EP1075267A2 - Traitement de la maladie caeliaque - Google Patents

Traitement de la maladie caeliaque

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Publication number
EP1075267A2
EP1075267A2 EP99917810A EP99917810A EP1075267A2 EP 1075267 A2 EP1075267 A2 EP 1075267A2 EP 99917810 A EP99917810 A EP 99917810A EP 99917810 A EP99917810 A EP 99917810A EP 1075267 A2 EP1075267 A2 EP 1075267A2
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Prior art keywords
gliadin
deamidation
substance
gluten
peptide
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EP99917810A
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German (de)
English (en)
Inventor
Hans Sjöström
Ove Nor N
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Kobenhavns Universitet
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Kobenhavns Universitet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/08—Oxides; Hydroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/10—Carbonates; Bicarbonates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/12—Magnesium silicate

Definitions

  • the present invention is based upon the finding that deamidation of glutamine residues in gliadin is important for immune activation and presumably tissue destruction in celiac disease.
  • the present invention thus relates to a method of treating celiac disease comprising in- terfering with the deamidation of disease eliciting glutamine residue (s) .
  • Celiac disease is a chronic intestinal disease with chronic diarrhoea, malnutrition and loss of weight as its most important manifestations. It is elicited by ingestion of components of wheat, rye, barley and possibly oats flour. The existing treatment is a life-long exclusion of flour from the diet, which affects the quality of life of CD individuals.
  • the incidence of the disease varies but a rate of 1 case of about every 300 born person in the population has been reported in several investigations. In some countries, e.g. Den- mark, the incidence is lower, but with increased investigations the gap tends to be reduced.
  • the pathogenesis of the disease has both genetic and environmental components. Monozygotic twins have a concordance rate of approximately 75%, suggesting an environmental component. About 10% of first degree relatives are affected. One genetic component is the HLA association of the disease; more that 90% of the patients have the DQA1*0501 and DQB1*02 alleles. However, there are more genetic components as the concordance rate between siblings with this particular DQ is only about 30%.
  • gliadins proteins that are the pathogenic agents. Gliadins can be classified into several groups ( ⁇ -type, ⁇ -type and ⁇ -type) and among these at least the ⁇ - and the ⁇ -type gliadins are known to cause the disease.
  • Each subcultivar of wheat contains about 30 different a- and ⁇ -gliadins, with some variations in amino acid sequences within each type. Both types are characterised by insolubility in neutral aqueous solutions, a molecular mass around 40 kDa and an N-terminal half built up of repeating sequences being very rich in glutamines and proli- nes.
  • T cells in the small intestinal mucosa that recognize the gliadins are characteristic of individuals with CD.
  • T cells can be isolated, propagated in vi tro and cloned. It has been demonstrated that presentation of gliadin to these cells is almost exclusively restricted to DQ2. Evidence is accumulating that there is a crucial role for these T cells in the immunopathogenesis of the disease (Scott et al . , 1997).
  • vi tro proliferation of gliadin-specific, HLA- DQ2 -restricted T cell clones from the small intestine of celiac disease patients can be initiated by the addition of a water soluble form of gliadin heated at low pH and a HLA-DQ2 carrying antigen presenting cell.
  • the proliferation is registered by the incorporation of radioactive thymidine .
  • the part of the structure of the gliadins (the toxic sequence (s) ) recognised by such T cells has, however, been elusive and this has hampered the understanding of the pathogenesis of the disease and thereby the possibility to develop interfering therapeutics.
  • the present invention relates to interfering with the deamidation of at least one glutamine residue in a gliadin molecule.
  • One main principle is to modify the gliadins of the food, e.g. the gliadin of the wheat flour making deamidation impossible. This might be achieved by deamidation of sensitive amides in glutamines followed by chemical derivation of the generated carboxyl groups to remove or modify the nega- tive charge either directly in an enzymatic reaction or by deamidation followed by derivation. It might also be possible to generate gliadins which cannot be deamidated by genetic engineering of wheat .
  • Another main principle is to interfere with the processing of gliadins in the preparative phase of the food or when the gliadins are processed by the CD individual.
  • Neutralisation of the acidic environment in the stomach by any procedure leading to decreased secretion of hydrochloric acid by the parietal cells, such as by administration of a proton pump inhibitor, a histamine 2 receptor antagonist, an anticholinergic agent, or a passive therapeutic agent like an acid neutralising agent, is likely to prohibit or minimize a deamidation due to the low pH of the stomach.
  • the substance to be used is capable of increasing the pH in the gastroduodenal tract of a subject. With the available knowledge, pH should be increased above 3.0. Further or alternatively, possible deamidase producing microorganisms may be reduced or eradicated by antibiotic treatment.
  • the invention relates to the use of a substance which is capable of eliminating or decreasing the amount of deamidating bacteria in the gastroduodenal tract of a subject.
  • substances that interfere with the effects of such an enzyme should be used either alone or as an additional supplement.
  • the invention also relates to the use of a substance which is capable of inhibiting at least one deamidating enzyme in the gastroduodenal tract of a subject .
