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Non-dietary therapeutic clinical trials in coeliac disease
European Journal of Internal Medicine 23 (2012) 9–14
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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Review article
Non-dietary therapeutic clinical trials in coeliac disease☆ Laura Crespo Pérez a,⁎, Gemma Castillejo de Villasante b, Ana Cano Ruiz a, Francisco León c a b c
Hospital Universitario Ramón y Cajal, Madrid, Spain Hospital Universitario San Joan, Reus, Spain Centocor, Malvern, Pennsylvania, USA
a r t i c l e
i n f o
Article history: Received 30 July 2011 Received in revised form 29 August 2011 Accepted 30 August 2011 Available online 29 September 2011 Keywords: Coeliac disease Treatment Gluten-free diet Larazotide Glutenases
a b s t r a c t Coeliac disease is a permanent immunological intolerance to gluten proteins in genetically predisposed individuals. The only management is life-long strict adherence to a gluten-free diet. Unfortunately, compliance with gluten-free diet is very difficult in practice due to the widespread presence of gluten in Western diets. For this reason, about 50% of coeliacs following a gluten-free diet continue to suffer from symptoms and present with autoantibodies and/or villous atrophy while on a gluten-free diet. It is therefore important to explore new therapies to improve the management of coeliac disease. To date, five experimental therapies have been tested in randomized and controlled clinical trials. Larazotide acetate reduces the para-cellular passage of gluten to the lamina propria by preventing the opening of intercellular tight junctions. The endopeptidases ALV003 and AN-PEP break down gluten to produce less or non-toxic peptide fragments. A therapeutic vaccine is being tested with the aim of developing gluten tolerance. Finally, infection with the nematode Necator americanus and treatment with the CCR9 antagonist Traficet-EN have also been reported. While substantial progress has been made in the last few years, it is important to remember that all these investigational therapies are in research stage and are generally being considered as “adjunctive” therapies to the gluten-free diet and not as substitutes of the gluten-free diet at this point in time. © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction Coeliac disease is one of the most prevalent genetically-determined clinical conditions [1,2]. It is characterized by an excessive immunological reaction against dietary gluten, reaction that starts in the small intestine. This reaction triggers an autoimmune response against several auto-antigens, and results in the development of intestinal and extraintestinal manifestations [3–6]. Several epidemiological studies have estimated a prevalence of one case per 130–400 individuals in the general population [3,6], with lower prevalence in Asian countries. Coeliac disease may start in the first years of life and remain undiagnosed until adulthood (the average diagnostic delay in the USA is 11 years). In other cases, the true onset takes place in adult life [7]. The most frequent clinical presentation nowadays is with concomitant intestinal and extra-intestinal manifestations, up to 15 times more common than the presence of intestinal symptoms in isolation [3].
☆ No grant support. Conflicts of interest: LCP, GCV and ACR have been investigators in clinical studies sponsored by Alba Therapeutics. FL is an ex-employee of Alba Therapeutics who does not currently own stock or stock options of the company. ⁎ Corresponding author at: Hospital Universitario Ramón y Cajal, Madrid, Carretera de Colmenar, Km. 9100, 28034, Madrid, Spain. Tel.: +34 913368354, +34 913368771; fax: +34 913368354. E-mail address: [email protected] (L. Crespo Pérez).
Coeliac disease is heavily under-diagnosed, as for every patient diagnosed there are 5–10 patients who are not [8–10]. The discovery and introduction in clinical practice of sensitive and specific serological tests (anti-endomysial -EMA- and anti-tissue transglutaminase antibodies -anti-tTG-) [6] have increased the diagnosis rates. It is crucial to diagnose this condition early on, since death associated with coeliac disease is 2–4 times greater than in the general population, mainly associated with an increase in T- and B-cell lymphomas and, to a lesser extent, other digestive tumors [3–4]. 2. Pathogenesis of coeliac disease and “points of attack” Coeliac disease has a multi-factorial pathogenesis. Genetic, immunological and environmental factors (gluten, intestinal infections) are involved [3,6]. Coeliac disease presents one of the closest associations described with the HLA region. In most populations, N90% of patients express the HLA-DQ2 heterodimer encoded by alleles DQA1*0501 and DQB1*02. The remainder express the HLA-DQ8 heterodimer encoded by alleles DQA1*03 and DQB1*0302 [3,6]. The predominant role of HLA DQ2 is explained by the fact that the gluten peptides modified by the tissue transglutaminase enzyme have an increased affinity for the DQ2 molecules of the antigen-presenting cells [2–6]. Gluten is a complex mixture of polypeptides present in cereals such as wheat, barley, rye and oats. It consists of two fractions: an alcoholsoluble fraction called gliadin, hordein, secalin or avenin depending on
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the cereal of origin (wheat, barley, rye and oats, respectively) and an insoluble fraction called glutenin [3,6,11]. Given the high content in proline and glutamine residues of gluten proteins, they are highly resistant to digestion by gastrointestinal enzymes since they lack major cleaving points for such proteases [3,6]. When these incompletely-processed peptides reach the lamina propria, they are an adequate substrate for the tissue transglutaminase enzyme (tTG), which deamidates and transforms glutamine residues into glutamic acid, producing negatively-charged peptides that are presented by HLA Class II DQ2 and DQ8 molecules [11–13]. One of these peptides, known as 33-mer, has a highly immunogenic sequence that is recognized by T-cells of the intestinal mucosa, and in all there are more than 200 immunogenic peptides in gluten [13]. They trigger the activation of CD4 T helper cells in the lamina propria, leading to intestinal inflammation and ultimately hyperplasia of the crypts and atrophy of the intestinal villi [14]. It is not fully understood how gluten peptides reach the lamina propria of the gut. There is some initial evidence that this can occur by epithelial trans-cytosis [15] and by the para-cellular route through disassembled epithelial tight junctions [16]. Following antigen presentation of such immunogenic peptides to lamina propria CD4-positive T-cells (presumably in the lymph nodes), responding T-cells are primed towards a Th1 cytokine profile mainly mediated by secretion of IFN-γ [17], although other Th1/Th17 related cytokines have been related as well (IL-15 [18], IL-21, IL-23 and IL-17 [19]). The final result is intestinal damage with villous atrophy and hyperplasia of the crypts, with a reduced intestinal absorptive surface (Fig. 1). The experimental therapeutic approaches under investigation have complementary “points of attack”. In the future this will possibly
mean that coeliac disease may be treated by a combination of two or more drugs. To date, 5 of these experimental approaches have been tested in clinical trials. Larazotide acetate (AT-1001) is used to prevent the passage of gluten peptides across intestinal epithelial tight junctions by regulating para-cellular permeability. The peptidases ALV003 and AN-PEP are used to digest gluten peptides and prevent the formation of toxic peptides. A desensitizing vaccine (NexVax2) is being tested in an attempt to use 3 gluten peptides to induce T cell immune tolerance in HLA-DQ2 positive coeliac patients. Finally, studies have been reported for an antagonist of the CCR9 chemokine receptor on T-cells (CCX282B, Traficet-EN®), and for the use of the parasite Necator americanus to try to reduce reactivity against gluten. These potentially complementary approaches are depicted in Fig. 2 and summarized in Table 1. 3. Current management of coeliac disease: gluten-free diet A gluten-free diet is a diet that excludes all products containing gluten. In other words, all products made from flours of wheat, barley, rye and – due to frequent cross-contamination – oats. There is controversy over the safety of oats for coeliac subjects, as oats have lower prolamine content than the other three cereals widely recognized as toxic. Toxic effects of oats are only observed long-term and may be only triggered by contamination with other cereals. Regardless, most countries advise against including them in the gluten-free diet [20–22]. The main difficulty to adhere strictly to a gluten-free diet is that cereal flours are widely used in the food industry and are present in
Fig. 1. Pathogenesis of coeliac disease. Gluten peptides in the diet are highly resistant to breakdown by intestinal proteases. They are believed to cross the intestinal epithelium via trans-cellular (receptor-mediated) and para-cellular (across open tight junctions) routes. Once in the lamina propria, they are deamidated by the enzyme tissue transglutaminase (tTG2 or TG2), producing highly immunogenic epitopes. These epitopes are presented via HLA DQ2 or DQ8 by antigen-presenting cells (APC) to the CD4 positive T cells. As a result of this, lymphocyte activation leads to the production of inflammatory cytokines and an increase in intraepithelial lymphocytes (IEL), eventually leading to mucosal remodeling with hyperplasia of the crypts and atrophy of the villi. In addition, B cell activation and loss of tolerance lead to the production of anti-transglutaminase antibodies (anti-tTG). Adapted from: Green PH, Cellier C. Celiac disease. N Engl J Med 2007; 357: 1731–1743 and from Schuppan D, Junker Y, Barisani D. Celiac Disease: From pathogenesis to novel therapies. Gastroenterology 2009; 137:1912–1933 [3,6].
