U.S. patent application number 13/019386 was filed with the patent office on 2011-08-18 for use of proteases for gluten intolerance.
Invention is credited to Hiroki Ido, James F. Jolly, Hirotaka Matsubara, Kyoichi Nishio, Tetsuya Takahashi.
Application Number | 20110200574 13/019386 |
Document ID | / |
Family ID | 44355745 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110200574 |
Kind Code |
A1 |
Jolly; James F. ; et
al. |
August 18, 2011 |
USE OF PROTEASES FOR GLUTEN INTOLERANCE
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.
Inventors: |
Jolly; James F.; (St.
Charles, IL) ; Ido; Hiroki; (Aichi, JP) ;
Matsubara; Hirotaka; (Aichi, JP) ; Takahashi;
Tetsuya; (Gifu, JP) ; Nishio; Kyoichi; (Aichi,
JP) |
Family ID: |
44355745 |
Appl. No.: |
13/019386 |
Filed: |
February 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61300726 |
Feb 2, 2010 |
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Current U.S.
Class: |
424/94.2 |
Current CPC
Class: |
A61K 38/43 20130101;
A61K 38/4886 20130101; A61P 29/00 20180101; A61P 3/02 20180101;
C12Y 304/21063 20130101; C12Y 304/22002 20130101; A61K 38/2242
20130101; A61P 1/04 20180101; A61P 3/00 20180101; A61K 38/1703
20130101; A61K 38/482 20130101; A61P 1/14 20180101; A61P 43/00
20180101; A61K 38/4873 20130101; A61K 38/24 20130101; A61P 1/00
20180101; C12Y 304/24039 20130101; A61K 38/482 20130101; A61K
2300/00 20130101; A61K 38/4873 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/94.2 |
International
Class: |
A61K 38/54 20060101
A61K038/54; A61P 3/00 20060101 A61P003/00 |
Claims
1. An enzyme cocktail comprising: a composition selected from the
group consisting essentially of GDEP; GDEP-LGG; GDEP-M; GDEP-LNA;
GDEP-2A; GDEP-AH, papain, Aorsin; an acidic serine protease
polypeptide having an amino acid sequence at least about 80%
homologous to Aorsin, or a fragment thereof; CPY; an enzyme having
an amino acid sequence at least about 80% homologous to CPY, or a
fragment thereof; a semi-alkali protease; and a preparation from
Penicillium citrimum; wherein the enzyme cocktail is capable of
cleaving a gluten oligopeptide.
2. The enzyme cocktail of claim 1, wherein the enzyme cocktail
comprises GDEP-LGG, papain, a semi-alkali protease and a
preparation from Penicillium citrimum.
3. The enzyme cocktail of claim 1, wherein the semi-alkali protease
is Protease P.
4. The enzyme cocktail of claim 1, wherein the papain has been
activated by a reductant.
5. The enzyme cocktail of claim 1, wherein the gluten oligopeptide
is a 33-mer peptide fragment of .alpha.-gliadin.
6. The enzyme cocktail of claim 5, wherein the 33-mer peptide
fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1).
7. The enzyme cocktail of claim 1, wherein the enzyme cocktail is
stable in acid conditions.
8. The enzyme cocktail of claim 1, wherein the enzyme cocktail is
formulated in a pharmaceutically acceptable excipient.
9. A method of treating gluten intolerance or reducing gluten
exposure in a human subject comprising: providing the subject with
a therapeutically effective amount of an enzyme cocktail, wherein
the enzyme cocktail is capable of cleaving a gluten oligopeptide at
acidic conditions.
10. The method of claim 9, wherein the enzyme cocktail comprises a
composition selected from the group consisting essentially of GDEP;
GDEP-LGG; GDEP-M; GDEP-LNA; GDEP-2A; GDEP-AH; papain; Aorsin; an
acidic serine protease polypeptide having an amino acid sequence at
least about 80% homologous to Aorsin, or a fragment thereof, CPY;
an enzyme having an amino acid sequence at least about 80%
homologous to CPY, or a fragment thereof; a semi-alkali protease;
and a preparation from Penicillium citrimum.
