EP2277046A2 - Analogues peptidiques du gluten non inflammatoires utilisés comme biomarqueurs de la maladie coeliaque - Google Patents

Analogues peptidiques du gluten non inflammatoires utilisés comme biomarqueurs de la maladie coeliaque

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Publication number
EP2277046A2
EP2277046A2 EP09746974A EP09746974A EP2277046A2 EP 2277046 A2 EP2277046 A2 EP 2277046A2 EP 09746974 A EP09746974 A EP 09746974A EP 09746974 A EP09746974 A EP 09746974A EP 2277046 A2 EP2277046 A2 EP 2277046A2
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EP
European Patent Office
Prior art keywords
peptide
gluten
mer
analog
gluten peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP09746974A
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German (de)
English (en)
Inventor
Michael Thomas Bethune
Chaitan Khosla
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Leland Stanford Junior University
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Leland Stanford Junior University
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Publication of EP2277046A2 publication Critical patent/EP2277046A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Clinical symptoms of celiac sprue include fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, as well as a substantially enhanced risk for the development of osteoporosis and intestinal malignancies (lymphoma and carcinoma).
  • the disease has an incidence of approximately 1 in 200 in most populations.
  • glutenases oral enzyme therapies
  • Other types of drugs are also being considered for treatment of celiac sprue.
  • a related disease is dermatitis herpetiformis, which is a chronic eruption characterized by clusters of intensely pruritic vesicles, papules, and urticaria-like lesions.
  • IgA deposits occur in almost all normal-appearing and perilesional skin.
  • Asymptomatic gluten-sensitive enteropathy is found in 75 to 90% of patients and in some of their relatives. Onset is usually gradual. Itching and burning are severe, and scratching often obscures the primary lesions with eczematization of nearby skin, leading to an erroneous diagnosis of . eczema. Strict adherence to a gluten-free diet for prolonged periods may control the disease in some patients, obviating or reducing the requirement for drug therapy.
  • Dapsone, sulfapyridine and colchicines are sometimes prescribed for relief of itching, although the underlying disease is unaffected by these drugs.
  • the above-mentioned therapies are also expected to be useful for the treatment of dermatitis herpetiformis.
  • the environmental trigger of celiac sprue is dietary gluten from common food grains such as wheat, rye, and barley. Duodenal digestion of ingested gluten releases proteolytically resistant, immunotoxic peptide fragments, such as the immunodominant 33- mer from ⁇ -gliadin (Shan et al. (2002) Science 297: 2275-2279).
  • peptides traverse the mucosal epithelium by unknown mechanisms and are deamidated at specific glutamine residues by an endogenous enzyme, tissue transglutaminase (tTG) (Molberg et al. (1998) Nat Med 4: 713-717; Arentz-Hansen et al. (2000) J Exp Med 191 : 603-612).
  • tTG tissue transglutaminase
  • Deamidated peptides bind with high affinity to the primary genetic determinant of celiac sprue, human leukocyte antigen (HLA) DQ2 (Quarsten et al. (1999) Eur J Immunol 29: 2506-2514; Kim et al.
  • CD4 + T cells Upon encountering DQ2-gluten complexes on the surface of antigen presenting cells (APC), gluten-specific, DQ2-restricted CD4 + T cells are activated to induce a Th1 response comprising the secretion of pro-inflammatory cytokines, such as IFN- ⁇ , and the recruitment of CD8 + intraepithelial lymphocytes, ultimately causing mucosal damage (Jabri et al. (2005) Immunol Rev 206: 219-231 ). Additionally, CD4 + T cells give help to a humoral immune response comprising production of both gluten- specific antibodies and TG2-specific autoantibodies (Sollid LM (2002) Nat Rev Immunol 2: 647-655).
  • EP-B2 the cleavage sites for EP-B2 in the immunodominant peptide, 33-mer, are coincident with those glutamine residues that are selectively deamidated by tTG (Bethune et al (2006) Chem Biol 13: 637-647), suggesting EP-B2-catalyzed cleavage of gluten may interfere specifically with this critical step in disease progression.
  • the therapeutic promise of EP-B2 is further underscored by its digestion of gluten in vivo (see Gass et al. (2006) J Pharmacol Exp Ther 318: 1178-1186) and by its ability to protect a gluten-sensitive rhesus macaque against gluten-induced clinical relapse when dosed orally (Bethune et al. (2008) PLoS ONE 3: e1857).
  • T cell proliferation assays are more sensitive, but they require invasive procedures (e.g. withdrawal of a small intestinal biopsy or relatively large quantities of blood to harvest adequate numbers of peripheral blood mononuclear cells) and are deemed to be too expensive for routine use.
  • invasive procedures e.g. withdrawal of a small intestinal biopsy or relatively large quantities of blood to harvest adequate numbers of peripheral blood mononuclear cells
  • the present invention addresses this emerging but unmet medical need.
  • enteropathic disease which diseases include, without limitation, celiac sprue, Crohn's disease and irritable bowel syndrome, and inflammation associated with intestinal infection, e.g. infection with rotavirus, giardia, enteroaggregative E. coli, Cryptosporidium, and the like.
  • the methods are utilized for monitoring ongoing therapeutic regimens for such enteropathic diseases.
  • the methods of the invention are used in determining the efficacy of a therapy for treatment of an enteropathic disease, either at an individual level, or in the analysis of a group of patients, e.g. in a clinical trial format. Such embodiments typically involve the comparison of two or more time points for a patient or group of patients. The patient status is expected to differ between the two time points as the result of administration of a therapeutic agent, therapeutic regimen, or challenge with a disease- inducing agent to a patient undergoing treatment.
  • the efficacy of therapy in a patient with an enteropathic disease is assessed by detecting the ability of the patient to metabolize an orally administered gluten peptide analog, where the term "metabolize” refers to proteolytic cleavage of gluten peptides and peptide analogs; or refers to the ability of the gut to maintain normal permeability.
  • Such gluten analog peptides are substantially similar to native gluten peptides in susceptibility to proteolytic cleavage; but are substantially decreased in pathogenic properties of the native gluten peptide, i.e. are not presented by the DQ MHC protein; and are not a substrate for deamidation by tissue transglutaminase.
  • these peptides are not presented to gluten-specific T cells and are consequently non-inflammatory.
  • Such analog peptides can be safely administered at a higher dose than native gluten proteins or peptides.
  • Gluten analog peptide compositions are also a feature of the invention.
  • the patient metabolism of the peptide may be monitored in a variety of ways.
  • the peptide analog may be labeled, e.g. with an isotopic, fluorescent, etc. label, and the appearance of labeled amino acids that result from metabolism of the peptide is detected in a patient sample over a period of time following oral administration, e.g. in urine, plasma, breath, saliva, etc.
  • the level of labeled or unlabeled peptide itself (or a partially proteolyzed metabolite) in a bodily fluid may be determined by various methods, including immunoassays.
  • the determination of the presence of the peptide is used in determining peptide cleavage, or for assessing the extent of leakiness of the epithelial barrier of the small intestine in the context of celiac sprue, or other inflammatory bowel diseases where mucosal leakiness is elevated.
  • the methods of the invention are used to diagnose celiac sprue.
  • the enhanced permeability of the epithelial barrier in patients with active disease is correlated to more established measures of diagnosis such as tTG auto-antibodies and upper intestinal villus atrophy.