  • ⁇ -gliadin 36 A distinct ⁇ -type gliadin ( ⁇ -gliadin 36) was purified from gliadin of the strain Kadett. It was digested with chymotryp- sin followed by heating at low pH. Fragments capable of stimulating a T-cell clone in vi tro were purified by chromato- graphies on Sephadex G-50, Superdex HR 10/30 and MonoQ.
  • Electrospray ionization mass spectrometry of methyl-esteri- fied peptides of different purified fragments demonstrated partial deamidations in many different positions with some positions being more prone to deamidation.
  • T cell lines from six patients as well as 25 T cell clones from five patients were tested with chymotrypsin digested gliadin either untreated or treated by heating at low pH. All T cell lines and most clones displayed weak reactivity against the untreated gliadin, while the reactivity was markedly enhanced by heating at low pH. Thus, deamidation appears to be important for recognition by most T cells from the small intestinal mucosa of CD patients.
  • the deamidation might either occur as part of the normal processing of baked wheat-containing food before or after it has been ingested or be specific for CD.
  • Deamidation under normal conditions might occur during a. fermentation of the bread, b. baking of the bread, c. exposure to the hydrochloric acid of the stomach, d. exposure to deamidating enzymes during digestion in the gastrointestinal tract, e.g. exposure to deamidating enzymes in the intestinal wall.
  • a disease-specific deamidation might occur as a result of bacterial infection in the gastrointestinal tract or a change in the effects of a deamidating enzyme in the digestive secretions or intestinal wall.
  • Gliadin was treated with yeast under conditions corresponding to the normal fermentation of a dough. No significant T-cell proliferation stimulatory activity could be detected.
  • a piece of bread was made of flour, water and yeast according to standard procedures. It was homogenised and gliadin prepared according to standard procedures. No significant T cell proliferation stimulatory activity could be detected.
  • mice with low hydrochloric acid secretion mice with low hydrochloric acid secretion
  • mice with low hydrochloric acid secretion mice with low hydrochloric acid secretion
  • mice with a substance which inhibits or reduces the secretion of gastric hydrochloric acid mice with low hydrochloric acid secretion
  • mice with low hydrochloric acid secretion mice with low hydrochloric acid secretion
  • mice with a substance which inhibits or reduces the secretion of gastric hydrochloric acid mice with a substance which inhibits or reduces the secretion of gastric hydrochloric acid
  • tissue trans- glutaminase also displays protein deamidase activity.
  • this enzyme is localised in subepithelial myofibro- blasts and secreted from here to the subepithelial reticular sheet located just beneath the basal lamina of the intestinal epithelium.
  • demidating enzymes occur in microorganisms .
  • Transglutaminases have been identified and characterised from Streptoverticillium (Kanaji et al . , 1993) and from Bacillus subtilis (Kobyashi et al . , 1998).
  • two protein deamidating enzymes peptidoglutaminase I and II, have been isolated from Bacillus circulans . It might be that enzymes of this type from intestinal bacteria cause the deamidation and that such bacteria constitute a predisposing environmental factor. This possibility is tested by analyses of deamidating activities of intestinal juice under normal and disease conditions. If deamidation activity is found, attempts to isolate and type the responsible micro- organism will be undertaken.
  • a synthetic peptide without basic or acidic residues (as is the case with the toxic sequence) has a net charge at neutral pH close to zero. This means that it is not retarded on an ion exchanger. Generation of glutamate residues results in one negative charge per deamidated glutamine.
  • a sensitive system using MonoQ-chromatography on the Pharmacia SMART system which allows the demonstration of peptides with one, two or three deamidated glutamines has been developed.
  • Deamidation can be demonstrated by the cell proliferative test using T cell clones from the diseased mucosa as originally used for the demonstration of the deamidation. Once biochemical evidence suggests that deamidation takes place, modified peptides can be tested using T cells.
  • Deamidation can also be followed by a quantitative analysis of the liberated ammonia. This can be done by a commercially available test from Boehringer Mannheim, Germany (cat.no. 1 112 732) . The test is based on spectrophotometric analysis at 340 nm of the disappearance of added NADH after further addition of 2-oxoglutarate and glutamate dehydrogenase to the sample .
  • Deamidation will be searched for either by looking for deami- dase activities using in vi tro enzymatic assays with a distinct peptide as substrate in combination with the MonoQ system or using monoclonal antibodies as monitoring system.
  • Deamidase activity will be looked for in pancreatic secretions, intestinal juice and intestinal wall under normal and patho- logical conditions. The origin and nature of a detected activity will be characterised (normally/pathologically occurring, origin of production (pancreas, intestinal wall, bacteria) ) .
  • deamidated residues in gliadin will be mo- nitored by monoclonal antibodies specific for the deamidation all through the gliadin processing (fermentation, baking, processing in the stomach, processing in the intestinal lumen and processing in the intestinal wall) .
  • One main principle is to modify the gliadins in the food.
