Use of proteases for gluten intolerance

a technology of proteases and gluten, applied in the direction of peptide/protein ingredients, drug compositions, metabolic disorders, etc., can solve the problems of inability of the intestine to absorb nutrients, negative test results, and inconclusive results

Inactive Publication Date: 2011-08-18
AMANO ENZYME USA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]Certain embodiments of the present technology provide an enzyme cocktail comprising a gluten degrading enzyme preparation and papain. The enzyme cocktail of the present technology is capable of cleaving a gluten oligopeptide, such as a 33-mer peptide fragment of α-gliadin. The enzyme cocktail may comprise activated papa

Problems solved by technology

It stimulates a T-cell immune response in susceptible subjects, resulting in an inflammation that damages the intestinal wall.
This, in turn, impairs the ability of the intestine to absorb nutrients, leading to malnutrition and a variety of other symptoms.
However, most gluten intolerant, or gluten sensitive, people test ne

Method used

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  • Use of proteases for gluten intolerance
  • Use of proteases for gluten intolerance
  • Use of proteases for gluten intolerance

Examples

Experimental program
Comparison scheme
Effect test

example 1

33-mer Peptide Degradation by GDEP-LGG

[0137]A 33-mer peptide (1 mg / ml; LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1)) was incubated with GDEP-LGG at various concentrations (0 mg / ml; 0.1 mg / ml, 0.5 mg / ml, and 1.0 mg / ml) in the presence of simulated intestinal fluid (SIF; pH 6.8) from U.S. Pharmacopeia. A 30 μL reaction volume comprised 10 μL 33-mer peptide, 10 μL enzyme solution, and 10 μL 3× SIF buffer. The reactions were carried out for about 15, 30, 60, 120, and 240 minutes at about 37° C. and, then, 5 min at 90° C. to stop the enzyme reaction.

[0138]Samples of enzyme-treated 33-mer were analyzed by enzyme-linked immunosorbent assay (“ELISA”) using a HRP-conjugated polyclonal antibody developed against the 33-mer peptide.

[0139]The results of the time-course and dose-response analysis of 33-mer peptide degradation by GDEP-LGG are shown in FIG. 1. Incubation with GDEP-LGG (at all concentrations) for about 120 minutes reduced the antigenicity of the 33-mer by at least about 70%.

example 2

33-mer Peptide Degradation by Components of GDEP-LGG

[0140]Individual components of GDEP-LGG, including Oryzin, NP I, and NP II, were tested for enzymatic activity against the 33-mer. The 33-mer peptide (1 mg / ml) was incubated with 0.1 mg / ml enzyme solution comprising GDEP-LGG; 3X GDEP-LGG (i.e., 0.3 mg / ml); Oryzin; NP I; NP II; or combinations of Oryzin, NP I, and NP II (at ratios of 1:1:1 and 2:1:4) in the presence of simulated intestinal fluid (SIF; pH≈6.8). A 30 μL reaction volume comprised 10 μl 33-mer peptide, 10 μL enzyme solution, and 10 μL 3× SIF buffer. The reactions were carried out for about 20, 60, 120, and 240 minutes at about 37° C. and, then, 5 min at 90° C. to stop the enzyme reaction.

[0141]Samples of enzyme-treated 33-mer were analyzed as in Example 1. Degradation of the 33-mer by GDEP-LGG and its individual component enzymes is shown in FIG. 2. Incubation with GDEP-LGG (at both concentrations tested) for about 60 minutes resulted in degradation of about 90% of the ...

example 3

Comparison of GDEP-LGG and Other Digestive Proteases

[0144]The 33-mer peptide (1 mg / ml) was incubated with 0.25 mg / ml enzyme solution (containing pepsin, proteinase K, or GDEP-LGG) in the presence of simulated intestinal fluid (SIF; pH≈6.8) or simulated gastric fluid (SGF; pH≈2.0). A 40 μL reaction volume comprised 10 μL 33-mer peptide, 10 μL enzyme solution, and 20 μL 2× SIF or SGF buffer. The reactions were carried out for about 15 hours at 37° C. and, then, 5 min at 90° C.

[0145]Samples of enzyme-treated 33-mer were analyzed by reverse phase high pressure liquid chromatography (“HPLC”) using an Agilent 1100 HPLC system. Samples were loaded onto an Eclipse SB-C18 (ID2.1 mm×150 mm, 3.5 um) column. The mobile phase comprised of (A) distilled water with 0.1% formic acid and (B) acetonitril with 0.1% formic acid was used to elute targets in gradient mode (0 min, 2% B→10 min, 25% B→40 min, 40% B→45 min, 100% B→50 min, 100% B). Flow rate was set at 0.2 mL / min and the injection volume was ...

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Abstract

The present technology relates to an enzyme composition. The enzyme composition may be used to treat gluten intolerant subjects, including suffering from non-Celiac gluten intolerance and/or non-Celiac gluten sensitivity. The enzyme composition may also be used to reduce gluten exposure in certain individuals. For example, the enzyme composition may also be used as a prophylactic to reduce exposure to gluten oligopeptides.

Description

RELATED APPLICATIONS[0001]This application claims priority to and benefit from U.S. provisional patent application No. 61 / 300,726, filed on Feb. 2, 2010, which is hereby incorporated by reference in its entirety.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002][Not Applicable][0003][MICROFICHE / COPYRIGHT REFERENCE][0004][Not Applicable]BACKGROUND OF THE INVENTION[0005]Gluten is a common complex of proteins found in certain grass-related grains, including wheat, barley, and rye. Gluten is a mixture of proteins comprising gliadin and glutenin. A 33-mer peptide derived from α-2 gliadin (residues 57-89), which is not digested in the human digestive system, has been identified as an initiator of the inflammatory response to gluten in, for example, Celiac disease. The 33-mer derived from α-2 gliadin is particularly rich in proline and glutamine residues. It stimulates a T-cell immune response in susceptible subjects, resulting in an inflammation that damages the intestinal wall. This, in t...

Claims

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Application Information

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IPC IPC(8): A61K38/54A61P3/00
CPCA61K38/482A61K38/4873C12Y304/24039A61K38/4886A61K38/24C12Y304/21063A61K38/1703A61K38/2242A61K2300/00A61P1/00A61P1/04A61P1/14A61P3/00A61P3/02A61P29/00A61P43/00C12Y304/22002A61K38/43
Inventor JOLLY, JAMES F.IDO, HIROKIMATSUBARA, HIROTAKATAKAHASHI, TETSUYANISHIO, KYOICHI
Owner AMANO ENZYME USA CO LTD
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