  • the sequence of a 33-residue immunotoxic gluten peptide derived from ⁇ 2-gliadin is modified so that three key glutamine (Q) residues that are selectively deamidated by TG2 are substituted with non-substrate amino acids, e.g. by asparagine (N) or histidine (H). Labeled or unlabeled biomarker peptides can safely be administered orally to celiac sprue patients in conjunction with a drug or placebo.
  • a patient sample is obtained prior to treatment, as a control, and compared to samples from the same patient following treatment.
  • the CYP3A function is assessed over long periods of time to monitor patient status.
  • FIG. 1 Synthetic gluten and biomarker peptide sequences. Sequences are shown for the native 33-mer (designated QQQ-33-mer) derived from (2-gliadin, the synthetically deamidated 33-mer (EEE-33-mer), the biomarkers NNN-33-mer and HHH-33-mer, and a non-inflammatory control peptide of unrelated sequence derived from myoglobin. Bonds that are scissile to EP-B2-mediated cleavage are designated by arrowheads. Glutamine residues that are selectively deamidated by TG2 or synthetically replaced in the biomarker peptides are in bold. Leucine residues that are isotope-labeled for in vivo experiments are underlined.
  • TAME internal standard T
  • intact peptide peak 5
  • minimally processed peptide lacking only the N-terminal LQ peak 4
  • other major digestion products are indicated for each peptide trace overlay
  • g-j Integrated area-under-the-curve analysis for intact peptides
  • g QQQ-33-mer
  • h NNN-33-mer
  • i HHH-33-mer
  • j myoglobin peptide showing dose- and time-dependency of EP-B2- mediated digestion.
  • Each peptide 300 ⁇ M was digested in vitro with pepsin supplemented with specified concentrations of EP-B2, and reaction products were analyzed by HPLC.
  • Biomarkers are non-inflammatory in the context of celiac sprue, (a) Specific activity of transglutaminase 2 (TG2; 5 ⁇ M) in the presence of 100 ⁇ M gluten peptide QQQ- 33-mer, biomarkers NNN-33-mer or HHH-33-mer, control myoglobin peptide, or no peptide. Means ⁇ s.d. for triplicate assays are shown. Data are representative of 3 independent experiments. Statistical comparisons were performed with respect to samples containing QQQ-33-mer. *** p ⁇ 0.001.
  • Enteropathic inflammatory disease is clinically monitored by measuring the metabolism and digestion kinetics of gluten peptide analogs.
  • such substances are orally administered as a solution, capsule, enteric formulation, etc.
  • therapeutic drug or “therapeutic regimen” refers to an agent used in the treatment or prevention of a disease or condition, particularly an enteropathic condition for the purposes of the present invention. Of interest are clinical trials using such therapies, and monitoring of patients undergoing such therapy.
  • the therapy involves treatment of celiac sprue patients with glutenase. In other embodiments, the therapy involves treatment of celiac sprue patients with a permeabilibity modifying agent. Assessment of treatment may utilize a gluten challenge. In some embodiments, 1-14 days of a moderate dose (at least about 1 g/day, at least about 5 g/day, at least about 10 g/day, or more) of oral gluten is utilized for this for this purpose. Patients may be control patients that have not been treated, or patients subject to a clinical regimen of interest, e.g. dietary restriction of gluten, treatment with permeability modifier, treatment with glutenase, and the like.
  • a "patient,” as used herein, describes an organism, including mammals, from which samples are collected in accordance with the present invention.
  • Mammalian species that benefit from the disclosed systems and methods for therapeutic drug monitoring include, and are not limited to, apes, chimpanzees, orangutans, humans, monkeys; and domesticated animals (e.g., pets) such as dogs, cats, mice, rats, guinea pigs, and hamsters.
  • pharmacokinetics refers to the mathematical characterization of interactions between normal physiological processes and a therapeutic drug over time (i.e., body effect on drug). Certain physiological processes (absorption, distribution, metabolism, and elimination) will affect the ability of a drug to provide a desired therapeutic effect in a patient. Knowledge of a drug's pharmacokinetics aids in interpreting drug blood stream concentration and is useful in determining pharmacologically effective drug dosages
  • sample refers to a sample from an animal, most preferably a human, seeking diagnosis or treatment of a disease, e.g. an enteropathic disease.
  • Samples of the present invention include, without limitation, urine, saliva, breath, and blood, including derivatives of blood, e.g. plasma, serum, etc.
  • Sample analysis Patient samples are analyzed to determine the metabolism of a gluten peptide analog, usually an orally administered gluten peptide analog. Sample may be quantitatively analyzed for the presence of the substrate and/or its metabolites by any suitable assay, which are well-known in the art. Methods of analysis include liquid chromatography-mass spectroscopy (see Kanazawa et al. (2004) J.
  • the biological sample is patient urine.
  • concentration of the peptide and its metabolites, i.e. amino acids, dipeptides, etc. can be monitored in a 6- hour urine collection.
  • Conditions of interest for monitoring methods of the present invention include a variety of enteropathic conditions, particularly inflammatory chronic conditions.
  • a patient is diagnosed as having an enteropathic condition, for which treatment is contemplated.
  • the patient may be initially tested for activity prior to treatment, in order to establish a baseline level of activity.
  • the patient may be released from a treatment regimen for a period of time sufficient to induce an enteropathic state, in which state the patient is tested for activity in order to establish a baseline level of activity.
  • Enteropathic conditions of interest include, without limitation, celiac sprue, herpetiformis dermatitis, irritable bowel syndrome (IBS); Crohn's Disease; and inflammation associated with intestinal infection.
  • Celiac sprue is an immunologically mediated disease in genetically susceptible individuals caused by intolerance to gluten, resulting in mucosal inflammation, which causes malabsorption. Symptoms usually include diarrhea and abdominal discomfort. Onset is generally in childhood but may occur later. No typical presentation exists. Some patients are asymptomatic or only have signs of nutritional deficiency. Others have significant Gl symptoms.
  • Celiac sprue can present in infancy and childhood after introduction of cereals into the diet. The child has failure to thrive, apathy, anorexia, pallor, generalized hypotonia, abdominal distention, and muscle wasting. Stools are soft, bulky, clay-colored, and offensive. Older children may present with anemia or failure to grow normally. In adults, lassitude, weakness, and anorexia are most common. Mild and intermittent diarrhea is sometimes the presenting symptom. Steatorrhea ranges from mild to severe (7 to 50 g fat/day). Some patients have weight loss, rarely enough to become underweight. Anemia, glossitis, angular stomatitis, and aphthous ulcers are usually seen in these patients. Manifestations of vitamin D and Ca deficiencies (eg, osteomalacia, osteopenia, osteoporosis) are common. Both men and women may have reduced fertility.
  • vitamin D and Ca deficiencies eg, osteomalacia, osteopenia, osteoporosis
  • These markers can also be used to screen populations with high prevalence of celiac sprue, including 1st- degree relatives of affected patients and patients with diseases that occur at a greater frequency in association with celiac sprue. If either test is positive, the patient may have a diagnostic small-bowel biopsy performed. If both are negative, celiac sprue is unlikely. Other laboratory abnormalities often occur and may be sought. These include anemia (iron-deficiency anemia in children and folate-deficiency anemia in adults); low albumin, Ca, K, and Na; and elevated alkaline phosphatase and PT. Malabsorption tests are sometimes performed, although they are not specific for celiac sprue.
  • common findings include steatorrhea of 10 to 40 g/day and abnormal D- xylose and (in severe ileal disease) Schilling tests.