  • One embodiment of the invention is thus a method of treating celiac disease comprising interfering with the deamidation of at least one glutamine residue in a gliadin molecule, comprising prohibiting or minimizing deamidation of at least one, preferably all relevant, glutamine residues by derivation of a gliadin in wheat flour.
  • This may be done by bulk deamidation of wheat flour either by non-en- zymatic deamidation using mild alkali or acid treatment, or enzymes (transglutaminase, proteases or peptidoglutaminase) (Hamada JS, 1994) .
  • Blocking might be achieved by chemical or enzymatic derivation.
  • the use of transglutaminase may allow the chemical derivation of glutamine amides in a single reac- tion.
  • the deamidation of at least one glutamine residue in a gliadin molecule may be interfered with and a patient having or suspected of having celiac disease may be treated by admini- stering at least one of the following substances:
  • the pH in the gastroduodenal tract may be increased by admi- nistrering an antacidum, such as aluminiumoxide hydrate, magnesium carbonate, magnesium hydroxide, magnesium oxide, dihy- droaluminium sodium carbonate, magnesium aluminium silicate, aluminium aminoacetate, calcium carbonate or combinations of these substances.
  • an antacidum such as aluminiumoxide hydrate, magnesium carbonate, magnesium hydroxide, magnesium oxide, dihy- droaluminium sodium carbonate, magnesium aluminium silicate, aluminium aminoacetate, calcium carbonate or combinations of these substances.
  • these substances or combinations of substances will be administered in combinations and dosages commonly used for anti-ulcer treatment and the effect tested by a clinical trial essentially as outlined below with respect to a proton pump inhibitor.
  • a substance which is capable of inhibiting or reducing the secretion of gastric acid from parietal cells may be administered in order to increase the pH in the ga- stroduodenal tract.
  • substances suitable for this purpose are anticholinergic agents such as butylscopolamine and propantheline, H-receptor antagonists such as ranitidine, cimetidine, famotidine and nizatidine, and proton pump inhibitors such as omeprazol, lansoprazol and pantoprazol .
  • these substances will be administered in dosages commonly used for anti-ulcer treatment and the effect tested by a clinical trial essentially as outlined in the following section with respect to a proton pump inhibitor.
  • a proton pump inhibitor such as omepra- zol, lansoprazol or pantoprazol in combination with monitoring of the morphology of the intestine and the clinical symptoms.
  • a prospective, open and uncontrolled pilot experiment consisting of 6 to 12 patients with CD in remission and some patients with so-called refractory CD will be performed.
  • the patients will be treated with omeprazol, 40 mg x 2 daily, under gluten provocation.
  • lansoprazol in a dosage of 30 mg x 1-2 or pantoprazol in a dosage of 40 mg x 1-2 may be used.
  • the total length of the trial period is planned to be at most 24 weeks .
  • the results will be evaluated as ear- liest after 6 patients.
  • the study has been accepted by the Danish Medicines Agency and a response from the local ethical committee is awaited within short. It is described in detail in Example 4.
  • Enzymes from the intestinal bacteria may cause deamidation. If it is established that this mechanism is of significance, the deamidase (s) from the intestinal microbiological flora should be reduced or eradicated by treatment with an antibiotic or antimicrobial agent, preferably by a non-absorbable and locally acting treatment.
  • an antibiotic or antimicrobial agent preferably by a non-absorbable and locally acting treatment.
  • the exact choice of antibiotic or antimicrobial agent will depend on the isolation and characterisation of the deamidase producing microorganism (s) . It is contemplated that combinations of tetracycline or amoxi- cillin with metronidazole or clarithromycin for 1-2 weeks will be useful.
  • the antibiotic or antimicrobial agent or combinations of agents will be administered in combinations and dosages commonly used for treating infection by Helicobacter pylori and the effect tested by a clinical trial essentially as outlined above with respect to a proton pump inhibitor.
  • deamidation is caused by an enzyme functioning during the normal processing, it will be possible - dependent on its nature and localisation - either to remove the enzyme or to interfere with its deamidating activity, e.g. by specific enzyme inhibitors.
  • a possible deamidase activity might be removed from baker's yeast by genetic engineering. The effect of the treatment will be tested by a clinical trial essentially as outlined above with respect to a proton pump inhibitor.
  • a chemical derivation has the advantage that it allows the blocking with a substance that does not allow degradation in the intestine (D-lysine, methanol) .
  • the reaction may either be performed on solubilised gluten or on a gluten suspension. Solubilisation without deamidation can be achieved by proteo- lytic treatment, e.g. Pronase E (Babiker et al . , 1996). It is thus possible that transamidation with a proper polar sub- stance similarly will solubilise gluten.
  • Tissue transglutaminase has earlier been used to introduce lysine (Iwami and Yasumoto, 1986) and lysyl dipeptides (Ikura et al . , 1985) into food proteins. This approach has the drawback of high cost of the tissue transglutaminase.
  • a microbial transglutaminase purified from Streptoverticillium (Ando et al . , 1989) is used in some other food industrial processing procedures. Transamidation with lysine or a lysine dipeptide will additionally increase the nutritional value of gluten (Friedman and Finot, 1990; Segura et al . , 1996). There are several studies on the metabolism of gammaglutamyl- lysine.