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Fig. 2. Mechanisms of action and sites of attack of the potential therapeutic alternatives under investigation in coeliac disease. Gluten peptides in the diet would cross the epithelium via para-cellular or trans-cellular pathways and reach the lamina propria where they are deamidated by the tissue transglutaminase enzyme (TG2), producing highly immunogenic epitopes. Larazotide acetate (AT-1001) is designed to reduce the paracellular transport of gluten peptides by regulating intestinal tight junctions. Glutenases like ALV003 and AN-PEP break down gluten fragments into small peptides proposed to be less or non-toxic. Other drugs currently under study are the antagonist of the CCR9 chemokine receptor on T-cells (CCX282B or Traficet-EN®), the therapeutic vaccine NexVax2 intended to induce immune tolerance in HLA-DQ2 positive patients, and the subcutaneous inoculation of Necator americanus.
numerous food products. In addition, labeling of food products is deficient in many countries. For these reasons, coeliac sufferers are regularly exposed to gluten contamination in the food and beverages they consume. This exposure and its consequences result in a limitation of social activities and/or a reduction in the variety of foods consumed. Moreover, specifically manufactured gluten-free products may be difficult to find, tend to be less flavorful and are more expensive than regular gluten containing products. It has been estimated that 30–50% of coeliac sufferers are not able to strictly follow a
gluten-free diet for a prolonged period of time and have recurrent symptoms and signs [20,23–24]. Eventually, non-compliance with the gluten-free diet, either willful or inadvertent, results in higher morbidity (anemia, osteopenia, repeated miscarriages, delayed intrauterine fetal growth, other autoimmune diseases) and mortality (due to the increased risk of neoplasms, mainly of the digestive tract and lymphomas) [3]. For these reasons, alternative treatments that can act in combination with the gluten-free diet are required in order to improve the quality of life of coeliac patients. These new therapeutic
Table 1 Major non-dietary therapeutic clinical trials in coeliac disease. Investigational agent
Phase of trials
Investigator-company country
Mechanism of action
Route of administration
Findings
Safety and tolerability
Larazotide acetate (AT-1001) ALV003
2b
Alba Therapeutics, USA
Oral
AN-PEP
2a
Alvine Pharmaceuticals, USA DSM, Holland
NEXVAX2
1
NECATOR AMERICANUS Traficet-EN® (CCX282B)
2a
Ameliorated anti-tTG and symptom development in gluten challenges Reduction in blood markers of immunological activation Reduction in intestinal deposits of anti-tTG IgA antibodies. Development of IFN-gamma-producing anti gluten T-cells Better tolerance and reduced symptoms in gluten challenges Limits migration of T cells from the blood flow to the intestinal mucosa
Comparable to placebo
2a
Prevents opening of intestinal epithelial tight junctions Combination of two different gluten degrading proteases Prolyl-endoprotease derived from Aspergillus niger Desensitizing vaccine with 3 gluten peptides Inhibits Th1 immune response by inducing a Th2 response Inhibits CCR9, a chemokine receptor for gut T cell homing
2a
NEXPep, ImmuSanT, Australia/USA Princess Adelaide University, Australia ChemoCentryx, GlaxoSmith-Kline, USA
Oral Oral SC injection Skin incision Oral
Comparable to placebo Comparable to placebo Gastrointestinal Symptoms Active infection with helminth Not reported
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approaches must be safe, effective and reasonably priced. To date, the objective of clinical trials has been to neutralize the effects of small amounts of gluten. 4. The intake of small amounts of gluten damages the intestinal mucosa The gluten-free diet poses a challenge for the coeliac patient. Motivation to comply is related to age (worse in adolescents) and the degree of symptoms (more in symptomatic). In addition, contamination of naturally gluten-free food products and of manufactured gluten-free products makes patients feel insecure about their diet. It is also difficult to determine the gluten contents of foods. Currently, ELISA techniques are used, employing monoclonal or polyclonal antibodies against wheat components. Recently, new analytical methods have been developed to detect barley and rye gluten. Unfortunately, comparison of the results obtained with different ELISA techniques reveals inconsistency in these methods (especially for low contents near to the toxic threshold). In northern European countries, amounts up to 100 parts per million (ppm) are permitted in gluten-free products designated apt for coeliac sufferers. A more conservative limit of 20 ppm is established in the United States and Southern Europe [22]. In a double-blind, placebocontrolled prospective study, Catassi et al. demonstrated that an intake of 50 mg of gluten per day for 3 months was sufficient to cause a significant decrease in the gut mucosa villous height/crypt depth ratio [23]. No clinical or serological correlation (IgA anti-transglutaminase) was found with these gluten traces. A recent review suggested that daily intakes of gluten of lower than 10 mg are unlikely to produce significant histological abnormalities [25]. With the threshold at less than 20 ppm of gluten in special food products for coeliac, an intake of less than 50 mg/day can be achieved, providing a sufficient safety margin [26]. In spite of these recommendations, approximately 50% (30–80%) of all coeliac subjects on a gluten-free diet have active disease at any given time in the US and Europe [20,24]. 