11. The method of claim 9, wherein the enzyme cocktail comprises
GDEP-LGG; and papain.
12. The method of claim 9, wherein the enzyme cocktail comprises
GDEP-LGG; papain; a semi-alkali protease; and a preparation from
Penicillium citrimum.
13. The method of claim 9, wherein the enzyme cocktail is
formulated in a pharmaceutically acceptable excipient.
14. The method of claim 9, wherein the enzyme cocktail is
formulated for oral delivery.
15. A formulation for use in treating gluten intolerance or
reducing gluten exposure, comprising: at least two enzyme
compositions selected from the group consisting essentially of
GDEP; GDEP-LGG; GDEP-M; GDEP-LNA; GDEP-2A; GDEP-AH; papain; Aorsin;
an acidic serine protease polypeptide having an amino acid sequence
at least about 80% homologous to Aorsin, or a fragment thereof;
CPY; an enzyme having an amino acid sequence at least about 80%
homologous to CPY, or a fragment thereof; a semi-alkali protease;
and a preparation from Penicillium citrimum; wherein the at least
two enzyme compositions are capable of digesting a gluten
oligopeptide in an in vitro gastrointestinal model
16. The formulation of claim 15, wherein the at least two enzyme
compositions are a GDEP-LGG and papain.
17. The formulation of claim 15, wherein the model comprises
incubating the at least two enzyme compositions with the gluten
oligopeptide in simulated gastric fluid at about 37.degree. C. for
about 120 minutes.
18. The formulation of claim 17, wherein the simulated gastric
fluid comprises gastric mucosa mucin, pepsin, gelatinase, amylase,
and lipase.
19. The formulation of claim 15, wherein the model comprises
incubating the at least two enzyme compositions with the gluten
oligopeptide in simulated intestinal fluid at about 37.degree. C.
for about 60 minutes.
20. The formulation of claim 19, wherein the simulated intestinal
fluid comprises one or more pancreatic enzymes and a bile salt.
21. The formulation of claim 20, wherein the one or more pancreatic
enzymes are selected from the group consisting essentially of
trypsin, chymotrypsin, amylase, and lipase.
22. The formulation of claim 15, wherein the model comprises:
incubating a mixture comprising the at least two enzyme
compositions, the gluten oligopeptide, gastric mucosa mucin,
pepsin, gelatinase, amylase, and lipase in acidic conditions at
about 37.degree. C. for about 60 to about 120 minutes; adding an
acid neutralizing substance to the mixture; and incubating the
mixture with trypsin, amylase, and lipase, and a bile salt in
neutral conditions at about 37.degree. C. for about 60 minutes.
23. The formulation of claim 15, wherein the gluten oligopeptide is
a 33-mer peptide fragment of .alpha.-gliadin.
24. The formulation of claim 23, wherein the 33-mer peptide
fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1).
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 .alpha.-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 .alpha.-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 turn, impairs the ability of the intestine to absorb
nutrients, leading to malnutrition and a variety of other
symptoms.
[0006] Gluten intolerance, or gluten sensitivity, is a collective
term which includes all kinds of sensitivity to gluten. A small
proportion of gluten intolerant people will test positive for
Celiac disease. The standard diagnostic for celiac disease is
villus atrophy detected in duodenal biopsies. In addition,
antibodies to tissue transglutaminase (tTG) and gliadin appear in
almost 100% of the patients with active Celiac disease, and the
presence of such antibodies, particularly of the IgA class, has
been used in diagnosis of the disease. The large majority of Celiac
patients express the HLA-DQ2 [DQA1*0501, DQB1*02] and/or DQ8
[DQA1*0301, DQB1*0302] molecules. Clinical symptoms of Celiac
disease include, for example, 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).
[0007] However, most gluten intolerant, or gluten sensitive, people
test negative (or inconclusive) for Celiac disease. Up to
approximately 15% of the population (or 1 in 7 people) are
non-Celiac gluten sensitive or non-Celiac gluten intolerant. These
subjects are non-Celiac gluten sensitive or non-Celiac gluten
intolerant in that they suffer symptoms and illness similar to
celiac disease patients without meeting diagnostic criteria for
celiac disease. Thus, non-Celiac gluten sensitivity ("NCGS"),
non-Celiac gluten intolerance ("NCGI"), or gluten related disease
("GRD"), refer to a condition or disorder in which individuals
suffer symptoms very similarly to people with Celiac disease, but
the diagnostic tests (including blood tests) which identify and
diagnose Celiac disease are negative or inconclusive. Thus, as much
as 15% of the US population (or 1 in 7 people) may have NCGS.