  • the suitability of wheat for baking bread is determined by interaction between glutenins and gliadins of the gluten. Derivatives of gluten can therefore be expected to change these properties.
  • derivatised gluten Once derivatised gluten has been generated, it will be tested in standardised baking experiments in collaboration with a flour producer. Having obtained detoxified gluten with preserved baking properties, approval for its use as a food with respect to non-toxicity will be obtained. Final- ly, the effect of its intake on the disease will be tested by a clinical trial essentially as outlined above with respect to a proton pump inhibitor.
  • Proliferation of the gut-derived DQ2 -restricted T cell clone 4.32 in response to synthetic peptides of a ⁇ -type gliadin were tested in the untreated form at 10 ⁇ M (black bars) or in the acid/heat-treated form at concentrations of 10, 1, 0.1 ⁇ M (open bars) .
  • the lower panel depicts the responses against the peptides 140-150 ;Pyl40 (SEQ ID NO: 14) and 140-150 ; Pyl40 , E148 (SEQ ID NO:15).
  • the concentrations of the peptides were as above, except that the 140-150;Pyl40,E148 peptide (SEQ ID NO:15) was also tested at 1 and 0.1 ⁇ M in the untreated form.
  • the peptide was incubated with TGase for various time periods and then analyzed by ion exchange chromatography. Depicted are the chromatographs of the peptide after 0, 30, 60 and 180 min incubation with TGase. The fractions of the 180 min chromatography were divi- ded, and one aliquot was tested for recognition by the DQ2- restricted T cell transfectant 60.6. Fractions 15 and 28 from the other aliquot were methyl-esterified and sequenced by ESI MS/MS to determine the number and positions of deamidated glutamine residues.
  • the peak corresponding to fractions 38-46 in the chromatographs contains mainly impurities in the Tris- HC1 buffer. Recognition of each of the fractions by DQ2-restricted T cell transfectant 60.6 was measured by quantification of IL-2 release.
  • the upper part denotes the amino acid sequence of gliadin peptide 134-153 (SEQ ID N0:1) prior to TGase treatment.
  • the lower part denotes the number and positions of deamidated glutamine residues in chromatography fractions 15 and 28 of peptide 134-153 (SEQ ID N0:1) after 180 min TGase treatment. The number and positions of deamidated glutamine residues in each fraction were assessed by ESI MS/MS analysis.
  • Peptide binding to DQ2 was measured in a cell free assay with purified HLA-molecules and binding affinity is given as IC 50 , i.e. the amount of gliadin peptide necessary for 50% inhibition of binding of a labeled indicator peptide.
  • the synthetic peptides were made with Gin to Glu substitutions in the same positions (140, 148 and 150) which were deamidated upon incubation of gliadin peptide with TGase ( Figure 6B) .
  • Recognition of peptides by the DQ2-restricted T cell transfectant 60.6 was measured by IL-2 release in response to peptide stimulation.
  • ⁇ -gliadin 36 A distinct ⁇ -type gliadin ( ⁇ -gliadin 36) was purified from gliadin of the wheat strain Kadett as described in Sj ⁇ str ⁇ m H et al . , 1992. Carboxyamidomethylation and chymotrypsin digestion (1:200 w/w, 24 h) was performed as described in Stone, RA et al . , 1989 followed by heating (98°C) in an acetic acid- HC1 solution, pH 1.8 for 60 min (acid/heat treatment) .
  • Fragments capable of stimulating a T-cell clone in vi tro were purified by successive chromatographies on Sephadex G-50, Superdex Peptide HR 10/30 and MonoQ equilibrated with 5 mM Tris/HCl buffer, pH 6.5 and developed with a gradient ending at 50 mM NaCl . The two latter were run on a SMART system (Pharmacia, Sweden) .
  • T cell clone 4.32 from CD patient 280 (Lundin KEA et al . , 1993) and T cell lines and 25 DQ2-restricted T cell clones from the CD patients 370, 372, 380, 387 (Molberg et al . , 1997), 410, 411, 412 (Molberg et al . , 1998A) were derived from small intestinal biopsies challenged in vi tro with pepsin and trypsin digested gliadin. Proliferative T cell assays using DR3 , DQ2 positive B lymphoblastoid cell lines as antigen presenting cells were performed as described elsewhere (Lundin KEA et al . , 1993).
  • MALDI-TOF Matrix-assisted laser desorption/ionization-time-of-flight
  • Partial sequence of the peptide was obtained by ESI MS/MS (Hunt DF et al . , 1986) and by C-terminal sequencing using carboxypeptidase Y followed by MALDI-TOF analysis.
  • the information on peptide mass, fragment masses and partial sequence matched the sequence of a ⁇ -type gliadin (Sugiyama T et al . , 1986) in the SWISSPROT database (amino acids 134-153 of GDB2_WHEAT; accession no. P08453) when accounting for modification of an N-terminal glutamine to a pyroglutamic acid and deamidation of 1, 2 or 3 of the other glutamines.