  • Conventional treatment is a gluten-free diet (avoiding foods containing wheat, rye, or barley). Gluten is so widely used that a patient needs a detailed list of foods to avoid. Patients are encouraged to consult a dietitian and join a celiac support group. The response to a gluten-free diet is usually rapid, and symptoms resolve in 1 to 2 months. Ingesting even small amounts of food containing gluten may prevent remission or induce disease.
  • Complications include refractory sprue, collagenous sprue, and the development of intestinal lymphomas. Intestinal lymphomas affect 6 to 8% of patients with celiac sprue, usually presenting in the patient's 50s. The incidence of other Gl malignancies (eg, carcinoma of the esophagus or oropharynx, small-bowel adenocarcinoma) increases. Adherence to a gluten-free diet can significantly reduce the risk of malignancy.
  • Gl malignancies eg, carcinoma of the esophagus or oropharynx, small-bowel adenocarcinoma
  • Dermatitis herpetiformis is a chronic eruption characterized by clusters of intensely pruritic vesicles, papules, and urticaria-like lesions. The cause is autoimmune. Diagnosis is by skin biopsy with direct immunofluorescence testing. Treatment is usually with dapsone or sulfapyridine.
  • Patients may have skin biopsy of a lesion and adjacent normal-appearing skin. IgA deposition in the dermal papillary tips is usually present and important for diagnosis. Patients should be evaluated for celiac sprue.
  • sulfapyridine may be used as an alternative for those who cannot tolerate dapsone.
  • Initial oral dosage is 500 mg bid, increasing by 1 g/day q 1 to 2 wk until disease is controlled. Maintenance dosage varies from 500 mg twice/wk to 1000 mg once/day. Colchicine is another treatment option. Treatment continues until lesions resolve.
  • Gluten peptide analogs of the present invention have properties of native gluten peptides, non-limiting examples of which are provided in Table 1 , but are modified to decrease the pathogenic properties of stable binding to MHC DQ proteins, and of acting as a substrate for tissue transglutaminase.
  • Native gluten peptides suitable for modification are at least about 14 amino acids in length, and not more than about 40 amino acids in length, and may be obtained by partial proteolytic digestion of a grain gluten or gliaden polypeptide, e.g. wheat, barley, oats, etc. as is known in the art, for example as set forth in Shan et al. (2005) J. Proteome Res. 4:1732-1741 (herein specifically incorporated by reference).
  • Such oligopeptides have the properties of being (i) resistant to digestion with mammalian pancreatic and gastric proteases, e.g. as described in detail in U.S. Patent no. 7,303,871 , and in Shan et al.
  • (ii) are substrates for deamidation by tissue transglutaminase, e.g. as described in Bethune et al. (2006) supra, (herein specifically incorporated by reference); and (iii) are presented by human MHC DQ, e.g. as described by Quarsten et al. (1999), supra., and Kim et al. (2004), supra., (each herein specifically incorporated by reference).
  • the amino acid sequence of a native gluten peptide is modified from the native peptide, e.g. a peptide selected from SEQ ID NO:1-SEQ ID NO:38 to reduce the pathogenicity without substantially changing the metabolic profile of the peptide.
  • Analog peptides of the invention are typically altered from a native peptide in the replacement of at least one, at least two, at least three, and not more than about five amino acid residues.
  • analog peptides are similarly resistant to gastrointestinal proteases, as compared to native gluten peptides, e.g. SEQ ID NO:38.
  • native gluten peptides e.g. SEQ ID NO:38.
  • an analog peptide of interest has at least about 75% of the resistance of the native gluten peptide, at least about 85% of the resistance, at least about 95% of the resistance.
  • Analog peptides of interest have significantly decreased activity as a tissue transglutaminase (tTG) substrate. Under test conditions, e.g. as set forth in the Examples, as set forth in Bethune et al. (2006) supra., etc., an analog peptides of interest has less than about 10% of tTG substrate activity; less than about 5% of tTG substrate activity; less than about 1 % of tTG substrate activity.
  • Analog peptides of interest have low affinity for DQ2, thereby minimizing presentation to T cells, which also correlates with having poor immunostimulatory capacity toward gluten-specific T cells derived from celiac patient intestinal biopsies. This minimizes the risk of inducing inflammation upon oral administration of the biomarker.
  • an analog peptides of interest has less than about 10% of T cell stimulatory or DQ binding affinity activity; less than about 5% of T cell stimulatory or DQ binding affinity activity; less than about 1% of T cell stimulatory or DQ binding affinity activity.
  • the criteria set forth above are accomplished by substituting one, two or three glutamine residues of a native gluten peptide, e.g. a peptide set forth in SEQ ID NO: 1 -SEQ ID NO:38, with amino acids other than glutamine and other than a negatively charged amino acid.
  • the substituting amino acid is a histidine, an asparagine, or a combination thereof.
  • Glutamine residues susceptible to deamidation by tTGase can be experimentally determined, as described above and as known in the art.
  • the deamidated glutamine is the underlined residue in the motif PQPQLPY.
  • the deamidated glutamine is present in the motif QXP, where Q is a glutamine targeted by tTG and X is an amino acid intervening between Q and P.
  • the deamidated glutamine is replaced by X, where X is an amino acid other than glutamine.
  • analog peptides without limitation, are provided in Table 2, where one or more X residues in a given peptide are an amino acid other than glutamine, preferably a neutral or positively charged amino acid, and which may be histidine or asparagine.
  • X residues in a given peptide are an amino acid other than glutamine, preferably a neutral or positively charged amino acid, and which may be histidine or asparagine.
  • X residues in a given peptide are an amino acid other than glutamine, preferably a neutral or positively charged amino acid, and which may be histidine or asparagine.
  • the analog peptide consists or comprises of the sequence set forth in SEQ ID NO:76, where each X is histidine, asparagine, or a combination thereof, i.e. one histidine substitution and two asparagine substitutions; three histidine substitutions; three asparagine substitutions; and the like.
  • the peptide is LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77)
  • Analog peptides optionally comprise one, two or more labeled amino acids, where the label provides for specific detection of the peptide and its metabolites.
  • Preferred labels are non-radioactive, biogenic moieties, e.g. 13 C, 2 H, etc.
  • Other labels of interest include radiography moieties (e.g. heavy metals and radiation emitting moieties), positron emitting moieties, magnetic resonance contrast moieties, and optically visible moieties, e.g., fluorescent or visible-spectrum dyes, quantum dots, visible particles, FRET pairs; etc.
  • radiography moieties e.g. heavy metals and radiation emitting moieties
  • positron emitting moieties positron emitting moieties
  • magnetic resonance contrast moieties e.g., magnetic resonance contrast moieties
  • optically visible moieties e.g., fluorescent or visible-spectrum dyes, quantum dots, visible particles, FRET pairs; etc.
  • Isotopes that decay by electron capture and/or ⁇ emission are used in SPECT, and include 125 I, 128 I, 123 I and 99m Tc.
  • Optically visible moieties include fluorescent dyes, or visible-spectrum dyes, visible particles, and other visible labeling moieties. Fluorescent dyes such as ALEXA dyes, fluorescein, coumarin, rhodamine, bodipy Texas red, and cyanine dyes, are useful when sufficient excitation energy can be provided to the site to be inspected visually. Acceptable dyes include FDA-approved food dyes and colors, which are non-toxic, although pharmaceutically acceptable dyes which have been approved for internal administration are preferred. Methods of the Invention
  • the ability of an individual to metabolize a gluten peptide analog via an intestinal route is analyzed by administering an oral dose of a gluten peptide analog to an individual suffering from an enteropathic disorder, and quantitating the presence of the gluten peptide analog and/or its metabolite(s) in at least one patient sample.
