Title: Pharmaceutical Modification of a Gluten-degrading Enzyme Derived From Oral Bacteria
Ghassan Darwish (Presenter)
Eva Helmerhorst, Boston University
Guoxian Wei, Boston University
Objectives: Detoxification of gluten immunogenic epitopes is a strategy for the treatment of celiac disease. Previous studies have demonstrated that the epitopes can be degraded by subtilisin enzyme derived from Rothia mucilaginosa, a natural colonizer of the oral cavity. But the enzyme loses its activity under acidic conditions, as encountered in the stomach. The objective of this study is to protect and maintain enzyme activity by exploring pharmaceutical PEGylation and microencapsulation techniques.
Methods: PEGylation was performed by mixing subtilisin-A (Sub-A) with methoxypolyethylene glycol (mPEG-amine, 5 kDa) in borate buffer (pH 9.0). After stirring, the mixture was quenched with potassium phosphate buffer (pH 6.9), dialyzed to remove no-PEGylated mPEG-amine, followed by lyophilization. The extent of mPEG modification was assessed by the trinitrobenzenesulfonic acid (TNBSA) method. The PEGylated Sub-A (mPEG-Sub-A) was further micro-encapsulated by polylactic glycolic acid (PLGA) using emulsification techniques. The activity of the modified enzyme was evaluated at pH 3.0 using the paranitroanilide-derivatized substrate Suc-AAPF-pNA. The efficacy of detoxifying immunogenic gluten epitopes was assessed using an R5 antibody-based ELISA.
Results: PEGylated subtilisin-A (mPEG-Sub-A) was obtained at a 73% yield. The concentration of Sub-A in mPEG-sub-A was 34% (w/w). The extent of mPEG modification was 55% (~6 of 10 primary amines in Sub-A were PEGylated). For PLGA microencapsulation, the yield of mPEG-Sub-A was 43%. The Sub-A concentration in PLGA-mPEG-Sub-A was 8% (w/w). The PLGA-mPEG-Sub-A showed significantly increased protection against exposure to acid conditions in vitro. A pilot in vivo experiment (n=3) showed that gluten immunogenic epitopes decreased by 39% in the stomach of the mice fed with chow containing PLGA-mPEG-Sub-A (0.2mg Sub-A / g chow) compared to levels in mice fed with chow supplemented with unmodified Sub-A.
Conclusions: Pharmaceutical modification of subtilisins can protect enzyme activity under acidic conditions and such enzymes can be considered a promising modality for future applications to treat celiac disease. These studies were funded by NIH/NIAID grant AI101067, the BU Ignition Award, and the BU CTSI Award.