  • the peptides were next tested for binding to DQ2 in a cell free binding assay (Johansen BH et al . , 1994).
  • Peptide 138- 152;Pyl38,E148 (SEQ ID N0:11) bound significantly better than the 138-152;Pyl38 (SEQ ID NO:10) peptide (IC 50 values 6.2 ⁇ M vs. 61.4 ⁇ M) indicating deamidation to be relevant for binding to DQ2.
  • Alignment of the 141-150 sequence with the DQ2 binding motif (Johansen BH et al . , 1996; van de Wal Y et al . , 1996; Vartdal F et al .
  • the immuno-relevance of the 134-153 peptide was analyzed by testing a further 10 polyclonal gut-derived T cell lines derived from seven patients. None of the T cell lines responded positively to untreated peptide whereas T cells from two patients recognized the acid/heat treated peptide. The same T cell lines also responded to the 140- 150;Pyl40,E148 peptide (SEQ ID NO:15) but not to the untreated 140-150;Pyl40 peptide (SEQ ID N0:14) suggesting the 140-150;Pyl40,E148 epitope (SEQ ID NO: 15) to be a common epitope for intestinal T cells of different CD patients (Table 2) .
  • T cell lines from six patients as well as 25 T cell clones from five patients were tested with chymotrypsin digested gliadin either untreated or acid/heat treated. All T cell lines and most clones displayed weak reactivity against the untreated gliadin, while the reactivity was markedly enhanced by acid/heat-treatment (Fig. 3) . Thus deamidation appears to be important for recognition of gliadin by most T cells from the small intestinal mucosa of CD patients.
  • gliadin could theoretically take place ex vivo (i.e. during the fermentation or during the baking process) or in vivo .
  • Experiments failed to support the former possibility whereas there has been evidence for the latter.
  • Incubation at 37°C, pH 1.8 for 15 minutes of chymotrypsin digested gliadin created the epitope recognized by 4.32.
  • Similar results were obtained with one T cell line (CD380-R) and one clone (CD387.9-E) that recognize other DQ2-restricted gliadin epitopes. This suggests that sufficient deamidation can occur in the acidic environment of the stomach.
  • the deamidation could also be caused by enzymatic processes.
  • tissue transglutami- nase can deamidate gliadins and create the epitope described here (Example 2) .
  • enzymes of intestinal bacteria may cause the deamidation (Ogawa M et al . , 1978) and such bacteria may constitute a predisposing environmental factor in CD.
  • Negatively charged residues are preferred as anchors for binding of peptides to DQ2 in the positions P4 , P6 and P7 (Johansen et al . , 1996; van de Wal et al . , 1996; Vartdal F. et al . , 1996) .
  • No known motifs of other human class II al- leles demonstrate such a clear preference for negatively charged residues at P7 as DQ2 (Rammensee HG et al . , 1995). Proteins with a high content of negatively charged amino acids should hence be particularly likely sources of DQ2 -restricted epitopes.
  • Native gliadins have a very low content of negatively charged residues (usually ⁇ 2% of the residues) .
  • gliadins have a very high content of glutamine (usu- ally > 35% of the residues) . This could explain the drastic effect of deamidation for creation of DQ2-restricted T cell epitopes, and provides an explanation as to why gliadins are involved in DQ2 -restricted T cell recognition.
  • the present results furthermore indicate that recognition of deamidated gliadins is central in the pathogenesis of CD. Thus interference with processes leading to deamidation of gliadin is possibly a target for therapy of CD.
  • T cells can recognize epitopes only after their modification has implications for immunology in general and T cell mediated diseases in particular.
  • the methods traditionally employed for the characterization of T cell antigens are unlikely to report modified epitopes.
  • the present observations illustrate that taking the possibility of antigen modi- fication into consideration can lead to novel and important findings .
  • gliadin Digestion of crude gliadin (Sigma St. Louis, MO; G-3375) with pepsin (Sigma; P-7012) and trypsin (Sigma; T-7418) was done as described in Lundin KE et al . , 1993.
  • ⁇ -chymotrypsin Sigma; C-3142 digestion of gliadin from the wheat strain Kadett was performed at 200:1 (w/w) in 0.1 M NH 4 HC0 3 with 2 M urea at 37°C for 24h and stopped by incubation in boiling water for 5 min.
  • Synthetic gliadin peptides quality controlled by HPLC (> 80% purity) and MS were purchased from Research Genetics (Huntsville, AL, USA) .
  • PT-gliadin specific, gut-derived-T cell lines were established from small intestinal biopsies essentially as described in Lundin KE et al . , 1993, with the exception that isolation of some T cells from the gliadin-challenged biopsy material was done without enrichment for CD25 expressing cells.
  • Gut-derived T cell lines of three DQ2+CD patients (CD370, 380 and 387) and two DQ8+ patients (CD282 and CD360) on a gluten free diet and three untreated DQ2+ patients (CD410, 411 and 412) were tested.