  • the method comprises identifying a patient as having an enteropathic disorder, e.g. by criteria described above for specific disease conditions; administering an oral dose of a gluten peptide analog to an individual identified as having an enteropathic disorder, and quantitating the presence of the gluten peptide analog and/or its metabolite(s) in at least one patient sample.
  • Patient samples include a variety of bodily fluids in which the gluten peptide analog and/or metabolites will be present, e.g. blood and derivatives thereof, urine, saliva, breath, etc.
  • the samples will be taken prior to administration of the peptide, and at suitable time points following administration, e.g. at 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 6 hours, etc., following administration.
  • the methods of the invention are used in determining the efficacy of a therapy for treatment of an enteropathic disease, either at an individual level, or in the analysis of a group of patients, e.g. in a clinical trial format.
  • Such embodiments typically involve the comparison of two time points for a patient or group of patients. The patient status is expected to differ between the two time points as the result of a therapeutic agent, therapeutic regimen, or disease challenge to a patient undergoing treatment.
  • Examples of formats for such embodiments may include, without limitation, testing peptide metabolism at two or more time points, where a first time point is a diagnosed but untreated patient; and a second or additional time point(s) is a patient treated with a candidate therapeutic agent or regimen.
  • An additional time point may include a patient treated with a candidate therapeutic agent or regimen, and challenged for the disease, particularly for celiac sprue and/or dermatitis herpetiformis, which may be challenged with administration of gluten.
  • a first time point is a diagnosed patient in disease remission, e.g. as ascertained by current clinical criteria, as a result of a candidate therapeutic agent or regimen.
  • a second or additional time point(s) is a patient treated with a candidate therapeutic agent or regimen, and challenged with a disease-inducing agent, particularly for celiac sprue and/or dermatitis herpetiformis, which may be challenged with administration of gluten.
  • each set of time points may correspond to a single patient, to a patient group, e.g. a cohort group, or to a mixture of individual and group data. Additional control data may also be included in such clinical trial formats, e.g. a placebo group, a disease-free group, and the like, as are known in the art. Formats of interest include crossover studies, randomized, double-blind, placebo-controlled, parallel group trial is also capable of testing drug efficacy, and the like. See, for example, Clinical Trials: A Methodologic Perspective Second Edition, S. Piantadosi, Wiley-lnterscience; 2005, ISBN- 13: 978-0471727811 ; and Design and Analysis of Clinical Trials: Concepts and Methodologies, S. Chow and J. Liu, Wiley-lnterscience; 2003; ISBN-13: 978-0471249856, each herein specifically incorporated by reference.
  • Specific clinical trials of interest include analysis of therapeutic agents for the treatment of celiac sprue and/or dermatitis herpetiformis, where a patient is identified as having celiac sprue by conventional clinical indicia.
  • a daily dose of 5-10 g gluten equivalent to 2-3 slices of bread
  • can induce malabsorption as measured by a 72-hour quantitative fecal fat collection or a D-xylose urinary test (PyIe, 2005), providing for a means to challenge the efficacy of a treatment.
  • a blinded crossover clinical trial format is utilized.
  • a patient alternates for a set period of time, e.g. one week, two weeks, three weeks, or from around about 7-14 days, or around about 10 days, between a test drug and placebo, with a 4-8 week washout period.
  • the patient is challenged with gluten during both alternating time periods with around about 1 g gluten, about 5 g. gluten, about 10 g. gluten, or more, usually not more than about 25 g gluten daily.
  • Subjects are tested with a gluten peptide analog, as described above, at the beginning and end of each alternating time period.
  • the duration of gluten challenge may be about 1 , about 3, about 5, about 7, about 10 days, about 14 days, and the like.
  • a randomized, double-blind, placebo-controlled, parallel group trial is used to test drug efficacy.
  • individuals identified as having celiac sprue who are on a gluten-free diet, undergo three sequential treatment periods, each of 1-14 day durations. Subjects will be assessed with the gluten peptide analog at entry and at the end of each treatment period. During the entire study, subjects will consume regular gluten-free meals plus drug or placebo as indicated. During the first treatment period (run-in), all subjects will receive placebo. During the second treatment period, the subjects will be randomized into drug or placebo groups. During the third treatment period, subjects will remain on the same (drug or placebo) treatment as in the second period.
  • the gluten peptide analog is administered at a dose that is sufficient to monitor the metabolism over time, which will vary with the specific peptide that is selected.
  • the dose may be at least about 1 mg, at least about 5 mg at least about 10 mg, at least about 25 mg, at least about 100 mg, at least about 500 mg, and not more than about 1 g.
  • the peptide may be administered in any conventional formulation, e.g. solution, suspension, tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stea
  • An alternative formulation is an aqueous solution containing 5 g lactulose, 2 g mannitol and 22.3 g glucose as an osmotic filler.
  • the lactulose-mannitol cocktail in this solution facilitates an independent assessment of intestinal permeability.
  • the oral formulations comprise enteric coatings, so that the active agent is delivered to the intestinal tract.
  • enteric coatings are created by coating a solid dosage form with a film of a polymer that is insoluble in acid environments, and soluble in basic environments.
  • Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate, methacrylate copolymers, and cellulose acetate phthalate.
  • enteric formulations comprise engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings and can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches.
  • the polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623): 410-414.
  • Drug delivery systems can also utilize a core of superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh et al. (2001 ) J Control Release 71(3):307-18.
  • SPH superporous hydrogels
  • SPHC SPH composite
  • the presence of the peptide may be determined by an affinity assay.
  • an antibody that specifically binds to the peptide may be used in a quantitative or semiquantitative assay.
  • Such antibodies are known in the art, e.g. see Moron et al. (2008) PLoS ONE 3(5): e2294. For such assays it is not necessary to label the peptide.
  • Other assays may utilize a labeled peptide, where the presence of the label is determined, e.g. by a chromatographic separation of peptide and free amino acids, by detection of cleavage through a peptide labeled with a FRET pair, and the like.
  • a labeled peptide where the presence of the label is determined, e.g. by a chromatographic separation of peptide and free amino acids, by detection of cleavage through a peptide labeled with a FRET pair, and the like.
  • databases of metabolic analyses will typically comprise analysis profiles of various individuals following a clinical protocol of interest etc., where such profiles are further described below.
  • the profiles and databases thereof may be provided in a variety of media to facilitate their use.
  • Media refers to a manufacture that contains the expression profile information of the present invention.
  • the databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer.
  • Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • Recorded refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the information of the present invention.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • input means input means
  • output means output means
  • data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.
  • kits thereof for practicing one or more of the above- described methods.
  • the subject reagents and kits thereof may vary greatly.
  • Reagents of interest include reagents specifically designed for use in production of the above described analysis.
  • Kits may include a gluten peptide analog, reagents for analysis of the peptide and/or metabolites, and such containers as are required for sample collection.
  • the kits may further include a software package for statistical analysis of one or more phenotypes.
  • the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • a suitable medium or substrate e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • a computer readable medium e.g., diskette, CD, etc.
  • a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • This invention also provides rapid commercial test methods and devices that use at least one gluten analog peptide of the invention.
  • detection may utilize, for example, standard immunochromatographic technology with visible colorimetric readout.
  • Such assay devices may contain a single test membrane, or two or more test membranes.
  • the test membranes may be present within a cassette, each receiving fluid from a single application, such as through an aperture in a test cassette equipped with means such as tubules for distributing said fluid to each test membrane.
