The enzyme AN-PEP might possibly assist in digesting unintentionally ingested amounts of gluten in those who cannot tolerate gluten. However, demonstrating a treatment effect on (small) clinical deterioration induced by small amounts of gluten in the placebo group may be difficult. Therefore, in this proof of principle study, the enzyme was given to patients consuming large amounts of gluten in a relative small period of time. A two-week safety phase (AN-PEP + gluten) preceded the randomization for AN-PEP or placebo as requested by the medical ethical commission due to concerns about such a high dose of gluten consumption. Unfortunately, the primary aim of the study was not met as the placebo arm did not show any deterioration after 2 wk of gluten consumption. With hindsight, the study should possibly have been designed for a much longer period of time with many more patients.
The baseline characteristics were balanced between groups except for median age at diagnosis, which was 20 years higher in the AN-PEP compared to the placebo group. However, this is unlikely to have influenced the study outcome as no relationship between the age of diagnosis and the response to gluten was observed (data not shown).
The safety phase showed that AN-PEP treatment, when consumed with a high dose of about 7 g of gluten for 2 wk, was safe in patients and no severe adverse events were reported. The CD quality of life scores remained relatively high during 2 wk consumption of gluten and AN-PEP indicating that patients’ general well-being remained high. Serum antibodies of the sixteen patients did not increase when consuming AN-PEP with 7 g of gluten for 2 wk. Also, histology of the biopsies of the majority of patients (fourteen) showed no deterioration while two patients developed increased Marsh scores, however not accompanied by increased antibodies. The safety phase was subject to a so-called “ceiling effect” because patients entered the study on a GFD reflecting relatively healthy baseline values, limiting the ability to demonstrate any further improvement by AN-PEP.
Patients in the placebo group did not show significant deterioration on any of the measured clinical variables after a 2-wk gluten challenge, indicating that 2 wk of gluten challenge is insufficient to induce a clear clinical response in this population of celiac patients. Due to lack of response to gluten in the placebo arm, no treatment effect of AN-PEP could be detected. The measured serum levels of IgA-tTG, IgA-EM, and IgA/G-DGP-tTG antibodies are considered sensitive markers of CD and should be able to detect subtle immunogenic effects of gluten. Similarly, the CD-specific quality of life questionnaire is considered a CD-specific measure of quality of life and should also be able to pick up relevant changes in health[14
]. However, histological examination of small intestinal biopsies may be less reliable than CD-associated antibodies due to heterogeneous distribution of lesions, low grade histopathology, and intra- and interobserver variability[22
]. Interestingly, measures of clinical response to gluten (Marsh scores, antibody titres, quality of life scores) did not correlate in this study, which may in part be explained by the lack of response to gluten. The IgA antibody reactivity to small intestinal mucosa tTG has been considered to be an early marker for gluten-induced pathology in CD patients[23
]. It was observed that intestinal IgA-tTG deposits can be detected in latent CD patients in whom the mucosal villous architecture is still intact, and that the intensity of these mucosal deposits decreased after adherence to a GFD and increased after gluten consumption. Although numbers were low, mucosal IgA-tTG deposits increased in four patients on placebo and one on AN-PEP and decreased in one patient on AN-PEP, compared to baseline values, suggesting that AN-PEP may mitigate gluten exposure.
Some gastrointestinal-related symptoms, mostly mild and transient, were reported during gluten challenge and symptoms between the two groups were comparable suggesting no treatment-related effects on gastrointestinal symptoms. Besides the substantial gluten intake, emotional stress as a consequence of having to ingest gluten might have triggered some of the reported gastrointestinal complaints.
The celiac patients consumed approximately 7 g of gluten daily, which is about half of the average adult daily gluten intake in The Netherlands[24
]. Despite this high gluten dose, no substantial histological, serological, or symptom changes were observed with placebo after 2 wk. In another study[25
] in which adult CD patients consumed approximately 3.5 g/d of gluten from cracker biscuits for 2 wk, only few patients consuming gluten on placebo showed deterioration on histology, serology, and symptoms. Two other studies investigating a gluten challenge in adult patients, based on either lower gluten intake (2.5-5.0 g/d for at least 3 mo)[26
] or comparable gluten intake (4 slices of white bread daily; approximately 8 g/d)[27
] showed that a moderate gluten intake can be tolerated by some patients for several weeks-to-months without significant changes in symptoms[27
] and histology[26,27
]. The time to serological and mucosal relapse and recovery after gluten re-introduction and elimination, respectively, can be highly variable among adult CD patients from several weeks up to many years[27-30
]. Excluding 2 out of 16 patients that may have been more sensitive to gluten from the efficacy phase may, to a small extent, have caused sample bias by selecting patients being less sensitive to gluten. Nevertheless, the same population of patients that entered the efficacy phase was randomly allocated to the AN-PEP or placebo arm. Also attrition bias can be excluded since all patients remained in the study. The lack of substantial clinical response to gluten observed in this study indicates that a longer gluten challenge is likely necessary to induce a significant clinical response to gluten in the majority of patients. For the same reason a longer wash-out period should be considered. Moreover, unresponsiveness to gluten of patients being diagnosed for more than 10 years ago, suggests that future studies may benefit from selecting more recently diagnosed patients.
In conclusion, AN-PEP appeared to be safe in celiac patients. More patients and gluten challenge for a longer period of time seem to be required to induce significant clinical changes and to confirm whether the tendency of AN-PEP to reduce small bowel IgA-tTG deposits is of clinical significance. These results together with previous in vitro evidence that AN-PEP efficiently degrades gluten under simulated gastrointestinal conditions warrant confirmation in a larger trial.