  • DQ2-restricted T cell clones (370.E-14, 380. E-3, E-ll and E-27, 387.R-3,R-12 and R-16, 411.
  • V ⁇ and V ⁇ gene segments from the gliadin specific gut-derived T cell clone 4.32 were amplified from genomic DNA and cloned into the T cell receptor expression vectors pT ⁇ cass and pT ⁇ cass, respectively (Kouskoff V et al . , 1995) .
  • These vectors allow for functional expression of mu- rine or human variable region T cell receptor spliced to mu- rine constant regions (Madsen, L., Svejgaard, A. and Fugger, L. unpublished observations) .
  • T cells (5xl0 4 ) were added to 5xl0 4 APC (either HLA matched allogenic Epstein Barr virus transformed B-LCL irradiated 80 Gy or HLA matched allogenic PBMC irradiated 25 Gy) which had been incubated for 16-20h with complex antigen or 4h with peptides in a volume of 100 ⁇ l of RPMI 1640 (Gibco, Paisley, Scotland) supplemented with 15% pooled human serum. Assays were performed in 96 well U-bottom plates (Nunc, Roskilde, Denmark) and T cell proliferation was measured as 3 H thymidine incorporation 48-72 hours after antigenic stimulation.
  • Activation of the 60.6 T cell transfectant was measured as IL2 release following an 18 hour incubation of 2.5xl0 4 transfectant cells with 5xl0 4 DQ2+ B-LCL as APC.
  • the concentration of IL2 was quantified by time-resolved fluorometry, using Delfia reagents (Wallac, Finland) and murine anti-IL2 antibodies (JES6-1A12 and JES6-5H4, Pharmingen, CA, USA) .
  • Parallel assays using the original T cell clone and the transfectant confirmed that the chimeric T cell receptor expressed by the transfectant maintained its specificity both in terms of MHC restriction and peptide specificity (data not shown) .
  • gliadin (1-1000 ⁇ g/ml) , chymotrypsin-digested gliadin
  • gliadin/TGase mixtures were incubated with APC and tested in T cell assays as above.
  • Peptides 134-153 (SEQ ID NO:l), 134-153;E148 (SEQ ID NO : 4 ) , 134-153;E140,E148,E150 (SEQ ID NO:7) were synthesized with an extra N-terminal Y (i.e. 133-153, Y133 etc.) and radiolabeled with 125 I using the chloramine T method as described in Johansen B et al., 1996. TGase was 125 I-labeled as control.
  • Peptide ions were isolated in the ion trap and fragmented by collisions with He gas creating a series of N- and C-terminal fragments with differing peptide chain lengths. By comparison of the fragmentation patterns of the native peptide with the TGase treated fractions, mass increments of 15 Da could be assigned to methyl-esterified Glu residues in these fragment ions.
  • MALDI-TOF mass spectra were obtained on a Bruker Reflex mass spectrometer (Bruker-Franzen Analytik GmbH, Bremen, Germany) .
  • ESI tandem mass spectra were recorded on an ESQUIRE ion trap mass spectrometer (Bruker- Franzen Analytik) .
  • a panel of T cell lines and clones isolated from duodenal biopsies challenged ex vivo with pepsin-trypsin digested gliadin (PT-gliadin, an antigen known to provoke disease in vivo (Frazer AC, 1959) were tested. It was found that addition of TGase to proli erative assays enhanced reactivity and sensitivity to PT-gliadin for all these gut-derived T cells (data not shown) . None of the gut-derived T cells were reactive to TGase alone.
  • TGase was not dependent on type of antigen presenting cells (APC) as results with B lymphoblastoid cells lines (B- LCL) and T cell depleted peripheral blood mononuclear cells (PBMC) both demonstrated enhanced responses (10 fold and 30 fold) . Further, the effect of TGase on T cell recognition appeared to be specific for gliadin.
  • APC antigen presenting cells
  • B- LCL B lymphoblastoid cells lines
  • PBMC peripheral blood mononuclear cells
  • PBMC-derived T cell clones specific for PPD Purified Protein Derivative
  • TGase mediated antigen modification data not shown.
  • the TGase effect is restricted to gut-derived, gliadin specific T cells
  • TGase The most established property of TGase is to catalyze protein crosslinking via the formation of isopeptide bonds between Gin and Lys residues (Folk JE et al . , 1983; Aeschlimann D et al . , 1994). Gliadins are rich in Gin residues, but typically have very few Lys residues (Wieser H, 1995) . Nonetheless, gliadin-gliadin complexes are known to form in the presence of TGase (Larre C et al . , 1993) .
  • TGase could enhance gut-derived T cell recognition of gliadin by catalyzing the formation of gliadin complexes.
  • DQ2 -restricted T cell clone no increase in PT-gliadin specific proliferation of PBMC derived T cells from six CD-patients (Gjertsen HA et al . , 1994) was detected upon addition of TGase (data not shown) . This argues that increased uptake of gliadin mediated by TGase cannot account for the observed enhanced proliferation of gut-derived T cells.