  • kits which comprise one or more test membrane strips comprising binding partners for the antigens used in the assay method.
  • Assay devices may include puncture or other physical means known to the art, e.g. finger prick devices.
  • Preferred binding partners for gluten analog peptides are antibodies specific thereto.
  • a first antibody may bind to the gluten analog peptide of the invention to form a complex, and a second antibody may bind to the complex. Either the first or second antibody may be labeled, and either the first or second antibody may be immobilized on a substrate such as a test membrane for ease of detection.
  • This invention further provides an assay system comprising: (1 ) a cassette; (2) a test membrane housed within the cassette; (3) first antibodies specific to one or more gluten analog peptide(s), said first antibodies being capable of binding to the corresponding peptides to form first antibody-antigen complexes; (4) binding partners specific to such complex; and (5) labels attached to said first antibodies or said binding partners.
  • the binding partners capable of binding to the complexes may be second antibodies specific to said gluten analog peptide; or they may be second antibodies specific to the first antibodies.
  • the binding partners may also be detection antigens capable of binding specifically to each of the first antibodies.
  • the binding partners for the complex, or the first antibodies may be immobilized on a substrate such as. a test membrane.
  • the first antibodies or the binding partners for the complex may be labeled.
  • the labels used are detectably different for detecting each antigen.
  • Immobilized antibodies or binding partners for the complexes may be laid down on the substrate in different patterns.
  • the assay system may be in the form of a cassette comprising all needed antibodies and antigens, or may be in the form of a kit which includes necessary antigens and/or antibodies or other reagents as separate reagents.
  • the kit includes a cassette comprising all needed antibodies; and a predetermined dose of gluten analog peptide, which may be in a form suitable for ingestion.
  • a preferred cassette comprises a sample aperture for introducing sample fluid into the assay, preferably with a sample pad positioned beneath the sample aperture.
  • the cassette also comprises a substrate such as a test membrane for immobilizing antibodies and/or antigens.
  • a filter may be positioned downstream from the sample aperture.
  • the cassette also preferably comprises a test window positioned above the point on the test membrane wherein labeled first antibodies, labeled second antibodies, or labeled detection antigens are immobilized. The human eye or a detection device may be used to view test results through the test window.
  • the cassette may also comprise control peptides. These may or may not be immobilized on the test membrane and may or may not be labeled.
  • the cassette also comprise binding partners for the control antigens. The binding partners may be labeled and may be immobilized on the test membrane.
  • the cassette also comprises a control window positioned above the test membrane at the point where the control antigens or their binding partners are immobilized, so that results can be viewed through the control window by the human eye or a detection device.
  • biomarker for disease management and clinical drug development was inspired by the physical, chemical and biological properties of immunotoxic gluten peptides such as the 33-mer from ⁇ -gliadin. Specifically, we sought to engineer a gluten peptide analogue that mimics the 33-mer with respect to some criteria but can be differentiated from the natural product with respect to others. Like the 33-mer, the biomarker must be resistant to gastrointestinal proteases, so that it is not rapidly cleared from the stomach or intestinal lumen. Also like the 33-mer, it must be efficiently proteolyzed by therapeutic glutenases, so that its amino acid metabolites are rapidly assimilated into the bloodstream in a glutenase dose dependent manner.
  • the biomarker must be able to penetrate across the intestinal epithelium.
  • the biomarker must neither be recognized by human TG2 nor HLA-DQ2. Consequently, it must be unable to stimulate disease-specific T cells so as to elicit an inflammatory response.
  • proEP-B2 rapidly degraded the 33-mer (Figure 2D), as well as the derivative biomarkers (Figure 2E,F) in a time- and dose-dependent manner (Figure 2G 1 H 1 I).
  • pepsin alone catalyzed nearly complete cleavage of the myoglobin control peptide within 10 min ( Figure 2J).
  • the major products of biomarker digestion identified by LC-MS were similar to those produced by 33-mer cleavage ( Figure 2K), indicating that the substitutions in these 33-mer analogues did not substantially alter the sites of susceptibility to EP-B2-mediated digestion.
  • Biomarkers are non-inflammatory in the context of celiac sprue. For biomarkers to be administered safely to celiac sprue patients, they must not be deamidated by TG2, bind HLA DQ2, or stimulate a strong immune response by pre-existing gluten-specific T cells. To determine the capacity for gluten peptide-based biomarkers to elicit an inflammatory T cell response in celiac sprue patients, these characteristics were tested in vitro and compared to the immunodominant 33-mer gluten peptide.
  • each biomarker for HLA-DQ2 was determined using a peptide exchange assay in which fluorescein-labeled peptides were incubated with soluble HLA-DQ2 molecules at pH 5.5, 37 0C to simulate the endocytic environment. After 45 h, DQ2-bound and free fluorescein- labeled peptides were separated by high-performance size exclusion chromatography (HPSEC) and the ratio of their peak heights determined by fluorometry.
  • HPSEC high-performance size exclusion chromatography
  • Biomarker transepithelial transport parallels gluten peptide transport under basal and inflammatory conditions.
  • noninflammatory gluten peptide-based biomarkers are useful tools for understanding the factors and mechanisms that modulate intestinal permeability of immunogenic dietary peptides, as well as for practical applications related to the diagnosis of celiac sprue and its treatment with modulators of epithelial permeability.
  • these biomarkers must be similar to inflammatory gluten peptides in terms of their transport and transepithelial stability across healthy and inflamed mucosa.
  • the T84 epithelial cell line was used to model the intestinal epithelium because its responsiveness to IFN- ⁇ , the major inflammatory cytokine present in celiac lesions, has been extensively studied. Additionally, the effect of IFN- ⁇ on the intact transport of the 33-mer and other gluten peptides across T84 epithelial monolayers has recently been described. To simulate transport under healthy and inflammatory states, media alone or media containing IFN- ⁇ was incubated for 48 hours on the basolateral side of T84 epithelial cells cultured on transwell supports, and the apical- to-basolateral flux of Cy5-labeled 33-mer and biomarkers was measured thereafter.
  • the flux of Cy5-33-mer and both Cy5-labeled biomarker peptides was ⁇ 6 pmol/cm2/h under basal conditions. Following exposure of T84 monolayers to IFN- ⁇ , the flux of all 3 peptides was increased ⁇ 10-fold. No significant difference in flux was observed between 33-mer and either biomarker under basal or simulated inflammatory conditions. Some processing of the 33-mer may occur upon its transport across the intestinal epithelium. Therefore, to evaluate the stability of apical and translocated Cy5-labeled biomarkers, the apical and basolateral media were analyzed by LC-MS immediately after peptide addition to the apical chamber and 1O h thereafter.
  • both biomarkers were highly stable in the presence of epithelial cells, and were translocated intact to a similar extent as the 33-mer under basal and simulated inflammatory conditions.
  • catheterized rats were administered 20 mg [ 13 C3]-HHH-33-mer biomarker by oral gavage following 2 days of daily intravenous treatment with vehicle or IFN- ⁇ .
  • the level of intact peptide in peripheral plasma 60 min after peptide administration was determined by 3Q LCMS/ MS.
  • biomarker was detected in the plasma of only 1 of 8 control rats administered oral biomarker.
  • Pretreatment with IFN- ⁇ did not elicit a general increase in biomarker transport in the test animal group relative to controls, as only 1 of 4 IFN- ⁇ -treated animals exhibited plasma biomarker.