[0008] Moreover, 15-30 million people in United States buy
gluten-free products in an attempt to reduce gluten exposure for a
variety of reasons.
[0009] U.S. Pat. No. 7,320,788 ("Shan") has proposed administering
certain enzymes, termed "glutenases," to Celiac or dermatitis
herpetiformis patients. According to Shan, glutenases include
endoproteases found in wheat, barley, and rye, such as an
endoprotease from Hordeum vulgare; prolyl endopeptidases ("PEP"),
and specifically PEP from Flavobacierium meningoscepticum (Genbank
ID #D10980) and Myxococcus xanthus (Genbank ID #AF 127082); and
brush border enzymes that catalyze the removal of dipeptides,
including dipeptidyl peptidase IV and dipeptidyl carboxypeptidase.
Shan also mentions that the X-Pro dipeptidase from Aspergillus
oryzae (GenBank ID #BD191984) and the carboxypeptidase from
Aspergillus saitoi (GenBank ID #D25288) can improve gluten
digestion in the Celiac intestine. Shan teaches that the
oligopeptides such as Gly-Pro-pNA, Z-Gly-Pro-pNA, and Hip-His-Leu
can be used to determine whether a candidate enzyme will digest a
toxic gluten oligopeptide. In fact, Shan teaches that the dose of a
PEP enzyme is determined by the amount of that enzyme required to
hydrolyze 1 micromol Z-Gly-Pro-pNA.
[0010] U.S. Pat. No. 7,534,426 ("Piper") is directed to methods of
determining the therapeutic efficacy of candidate glutenase enzyme
by detecting the ability of a candidate enzyme to digest one or
more selected oligopeptides. US Patent Application Publication No.
2008/0213245 ("Hausch") refers to enzyme treatment of foodstuffs
and also mentions screening methods that employ toxic oligopeptides
to identify active glutenases. Like Shan, both Piper and Hausch
mention PEPs for treatment of Celiac Sprue and/or dermatitis
herpetiformis and teach that the oligopeptide Z-Gly-Pro-pNA can be
used to determine whether a candidate enzyme will digest a toxic
gluten oligopeptide. U.S. Pat. No. 7,462,688 ("Khosla") is directed
to methods for diagnosing Celiac disease and/or dermatitis
herpetiformis by detecting toxic gluten oligopeptides, or T cells
and/or antibodies reactive thereto. Kholsa suggests treating such
patients by administering peptides that interfere with the binding
of toxic gluten oligopeptides to T cells and/or HLA molecules.
[0011] International Publication No. WO 2005/027953 ("Edens") is
directed to processes for the proteolytic hydrolysis of a peptide
or a polypeptide and mentions using proline specific
endopeptidases, such as PEP from Aspergillus species, to produce
food devoid of Celiac related epitopes. Likewise, U.S. Pat. No.
7,563,864 ("Marti") mentions an in vitro proteolytic protocol to
detoxify gluten employing pepsin, trypsin/chymotrypsin, elastase,
carboxypeptidase A, PEP, and rat brush border membrane enzymes.
Rizello et al. (Applied and Environmental Microbiology, July 2007,
p. 4499-4507) uses a combination of lactobacilli and proteases from
Aspergillus oryzae (supplied by BIO-CAT) to reduce gluten
concentration during food processing. Doumas et al. (Applied and
Environmental Microbiology, December 1998, p. 4809-4815) provides
the sequence of Aspergillus oryzae prolyl dipeptidyl peptidase
(DPPIV) and suggests that the DPPIV enzyme may be of importance in
industrial hydrolysis of wheat gluten-based substrates.
[0012] Ehren et al. (PLoS ONE 4(7):e6313) discuss a food-grade
enzyme preparation with modest gluten detoxification properties.
