Gluten Degradation Research Articles

Transport of wheat gluten exorphins A5 and C5 through an in vitro model of intestinal epithelium

The gluten exorphins (GEs) A5 and C5 are peptides with a potential opioid-like activity derived from enzymatic degradation of wheat gluten. In this study, a 70% Caco2/30% HT-29 co-culture layer was used as an in vitro model of human small intestine to investigate the transport of the two GEs and their resistance to brush border peptidases. Thirty, 150 or 600μg of the peptides, GE A5 and GE C5, were added to the apical chamber of the transwells. We previously demonstrated these amounts to be released during in vitro digestion of wheat-derived bread and pasta samples. The solutions in apical and basolateral chambers were analyzed for intact GEs or related fragments by UPLC/HRMS. The two peptides behaved differently since after 120min higher amount of intact GE A5 (0.55–24.60%) compared to GE C5 (0.20–1.00%) was recovered in the basolateral chamber, depending on the initial dose that was added to the transwell apical compartment. The GE C5 was strongly hydrolyzed by cellular peptidases. The fluxes of two GEs through the in vitro intestinal epithelium were concentration- and time-dependent. The transport inhibitors Wortmannin and Glycyl-sarcosine did not significantly affect the apical-to-basolateral fluxes of the two GEs. Differently, Cytochalasin D slightly increased the transepithelial transport of both GEs, and decreased the TEER values of the co-culture cells. These results suggest the paracellular route as a possible transport mechanism of both GEs. Overall, this study indicates the possibility that food-derived GEs can cross the human intestinal epithelium as intact peptides, and improves the knowledge for exploiting the opioid-like activity potentially associated with the ingestion of wheat-derived foods.

Production and application of barley malt extract with high peptidase activity for the degradation of gluten in wort

Special malt with high gluten-specific peptidase activity was prepared by germinating barley grain for 8 days at 18 °C and 48 % humidity. Endogenous malt peptidases were characterized and used to degrade gluten in barley wort. Peptidases of aqueous malt extracts degraded celiac-active peptides by cleaving peptide bonds involving proline residues. The peptidase activity was not affected by cultivar, area of cultivation, and high temperatures up to 80 °C during kiln drying. Temperatures up to 50 °C were tolerated without loss of full activity in aqueous solution. The presence of ethanol decreased the activity significantly. Cross-flow filtration, freeze-drying, and evaporation under reduced pressure at 50 °C were used to produce a concentrated enzyme-active malt extract. The latter method yielded the best results, and the obtained concentrate was used to study gluten degradation in barley wort as affected by concentration of the extract, incubation time, and temperature. The gluten content was determined by a competitive R5-ELISA. The addition of 10 % concentrated extract (grist-to-water ratio 1:2.5; 40.4 °Brix) to wort and incubation for 24 h at 50 °C resulted in a gluten-free wort.

Prolyl-specific peptidases for applications in food protein hydrolysis.

Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers.

SummaryBackground Aspergillus niger prolyl endoprotease (AN‐PEP) efficiently degrades gluten molecules into non‐immunogenic peptides in vitro.AimTo assess the efficacy of AN‐PEP on gluten degradation in a low and high calorie meal in healthy subjects.MethodsIn this randomised, double‐blind, placebo‐controlled, cross‐over study 12 healthy volunteers attended to four test days. A liquid low or high calorie meal (4 g gluten) with AN‐PEP or placebo was administered into the stomach. Via a triple‐lumen catheter gastric and duodenal aspirates were sampled, and polyethylene glycol (PEG)‐3350 was continuously infused. Acetaminophen in the meals tracked gastric emptying time. Gastric and duodenal samples were used to calculate 240‐min area under the curve (AUC 0–240 min) of ?‐gliadin concentrations. Absolute ?‐gliadin AUC 0–240 min was calculated using duodenal PEG‐3350 concentrations.Results AN‐PEP lowered α‐gliadin concentration AUC 0–240 min, compared to placebo, from low and high calorie meals in stomach (low: 35 vs. 389 μg × min/mL; high: 53 vs. 386 μg × min/mL; P < 0.001) and duodenum (low: 7 vs. 168 μg × min/mL; high: 4 vs. 32 μg × min/mL; P < 0.001) and absolute α‐gliadin AUC 0–240 min in the duodenum from low (2813 vs. 31 952 μg × min; P < 0.001) and high (2553 vs. 13 095 μg × min; P = 0.013) calorie meals. In the placebo group, the high compared to low calorie meal slowed gastric emptying and lowered the duodenal α‐gliadin concentration AUC 0–240 min (32 vs. 168 μg × min/mL; P = 0.001).ConclusionsAN‐PEP significantly enhanced gluten digestion in the stomach of healthy volunteers. Increasing caloric density prolonged gastric residence time of the meal. Since AN‐PEP already degraded most gluten from low calorie meals, no incremental effect was observed by increasing meal caloric density. ClinicalTrials.gov, Number: NCT01335503; www.trialregister.nl, Number: NTR2780.

Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

BackgroundDue to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP).MethodsFive commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays.FindingsThe digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.ConclusionCurrently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.

Sourdough fermentation of wheat flour does not prevent the interaction of transglutaminase 2 with α2-gliadin or gluten.

The enzyme transglutaminase 2 (TG2) plays a crucial role in the initiation of celiac disease by catalyzing the deamidation of gluten peptides. In susceptible individuals, the deamidated peptides initiate an immune response leading to celiac disease. Several studies have addressed lactic fermentation plus addition of enzymes as a means to degrade gluten in order to prevent adverse response in celiacs. Processing for complete gluten degradation is often harsh and is not likely to yield products that are of comparable characteristics as their gluten-containing counterparts. We are concerned that incomplete degradation of gluten may have adverse effects because it leads to more available TG2-binding sites on gluten peptides. Therefore, we have investigated how lactic acid fermentation affects the potential binding of TG2 to gluten protein in wheat flour by means of estimating TG2-mediated transamidation in addition to measuring the available TG2-binding motif QLP, in α2-gliadin. We show that lactic fermentation of wheat flour, as slurry or as part of sourdough bread, did not decrease the TG2-mediated transamidation, in the presence of a primary amine, to an efficient level (73%–102% of unfermented flour). Nor did the lactic fermentation decrease the available TG2 binding motif QLP in α2-gliadin to a sufficient extent in sourdough bread (73%–122% of unfermented control) to be useful for celiac safe food.

Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

Degradation of gluten in rye sourdough products by means of a proline-specific peptidase

The gluten content of rye sourdough during fermentation was monitored by a competitive ELISA based on the R5 antibody. Although a time-dependent decrease was found, the gluten content was not reduced below the threshold for gluten-free foods of 20 mg/kg, even after prolonged fermentation. Instead, Aspergillus niger prolyl endopeptidase (AN-PEP) extensively degraded gluten concentrations of up to 80,000 mg/kg in rye flour, rye sourdough, and sourdough starter under distinct temperatures and pH values. The enzyme did not lead to inactivation of the microorganisms in the sourdough starter. Gluten-free rye flour alone or in combination with sourdough starter was used to produce gluten-free bread, which was evaluated for its sensory properties. Whereas gluten-free sourdough bread had poor sensory attributes compared to a conventional rye bread used as reference, the replacement of sourdough by egg proteins yielded gluten-free bread comparable to the reference and with higher sensory quality than bread prepared from naturally gluten-free ingredients. Therefore, the feasibility of producing high-quality bread from originally gluten-containing cereals such as rye by means of treatment with AN-PEP has been shown. Rye products rendered gluten-free in this manner have the potential to increase the choice of high-quality gluten-free foods for celiac patients.