  • the level of plasma biomarker in this IFN- ⁇ -treated animal (101.7 nM) was ⁇ 10-fold higher than that in the control animal exhibiting detectable plasma biomarker (9.5 nM), a difference similar to that caused by IFN- ⁇ in vitro.
  • 33-mer dosed together with [ 13 C3]-HHH-33-mer was detected in the plasma of both animals exhibiting plasma biomarker, but not in other animals, suggesting this inflammatory gluten peptide and its non-inflammatory counterpart are transported in parallel in vivo.
  • the labeled biomarker was clearly detected (2.3 ⁇ 0.1 nM) in peripheral blood 60 min after administration, similar to the extent and rate of absorption reported for the 33-mer gluten peptide.
  • intragastrically administered labeled biomarker was again detected in the peripheral blood of this gluten-sensitive macaque (2.2 ⁇ 0.5 nM), but was not detected in samples from two identically dosed healthy controls.
  • Celiac sprue affects up to 1% of many human populations, but despite the wide prevalence and serious manifestations of the disease, the only treatment available remains a life-long gluten-free diet. Compliance with this burdensome dietary treatment is poor, and recurrent exposure to gluten causes chronic inflammation, increased morbidity, and more serious health effects over time. Moreover, in asymptomatic celiac sprue patients, disease management is especially difficult, as invasive histological evaluations are the only reliable way to assess response to a gluten-free diet. Finally, the development of non-dietary treatment alternatives to the gluten-free diet requires a long-term gluten challenge in celiac patients, which is inherently problematic. Therefore, novel tools for monitoring compliance with the gluten-free diet, and safely evaluating non-dietary treatments in vivo are needed.
  • Celiac sprue is uniquely suited for this biomarker strategy because we know the environmental trigger and have structural information about its binding mode to the primary genetic determinant for the disease. Additionally, gluten peptides are extraordinarily stable in the relevant physiological compartment, making them ideal scaffolds for drug and biomarker design.
  • the 33-mer from ⁇ 2-gliadin was used as a scaffold for biomarker design because its metabolism in the presence and absence of glutenases has been extensively characterized and because its intact transepithelial translocation has been demonstrated.
  • Other examples of disease-relevant gluten peptide sequences that are suitable for biomarker design include the 26-mer from ⁇ 5-gliadin and the p31-49 peptide that stimulates an innate immune response in mucosal biopsies from celiac sprue patients.
  • Oral protease therapy is one of the more promising non-dietary treatments being developed for celiac sprue, but few studies have been conducted in vivo. Celiac patients administered an undefined enzyme mixture from animal digestive extracts showed modest improvement in a clinical trial. More recently, clinical efficacy of oral EP-B2 was demonstrated in a gluten sensitive rhesus macaque. These studies relied on histological, clinical, and serological readouts, complex parameters which require weeks to register a response and which are indirect measures of glutenase-mediated gluten detoxification. By contrast, in the present study, the metabolism of [ 13 C3]-HHH-33-mer in rats dosed with EP- B2 provided an immediate and direct readout for glutenase activity in vivo.
  • biomarkers are a useful clinical tool for monitoring adherence to a gluten-free diet as well. Notwithstanding the effect of a gluten-free diet on intestinal permeability, epithelial uptake of gluten remains altered in treated celiac patients with respect to healthy controls. This is likely related to the 7.6-fold higher levels of IFN- ⁇ present in treated patients relative to healthy controls. In our experiments, both biomarkers were translocated intact across epithelial monolayers to a 10-fold greater extent following preincubation of the cells with IFN- ⁇ .
  • these peptides may be used as a screening tool for celiac sprue, en route to a diagnosis, as well as for other inflammatory bowel diseases in which intestinal IFN- ⁇ levels and mucosal leakiness of antigenic peptides and proteins are elevated.
  • Cy5-NHS ester was purchased from Amersham Biosciences (Piscataway, NJ). lsotopelabeled amino acids were purchased from Cambridge Isotope Laboratories (Andover, MA). Recombinant IFN- ⁇ was purchased from Peprotech, Inc. (Rocky Hill, NJ). Cell culture media, antibiotics, human serum, 5'(and 6') carboxyfluorescein, and fluorescently labeled dextrans were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA).
  • Gluten flour was purchased from Bob's Red Mill (Milwaukie, OR). Thrombin was purchased from Novagen (Madison, Wl). Protease inhibitor cocktail set 1 was purchased from Calbiochem (San Diego, CA). Pepsin, trypsin, chymotrypsin, elastase, carboxypeptidase A, vancomycin, and N ⁇ -p-tosyl-L-arginine methyl ester hydrochloride (TAME) were purchased from Sigma-Aldrich (St. Louis, MO). Brush border membranes were purified from rat intestine as previously described and stored at -8O 0 C until use.
  • the recombinant proenzyme precursor of barley endoprotease EP-B234, Flavobacterium meningosepticum PEP44 and human TG236 were expressed in E. coli and purified as previously described.
  • Recombinant soluble DQ2 heterodimer- ⁇ l gliadin peptide fusion molecules were prepared and purified in insect cells as previously described.
  • peptides were purified over a reverse-phase C18 column by HPLC using a water/acetonitrile gradient in the presence of 0.1 % trifluoroacetic acid, lyophilized, and stored at -2O 0 C.
  • purified peptides were labeled at their amino terminus with Cy5-NHS ester in DMSO according to the manufacturer's instructions, repurified by HPLC, lyophilized, and stored at -20 0 C. The correct mass of all peptides was confirmed by LC-MS.
  • Working stocks were stored at 4 0 C and their integrity confirmed periodically by RP-HPLC.
  • DQ2 homozygous antigen-presenting cells (CD114, an Epstein Barr virus-transformed B lymphoblastoid cell line) were grown in APC media (RPMI supplemented with antibiotics and 5% (v/v) fetal bovine serum). Every other day, CD114 were split to 0.4 x 106 cells/ml.
  • T cell proliferation assays were performed in T cell media (RPMI 1640 supplemented with antibiotics and 10% (v/v) human serum). All cells were grown and assayed at 37 0 C with 5% atmospheric CO 2 .
  • Samples were heat-deactivated at 95 0 C for 5 min, diluted 1:5 in HPLC solvent A (95% H 2 O, 5% acetonitrile, 0.1% trifluoroacetic acid) supplemented with an internal standard (TAME), and analyzed by reverse-phase HPLC. Samples (50 ⁇ l) were separated over a C18 column (Grace Vydac, Hesperia, CA) using a water-acetonitrile gradient in the presence of 0.1% TFA. The absorbance at 215 nm was monitored. The area-under-the-curve for each intact peptide was calculated and normalized to the area under the curve for the internal standard, TAME.
  • HPLC solvent A 95% H 2 O, 5% acetonitrile, 0.1% trifluoroacetic acid
  • TAME internal standard
  • Recombinant prolyl endopeptidase from Flavobacterium meningosepticum was supplemented at 1.2 U/ml when added. Simulated duodenal digests were performed at 37 0 C. Samples were collected at 0, 10, 30, and 60 min and processed for HPLC as described. The area-under-the-curve for each intact peptide (together with the minimally processed -LQ product) was calculated and normalized to the internal standard. To identify digestion products, select samples were analyzed by LC-MS.
  • Samples (50 ⁇ l) processed for HPLC as described were injected on a reverse- phase C18 HPLC system (Waters Corporation, Milford, MA) coupled to a UV/Vis detector and a ZQ single quadrupole mass spectrometer with an electrospray ionization source. Samples were eluted with a wateracetonitrile gradient in the presence of 0.1% formic acid. Absorbance at 214/254 nm and total ion current were monitored, and spectra corresponding to major absorbance peaks were examined. Transglutaminase deamidation assay. Coupled spectrophotometric assays for TG2 activity in the presence of each peptide were performed as previously described36.