5. Therapeutic clinical trials in coeliac disease 5.1. Inhibition of intestinal paracellular permeability: larazotide acetate (AT-1001) The permeability of the intestinal epithelium is partly regulated by the tight junctions, dynamic intercellular points of contact between the enterocytes (intestinal epithelial cells), closely linked to the underlying cellular cytoskeleton [16]. Tight junctions regulate the passage of fluids and molecules through the narrow space between epithelial cells (para-cellular space). In normal conditions, harmful bacteria and dietary antigens are prevented from passing through tight junctions (nutrients enter the body through trans-cellular mechanisms via cell surface receptors or passive diffusion across the cell membrane). In coeliac disease and other immune intestinal diseases, inflammatory cytokines trigger the opening of tight junctions and increase paracellular permeability [16], potentially allowing the entry of harmful gluten peptides into the lamina propria. Larazotide acetate (AT-1001, developed by Alba Therapeutics [27]) is an octapeptide derived from a cholera toxin, ZOT [28–29], that prevents the opening of intestinal epithelial tight junctions by acting on the cytoskeleton. Larazotide acetate is expected to reduce the paracellular transport of gluten to the lamina propria, ameliorating the activation of the pathological immune cascade. Six clinical trials have been completed with larazotide acetate. Three of them were Phase 1 trials: two trials in healthy individuals (one single-dose and other multiple dose safety studies) and a third safety study in subjects with coeliac disease. In all studies, the safety profile was comparable to placebo. Subsequently, three Phase 2 studies have been completed. A Phase 2a trial was a randomized, placebo-
controlled, double-blind trial with 86 coeliac patients in remission with no symptoms or detectable autoantibodies [27]. The patients took a gluten challenge of 800 mg gluten 15 min after administration of AT-1001 or placebo, three times a day for two weeks (approximately 2.5 g of gluten per day). AT-1001 was well-tolerated at all the doses tested (0.25 to 8 mg) and showed safety and tolerability comparable to placebo. While the primary endpoint of intestinal paracellular permeability was not met, the symptoms triggered by the 2-week gluten challenge were significantly ameliorated by larazotide acetate [27]. In addition, two larger Phase 2b studies have been carried out. In the first Phase 2b study (randomized, placebo-controlled, double-blind) different doses of larazotide (1, 4 and 8 mg) were compared against placebo. The study enrolled 184 coeliac patients in remission to be subjected to a 6-week gluten challenge of 900 mg administered 3 times a day. The results have been presented in abstract form and are submitted [30]. Again, larazotide acetate could not demonstrate statistically significant efficacy in the reduction in intestinal permeability, though a favorable trend was observed [30]. Importantly, larazotide acetate did result in a beneficial reduction in the signs (anti-tissue transglutaminase antibodies, anti-tTG IgA) and symptoms of the gluten challenge [30] and the safety profile was satisfactory. The second Phase 2b study was a randomized, placebo-controlled, double-blind trial in coeliac patients with active disease who initiated a strict gluten-free diet at the beginning of the study and were treated for two months. The 3 arms studied were larazotide acetate 4 mg three times a day, larazotide acetate 8 mg three times a day and a placebo group. The results of this study have not been made public to date. A new large (320 patients) Phase 2b study has been announced by Alba Therapeutics and partner Cephalon, to study 0.5, 1 and 2 mg per day of larazotide acetate in coeliac subjects who are non-responsive to a gluten-free diet (ClinicalTrials.gov identifier: NCT01396213). 5.2. Enzyme therapy: endopeptidases (ALV003 and AN-PEP) When gluten polypeptides are hydrolyzed they lose the capacity to stimulate the intestinal immune system and damage the intestine. ALV003 (developed by Alvine Pharmaceuticals) is a combination of two different gluten-targeting proteases (“glutenases”) with complementary substrates: a cysteine-endoprotease derived from germinated barley seeds and a prolyl-endopeptidase from Sphingomonas capsulatum [31]. Both are active in acidic medium and their glutenase activity is maximized in a 1:1 formulation administered orally. A Phase 1 clinical trial was conducted with 20 coeliac patients in remission who were randomized to receive a diet containing gluten (16 g a day for 3 days) pre-treated with ALV003 (10 patients) versus a diet containing gluten pre-treated with placebo (10 patients). The group given ALV003-treated gluten presented a reduction in markers of immunological activation (IFN-γ production measured with the ELISpot technique) [31]. A subsequent Phase 1b study showed that ALV003 is very effective, in the stomach, in breaking down foods with high gluten content [32]. Alvine Pharmaceuticals has recently conducted a larger 6-week Phase 2a study of ALV003 administered to coeliac subjects exposed to a gluten challenge. This study was a randomized, placebo-controlled, double-blind trial in well-controlled coeliac individuals instructed to take 6 g a day of gluten. The results have not been made public at the time of this writing. A second prolyl-endoprotease called AN-PEP is being developed by an alimentary company, DSM. AN-PEP is derived from Aspergillus niger [33]. In an interventional, cross-over, randomized, doubleblind, placebo-controlled clinical trial, AN-PEP's ability to detoxify 8 g of gluten contained in a commercial food product was tested. The primary objective of the study was the change in histology, and other endpoints measured included peripheral blood T-cells and coeliac antibodies. The study was conducted in 14 patients who ate food prepared with gluten together with AN-PEP for 2 weeks. After a
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second 2-week washout period (in which they were kept on a strict gluten-free diet), patients started the third 2-week period for which they were randomized to continue eating the same food containing gluten and AN-PEP, or gluten and placebo. The results have been communicated in abstract form and there was no significant effect of ANPEP on pre-specified endpoints although a post-hoc analysis showed a possible reduction in the intestinal deposits of anti-tTG IgA antibodies. The safety profile was satisfactory [33]. In a commentary, Donnelly et al. speculate that this trial studied a very large dose of gluten with a relatively brief wash-out period between periods while peptidases may be more appropriate for the digestion of minor quantities of gluten or in the pre-treatment of gluten-free products that contain a small amount of gluten [34]. There is currently a new clinical trial announced for AN-PEP in 12 subjects to understand the extent of the degradation of gluten in the stomach (ClinicalTrials.gov identifier: NCT01335503). In addition to these two company-sponsored clinical trials with proteases, there is a third protease mix being tested by an academic institution, Stanford University. The group led by Prof. Khosla is conducting a Phase I + II clinical trial with STAN1 (a mix of commerciallyavailable proteases) at the Heim Pal Children's Hospital in Hungary. The results are not available at the time of this writing. (ClinicalTrials.gov identifier: NCT00962182). 