Su2096 Gluten Degrading Enzyme Effectively Digests Gluten in the Stomach and Small Intestine of Healthy Volunteers

A significant proportion of the population does not tolerate gluten. Aspergillus niger prolyl endopeptidase (AN-PEP) enzyme efficiently degrades gluten molecules into non-immunogenic peptides in a dynamic, multi-compartmental gastrointestinal simulation model but its efficacy in vivo remains to be established. Aim of our study was to assess the efficacy of ANPEP on gastrointestinal breakdown of gluten in healthy subjects, as well as the effect of meal caloric density on AN-PEP efficiency. Methods: In this double-blind, randomized, placebo-controlled cross-over study 12 healthy subjects attended to four test days in random order. On each occasion they received a low (143 kCal) or high caloric (405 kCal) meal, containing 4g gluten, with AN-PEP or placebo via a triple-lumen nasoduodenal catheter. Acetaminophen was added to measure gastric emptying rate. One lumen, positioned in the stomach, was used for administration of the test meal and collection of gastric fluid. A second lumen was used for the continuous injection of the inert dilution marker polyethylene glycol 3350 (PEG3350) to enable the calculation of meal dilution by endogenous secretions, with the injection port positioned just distal to the pylorus. A third lumen, located at the tube tip 10 cm distal to the second port, was used for collection of duodenal content. Fluid samples were taken regularly during 4 hours after meal infusion. The presence of the DQ2.5glia-α3 epitopes was quantified using the Gluten-Tec® ELISA assay, which provides an accurate estimate of the actual α-gliadin content of the samples. Degradation of intact gluten proteins was measured by Western-Blot analysis. Duodenal PEG3350 concentrations and gastric acetaminophen concentrations were determined by high-performance liquid chromatography. The effect of AN-PEP on gluten degradation was measured by the difference in 240-minute Area Under the Curve (AUC) of relative and absolute gluten exposure between AN-PEP and placebo. Differences between combinations of treatment and meal were assessed using linear mixed models. Results: AN-PEP significantly reduced gliadin concentration and absolute output in the stomach and duodenum (as 240-min AUCs) in both low and high caloric meals, compared to placebo. Furthermore, in the high caloric meal with placebo the duodenal gliadin concentration was lower, compared to the low caloric meal with placebo. No differences were observed between gliadin concentrations of a low or high caloric meal containing AN-PEP (Table). The gastric emptying time of the high caloric meal was longer compared to the low caloric meal, both in the presence of placebo (p=0.014) and AN-PEP (p=0.100). Conclusion: AN-PEP addition to a gluten containing meal significantly enhances gluten digestion in healthy volunteers. Meal caloric density does not affect the efficacy of AN-PEP on gluten degradation. Gliadin concentration and absolute output in stomach and duodenum

Degradation of Gluten in Wheat Bran and Bread Drink by Means of a Proline-Specific Peptidase

Gluten-free wheat bran and bread drink were produced by degrading gluten with Aspergillus niger prolyl endopeptidase (AN-PEP). For this purpose, bran from native and germinated wheat grains as well as bread drink were mixed with AN-PEP and incubated at 50°C under different conditions. The amount of enzyme activity (1.2 • 10-4 - 8.7 • 10-1 U), the incubation time (0-72 h), and the pH value (1.0-9.0) were systematically altered. The gluten content was monitored by a competitive ELISA using the R5 antibody. Gluten in two wheat bran samples produced in the laboratory was degraded below the threshold for gluten-free foods of 20 mg/kg. This was not possible in a commercial wheat bran sample that had potentially been heat-treated leading to strong crosslinking of gluten by incorporation of gliadins into the glutenin fraction, possible formation of isopeptide crosslinks and, thus, poor digestibility. In contrast, gluten in bread drink was easily degraded after a short incubation time of 30 min and low AN-PEP activity. No significant differences of the quality parameters between treated and untreated products were found. Remarkably, bran from germinated grains was strongly enriched in nutritionally positive compounds such as dietary fiber and folates compared to native grains. Thus, wheat bran from germinated grains rendered gluten-free by treatment with AN-PEP can contribute to increasing the nutritional value of the gluten-free diet.