  • gliadin-specific T cells resident in peripheral blood and gut mucosa may recognize distinct epitopes and, most importantly, that TGase could be instrumental in the creation of gliadin epitopes recognized by gut-derived T cells.
  • PT-gliadin recognized by gut-derived T cells has been subjected to digestion at low pH, a condition known to faci- litate non-enzymatic deamidation of Gin residues (Hamada JS, 1994) .
  • TGase can catalyze substrates to react with water to be deamidated when amine acceptors are in deficit (Folk JE, 1983) .
  • Gin deamidations have been demonstrated with gliadin as a substrate (Larre C et al . , 1993) .
  • TGase mediated deamidation of gliadin could be important for recognition by gut-derived T cells was tested by analyzing the effect of TGase on gut-de- rived T cell recognition of a water-soluble chymotrypsin digested gliadin. Digestion with chymotrypsin was performed at neutral pH, thereby avoiding spontaneous non-enzymatic deamidation. Chymotrypsin-digested gliadin only weakly stimulated gut-derived T cells from six CD patients (four DQ2+ and two DQ8+) . However, it could be converted to an efficient antigen by incubating it in acid (pH 1.8) for 1 hour at 98°C. Notably, TGase treatment of the chymotrypsin-digested gliadin generated a more potent antigen for all of the gut-derived T cells than the acid/heat treatment ( Figure 4 and data not shown) .
  • Example 1 A detailed molecular analysis of the actions of TGase on gliadin focused on a recently characterized gliadin epitope recognized by gut-derived DQ2 -restricted T cells from three different CD patients (Example 1) .
  • This epitope was originally identified within a 20mer fragment isolated from a purified ⁇ -type gliadin. In its unmodified form this fragment was determined to have the sequence QQLPQPQQPQQSFPQQQRPF (SEQ ID N0:1), a sequence that matched the residues 134-153 of the SWISSPROT entry GDB2_WHEAT (accession No. P08453) .
  • Gut-derived T cell lines from two patients (CD370 and CD412) , as well as a T cell transfectant (60.6) expressing a chimeric T cell receptor containing variable regions cloned from the gliadin peptide-specific gut-derived T cell clone 4.32 recog- nized the acid/heat treated peptide 134-153 (SEQ ID N0:1) .
  • These T cells were used to demonstrate that TGase treatment transformed peptide 134-153 (SEQ ID NO:l) from a non-stimulatory to a potent T cell stimulatory peptide.
  • the proposed peptide-binding motif for DQ2 predicts a prefe- rence for negatively charged residues in relative position 7 (Johansen BH et al . , 1996; van de Wal Y et al . , 1996).
  • Testing of synthetic peptides in a cell free DQ2 binding assay demonstrated a 10-fold increase in binding affinity of peptides with a E148 substitution ( Figure 6A and Example 1) , indi- eating that position 148 could be accommodated into DQ2 pocket 7.
  • TGase might be involved in processing of gliadin prior to binding to DQ2 molecules.
  • DQ2 molecules There is an infiltrate of T cells in the same region, and ex vivo studies of gliadin challenged biopsies have demonstrated that some of these T cells are gliadin specific (Halstensen TS et al . , 1992) .
  • the subepithelial region appears to be a micro- environment where TGase mediated modification, DQ2 binding and T cell recognition of gliadin might occur.
  • the present data indicate that, in addition to generating gliadin fragments recognized by gut-derived T cells, TGase can also catalyze formation of complexes between gliadin T cell epitopes and TGase.
  • TGase can also catalyze formation of complexes between gliadin T cell epitopes and TGase.
  • autoantibody production to TGase does not involve break of T cell tolerance to the abundantly expressed TGase protein.
  • Chemically deamidated gluten is available as a food additive from several food companies, as acid hydrolysis of gluten is an accepted method to change the properties of gluten.
  • the Amylum Group (Belgium) thus prepares and sells SWP 050, a soluble wheat protein especially developed for its emulsifying properties, and it is recommended for use in meat, sauces and dressings.
  • Another product of The Amylum Group, SWP 100 is completely soluble above pH 5.5 and has a high emulsifying capacity and an excellent emulsion stability. This product is recommended for coffee creamers, soups, sauce, bread and meat.
  • Corresponding products can also be obtained from other gluten producers, e.g. Midwest grain products, Inc. Atchin- son, KS, USA and Kroner Starke, Germany.
  • Enzymatically digested gluten for calf feeding is industrially produced and available from The Amylum Group, Belgium (Solpro) and from Hyproca Dairy, The Netherlands (Novolat 80) .
  • the Amylum Group also has a corresponding product, SWP 500, for use as a food additive.
  • Microbial transglutaminase is used in the food industry to induce crosslinks between proteins and thereby give a better texture and improve the baking strength. It is produced as a commercial product (ActivaTM) for food by Ajinomoto, Japan.
  • the derivation of glutamine amide groups can be monitored by the analysis of liberated ammonia.
  • Ammonia is analysed by the quantification of the disappearance of NADH after the addition of 2-oxo-glutarate, NADH and glutamate dehydrogenase to the sample. The test is commercially available from Boehrin- ger Mannheim, Germany.
