ABCD Anti-inflammatory activity of the bradykinin B1R antagonist in a model of LPS-induced lung inflammation in the cynomolgus monkey T Bouyssou, B Jung, P Nickolaus, J Duan*, F Dai* and H Doods, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, * PharmaLegacy Laboratories Inc., Shanghai, China Introduction Total cell count in 1 mL BALF Neutrophil cell count in 1 mL BALF Results The non-human primate LPS model bridges the translation between pre-clinical rodent and LPS proof-of-concept studies in healthy volunteers [1}. The aim of the present study was to investigate the anti-inflammatory activity of a bradykinin B1R antagonist in this monkey model and to compare its efficacy to the anti-inflammatory activity of dexamethasone and also to the anti-inflammatory activity of a PDE4 inhibitor, a drug class, which besides inhaled corticosteroids, represents the gold standard therapy for the anti-inflammatory treatment of patients with chronic obstructive pulmonary disease (COPD) [2]. At 12 h post LPS challenge, BAL cells were significantly elevated (p < 0.05) over baseline (LPS: 20 x 106 vs naive: 2 x 106) which was mainly due to an accumulation of neutrophils (LPS: 80 % vs naive: 1.5 %). BALF protein was significantly increased (p < 0.05) 12 h after LPS challenge (LPS: 3 mg/ml vs naive: 0.9 mg/ml). Of all the cytokines measured, only BALF IL-8 was significantly increased (p < 0.05) 12 h after LPS challenge (LPS: 1100 pg/ml vs naive: 200 pg/ml). The number of neutrophils and other inflammatory parameters returned to baseline level one week after LPS challenge. All treatments, B1R antagonist, PDE4 inhibitor and dexamethasone, significantly reduced (p < 0.05) LPS-induced BAL neutrophilia (~80 % inhibition) and BALF protein (~90 % inhibition). The B1R-antagonist inhibited BALF IL-8 by 50 %, dexamethasone by 80 % and the PDE4 inhibitor by 100%. saline LPS dexa PDE4 B1R saline LPS dexa PDE4 B1R Fig. 1: Effect of dexamethasone, PDE4 inhibitor and B1R antagonist on BALF total cell count following LPS challenge in cynomolgus monkeys. Intratracheal nebulization of LPS led to an accumulation of inflammatory cells in the lungs 12 h later. (mean ± SEM, n=6 per group, * p < 0.05) Fig. 2: Effect of dexamethasone, PDE4 inhibitor and B1R antagonist on BALF neutrophils following LPS challenge in cynomolgus monkeys. Intratracheal nebulization of LPS led to an accumulation of neutrophils in the lungs 12 h later (mean ± SEM, n=6 per group, * p < 0.05) Methods A total of 6 naive cynomolgus monkeys (5.5 to 7 kg) were challenged once a week. In the first week, bronchoalveolar lavage (BAL) from the left lung (flushed with 10 mL PBS solution), was collected using a pediatric bronchoscope under anaesthesia (zoletil, xylazine). In the second week, BAL was performed 12 h after intra-tracheal LPS nebulization (20 µg/L for 5 min). In addition, the monkeys received the vehicle orally (0.5 % carboxymethyl cellulose, 5 mL/kg), 1 h before and 6 h after LPS. Using a cross-over design, in weeks 3, 4 and 5, the animals were challenged with LPS and they received at random the test compound orally: B1R antagonist (30 mg/kg, bid), PDE4 inhibitor (0.5 mg/kg, bid) or dexamethasone (1 mg/kg, bid). BAL was performed 12 h after LPS challenge. BALF total cell count, differential cell count, BALF protein, and BALF cytokines (IL-1α, IL-1ß, IL-8, IL-10 and IL-18) were measured. Total protein in 1 mL BALF IL-8 concentration in 1 mL BALF Conclusions These data show that a cross-over design in the model of LPS-induced lung inflammation in the cynomolgus monkey is meaningful because all inflammatory parameters returned to baseline one week after LPS challenge. In this study the B1R antagonist displayed an anti-inflammatory activity similar to dexamethasone and the PDE4 inhibitor. References saline LPS dexa PDE4 B1R saline LPS dexa PDE4 B1R Fig. 3: Effect of dexamethasone, PDE4 inhibitor and B1R antagonist on BALF total protein following LPS challenge in cynomolgus monkeys. Intratracheal nebulization of LPS led to a significant increase in lung vascular permeability (mean ± SEM, n=6 per group, * p < 0.05) Fig. 4: Effect of dexamethasone, PDE4 inhibitor and B1R antagonist on BALF IL-8 concentration following LPS challenge in cynomolgus monkeys. Intratracheal nebulization of LPS led, 12 h later, to a significant increase in BALF concentration of IL-8 (mean ± SEM, n=6 per group, * p < 0.05) [1] Plopper C.G, Hyde D.M. (2008): The non-human primate as a model for studying COPD and Asthma. Pulmonary Pharmacology & Therapeutics. 21(1):755-766. [2] Yawn BP (2012): Is “GOLD” standard for the management of COPD in clinical practice? Drug in context 195:1- 14