5.3. Therapeutic vaccine: Nexvax2 NexVax2 is a desensitizing or therapeutic “vaccine” under development by biotechnology companies NexPep and ImmuSanT. NexVax2 uses 3 gluten peptides with the goal of inducing a “tolerogenic” response in coeliac patients with HLA DQ2. NexVax2 has showed efficacy in mice with gluten-sensitive T-cells and transgenic for HLA DQ2 [35]. The phase 1 study, of recent completion, consisted of subcutaneous injections of increasing doses of up to 90 μg of Nexvax2 in 40 patients with well controlled coeliac disease, weekly for 3 weeks. The results have been communicated in abstract form. While there were some gluten-related gastrointestinal side effects, dose escalation could be completed and safety was acceptable. The investigators also reported the development of IFN-gamma-producing anti-gluten T cells in vaccinated subjects [36]. 5.4. Modulation of immunity with Necator americanus and probiotics This research project is based on the theory that the human immune system needs to be exposed to exogenous organisms in order to function correctly (“hygiene hypothesis”) [37]. The disappearance of certain microbial strains from the gut of subjects residing in developed countries could be the reason for the increased prevalence of immune-mediated illnesses such as coeliac disease, Crohn's disease, ulcerative colitis or asthma [37]. Survival of the microbes in the intestine is dependent on their ability to interfere with the immune response of the host. The mechanisms employed to do this are similar to those used to regulate autoimmune diseases. Researchers suspect that removal of microbes from the environment could lead predisposed individuals to develop autoimmune diseases [37]. Researchers at the Prince Adelaide University in Brisbane, Australia, postulated that the parasite Necator americanus would inhibit the Th1 immune response against gluten in coeliac subjects by inducing a Th2 response. A Phase 2a randomized, double-blind, placebo-controlled study was completed with a 3 to 5 day gluten challenge in 20 well-controlled coeliac subjects inoculated with Necator americanus or placebo. The results have been communicated in abstract form [38]. Parasite larvae were subcutaneously injected at two time points (ten larvae at time points 0 and 5 more larvae at 12 weeks). Patients in the placebo group were injected with a preparation containing a few drops of Tabasco sauce to mimic the irritation caused by the larvae. Patients hosting the live parasite tolerated the gluten challenge better and had better scores in the digestive symptom questionnaires
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compared with control subjects without the parasites [38]. They also had less mucosal inflammation, although these results were not statistically significant. At the end of the 21 weeks of the study, the volunteers were offered medication to remove the parasites but most patients chose to keep them [38]. In a related effort, Prof. Bai has announced a 20-patient clinical trial to study the effects of Bifidobacterium infantis, a probiotic with immune regulatory properties, in active celiac disease at the Dr. C. Bonorino Udaondo Gastroenterology Hospital, Buenos Aires, Argentina (ClinicalTrials.gov identifier: NCT01257620). 5.5. Immunomodulators: CCX282b (Traficet-EN®) CCR9 is expressed at high levels on lamina propria mononuclear cells in the small intestine [4], and its ligand CCL25/TECK is expressed on both lamina propria capillaries and on small intestine enterocytes. This chemokine receptor controls the trafficking of T-cells selectively migrating to the intestinal tract [40]. CCX282B (Traficet-EN®), in development by ChemoCentryx and Glaxo Smith Kline for Crohn's and coeliac disease, inhibits the CCR9 receptors of T-cells limiting their migration from the blood flow to the intestinal mucosa [6,39]. Traficet-EN® is administered via oral route. A gluten challenge study was carried out in 2008 using Traficet-EN® in 90 patients (Phase 2, randomized, double blind, placebo-controlled study). The results have not been made public to date. In a review, Solid and Khosla discuss a potential concern for intestinal infections if the CCR9 antagonists are effective given the non-antigen-specific mode of action, and recommend careful monitoring [40]. 6. Summary Coeliac disease is a highly prevalent autoimmune disease triggered by dietary gluten, with no approved medication. Coeliac disease leads to morbidity and increased mortality. Gluten is “everywhere” and is very hard to eliminate from the diet. While a strict gluten-free diet eliminates most of the risk for complications of coeliac disease, at any given time approximately 50% of subjects have active disease, mostly due to non-compliance, either willful or inadvertent. While substantial progress has been made in the last few years in the development of experimental medications for coeliac disease, all the new proposed therapies remain in early research stage, and no Phase 3 trial has ever been conducted in coeliac disease. Further progress is needed in the design of clinical trials and the use of surrogate endpoints in coeliac disease [40]. The first medication(s) for coeliac disease will likely be adjunct to the gluten-free diet and the importance of strict adherence to this difficult diet cannot be overemphasized at this time. Learning points • Despite a high rate of non-responsiveness to the gluten-free diet, there is no pharmacological approach approved for coeliac disease, a highly prevalent chronic autoimmune disease. • Clinical research for coeliac disease medications is in its infancy and only a handful of compounds have reached Phase 2 studies. Conflicts of interest Francisco Leon is an ex-employee of Alba Therapeutics, a biotechnology company working in coeliac disease. He does not currently own stock or stock options in the company. The rest of the authors have been investigators in clinical trials sponsored by Alba Therapeutics. References [1] Di Sabatino A, Corazza GR. Coeliac disease. Lancet 2009;373:1480–93. [2] Farrel RF, Nelly CP. Celiac sprue. N Engl J Med 2002;346:180–8.