Ehren et al. note that aspergillopepsin from Aspergillus niger
markedly enhances gluten digestion. Ehren et al. employed peptidase
P from Aspergillus oryzae, which contains the exopeptidase DPPIV,
to augment the extent to which ASP hydrolyzes gluten. Ehren et al.
teach that DPPIV alone is unable to detoxify gluten oligopeptides,
including a synthetic 33-mer from .alpha.2-gliadin or the 26-mer
from .gamma.5-gliadin and that DPPIV is ineffective at low pH and,
therefore, would not be effective in vivo in the absence of an
antacid adjuvant.
[0013] "Oral Papain in Gluten Intolerance" ("Messer et al.")
mentions that the treatment of a patient regarded as having celiac
disease with oral papain resulted in the patient being able to
consume a gluten-containing diet with no further symptoms of celiac
disease. Australian Patent Application No. 2008100719 ("Cornell and
Stelmasiak") mentions compositions and methods for the prophylaxis
or treatment of celiac disease. Cornell et al. mentions
compositions and methods that include an extract of papaya resin or
a functional analogue thereof. "Papaya latex enzymes capable of
detoxification of gliadin" ("Cornell et al.") mentions that the
activity of papaya is due largely to caricain, and to a lesser
extent chymopapain and glutamine cyclotransferase.
BRIEF SUMMARY OF THE INVENTION
[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 .alpha.-gliadin. The enzyme cocktail may comprise
activated papain. For example, papain can be activated by a
reductant. The enzyme cocktail may be formulated for oral delivery
and/or contained in a formulation that contains an enteric coating.
The enzyme cocktail may be contained in a formulation that includes
a pharmaceutically acceptable carrier, such as a solid, a capsule,
or a liquid.
[0015] Certain embodiments of the present technology provide an
enzyme cocktail comprising a gluten degrading enzyme preparation
and an acidic serine protease polypeptide having an amino acid
sequence at least about 80% homologous to Aorsin, or a fragment
thereof. The enzyme cocktail of the present technology is capable
of cleaving a gluten oligopeptide, such as a 33-mer peptide
fragment of .alpha.-gliadin. The enzyme cocktail may further
comprise papain (including activated papain, chymopapain, and/or
purified papain), carboxypeptidase Y ("CPY"), and/or an enzyme
having an amino acid sequence at least about 80% homologous to CPY,
or a fragment thereof. The CPY may be Saccharomyces cerevisiae CPY,
Aspergillus niger CPY, Schizosaccharomyces pombe CPY, or
Aspergillus fumigatus CPY. The enzyme cocktail may be formulated in
a pharmaceutically acceptable excipient. The enzyme cocktail may be
formulated for oral delivery and/or contained in a formulation that
contains an enteric coating. The enzyme cocktail may be contained
in a formulation that includes a pharmaceutically acceptable
carrier, such as a solid, a capsule, or a liquid.
[0016] Certain embodiments of the present technology provide a
formulation for use in reducing gluten exposure or treating gluten
intolerance comprising an enzyme composition capable of cleaving an
immunogenic gluten oligopeptide into non-toxic fragments in vitro.
The enzyme composition may include gluten degrading enzyme
preparation; papain (including activated papain, chymopapain,
and/or purified papain); Aorsin; an acidic serine protease
polypeptide having an amino acid sequence at least about 80%
homologous to Aorsin, or a fragment thereof; CPY; an enzyme having
an amino acid sequence at least 80% homologous to CPY; a protease
from Aspergillus melleus; and/or a preparation from Penicillium
citrimum. The formulation may be capable of cleaving at least about
70%, at least about 80%, or at least about 90% of the immunogenic
gluten oligopeptide into non-toxic fragments in vitro. The
formulation may include a pharmaceutically acceptable carrier, such
as a solid, a capsule, or a liquid.