  • each peptide 100 ⁇ M was added to a 200 mM MOPS, pH 7.2 buffer containing 5 mM CaCI2, 1 mM Na4EDTA, 10 mM ⁇ -ketoglutarate, and 250 ⁇ M NADH.
  • Glutamate dehydrogenase was added to a final concentration of 0.036 U/ ⁇ l, and this incomplete reaction mixture was incubated at room temperature for 10 min to stabilize the initial absorbance at 340 nm.
  • Peptide exchange assays for determining the equilibrium occupancy of each peptide on HLA-DQ2 were modified from previously described methods. Briefly, recombinant DQ2- ⁇ l-gliadin peptide fusions (35 ⁇ M) were treated with 0.15 U/ ⁇ l thrombin in phosphate-buffered saline (PBS), pH 7.4 supplemented with 0.02% (w/v) NaN 3 for 2 h at 4 0 C, after which protease inhibitor cocktail was added.
  • PBS phosphate-buffered saline
  • Thrombin-cleaved DQ2- ⁇ l- gliadin peptide complexes (9.4 ⁇ M) were incubated with 0.185 ⁇ M fluorescein-conjugated peptides in a citrate- PBS buffer, pH 5.5 (24 mM sodium citrate, 55 mM Na 2 HPO 4 , 75 mM NaCI, 0.02% (w/v) NaN 3 ) for 45 h at 37 0 C.
  • binding reactions were diluted 1 :5 in PBS, pH 7.4 supplemented with 0.02% (w/v) NaN 3 and 12.5 ⁇ l was injected on an HPSEC system coupled to a fluorescence detector (Shimadzu, Columbia, MD).
  • DQ2-bound and unbound fluorescent peptides were separated using a BioSep 3000 size exclusion column (Phenomenex, Torrance, CA) with a flow rate of 1 ml/min PBS, pH 7.4 supplemented with 0.02% (w/v) NaN 3 .
  • the detector was set to monitor excitation at 495 nm and emission at 520 nm.
  • the bound/free ratio for each peptide was calculated by dividing the measured peak height for bound peptide by that for the free peptide.
  • T cell proliferation assay The T cell proliferation assay was modified from previously described methods, as follows. Briefly, the 33-mer, NNN-33-mer, and HHH-33-mer peptide stocks (250 ⁇ M) were deamidated by treatment with 100 ⁇ g/ml TG2 in 100 mM Tris, pH 7.4 for 2 hours at 37 0 C in the presence of 2 mM CaCI 2 . Antigen-presenting cells (CD114, 60,000 cells/well) were irradiated (80 Gy) and incubated overnight at 37°C in U-bottom, 96- well plates with various concentrations of native or TG2-deamidated peptides in T cell media.
  • Vancomycin was added as a non-absorbable dosing internal standard, as previously described. Animals consumed the test meal completely within 60 min, and animals were euthanized 30 min thereafter (i.e. 90 min after meal administration). The gastric, duodenal, jejunal, and ileal contents were collected immediately and stored at -80 0 C as previously described. Peripheral plasma samples were collected via cardiac puncture at the time of euthanasia and stored at -80 0 C.
  • Samples were heat-deactivated for 5 min at 95 0C, supplemented with ethanol to a final concentration of 70% (v/v), and centrifuged for 10 min at 16,100 x g.
  • Syringe-filtered (0.45 ⁇ m) supernatants 100 ⁇ l were diluted 1 :5 in 95% H2O, 5% acetonitrile, 0.1% trifluoroacetic acid supplemented with an internal standard (TAME), and analyzed by reverse-phase HPLC.
  • Samples (50 ⁇ l) were separated over a C18 column (Grace Vydac, Hesperia, CA) using a water acetonitrile gradient in the presence of 0.1% TFA. The absorbance at 215 nm was monitored. Intestinal flushes were thawed on ice and centrifuged for 10 min at 4 0 C, 3100 x g.
  • Supernatants were heat- deactivated and processed for HPLC as described for gastric contents.
  • gliadin-coated plates were washed twice with washing buffer (PBS, pH 7.2 containing 0.05% Tween-20) then blocked with blocking buffer (5% (w/v) nonfat milk in PBS, pH 7.2) for 2 h at room temperature.
  • assay buffer 3% (w/v) bovine serum albumin in PBS, pH 7.2.
  • G12-HRP monoclonal antibody- horseradish peroxidase conjugate Biomedal, Seville, Spain
  • Mixtures were incubated with gentle agitation for 2 h at room temperature, and then added to plate wells in triplicate. After 30 min incubation at room temperature, wells were washed 5 times with washing buffer, and TMB liquid substrate solution (Sigma) was added to wells.
  • A450 A450min + (A 45O max - A 45O mJnV[I + (x/IC 50 )n] where x is the peptide concentration, IC 50 is the 33-mer concentration at which competition is half-maximal, and n is the Hill slope.
  • concentration of peptides containing the sequence QPQLPY in gastric samples was determined by comparison to the linear portion of the 33-mer standard curve.
  • samples were heat-deactivated for 5 min at 95 0 C and centrifuged for 10 min at 16,100 x g.
  • syringe-filtered (0.45 ⁇ m) gastric supernatants, or plasma samples were depleted of larger proteins by addition of acetonitrile.
  • Samples were mixed with an equal volume of cold acetonitrile containing 0.1% formic acid and 200 nM NNN-33-mer as an internal standard. Samples were vortexed, incubated for 2 h at 4 0 C, and centrifuged for 10 min at 4 0 C, 16,100 x g.
  • the transitions monitored were m/z 632.72+ ⁇ 229.2+ (35V cone voltage, 25eV collision energy.) for the quantification assay and m/z 632.72+ ⁇ 129.2+ (35V cone voltage, 33eV collision energy) as a confirmatory transition.
  • the transitions monitored were m/z 968.64+ ⁇ 263.4+ (32V cone voltage, 32eV collision energy) for the quantification assay and m/z 968.64+ ⁇ 226.0+ (40V cone voltage, 5OeV collision energy) as a confirmatory transition.
  • cell monolayers were preincubated for 48 h with basolateral media containing either 0 or 600 U/ml recombinant IFN- ⁇ .
  • the translocation assay was performed by replacing media in both the apical and basolateral chambers with serum-free T84 media (1 :1::Dulbecco's Modified Eagle Medium:Ham's F12 media supplemented with antibiotics ), and adding 2 ⁇ M dextran (3,000 mol. wt.)-Alexa Fluor-488, 2 ⁇ M dextran (70,000 mol. wt.)-Texas Red, and 20 ⁇ M Cy5-labeled peptide to the apical chamber.
  • the slope of basolateral fluorescence units versus time was calibrated to the initial apical fluorescence and divided by the permeable support area (0.33 cm 2 ) to yield the transepithelial flux (pmol/cm 2 /h).
  • Samples 50 ⁇ l_ were analyzed by LC-MS as described for digests, except absorbance at 640 nm was monitored. Spectra corresponding to A 640 peaks were examined. Samples were also analyzed by HPSEC with fluorescence detection as described for DQ2- peptide binding analysis, except the detector was set to monitor excitation at 647 nm and emission at 665 nm.
  • the experiment was repeated four months later with FH45 and two healthy control macaques, HI48 and HK31 , on a gluten-containing diet.