  • the studies comprise a prospective, open, and uncontrolled pilot series of 6-12 patients with celiac disease in favour- sion as well as individual patients with so-called refractory celiac disease.
  • the patients are treated with omeprazol (40 mg x 2 daily) with gluten provocation, and the total study period for each patient is at the most 16 weeks.
  • the results are evaluated at the earliest when 6 patients have entered and at the latest when 12 patients have entered. If the studies indicate a positive effect of omeprazol, subsequent prospective, randomized, controlled and blinded studies are planned (specific protocol) .
  • the results are collected from two clinical, gastroenterologic centres (the University Hospitals in Copenhagen Rigshospitalet and Hvidovre Hospital) , 3-6 patients from either centre. The study is expected to begin on 1 May 1999 and will last 18 months.
  • T 0 : Patients in remission. Start with one tablet of omeprazol 40 mg x 2 (Losec®, Astra) for 3 days. On the 4th day, increasing gluten exposition is started, beginning with 1/2 slice of bread a day and doubling the amount of bread every 3rd day up to a total of 4 slices of white bread (Trier, 1998) .
  • the patients are started on a Losec® treatment (no provocation) .
  • Losec® is discontinued, and the protocol is followed unchanged for another 8 weeks .
  • Clinical recurrence Increasing symptoms: Moderate (bothering) to severe (preventing daily tasks) discomfort (stomach pain, diarrhoea, discomforting gases, other gastro-intestinal problems, unspecific complaints) ascribable to the gluten provocation.
  • the serum albumin concentration below 500 mg/litre (ref.: 540-800) .
  • the project medicine (Losec® tablets, Astra A/S) is labelled with the administration procedure, name, address and tele- phone number of the main investigator and Astra A/S. Counting of the remainder of the medicine is carried out at each medical control .
  • omeprazol treatment is continued if there is a beginning histological remission after 8 weeks, and a renewed biopsy is taken after 26 weeks.
  • Type I Normal mucosa .
  • Type II Shortened and broadened villi with a decreased crypt/villus ratio compared to type I, and increased cellu- larity of lamina propria.
  • Type III Very short, almost eradicated villi with an in- versed crypt/villus ration, cubic or pseudostratified epithelium, long twisted crypts, as well as tight mononuclear in- filtration of lamina propria.
  • Type IV Total villus atrophy.
  • Blood test values Hgb concentration, Se-iron, Se-albumin
  • Serological results Increase in gliadin and antibody titer against endomysium
  • Symptom development Change in the total score . Symptoms are assessed at a total score two days before beginning the study, and prior to each medical control (protocol) . Statistic evaluations are made non-parametrically.
  • T cells from the peripheral blood of coeliac disease patients recognize gluten antigens when presented by HLA-DR, -DQ, or -DP molecules. Scand. J. Immunol. 1994; 39:567-574.
  • HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity. 1997; 6:273-281.
  • Trier JS Diagnosis of celiac sprue. Gastroenterology 1998; 115:211-216.

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Abstract

L'invention concerne une méthode de traitement de la maladie caeliaque, qui consiste à contrecarrer la déamidation d'au moins un reste glutamine dans une molécule de gliadine ou de gluténine. Pour ce faire, on peut inhiber ou contrecarrer la déamidation d'au moins un reste glutamine, par la dérivation d'au moins un reste glutamine, par la dérivation d'au moins un reste glutamine dans une molécule de gliadine ou de gluténine de farine de blé, par déamidation chimique ou enzymatique du gluten, suivie de la dérivation chimique ou enzymatique du(des) groupe(s) carboxyle(s) généré(s). L'invention porte aussi sur une méthode permettant de contrecarrer la déamidation d'au moins un reste glutamine dans une molécule de gliadine ou de gluténine et donc de traiter la maladie caeliaque. Ledit procédé consiste à administrer, à un patient atteint d'une maladie caeliaque ou suspecté d'en être atteint, au moins une des substances suivantes: (a) une substance capable d'augmenter le pH dans le tractus gastroduodénal d'un sujet, par exemple un antacidum, un agent anticholinergique, des antagonistes du récepteur H2 ou un inhibiteur de la pompe à protons; (b) une substance capable d'éliminer la déamidation de bactéries dans le tractus gastroduodénal d'un sujet, par exemple un agent antibiotique ou antimicrobien; et/ou (c) une substance capable de contrecarrer l'effet d'au moins une enzyme de déamidation dans le tractus gastroduodénal d'un sujet.
EP99917810A 1998-05-06 1999-05-06 Traitement de la maladie caeliaque Withdrawn EP1075267A2 (fr)

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EP1563300B1 (fr) * 2002-11-20 2012-04-18 The Board Of Trustees Of The Leland Stanford Junior University Procede de diagnostic de la maladie coeliaque
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ITMI20062080A1 (it) 2006-10-30 2008-04-30 Consiglio Nazionale Ricerche Trattamento di farine di cereali per il consumo alimentare da parte di pazienti celiaci
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