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[23] Catassi C, Fabiani E, Iacono G, D'Agate C, Francavilla R, Biagi F, et al. A prospective, double blind, placebo controlled trial to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr 2007;85:160–6. [24] Lanzini A, Lanzarotto F, Villanacci V, Mora A, Bertolazzi S, Turini D, et al. Complete recovery of intestinal mucosa occurs very rarely in adult celiac patients despite adherence to gluten-free diet. Aliment Pharmacol Ther 2009;29:1299–308. [25] Akobeng AK, Thomas AG. Systematic review: tolerable amount of gluten for people with coeliac disease. Aliment Pharmacol Ther 2008;27:1044–52. [26] Gibert A, Espadaler M, Canela A, Sánchez A, Vaqué C, Rafecas M, et al. Consumption of gluten-free products: should the threshold value for trace amounts of gluten be 20, 100 or 200 ppm? Eur J Gastroenterol Hepatol 2006;18:1187–95. [27] Paterson BM, Lammers KM, Arrieta MC, Fasano A, Meddings JB. The safety, tolerance, pharmacokinetic and pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of concept study. Aliment Pharmacol Ther 2007;26:757–66. [28] Fasano A, Uzzau S, Fiore C, Margaretten K. The enterotoxic effect on zonula occludens toxin on rabbit small intestine involves the paracellular pathway. Gastroenterology 1997;112:839–46. [29] Marinaro M, Fasano A, De Magistris MT. Zonula occludens toxin acts as an adjuvant through different mucosal routes and induces protective immune responses. Infect Immun 2003;71:1897–902. [30] Kelly CP, Green PHR, Murray JA, Di Marino A, Colatrella A, Leffler DA, et al. Effect of larazotide acetate on the prevention of signs and symptoms of coeliac disease in patients undergoing a gluten challenge: a randomised placebo-controlled study. Gastroenterology 2009;136:M2048. [31] Pyle GG, Paaso B, Anderson BE, Allen DD, Marti T, Li Q, et al. Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin Gastroenterol Hepatol 2005;3:687–94. [32] Tye-Din JA, Anderson RP, Ffrench RA, Brown GJ, Hodsman P, Siegel M, et al. The effects of ALV003 pre-digestion of gluten on immune and symptoms in celiac disease in vivo. Clin Immunol 2010;134:289–95. [33] Tack GJ, Van de water J, Kooy-Winkelaar EM, van Bergen J, Meijer GA, von Blomberg BM, et al. Can prolyl-endoprotease enzyme treatment mitigate the toxic effect of gluten in coeliac patients? Gastroenterology 2010;138:S54. [34] Donnelly SC, Ellis HJ, Ciclitira PJ. Pharmacological and management strategies for coeliac disease. Expert Opin Pharmacother 2011;12:1731–44. [35] de Kauwe AL, Chen Z, Anderson RP, Keech CL, Price JD, Wijburg O, et al. Resistance to celiac disease in humanized HLA-DR3-DQ2-transgenic mice expressing specific anti-gliadin CD4+ T cells. J Immunol 2009;182:7440–50. [36] Brown GJ, Daveson J, Marjason JK, Ffrench RA, Smith D, Sullivan M, et al. A phase i study to determine safety, tolerability and bioactivity of Nexvax2® in HLA DQ2+ volunteers with celiac disease following a long-term, strict gluten-free diet. Gastroenterology 2011;140:S437–8. [37] Okada H, Kuhn C, Feillet H, Bach JF. The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update. Clin Exp Immunol 2010;160:1–9. [38] Daveson AJ, Jones DM, Gaze S, McSorley H, Clouston A, Pascoe A, et al. Effect of hookworm infection on wheat challenge in celiac disease–a randomised doubleblinded placebo controlled trial. PLoS One 2011;6:e17366. [39] Rodrigo L. Investigational therapies for celiac disease. Expert Opin Investig Drugs 2009;18:1865–73. [40] Sollid LM, Khosla C. Novel therapies for Coeliac Disease. J Intern Med 2011;296: 604–13.
Contents lists available at SciVerse ScienceDirect
European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim
Review article
Non-dietary therapeutic clinical trials in coeliac disease☆ Laura Crespo Pérez a,⁎, Gemma Castillejo de Villasante b, Ana Cano Ruiz a, Francisco León c a b c
Hospital Universitario Ramón y Cajal, Madrid, Spain Hospital Universitario San Joan, Reus, Spain Centocor, Malvern, Pennsylvania, USA
a r t i c l e
i n f o
Article history: Received 30 July 2011 Received in revised form 29 August 2011 Accepted 30 August 2011 Available online 29 September 2011 Keywords: Coeliac disease Treatment Gluten-free diet Larazotide Glutenases
a b s t r a c t Coeliac disease is a permanent immunological intolerance to gluten proteins in genetically predisposed individuals. The only management is life-long strict adherence to a gluten-free diet. Unfortunately, compliance with gluten-free diet is very difficult in practice due to the widespread presence of gluten in Western diets. For this reason, about 50% of coeliacs following a gluten-free diet continue to suffer from symptoms and present with autoantibodies and/or villous atrophy while on a gluten-free diet. It is therefore important to explore new therapies to improve the management of coeliac disease. To date, five experimental therapies have been tested in randomized and controlled clinical trials. Larazotide acetate reduces the para-cellular passage of gluten to the lamina propria by preventing the opening of intercellular tight junctions. The endopeptidases ALV003 and AN-PEP break down gluten to produce less or non-toxic peptide fragments. A therapeutic vaccine is being tested with the aim of developing gluten tolerance. Finally, infection with the nematode Necator americanus and treatment with the CCR9 antagonist Traficet-EN have also been reported. While substantial progress has been made in the last few years, it is important to remember that all these investigational therapies are in research stage and are generally being considered as “adjunctive” therapies to the gluten-free diet and not as substitutes of the gluten-free diet at this point in time. © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction Coeliac disease is one of the most prevalent genetically-determined clinical conditions [1,2]. It is characterized by an excessive immunological reaction against dietary gluten, reaction that starts in the small intestine. This reaction triggers an autoimmune response against several auto-antigens, and results in the development of intestinal and extraintestinal manifestations [3–6]. Several epidemiological studies have estimated a prevalence of one case per 130–400 individuals in the general population [3,6], with lower prevalence in Asian countries. Coeliac disease may start in the first years of life and remain undiagnosed until adulthood (the average diagnostic delay in the USA is 11 years). In other cases, the true onset takes place in adult life [7]. The most frequent clinical presentation nowadays is with concomitant intestinal and extra-intestinal manifestations, up to 15 times more common than the presence of intestinal symptoms in isolation [3].
☆ No grant support. Conflicts of interest: LCP, GCV and ACR have been investigators in clinical studies sponsored by Alba Therapeutics. FL is an ex-employee of Alba Therapeutics who does not currently own stock or stock options of the company. ⁎ Corresponding author at: Hospital Universitario Ramón y Cajal, Madrid, Carretera de Colmenar, Km. 9100, 28034, Madrid, Spain. Tel.: +34 913368354, +34 913368771; fax: +34 913368354. E-mail address: [email protected] (L. Crespo Pérez).