  • 50 mg of isotopically labeled biomarker was intragastrically administered to each animal. Animals were sedated and anesthetized prior to peptide inoculation and blood collections. Plasma samples were analyzed for intact biomarker by 3Q LC-MS/MS as described above.
  • the subject is given a dose of unlabeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where the dose may be 1 ; 10; 100 or 500 mg of peptide.
  • a urine sample is obtained from the subject, which sample is contacted with a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • the presence of the peptide is determined by localization of the dye label at the position where the second antibody is immobilized, which may for convenience be a shape of a "+", or other easily recognized symbol.
  • the subject is given a dose of unlabeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH- 33mer, SEQ ID NO:77), where the dose may be 1 ; 10; 100 or 500 mg of peptide.
  • a urine sample is obtained from the subject, which sample is contacted with a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • the presence of the peptide is determined by localization of the dye label at the position where the second antibody is immobilized, which may for convenience be a shape of a "+", or other easily recognized symbol.
  • the presence of the peptide in urine is indicative that the intestinal permeability is undesirably high, suggesting that an inflammatory response to gluten persists in the patient.
  • AT-1001 is an orally administered octapeptide zonulin receptor antagonist that appears to exert its inhibitory effect on gliadin-induced tight junction disassembly by blocking putative zonulin receptors on the luminal surface of the small intestine.
  • Pretreatment with the peptide fails to inhibit gliadin induced zonulin release, while administration of zonulin analogues or gliadin in the presence of AT-1001 fail to significantly affect intestinal permeability, confirming the effect of the molecule is specific to the zonulin receptor, in mitigating the effects of gluten ingestion in patients with celiac disease.
  • the subject is also given a dose of unlabeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where the dose may be 1 ; 10; 100 or 500 mg of peptide.
  • a urine sample is obtained from the subject, which sample is contacted with a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • the presence of the peptide is determined by localization of the dye label at the position where the second antibody is immobilized, which may for convenience be a shape of a "+", or other easily recognized symbol.
  • NexVax2 is a peptide-based vaccine to treat or prevent celiac disease.
  • the vaccine will include the gluten peptides most commonly recognized by T cells in people with celiac disease.
  • the subject is also given a dose of unlabeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where the dose may be 1 ; 10; 100 or 500 mg of peptide.
  • a urine sample is obtained from the subject, which sample is contacted with a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • the presence of the peptide is determined by localization of the dye label at the position where the second antibody is immobilized, which may for convenience be a shape of a "+", or other easily recognized symbol.
  • the subject is given a dose of unlabeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where the dose may be 1 ; 10; 100 or 500 mg of peptide to be ingested with normal meals.
  • LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF HHH-33mer, SEQ ID NO:77
  • a urine sample is obtained from the subject, which sample is contacted with a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • a dipstick comprising a dye antibody that selectively binds to the peptide; and an immobilized second antibody that selectively binds to the peptide or the peptide antibody complex.
  • the presence of the peptide is determined by localization of the dye label at the position where the second antibody is immobilized, which may for convenience be a shape of a "+", or other easily recognized symbol.
  • the presence of the peptide in urine is indicative that the peptide is not metabolized, and is indicative that intestinal permeability is undesirably high, and is indicative that the patient's diet and/or therapeutic regimen should be adjusted accordingly.
  • the subject is given a dose of labeled peptide consisting of the amino acid sequence LQLQPFPQPHLPYPQPHLPYPQPHLPYPQPQPF (HHH-33mer, SEQ ID NO:77), where a highly fluorescent label is conjugated to the amino terminus.
  • the dose may be 1 ; 10; 100 or 500 mg of peptide to be ingested with normal meals.
  • the peptide is conjugated with a fluorescent label and a quencher, such that the fluorescence is quenched in the intact peptide, and is unquenched in a proteolytically cleaved peptide.
  • a urine sample is obtained from the subject.
  • the peptide is labeled with a fluorescent dye and quencher pair
  • the sample is analyzed for the presence of fluorescence. Fluorescence is indicative that the peptide is cleaved and is indicative that the treatment is appropriately metabolizing gluten.
  • the sample is separated by chromatography to determine whether the label is associated with free amino acids or with peptide.
  • the presence of the label at a position corresponding to the peptide in urine is indicative that the peptide is not metabolized, and is indicative that intestinal permeability is undesirably high, and is indicative that the therapeutic regimen should be adjusted accordingly.

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Abstract

L'invention permet de poser le diagnostic d'une maladie entéropathique ou d'évaluer la réponse d'un patient souffrant d'une maladie entéropathique à une thérapie, dans le cadre d'un essai clinique ou de la gestion à long terme de la maladie, en détectant la capacité du patient à métaboliser un analogue peptidique du gluten administré par voie orale. On peut surveiller le métabolisme peptidique de diverses manières. On peut avantageusement détecter l'apparence d'un métabolite peptidique dans un prélèvement effectué chez le patient, p.ex. dans l'urine, le plasma, l'haleine, la salive, etc., sur une période de temps suivant l'administration orale. On peut facultativement marquer l'analogue peptidique du gluten, p.ex. à l'aide d'un marqueur isotopique, fluorescent ou autre.
EP09746974A 2008-05-16 2009-05-13 Analogues peptidiques du gluten non inflammatoires utilisés comme biomarqueurs de la maladie coeliaque Withdrawn EP2277046A2 (fr)

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AU2009321481B2 (en) 2008-11-30 2014-08-21 Immusant, Inc. Compositions and methods for treatment of celiac disease
US20140037661A1 (en) * 2011-02-08 2014-02-06 Phadia Ab Wheat Antigens and Peptides for Diagnosis of Wheat Induced Hypersensitivity
WO2014152233A1 (fr) * 2013-03-14 2014-09-25 Immusant, Inc. Procédé de provocation de gluten contrôlé par placebo
US10449228B2 (en) 2013-09-10 2019-10-22 Immusant, Inc. Dosage of a gluten peptide composition
ES2556177B1 (es) * 2014-07-09 2016-10-20 Universidad De Sevilla Detección de péptidos del gluten en fluidos humanos
WO2016054038A1 (fr) 2014-09-29 2016-04-07 Immusant, Inc. Utilisation de statut génétique hla pour évaluer ou sélectionner un traitement de la maladie cœliaque
EP3421995B1 (fr) * 2016-02-23 2022-08-31 Tanaka Kikinzoku Kogyo K.K. Dispositif d'analyse d'immunochromatographie pour détecter la gliadine, kit d'analyse d'immunochromatographie et procédé d'analyse d'immunochromatographie
WO2018005688A1 (fr) * 2016-06-28 2018-01-04 Immusant, Inc. Compositions et procédés de diagnostic de la maladie coeliaque à l'aide de cytokines/chimiokines circulantes
WO2019147990A1 (fr) * 2018-01-26 2019-08-01 The Regents Of The University Of California Méthodes et compositions concernant la perméabilité intestinale

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US7202216B2 (en) * 2002-05-14 2007-04-10 The Board Of Trustees Of The Leland Stanford Junior University Drug therapy for celiac sprue
US7462688B2 (en) * 2002-05-14 2008-12-09 The Board Of Trustees Of The Leland Stanford Junior University Peptides for diagnostic and therapeutic methods for celiac sprue
US7534426B2 (en) * 2004-04-26 2009-05-19 The Board Of Trustees Of The Leland Stanford Junior University Glutenase enzyme assays
US8034776B2 (en) * 2006-10-26 2011-10-11 Alba Therapeutics Corporation Materials and methods for the treatment of celiac disease

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