Interleukin-6 in LPS induced inflammation and coagulation

Sepsis and septic shock are characterized by excessive activation of the cytokine network, the coagulation system, and leukocytes. Bacterial cell wall components like endotoxin (lipopolysaccharide, LPS) may be responsible for most of the initial changes seen in sepsis. The ensuing activation of tissue factor (TF) induces activation of coagulation. Disseminated intravascular coagulation and overexuberant stimulation of the immune system may eventually lead to multiple organ failure. Animal models have shown that blockade of TF or FVIIa attenuated coagulation responses and prevented baboons in a lethal E. coli septic shock model from dying. Preliminary reports from a multicenter trial in 1690 patients indicate that infusion of the anticoagulant and antiinflammatory drug recombinant human activated Protein C significantly decreases mortality (relative risk reduction 19.4%) in septic patients (Bernard et al. Crit Care Med 2000;28:Suppl:A48:67). This decrease in mortality was accompanied by a more rapid decrease of D-dimer and interleukin-6 levels in the rhAPC treated patients compared to placebo (Kinasewitz et al. Crit Care Med 2000;28:Suppl:A48:68). This trial represents the first clinical study, in which a drug successfully reduced mortality of septic patients. Based on these encouraging data we hypothesize that other drugs with anticoagulant and antiinflammatory properties may also prove to be useful for the treatment of sepsis. Similar to rhAPC, an anti IL-6 antibody attenuated coagulation in endotoxin challenged chimpanzees, by an unknown mechanism. Therefore, we hypothesise that an anti IL-6 antibody will also attenuate coagulation in humans, and we plan to further characterise its mechanism of action. LPS-infusion in healthy subjects provides a powerful tool to safely study the influence of endotoxemia on the activation of the inflammation and coagulation cascades in humans, and to document possible effects of therapeutic or prophylactic interventions. Primary objective: A murine anti IL-6 antibody B-E8 has been expressed in hybridoma cells and been cloned successfully; it has been tested in various clinical trials without noticeable side effects. Hence, we plan to explore whether the anti IL-6 antibody B-E8 can block activation of the coagulation cascade induced by endotoxemia, in particular to investigate whether treatment with B-E8 can prevent the LPS mediated thrombin formation as measured by prothrombin fragment (F 1+2 ). Secondary objective: To find an optimal dose of B-E8 in a pilot study for LPS-induced coagulation, as measured by F1+2 -levels and C reactive protein levels. To explore whether aIL-6 Ab can attenuate LPS-induced coagulation and inflammation To study the role of IL-6 in activation induced apoptosis To compare the effect of an iv. bolus of 2ng/kg endotoxin with and without co-infusion of B-E8 on systemic coagulation and immunologic responses, in order to reveal a possible therapeutic role for B-E8 in preventing endotoxin-induced disseminated intravascular coagulation and inflammatory responses. The trial will be performed at a single center (Department of Clinical Pharmacology, University Hospital in Vienna). The trial consists of a pilot trial and a main trial. A double blind, randomised, placebo-controlled trial in two parallel groups will be conducted. The maximum number of subjects is 34. We estimate that ten subjects will be sufficient for the pilot trial. The sample size (n=24 in the main trial) is based on previous experience with heparin, hirudin, danaparoid, acenocoumarol and FFR-FVIIa in this setting. Based on the expected attenuation of prothrombin formation the sample size will be sufficient to detect a significant attenuation of thrombin formation by B-E8 (a=0.05, ß=0.8%). Healthy male subjects aged 19 to 35 years will be included. Primary: prothrombin fragment (F 1+2 ) levels Secondary: Coagulation: activation markers of thrombin and fibrin formation, and tissue factor -expression on and by monocytes Inflammation: Differential leukocyte counts, TNF, and Regulation of IL-6 and its receptor on protein and molecular levels Apoptosis: TNF-Receptor, Fas and BCL2 on the protein and molecular levels Enzyme immuno assays, flowcytometry, quantitative PCR LPS: (all subjects) 2 ng/kg LPS iv. bolus over 1-2 min Generic name: National Reference Endotoxin, E coli Manufacturer: United States Pharmacopeial Convention Inc., Rockville, MD, 20852 NaCl 0.9% (groups A & B): 80 mL/h over 12 hours Kochsalz <> 0.9% Infusionslösung B-E8: (pilot study and group A of main trial) Pilot trial: open dose-escalation trial in a max. of 10 healthy male subjects 10, 20, 40 and 80mg aIL-6 Ab We will start with 10mg aIL-6 Ab and will measure prothrombin fragment levels before and 4h after LPS infusion. First, two subjects will be challenged with LPS. If F1+2 -levels increase less than 2-fold (minimum increase observed previously) in both subjects we may decide to challenge more subjects with the same dose to gain more confidence. Otherwise we will continue with the two-fold higher dose (20mg and so on up to 80 mg) until F1+2 -levels increase less than 2-fold. Main trial: double blind, placebo controlled parallel group trial. When we will have shown that a particular dose of aIL-6 Ab has a clear anticoagulant effect (by attenuating the increase in F1+2 -levels) and suppresses inflammation (as measured by CRP-release), the main trial will be performed using an optimal dose of aIL-6 Ab. Manufacturer: Diaclone, Besancon, France Placebo: (group B) equal volume of physiological saline will be dosed.

Clinical trials show that interleukin-6 (IL-6) represents a predictive marker in human sepsis. Further, IL-6 has been proposed as a candidate mediator for endotoxin (LPS)-induced coagulation activation: in a primate model an aIL-6 antibody (aIL-6 Ab) almost abolished LPS-induced coagulation activation. Therefore, IL-6 may be an attractive target in human endotoxemia, particularly if IL-6 blockade also inhibited coagulation. To examine whether an aIL6 Ab (B-E8) attenuates LPS induced activation of coagulation in humans, 24 healthy volunteers were randomized to receive either 80 mg B-E8 or placebo iv. before bolus infusion of 2 ng/kg LPS. B-E8 had no influence on LPS- induced tissue factor-mRNA transcription, or downstream coagulation parameters (prothrombin fragment 1+2 and D-Dimer levels). Similarly, fibrinolytic activity (plasmin-antiplasmin complexes) and endothelial activation (E- selectin) were unaffected, while B-E8 effectively decreased IL-6 bioactivity as measured by CRP-levels. In conclusion, IL-6 does not appear to mediate early phase LPS-induced coagulation activation in humans. Further primate models may not be reliable models to predict the efficacy of drugs for disseminated intravascular coagulation in man.