Use of fungal proteases and selected sourdough lactic acid bacteria for making wheat bread with an intermediate content of gluten

This study was aimed at combining the highest degradation of gluten during wheat flour fermentation with good structural and sensory features of the related bread. As estimated by R5-ELISA, the degree of degradation of immune reactive gluten was ca. 28%. Two-dimensional electrophoresis and RP-FPLC analyses showed marked variations of the protein fractions compared to the untreated flour. The comparison was also extended to in vitro effect of the peptic/tryptic-digests towards K562 and T84 cells. The flour with the intermediate content of gluten (ICG) was used for bread making, and compared to whole gluten (WG) bread. The chemical, structural and sensory features of the ICG bread approached those of the bread made with WG flour. The protein digestibility of the ICG bread was higher than that from WG flour. Also the nutritional quality, as estimated by different indexes, was the highest for ICG bread.

10.5937/ratpov50-4482 = Functions of LR genes beside reduction of Puccinia triticina

Wheat leaf rust race specific resistance controlling genes (Lr) caused 0.01-0.03 decrease of SAGR = divided first two internodes with stem length. In addition, nitrogen transport in upper parts by those genes was 3-19% higher in comparison to near isogenic lines with nonspecific Lr 22a and Lr 22b genes. RAR (divided root nitrogen content with sum of up ground and root ones) values of Lr16, Lr29 and Lr37 between Lr1, Lr15 or Lr19 and nonspecific genes were explained by hydrolytic stability of endo-proteases responsible for cleaving of glutens disulphide bridges. According to RAR, Lr 34 seemed to be specific Lr gene but increased SAGR as was by nonspecific Lr genes focused SUTs as adequate for its conformity. Two different drought and heat stress respond systems were linked to Lr genes; one based on gluten degradation consequential through photosynthesis decrease and another on accelerated transfer of starch products and water in stem.

Heat-Induced Cross-Linking and Degradation of Wheat Gluten, Serum Albumin, and Mixtures Thereof

Some wheat-based food systems, such as cakes, cookies, and egg noodles, contain mixtures of animal and plant (gluten) proteins and are processed under (mildly) alkaline conditions. Although changes in these proteins during processing can affect end product quality, they have seldom been studied. This study investigated protein cross-linking and degradation during heating (0-120 min, pH 8.0, 50-130 °C) of (mixtures of) wheat gluten and bovine serum albumin (BSA). The decrease in protein extractabilities in sodium dodecyl sulfate containing buffer under (non)reducing conditions and the levels of (cross-linked) amino acids were measured. No indications for polymerization at 50 °C were found. Below 100 °C, BSA polymerized more readily than wheat gluten. Above 100 °C, the opposite was observed. The kinetics of heat-induced polymerization of a 1:1 gluten-BSA mixture were similar to that of isolated gluten, implying that gluten decelerated BSA denaturation. Severe heating (130 °C, >15 min) induced degradation reactions in gluten but not in BSA. At all conditions used in this study, disulfide (SS) bonds contributed to the extractability loss. In addition, above 110 °C, β-elimination of cystine led to non-SS cross-links. Intramolecular SS bonds more often transformed in intermolecular non-SS bonds in BSA than in gluten.

Recent Research on Folate Intake, Rice Products, Fiber Intake, and Enzyme Degradation of Gluten

Recent research on nutrition and health is discussed, including the impact of folate intake during pregnancy on the incidence of autism, developmental delays in children, and typical child development. Recent studies are also providing insight into the health benefits of rice products, their arsenic content, and their impact on chronic diseases. Two European reports indicate higher dietary fiber intakes are associated with lower overall and specific disease mortality. The results support recommendations for high dietary fiber intake for health maintenance. Another study focused on death from ischemic heart disease (IHD), finding an association between increased dietary fiber intake and decreased risk of IHD. Recent studies also have shown that gluten and its resulting peptides can be degraded by prolyl endopeptidases. Thus, gluten-containing materials could potentially be rendered less problematic for most celiac patients through the use of these enzymes in the production of foods intended for this popula...

Celiac Disease: Advances in Treatment via Gluten Modification

Celiac disease (CD) is an autoimmune enteropathy that occurs in genetically susceptible individuals carrying the prerequisite genetic markers HLA DQ2 or DQ8. These genetic markers are present in approximately 30% of the population, and the worldwide prevalence of CD is estimated to be approximately 1%-2%. Currently a gluten-free diet is the only treatment for CD, but novel therapies aimed at gluten modification are underway. This review will discuss gluten-based therapies including wheat alternatives and wheat selection, enzymatic alteration of wheat, oral enzyme supplements, and polymeric binders as exciting new therapies for treatment of CD.

