIIa, has also been shown to exhibit many physiological effects in systemic inflammation, which differ from those of many anti-coagulants including APC. Besides its role in triggering the coagulation pathway, the TF-FVIIa complex per se activates PAR2, which plays a crucial role in inflammatory responses, fibrosis and tissue remodeling [31, 32]. In a mouse model, activation of PAR2 in the lung resulted in airway constriction, pulmonary edema and increased lung vascular and epithelial permeability [33]. Lung PAR2 activation also induced acidosis, hypoxemia, hypercapnia and increase leukocyte infiltration, suggesting that PAR2 activation can trigger a variety of inflammatory responses associated with lung injury [33]. Similarly, recent studies provide evidence that TF/FVIIa-mediated FXa activation in the lung drives fibrotic responses via a PAR1-dependent signaling mechanism [34, 35]. Consequently, the effectiveness of anti-coagulants as anti-inflammatory agents may be largely dependent on the relevance of particular pro-inflammatory signaling processes at the systemic and local levels under different pathological conditions. Thus, the enthusiasm for employing novel anti-coagulants as therapeutic approaches for ALI/ARDS should not be tempered by the lack of clinical effectiveness of a particular anti-coagulant in the treatment of these inflammation-associated debilitating diseases.

Identification of an increased incidence of hematuria at the 0.10 mg/kg dose level supported an acceptable safe dose of ALT-801 at 0.08 mg/kg in patients with sepsis-induced ALI/ARDS in this Phase I trial. Pharmacokinetic analysis indicated that ALT-836 administrated at 0.06 to 0.1 mg/kg could provide sufficient serum antibody levels to inhibit elevated TF activity reported in ALI/ARDS patients. Taken together, these results suggest that evaluation of the clinical activity of this antibody in patients with ALI/ARDS is warranted. Thus, a follow-up randomized, double-blind, placebo-controlled trial with a target enrollment of 150 patients is currently being conducted to further assess the safety and efficacy of single and multiple dose ALT-836 treatment in patients with sepsis-induced ALI/ARDS (ClinicalTrials.gov ID: NCT00879606).