Chapter ten - Oral Enzyme Therapy for Celiac Sprue
Introduction
Celiac sprue is a complex inflammatory disease of the small intestine involving both genetic and environmental factors. It is unique among chronic bowel disorders in that a specific dietary component—gluten from wheat and related protein content from rye and barley—has been identified as the proximate environmental cause of inflammation (Dicke et al., 1953). Gluten is a heterogeneous mixture of water insoluble storage proteins comprising monomeric gliadins and polymeric glutenin aggregates up to 10 MDa in size (Wieser, 2007). Both gliadins and glutenins contain repetitive sequences that are rich in proline (Pro; 15%) and glutamine (Gln; 35%), residues that are not preferred substrates for any human digestive enzyme. Consequently, gluten is relatively resistant to gastrointestinal proteolysis, releasing metastable Pro/Gln-rich peptides up to 30–40 amino acids in length into the gut lumen following digestion (Fig. 10.1; Shan et al., 2002, Shan et al., 2005b). Intestinal transglutaminase 2 (TG2) deamidates some of these peptides at specific Gln residues (Molberg et al., 1998, Shan et al., 2002), thereby increasing their affinity for human leukocyte antigen (HLA) DQ2 (Quarsten et al., 1999), a major histocompatibility class II molecule associated with over 90% of celiac patients (Sollid et al., 1989). Deamidated gluten peptide–DQ2 complexes on the surface of antigen-presenting cells (APCs) elicit a potent Th1 inflammatory response from gluten-specific intestinal T cells (Nilsen et al., 1995, Nilsen et al., 1998). Ultimately, this inflammatory response causes destruction of the intestinal architecture, malabsorption of nutrients, and, in many patients, numerous secondary symptoms including diarrhea, wasting, and anemia (Alaedini and Green, 2005). Complete and life-long adherence to a gluten-free diet reverses the signs and symptoms of celiac sprue in most patients. However, the ubiquity of gluten in human diets renders this an extraordinarily difficult prescription to follow, leading to frequent lapses and chronic morbidity in many patients (Ciacci et al., 2002, Cornell et al., 2005, Mayer et al., 1991, Pietzak, 2005). Nondietary therapies that detoxify ingested gluten would therefore improve quality of life considerably for the 1–2% of people afflicted with this life-long disease.
A number of therapeutic alternatives to the gluten-free diet are under development, including enzymatic detoxification of gluten, inhibition of TG2 to prevent peptide deamidation, antagonism of deamidated peptide binding to HLA-DQ2, or suppression of deleterious immune responses (reviewed in Schuppan et al., 2009). In this chapter, we focus on enzymatic detoxification of gluten by oral administration of enzymes capable of digesting gluten (i.e., glutenases).
Oral enzyme therapy is attractive for several reasons:
First, it follows the lead of the established gluten-free diet by targeting the exogenous pathogen, gluten, rather than targeting endogenous effectors. During clinical development, gluten intake and glutenase dose can be titrated in a complementary, dynamic, and individualized manner, thereby enabling systematic analysis of drug pharmacodynamics.
Second, by targeting ingested gluten, oral enzyme therapy avoids the shortcomings of strategies that target gluten at the level of exposure, including the gluten-free diet, selection or genetic engineering of less toxic grains (Molberg et al., 2005, Spaenij-Dekking et al., 2005), or pretreatment of gluten with glutenases, analogous to pretreatment of dairy products with lactase (De Angelis et al., 2006). Because the same proteins that cause toxicity in patients impart the desirable viscoelastic properties of dough, genetic or enzymatic removal of toxic epitopes from gluten will likely reduce baking quality. More importantly, such strategies do not address inadvertent exposure to ubiquitous gluten.
Third, the absence of a bona fide animal model for gluten sensitivity precludes preclinical safety and efficacy testing of drugs targeting endogenous effectors (Bethune and Khosla, 2008). By contrast, oral enzyme therapy can be evaluated in vivo using healthy or existing gluten-sensitive animal models (Bethune et al., 2008, Gass et al., 2006).
Fourth, glutenases that incompletely detoxify gluten as monotherapies can be coadministered with enzymes of complementary specificity to synergistic benefit (Gass et al., 2007a, Siegel et al., 2006).
This chapter describes methods for preclinical evaluation of oral enzyme therapy for celiac sprue. In Section 2, we provide strategies for identifying novel glutenase candidates. In Section 3, we provide protocols for characterizing the enzymatic properties of these candidates using gluten-derived and surrogate substrates. In Section 4, we describe methods for gram-scale production and formulation of promising candidates for delivery to alimentary sites of action. Finally, in Section 5, we discuss prospects for evolving next-generation glutenases and delivering them to the gut by various mechanisms.
Section snippets
Lead Identification
Oral enzyme therapy requires proteases that can digest the uniquely Pro- and Gln-rich sequences constituting immunogenic gluten epitopes. Moreover, these enzymes must be stable and active in the harsh environments of the stomach and/or upper small intestine. In this section, we describe strategies for identifying novel glutenases for oral enzyme therapy. These strategies conform to two general approaches. The first approach is to rationally select glutenase candidates based on therapeutically
Assay Development
A bona fide animal model for celiac sprue has not been discovered or engineered. Assays for glutenase efficacy are therefore conducted in vitro, ex vivo, and in surrogate animal models (e.g., in gluten-tolerant animals or in incomplete animal models of gluten sensitivity). Primary assays utilize biochemical tools such as spectrophotometry, reversed-phase HPLC, and liquid chromatography-assisted mass spectrometry (LC-MS) to identify disease-relevant gluten peptides and to preliminarily assess
Gram-Scale Production and Formulation
Lead glutenases that show promise in early (i.e., in vitro) experiments must be evaluated in more advanced gastrointestinal models and/or animal surrogates prior to human clinical trials. Such studies require gram-scale quantities of glutenase, necessitating high-yield production methods. In this section, we describe the heterologous expression, purification, formulation, and storage of gram-scale quantities of recombinant glutenases.
Lead Optimization for Next-Generation Proteases
In previous sections, we described strategies for identifying and characterizing existing proteases for use in oral enzyme therapy. In this section, we discuss strategies for improving on these enzymes, either by simply coadministering them as a combination glutenase or by augmenting their therapeutically desirable properties through protein engineering. Additionally, we discuss potential modes of delivery for enzyme therapy, oral, and otherwise.
Summary and Outlook
Since the identification in 1953 of gluten as the primary environmental trigger of celiac sprue (Dicke et al., 1953), we have gained an expansive understanding of the genetic, immunological, and biochemical basis for the pathogenesis of this complex disease. Nonetheless, over this same timespan, the prevalence of celiac sprue has increased fourfold to 1–2% of humans (Lohi et al., 2007, Rubio-Tapia et al., 2009) and the only treatment available to this growing population remains a life-long
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