Suspect Sweetener: Arsenic Detected in Organic Brown Rice Syrup

Vol. 120, No. 5 News | Science SelectionsOpen AccessSuspect Sweetener: Arsenic Detected in Organic Brown Rice Syrupis accompanied byArsenic, Organic Foods, and Brown Rice Syrup Wendee Holtcamp Wendee Holtcamp Search for more papers by this author Published:1 May 2012https://doi.org/10.1289/ehp.120-a204aCited by:2View Article in:中文版AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit Organic brown rice syrup (OBRS) is a sweetener frequently used as an alternative to high-fructose corn syrup in organic and health food products. In a study of children’s foods, a team of researchers discovered high levels of arsenic in toddler formula products that contained OBRS. Given evidence that arsenic accumulates in rice at high levels, the researchers suspected OBRS was the source of the arsenic in the formulas—a suspicion corroborated by additional tests of several products both with and without the sweetener [EHP 120(5):623–626; Jackson et al].The researchers tested 3 commercially available brown rice syrups, 29 cereal bars (18 with OBRS), 3 high-performance “energy shot blocks” with OBRS, 15 infant formulas without the sweetener, and 2 toddler formulas with it. The researchers measured inorganic arsenic as well as the organic chemical species dimethylarsenate (DMA) and monomethylarsenate (MMA), which are generally believed to be less toxic than inorganic arsenic.The two toddler formulas tested are the only ones known to be made with OBRS.© Getty ImagesThe OBRS-sweetened toddler formulas (one soy-based, one milk-based) had about 20 times the total arsenic concentrations of the non-OBRS infant formulas. Samples of prepared milk-based toddler formula had inorganic arsenic concentrations just below the current Environmental Protection Agency (EPA) drinking water standard of 10 µg/L, whereas inorganic arsenic in samples of soy-based toddler formula tested 1.5–2.5 times above the EPA standard.In the brown rice syrups tested, inorganic arsenic made up 80–90% of the arsenic content of two of the syrups and half the arsenic content of the third. All three also contained DMA, with lesser amounts of MMA. All 29 cereal bars tested contained some arsenic, but those without any rice-based ingredients had the lowest levels. Nearly 60% of the others contained inorganic arsenic. The three energy shot blocks contained enough inorganic arsenic that if an individual were to consume the manufacturer-recommended four servings during a two-hour workout, they would ingest the equivalent of drinking a liter of water containing 10 µg/L arsenic; total arsenic would be twice that.The two toddler formulas tested were the only ones the researchers could find that contained OBRS. However, they estimate that approximately half of all cereal and energy bars contain OBRS or other rice products, and they conclude that food containing OBRS may be a major pathway to arsenic exposure for some people. Although the health effects of low-level arsenic exposure over limited durations are unknown, childhood exposure may affect lifelong health and should be limited. The authors therefore urge U.S. regulators to establish limits for arsenic levels in food, particularly infant and toddler formulas.FiguresReferencesRelatedDetailsCited by Jones J (2012) Recent Research on Folate Intake, Rice Products, Fiber Intake, and Enzyme Degradation of Gluten, Cereal Foods World, 10.1094/CFW-57-5-0235, 57:5, (235-238), Online publication date: 1-Sep-2012. Li Y, Carrillo J, Ding Y, He Y, Zhao C, Liu J, Liu G, Zan L, Song J and Fugmann S (2015) Transcriptomic Profiling of Spleen in Grass-Fed and Grain-Fed Angus Cattle, PLOS ONE, 10.1371/journal.pone.0135670, 10:9, (e0135670) Related articlesArsenic, Organic Foods, and Brown Rice Syrup16 February 2012Environmental Health Perspectives Vol. 120, No. 5 May 2012Metrics About Article Metrics Publication History Originally published1 May 2012Published in print1 May 2012 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Update on celiac disease &amp;ndash; etiology, differential diagnosis, drug targets, and management advances