Coeliac disease is heavily under-diagnosed, as for every patient diagnosed there are 5–10 patients who are not [8–10]. The discovery and introduction in clinical practice of sensitive and specific serological tests (anti-endomysial -EMA- and anti-tissue transglutaminase antibodies -anti-tTG-) [6] have increased the diagnosis rates. It is crucial to diagnose this condition early on, since death associated with coeliac disease is 2–4 times greater than in the general population, mainly associated with an increase in T- and B-cell lymphomas and, to a lesser extent, other digestive tumors [3–4]. 2. Pathogenesis of coeliac disease and “points of attack” Coeliac disease has a multi-factorial pathogenesis. Genetic, immunological and environmental factors (gluten, intestinal infections) are involved [3,6]. Coeliac disease presents one of the closest associations described with the HLA region. In most populations, N90% of patients express the HLA-DQ2 heterodimer encoded by alleles DQA1*0501 and DQB1*02. The remainder express the HLA-DQ8 heterodimer encoded by alleles DQA1*03 and DQB1*0302 [3,6]. The predominant role of HLA DQ2 is explained by the fact that the gluten peptides modified by the tissue transglutaminase enzyme have an increased affinity for the DQ2 molecules of the antigen-presenting cells [2–6]. Gluten is a complex mixture of polypeptides present in cereals such as wheat, barley, rye and oats. It consists of two fractions: an alcoholsoluble fraction called gliadin, hordein, secalin or avenin depending on
0953-6205/$ – see front matter © 2011 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2011.08.030
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the cereal of origin (wheat, barley, rye and oats, respectively) and an insoluble fraction called glutenin [3,6,11]. Given the high content in proline and glutamine residues of gluten proteins, they are highly resistant to digestion by gastrointestinal enzymes since they lack major cleaving points for such proteases [3,6]. When these incompletely-processed peptides reach the lamina propria, they are an adequate substrate for the tissue transglutaminase enzyme (tTG), which deamidates and transforms glutamine residues into glutamic acid, producing negatively-charged peptides that are presented by HLA Class II DQ2 and DQ8 molecules [11–13]. One of these peptides, known as 33-mer, has a highly immunogenic sequence that is recognized by T-cells of the intestinal mucosa, and in all there are more than 200 immunogenic peptides in gluten [13]. They trigger the activation of CD4 T helper cells in the lamina propria, leading to intestinal inflammation and ultimately hyperplasia of the crypts and atrophy of the intestinal villi [14]. It is not fully understood how gluten peptides reach the lamina propria of the gut. There is some initial evidence that this can occur by epithelial trans-cytosis [15] and by the para-cellular route through disassembled epithelial tight junctions [16]. Following antigen presentation of such immunogenic peptides to lamina propria CD4-positive T-cells (presumably in the lymph nodes), responding T-cells are primed towards a Th1 cytokine profile mainly mediated by secretion of IFN-γ [17], although other Th1/Th17 related cytokines have been related as well (IL-15 [18], IL-21, IL-23 and IL-17 [19]). The final result is intestinal damage with villous atrophy and hyperplasia of the crypts, with a reduced intestinal absorptive surface (Fig. 1). The experimental therapeutic approaches under investigation have complementary “points of attack”. In the future this will possibly
mean that coeliac disease may be treated by a combination of two or more drugs. To date, 5 of these experimental approaches have been tested in clinical trials. Larazotide acetate (AT-1001) is used to prevent the passage of gluten peptides across intestinal epithelial tight junctions by regulating para-cellular permeability. The peptidases ALV003 and AN-PEP are used to digest gluten peptides and prevent the formation of toxic peptides. A desensitizing vaccine (NexVax2) is being tested in an attempt to use 3 gluten peptides to induce T cell immune tolerance in HLA-DQ2 positive coeliac patients. Finally, studies have been reported for an antagonist of the CCR9 chemokine receptor on T-cells (CCX282B, Traficet-EN®), and for the use of the parasite Necator americanus to try to reduce reactivity against gluten. These potentially complementary approaches are depicted in Fig. 2 and summarized in Table 1. 3. Current management of coeliac disease: gluten-free diet A gluten-free diet is a diet that excludes all products containing gluten. In other words, all products made from flours of wheat, barley, rye and – due to frequent cross-contamination – oats. There is controversy over the safety of oats for coeliac subjects, as oats have lower prolamine content than the other three cereals widely recognized as toxic. Toxic effects of oats are only observed long-term and may be only triggered by contamination with other cereals. Regardless, most countries advise against including them in the gluten-free diet [20–22]. The main difficulty to adhere strictly to a gluten-free diet is that cereal flours are widely used in the food industry and are present in
Fig. 1. Pathogenesis of coeliac disease. Gluten peptides in the diet are highly resistant to breakdown by intestinal proteases. They are believed to cross the intestinal epithelium via trans-cellular (receptor-mediated) and para-cellular (across open tight junctions) routes. Once in the lamina propria, they are deamidated by the enzyme tissue transglutaminase (tTG2 or TG2), producing highly immunogenic epitopes. These epitopes are presented via HLA DQ2 or DQ8 by antigen-presenting cells (APC) to the CD4 positive T cells. As a result of this, lymphocyte activation leads to the production of inflammatory cytokines and an increase in intraepithelial lymphocytes (IEL), eventually leading to mucosal remodeling with hyperplasia of the crypts and atrophy of the villi. In addition, B cell activation and loss of tolerance lead to the production of anti-transglutaminase antibodies (anti-tTG). Adapted from: Green PH, Cellier C. Celiac disease. N Engl J Med 2007; 357: 1731–1743 and from Schuppan D, Junker Y, Barisani D. Celiac Disease: From pathogenesis to novel therapies. Gastroenterology 2009; 137:1912–1933 [3,6].
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Fig. 2. Mechanisms of action and sites of attack of the potential therapeutic alternatives under investigation in coeliac disease. Gluten peptides in the diet would cross the epithelium via para-cellular or trans-cellular pathways and reach the lamina propria where they are deamidated by the tissue transglutaminase enzyme (TG2), producing highly immunogenic epitopes. Larazotide acetate (AT-1001) is designed to reduce the paracellular transport of gluten peptides by regulating intestinal tight junctions. Glutenases like ALV003 and AN-PEP break down gluten fragments into small peptides proposed to be less or non-toxic. Other drugs currently under study are the antagonist of the CCR9 chemokine receptor on T-cells (CCX282B or Traficet-EN®), the therapeutic vaccine NexVax2 intended to induce immune tolerance in HLA-DQ2 positive patients, and the subcutaneous inoculation of Necator americanus.
numerous food products. In addition, labeling of food products is deficient in many countries. For these reasons, coeliac sufferers are regularly exposed to gluten contamination in the food and beverages they consume. This exposure and its consequences result in a limitation of social activities and/or a reduction in the variety of foods consumed. Moreover, specifically manufactured gluten-free products may be difficult to find, tend to be less flavorful and are more expensive than regular gluten containing products. It has been estimated that 30–50% of coeliac sufferers are not able to strictly follow a
gluten-free diet for a prolonged period of time and have recurrent symptoms and signs [20,23–24]. Eventually, non-compliance with the gluten-free diet, either willful or inadvertent, results in higher morbidity (anemia, osteopenia, repeated miscarriages, delayed intrauterine fetal growth, other autoimmune diseases) and mortality (due to the increased risk of neoplasms, mainly of the digestive tract and lymphomas) [3]. For these reasons, alternative treatments that can act in combination with the gluten-free diet are required in order to improve the quality of life of coeliac patients. These new therapeutic
Table 1 Major non-dietary therapeutic clinical trials in coeliac disease. Investigational agent
