Safety and efficacy of bovine calf intestinal alkaline phosphatase, a novel therapeutic drug for LPS-mediated diseases

Abstract

Alkaline phosphatases are dimeric, membrane-bound ectoenzymes and can be found in many tissues throughout the body where they serve various physiological functions. In mammals, four isoforms can be distinguished, including placental (PLAP), germ cell (GCAP), intestinal (IAP) and the bone-liver-kidney (BLK) isotypes. In 1997 it was shown that human placental alkaline phosphatase (HPLAP) dephosphorylates and thereby detoxifies LPS at physiological pH levels. LPS is a constituent of the outer membrane of Gram-negative bacteria and is responsible for much of the clinical symptoms associated with Gram-negative sepsis. Despite all innovations in the technical management of critically ill patients and the efforts made in developing and exploring new therapies, sepsis still continues to be one of the leading causes of mortality and morbidity in hospitals with mortality rates of 35-50%. Many therapies against this LPS-mediated disease aimed at neutralizing or antagonizing the production of pro-inflammatory cytokines and mediators or LPS itself. However, none of these trials have been proven effective. This thesis describes pre-clinical and clinical studies that were initiated in order to investigate if bovine calf intestinal alkaline phosphatase (BIAP) can be developed as a novel therapeutic agent in the treatment of Gram-negative sepsis and other LPS-mediated diseases. The main goal of the initial studies was to provide Proof of Principle data on the efficacy of BIAP. The observation that BIAP significantly reduced mortality in mice that had received a lethal doses of E.coli bacteria warranted for further study in a LPS model. For this purpose, piglets were administered LPS with or without BIAP. The results of this study show that BIAP attenuated TNF? levels up to 80%. In addition, changes in hematological parameters were antagonized. In vitro, using macrophages and epithelial cells, BIAP reduced the production of cytokines and nitric oxide after LPS stimulation. The above mentioned results indicate that BIAP may be beneficial in Gram-negative sepsis. However, before BIAP can be applied as therapeutic, safety and tolerability of its administration had to be investigated. For this purpose, single as well as multiple-dosing studies were performed in different animal species as well as humans. The results show that BIAP administration resulted in big differences in immune reactivity towards the protein. Single dosing of BIAP was safe and also multiple dosing of BIAP to dogs and piglets did not result in any adverse effect. However, mice that received multiple doses of BIAP reacted with antibody production and serum sickness. These reactions could be prevented by pretreatment of mice with BIAP in their drinking water. Besides studying safety and tolerability, the pharmacokinetic profile of BIAP was investigated. BIAP clerarance showed to be dose-independent and biphasic with a fast initial distribution phase, followed by a much slower clearance. In conclusion, based on efficacy of the protein as well as safety and tolerability after its administration, BIAP potentially encompasses a novel therapeutic drug in the treatment of LPS-mediated diseases. Results of a clinical phase II in sepsis patients, which is currently ongoing, will learn us if BIAP is indeed beneficial in Gram-negative sepsis.