[0017] Certain embodiments of the present technology provide a
formulation for use in reducing gluten exposure or treating gluten
intolerance. The formulation is capable of digesting a gluten
oligopeptide in an in vitro gastrointestinal model and includes at
least two enzyme compositions. The enzyme compositions may be
gluten degrading enzyme preparation; papain (including activated
papain, chymopapain, and/or purified papain); Aorsin; an acidic
serine protease polypeptide having an amino acid sequence at least
about 80% homologous to Aorsin, or a fragment thereof; CPY; an
enzyme having an amino acid sequence at least 80% homologous to
CPY; a protease from Aspergillus melleus; and/or a preparation from
Penicillin citrimum. The in vitro gastrointestinal model may
include incubating the enzyme compositions with a gluten
oligopeptide in simulated gastric fluid at about 37.degree. C. for
a period representative of in vivo contact with gastric fluids
and/or incubating the enzyme compositions with a gluten
oligopeptide in simulated intestinal fluid at about 37.degree. C.
for a period representative of in vivo contact with intestinal
fluids. The simulated gastric fluid may include gastric mucosa
mucin, pepsin, gelatinase, amylase, and/or lipase. The simulated
intestinal fluid may include one or more pancreatic enzymes and a
bile salt. The pancreatic enzymes may be trypsin, chymotrypsin,
amylase, and/or lipase. The gluten oligopeptide may be a 33-mer
peptide fragment of .alpha.-gliadin, such as a peptide having the
aminoacid sequence LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID
NO:1).
[0018] Certain embodiments of the present technology provide a
method of treating gluten intolerance in a human subject comprising
providing the subject with a therapeutically effective amount of an
enzyme cocktail that is capable of cleaving a gluten oligopeptide
at acidic conditions. The enzyme cocktail may include a gluten
degrading enzyme preparation; papain (including activated papain,
chymopapain, and/or purified papain); Aorsin; an acidic serine
protease polypeptide having an amino acid sequence at least about
80% homologous to Aorsin, or a fragment thereof; CPY; an enzyme
having an amino acid sequence at least 80% homologous to CPY; a
protease from Aspergillus melleus; and/or a preparation from
Penicillin citrimum. The enzyme cocktail may include a composition
derived from Aspergillus oryzae. The enzyme cocktail may be
formulated in a pharmaceutically acceptable excipient. The enzyme
cocktail may be formulated for oral delivery and/or contained in a
formulation that contains an enteric coating. The enzyme cocktail
may be contained in a formulation that includes a pharmaceutically
acceptable carrier, such as a solid, a capsule, or a liquid.
[0019] Certain embodiments of the present technology provide a
method of reducing gluten exposure in a subject, comprising
providing the subject with an enzyme cocktail that is capable of
cleaving a gluten oligopeptide at acidic conditions. The enzyme
cocktail may include a gluten degrading enzyme preparation; papain
(including activated papain, chymopapain, and/or purified papain);
Aorsin; an acidic serine protease polypeptide having an amino acid
sequence at least about 80% homologous to Aorsin, or a fragment
thereof; CPY; an enzyme having an amino acid sequence at least 80%
homologous to CPY; a protease from Aspergillus melleus; and/or a
preparation from Penicillium citrimum. The enzyme cocktail may
include a composition derived from Aspergillus oryzae. The enzyme
cocktail may be formulated in a pharmaceutically acceptable
excipient. The enzyme cocktail may be formulated for oral delivery
and/or contained in a formulation that contains an enteric coating.
The enzyme cocktail may be contained in a formulation that includes
a pharmaceutically acceptable carrier, such as a solid, a capsule,
or a liquid.
[0020] Certain embodiments of the present technology provide a
method of assessing the efficacy of an enzyme composition,
comprising the steps of (i) incubating a mixture comprising a
candidate enzyme composition and a gluten oligopeptide in simulated
gastric fluid at about 37.degree. C. for a period representative of
in vivo contact with gastric fluids; (ii) adding an acid
neutralizing substance to the mixture; (iii) incubating the mixture
in simulated intestinal fluid at about 37.degree. C. for a period
representative of in vivo contact with intestinal fluids; and (iv)
determining the amount of intact gluten oligopeptide in the
mixture. The period representative of in vivo contact with gastric
fluids may be about 120 minutes. The period representative of in
vivo contact with intestinal fluids may be about 60 minutes. The
simulated gastric fluid may include gastric mucosa mucin, pepsin,
gelatinase, amylase, and lipase. The simulated intestinal fluid
comprises one or more pancreatic enzymes and a bile salt. The
pancreatic enzymes may be trypsin, chymotrypsin, amylase, and/or
lipase. The gluten oligopeptide may be a 33-mer peptide fragment of
.alpha.-gliadin, such as a peptide having the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1).