Phase of trials
Investigator-company country
Mechanism of action
Route of administration
Findings
Safety and tolerability
Larazotide acetate (AT-1001) ALV003
2b
Alba Therapeutics, USA
Oral
AN-PEP
2a
Alvine Pharmaceuticals, USA DSM, Holland
NEXVAX2
1
NECATOR AMERICANUS Traficet-EN® (CCX282B)
2a
Ameliorated anti-tTG and symptom development in gluten challenges Reduction in blood markers of immunological activation Reduction in intestinal deposits of anti-tTG IgA antibodies. Development of IFN-gamma-producing anti gluten T-cells Better tolerance and reduced symptoms in gluten challenges Limits migration of T cells from the blood flow to the intestinal mucosa
Comparable to placebo
2a
Prevents opening of intestinal epithelial tight junctions Combination of two different gluten degrading proteases Prolyl-endoprotease derived from Aspergillus niger Desensitizing vaccine with 3 gluten peptides Inhibits Th1 immune response by inducing a Th2 response Inhibits CCR9, a chemokine receptor for gut T cell homing
2a
NEXPep, ImmuSanT, Australia/USA Princess Adelaide University, Australia ChemoCentryx, GlaxoSmith-Kline, USA
Oral Oral SC injection Skin incision Oral
Comparable to placebo Comparable to placebo Gastrointestinal Symptoms Active infection with helminth Not reported
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approaches must be safe, effective and reasonably priced. To date, the objective of clinical trials has been to neutralize the effects of small amounts of gluten. 4. The intake of small amounts of gluten damages the intestinal mucosa The gluten-free diet poses a challenge for the coeliac patient. Motivation to comply is related to age (worse in adolescents) and the degree of symptoms (more in symptomatic). In addition, contamination of naturally gluten-free food products and of manufactured gluten-free products makes patients feel insecure about their diet. It is also difficult to determine the gluten contents of foods. Currently, ELISA techniques are used, employing monoclonal or polyclonal antibodies against wheat components. Recently, new analytical methods have been developed to detect barley and rye gluten. Unfortunately, comparison of the results obtained with different ELISA techniques reveals inconsistency in these methods (especially for low contents near to the toxic threshold). In northern European countries, amounts up to 100 parts per million (ppm) are permitted in gluten-free products designated apt for coeliac sufferers. A more conservative limit of 20 ppm is established in the United States and Southern Europe [22]. In a double-blind, placebocontrolled prospective study, Catassi et al. demonstrated that an intake of 50 mg of gluten per day for 3 months was sufficient to cause a significant decrease in the gut mucosa villous height/crypt depth ratio [23]. No clinical or serological correlation (IgA anti-transglutaminase) was found with these gluten traces. A recent review suggested that daily intakes of gluten of lower than 10 mg are unlikely to produce significant histological abnormalities [25]. With the threshold at less than 20 ppm of gluten in special food products for coeliac, an intake of less than 50 mg/day can be achieved, providing a sufficient safety margin [26]. In spite of these recommendations, approximately 50% (30–80%) of all coeliac subjects on a gluten-free diet have active disease at any given time in the US and Europe [20,24]. 5. Therapeutic clinical trials in coeliac disease 5.1. Inhibition of intestinal paracellular permeability: larazotide acetate (AT-1001) The permeability of the intestinal epithelium is partly regulated by the tight junctions, dynamic intercellular points of contact between the enterocytes (intestinal epithelial cells), closely linked to the underlying cellular cytoskeleton [16]. Tight junctions regulate the passage of fluids and molecules through the narrow space between epithelial cells (para-cellular space). In normal conditions, harmful bacteria and dietary antigens are prevented from passing through tight junctions (nutrients enter the body through trans-cellular mechanisms via cell surface receptors or passive diffusion across the cell membrane). In coeliac disease and other immune intestinal diseases, inflammatory cytokines trigger the opening of tight junctions and increase paracellular permeability [16], potentially allowing the entry of harmful gluten peptides into the lamina propria. Larazotide acetate (AT-1001, developed by Alba Therapeutics [27]) is an octapeptide derived from a cholera toxin, ZOT [28–29], that prevents the opening of intestinal epithelial tight junctions by acting on the cytoskeleton. Larazotide acetate is expected to reduce the paracellular transport of gluten to the lamina propria, ameliorating the activation of the pathological immune cascade. Six clinical trials have been completed with larazotide acetate. Three of them were Phase 1 trials: two trials in healthy individuals (one single-dose and other multiple dose safety studies) and a third safety study in subjects with coeliac disease. In all studies, the safety profile was comparable to placebo. Subsequently, three Phase 2 studies have been completed. A Phase 2a trial was a randomized, placebo-
controlled, double-blind trial with 86 coeliac patients in remission with no symptoms or detectable autoantibodies [27]. The patients took a gluten challenge of 800 mg gluten 15 min after administration of AT-1001 or placebo, three times a day for two weeks (approximately 2.5 g of gluten per day). AT-1001 was well-tolerated at all the doses tested (0.25 to 8 mg) and showed safety and tolerability comparable to placebo. While the primary endpoint of intestinal paracellular permeability was not met, the symptoms triggered by the 2-week gluten challenge were significantly ameliorated by larazotide acetate [27]. In addition, two larger Phase 2b studies have been carried out. In the first Phase 2b study (randomized, placebo-controlled, double-blind) different doses of larazotide (1, 4 and 8 mg) were compared against placebo. The study enrolled 184 coeliac patients in remission to be subjected to a 6-week gluten challenge of 900 mg administered 3 times a day. The results have been presented in abstract form and are submitted [30]. Again, larazotide acetate could not demonstrate statistically significant efficacy in the reduction in intestinal permeability, though a favorable trend was observed [30]. Importantly, larazotide acetate did result in a beneficial reduction in the signs (anti-tissue transglutaminase antibodies, anti-tTG IgA) and symptoms of the gluten challenge [30] and the safety profile was satisfactory. The second Phase 2b study was a randomized, placebo-controlled, double-blind trial in coeliac patients with active disease who initiated a strict gluten-free diet at the beginning of the study and were treated for two months. The 3 arms studied were larazotide acetate 4 mg three times a day, larazotide acetate 8 mg three times a day and a placebo group. The results of this study have not been made public to date. A new large (320 patients) Phase 2b study has been announced by Alba Therapeutics and partner Cephalon, to study 0.5, 1 and 2 mg per day of larazotide acetate in coeliac subjects who are non-responsive to a gluten-free diet (ClinicalTrials.gov identifier: NCT01396213). 5.2. Enzyme therapy: endopeptidases (ALV003 and AN-PEP) When gluten polypeptides are hydrolyzed they lose the capacity to stimulate the intestinal immune system and damage the intestine. ALV003 (developed by Alvine Pharmaceuticals) is a combination of two different gluten-targeting proteases (“glutenases”) with complementary substrates: a cysteine-endoprotease derived from germinated barley seeds and a prolyl-endopeptidase from Sphingomonas capsulatum [31]. Both are active in acidic medium and their glutenase activity is maximized in a 1:1 formulation administered orally. A Phase 1 clinical trial was conducted with 20 coeliac patients in remission who were randomized to receive a diet containing gluten (16 g a day for 3 days) pre-treated with ALV003 (10 patients) versus a diet containing gluten pre-treated with placebo (10 patients). The group given ALV003-treated gluten presented a reduction in markers of immunological activation (IFN-γ production measured with the ELISpot technique) [31]. A subsequent Phase 1b study showed that ALV003 is very effective, in the stomach, in breaking down foods with high gluten content [32]. Alvine Pharmaceuticals has recently conducted a larger 6-week Phase 2a study of ALV003 administered to coeliac subjects exposed to a gluten challenge. This study was a randomized, placebo-controlled, double-blind trial in well-controlled coeliac individuals instructed to take 6 g a day of gluten. The results have not been made public at the time of this writing. A second prolyl-endoprotease called AN-PEP is being developed by an alimentary company, DSM. AN-PEP is derived from Aspergillus niger [33]. In an interventional, cross-over, randomized, doubleblind, placebo-controlled clinical trial, AN-PEP's ability to detoxify 8 g of gluten contained in a commercial food product was tested. The primary objective of the study was the change in histology, and other endpoints measured included peripheral blood T-cells and coeliac antibodies. The study was conducted in 14 patients who ate food prepared with gluten together with AN-PEP for 2 weeks. After a