Celiac disease (CD) is an immune-mediated enteropathy triggered by exposure to wheat gluten and similar proteins found in rye and barley that affects genetically susceptible persons. This immune-mediated enteropathy is characterized by villous atrophy, intraepithelial lymphocytosis, and crypt hyperplasia. Once thought a disease that largely presented with malnourished children, the wide spectrum of disease activity is now better recognized and this has resulted in a shift in the presenting symptoms of most patients with CD. New advances in testing, both serologic and endoscopic, have dramatically increased the detection and diagnosis of CD. While the gluten-free diet is still the only treatment for CD, recent investigations have explored alternative approaches, including the use of altered nonimmunogenic wheat variants, enzymatic degradation of gluten, tissue transglutaminase inhibitors, induction of tolerance, and peptides to restore integrity to intestinal tight junctions.

Safety, Tolerability, and Activity of ALV003: Results from Two Phase 1 Single, Escalating-Dose Clinical Trials

Celiac disease is the most common hereditary autoimmune disease in humans. The only treatment option for non-refractory celiac disease patients is adherence to a strict life-long gluten-free diet, which often fails to normalize small bowel histology. ALV003 is a mixture of two proteases that degrades gluten and is in clinical development as an oral therapy for patients with celiac disease. The safety, tolerability, and activity of ALV003 were assessed in two phase 1 clinical trials. In study 1 (N = 28) the study drug was administered in the fasted state; in study 2 (N = 53) the study drug was administered together with a gluten-containing meal. Both studies were single-dose, single-blind, placebo-controlled, cross-over trials. ALV003 was dosed at escalating dose levels by cohort (100, 300, 900, and 1,800 mg) and gastric samples were aspirated using a nasogastric tube. Adverse events, serum drug levels, and anti-drug antibody titers were measured. Gastric samples were assessed for ALV003 enzymatic activity over time (gastric pharmacokinetics) and gluten degradation (gastric pharmacodynamics). All doses were well tolerated, and no serious adverse events or allergic reactions were observed. Gastric aspirates collected 30 min following a meal showed that 100 and 300 mg ALV003 degraded 75 ± 10% (N = 8) and 88 ± 5% (N = 8), respectively, of one gram of wheat bread gluten. ALV003 is an orally active protease that appears to be stable in the fed stomach and degrades dietary gluten in this compartment. Single doses of oral ALV003 were not associated with serious adverse reactions.

Quantification of ALV003-Mediated Gluten Degradation Using Mass Spectrometry

Quantification of ALV003-Mediated Gluten Degradation Using Mass Spectrometry

Peripheral and Mucosal B Cells From Celiac Disease Patients Show Increased Expression of CXCR3 and IgA Switch Markers

ponding to subdivisions of the 33mer based on the ability of ALV001 to cut several times within this gluten epitope. RESULTS: Mass spectrometry detected 33mer and all five peptide fragments resulting from degradation of the 33mer by ALV001. A large linear range was observed, with detection as low as 1 ng/ml of the peptides of interest. Intraday coefficient of variability was typically less than 5%. ALV001 cut within the 33mer epitope in the context of wheat gluten, releasing all five fragments that initially rose in concentration but plateaued as ALV001-mediated degradation slows. Using a clinical dose (150 mg, 0.3 mg/ml), ALV001 degraded 98% (10 minutes) and 99.5% (30 minutes) of the 33mer epitope. ALV001 and ALV002 functioned cooperatively; ALV002 degraded the five relatively stable peptide fragments as ALV001 activity plateaued. SUMMARY: A mass spectrometry assay was established to quantify the ability of ALV003 to degrade 33mer within wheat bread gluten in the context of a complex meal. Using this methodology, it was possible to distinguish separate but complementary contributions of ALV001 and ALV002 to 33mer degradation. The data support a model for ALV003-mediated gluten degradation in which ALV001 rapidly cleaves gluten adjacent to glutamine residues, releasing small proline-rich peptide fragments that serve as substrates for the prolyl endopeptidase ALV002. Data from a simulated gastric environment suggest that ALV003 should degrade ingested gluten in patients with celiac disease.