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second 2-week washout period (in which they were kept on a strict gluten-free diet), patients started the third 2-week period for which they were randomized to continue eating the same food containing gluten and AN-PEP, or gluten and placebo. The results have been communicated in abstract form and there was no significant effect of ANPEP on pre-specified endpoints although a post-hoc analysis showed a possible reduction in the intestinal deposits of anti-tTG IgA antibodies. The safety profile was satisfactory [33]. In a commentary, Donnelly et al. speculate that this trial studied a very large dose of gluten with a relatively brief wash-out period between periods while peptidases may be more appropriate for the digestion of minor quantities of gluten or in the pre-treatment of gluten-free products that contain a small amount of gluten [34]. There is currently a new clinical trial announced for AN-PEP in 12 subjects to understand the extent of the degradation of gluten in the stomach (ClinicalTrials.gov identifier: NCT01335503). In addition to these two company-sponsored clinical trials with proteases, there is a third protease mix being tested by an academic institution, Stanford University. The group led by Prof. Khosla is conducting a Phase I + II clinical trial with STAN1 (a mix of commerciallyavailable proteases) at the Heim Pal Children's Hospital in Hungary. The results are not available at the time of this writing. (ClinicalTrials.gov identifier: NCT00962182). 5.3. Therapeutic vaccine: Nexvax2 NexVax2 is a desensitizing or therapeutic “vaccine” under development by biotechnology companies NexPep and ImmuSanT. NexVax2 uses 3 gluten peptides with the goal of inducing a “tolerogenic” response in coeliac patients with HLA DQ2. NexVax2 has showed efficacy in mice with gluten-sensitive T-cells and transgenic for HLA DQ2 [35]. The phase 1 study, of recent completion, consisted of subcutaneous injections of increasing doses of up to 90 μg of Nexvax2 in 40 patients with well controlled coeliac disease, weekly for 3 weeks. The results have been communicated in abstract form. While there were some gluten-related gastrointestinal side effects, dose escalation could be completed and safety was acceptable. The investigators also reported the development of IFN-gamma-producing anti-gluten T cells in vaccinated subjects [36]. 5.4. Modulation of immunity with Necator americanus and probiotics This research project is based on the theory that the human immune system needs to be exposed to exogenous organisms in order to function correctly (“hygiene hypothesis”) [37]. The disappearance of certain microbial strains from the gut of subjects residing in developed countries could be the reason for the increased prevalence of immune-mediated illnesses such as coeliac disease, Crohn's disease, ulcerative colitis or asthma [37]. Survival of the microbes in the intestine is dependent on their ability to interfere with the immune response of the host. The mechanisms employed to do this are similar to those used to regulate autoimmune diseases. Researchers suspect that removal of microbes from the environment could lead predisposed individuals to develop autoimmune diseases [37]. Researchers at the Prince Adelaide University in Brisbane, Australia, postulated that the parasite Necator americanus would inhibit the Th1 immune response against gluten in coeliac subjects by inducing a Th2 response. A Phase 2a randomized, double-blind, placebo-controlled study was completed with a 3 to 5 day gluten challenge in 20 well-controlled coeliac subjects inoculated with Necator americanus or placebo. The results have been communicated in abstract form [38]. Parasite larvae were subcutaneously injected at two time points (ten larvae at time points 0 and 5 more larvae at 12 weeks). Patients in the placebo group were injected with a preparation containing a few drops of Tabasco sauce to mimic the irritation caused by the larvae. Patients hosting the live parasite tolerated the gluten challenge better and had better scores in the digestive symptom questionnaires
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compared with control subjects without the parasites [38]. They also had less mucosal inflammation, although these results were not statistically significant. At the end of the 21 weeks of the study, the volunteers were offered medication to remove the parasites but most patients chose to keep them [38]. In a related effort, Prof. Bai has announced a 20-patient clinical trial to study the effects of Bifidobacterium infantis, a probiotic with immune regulatory properties, in active celiac disease at the Dr. C. Bonorino Udaondo Gastroenterology Hospital, Buenos Aires, Argentina (ClinicalTrials.gov identifier: NCT01257620). 5.5. Immunomodulators: CCX282b (Traficet-EN®) CCR9 is expressed at high levels on lamina propria mononuclear cells in the small intestine [4], and its ligand CCL25/TECK is expressed on both lamina propria capillaries and on small intestine enterocytes. This chemokine receptor controls the trafficking of T-cells selectively migrating to the intestinal tract [40]. CCX282B (Traficet-EN®), in development by ChemoCentryx and Glaxo Smith Kline for Crohn's and coeliac disease, inhibits the CCR9 receptors of T-cells limiting their migration from the blood flow to the intestinal mucosa [6,39]. Traficet-EN® is administered via oral route. A gluten challenge study was carried out in 2008 using Traficet-EN® in 90 patients (Phase 2, randomized, double blind, placebo-controlled study). The results have not been made public to date. In a review, Solid and Khosla discuss a potential concern for intestinal infections if the CCR9 antagonists are effective given the non-antigen-specific mode of action, and recommend careful monitoring [40]. 6. Summary Coeliac disease is a highly prevalent autoimmune disease triggered by dietary gluten, with no approved medication. Coeliac disease leads to morbidity and increased mortality. Gluten is “everywhere” and is very hard to eliminate from the diet. While a strict gluten-free diet eliminates most of the risk for complications of coeliac disease, at any given time approximately 50% of subjects have active disease, mostly due to non-compliance, either willful or inadvertent. While substantial progress has been made in the last few years in the development of experimental medications for coeliac disease, all the new proposed therapies remain in early research stage, and no Phase 3 trial has ever been conducted in coeliac disease. Further progress is needed in the design of clinical trials and the use of surrogate endpoints in coeliac disease [40]. The first medication(s) for coeliac disease will likely be adjunct to the gluten-free diet and the importance of strict adherence to this difficult diet cannot be overemphasized at this time. Learning points • Despite a high rate of non-responsiveness to the gluten-free diet, there is no pharmacological approach approved for coeliac disease, a highly prevalent chronic autoimmune disease. • Clinical research for coeliac disease medications is in its infancy and only a handful of compounds have reached Phase 2 studies. Conflicts of interest Francisco Leon is an ex-employee of Alba Therapeutics, a biotechnology company working in coeliac disease. He does not currently own stock or stock options in the company. The rest of the authors have been investigators in clinical trials sponsored by Alba Therapeutics. References [1] Di Sabatino A, Corazza GR. Coeliac disease. Lancet 2009;373:1480–93. [2] Farrel RF, Nelly CP. Celiac sprue. N Engl J Med 2002;346:180–8.
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