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0021] FIG. 1 shows degradation of a 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) by GDEP-LGG
at various doses and times.
[0022] FIG. 2 shows degradation of a 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) by gluten
degrading enzyme preparation and its individual component enzymes,
Oryzin, NP I, and NP II.
[0023] FIG. 3 shows degradation of a 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) by gluten
degrading enzyme preparation and its individual component enzymes,
Oryzin, NP I, and NP II, and various combinations thereof.
[0024] FIG. 4 shows chromatographs of HPLC analysis with 280 nm and
210 nm (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with pepsin (FIG. 4A), proteinase K
(FIG. 4B), and GDEP-LGG (FIG. 4C).
[0025] FIG. 5 shows chromatographs of HPLC analysis with 280 nm and
210 nm (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with gluten degrading enzyme
preparation (FIG. 5A) and other Aspergillus-derived proteases,
GDEP-2A (FIG. 5B), GDEP-LNA (FIG. 5C), GDEP-M (FIG. 5D), and
Protease P (FIG. 5E).
[0026] FIG. 6 shows chromatographs of HPLC analysis with 280 nm and
210 nm (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with GDEP-M at a pH ranging from 3.0
to 6.0 (FIG. 6A-6D, 6H) and GDEP-LGG at a pH ranging from 4.0 to
6.0 (FIG. 6E-6G).
[0027] FIG. 7 shows chromatographs of HPLC analysis with 280 nm and
210 nm (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with GDEP-LGG alone (FIG. 7A) and
GDEP-LGG in the presence of PMSF (FIG. 7B), pepstatin (FIG. 7C),
and EDTA (FIG. 7D). FIG. 7 also shows chromatographs of HPLC
analysis (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with GDEP-M alone (FIG. 7E) and
GDEP-M in the presence of PMSF (FIG. 7F), pepstatin (FIG. 7G), and
EDTA (FIG. 7H).
[0028] FIG. 8 shows chromatographs of HPLC analysis with 280 nm and
210 nm (panels 1 and 2) and mass spectral analysis (panel 3) of a
33-mer peptide after treatment with various amounts of Aorsin A
(FIGS. 8A-8D) and Aorsin B (FIGS. 8E-8H).
[0029] FIG. 9 shows degradation of a 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) by
Aorsin.
[0030] FIG. 10 shows chromatographs of HPLC analysis (panels 1 and
2) and mass spectral analysis (panel 3) of a 33-mer peptide after
treatment with Carboxypeptidase Y ("CPY") at a pH ranging from 4.0
to 7.0 (FIG. 10A-10D) and with Myrococcus xamhus prolyl
endopeptidase ("MX-PEP") at a pH ranging from 4.0 to 7.0 (FIG.
10E-10H).
[0031] FIG. 11 shows chromatographs of HPLC analysis (panels 1 and
2) and mass spectral analysis (panel 3) of a 33-mer peptide after
treatment with various amounts of CPY (FIG. 11A-11D), MX-PEP (FIG.
11E-11H), and Aspergillus niger prolyl endopeptidase ("AN-PEP")
(FIG. 11I-11L). FIG. 11 also shows chromatographs of HPLC analysis
(panels 1 and 2) and mass spectral analysis (panel 3) of a 33-mer
peptide after treatment with CPY (FIG. 11M-11P), MX-PEP (FIG.
11Q-11T), and AN-PEP (FIG. 11U-11V).
[0032] FIG. 12 shows degradation of a 33-mer peptide (as assessed
by measuring the levels of intact 33-mer peptide by ELISA) by CPY
and the prolyl endopeptidases MX-PEP and AN-PEP (FIG. 12A). FIG. 12
also shows degradation of a 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) by GDEP-LGG
and CPY (FIG. 12B).
[0033] FIG. 12 also shows degradation of a 33-mer peptide (as
assessed by measuring the levels of intact 33-mer peptide by ELISA)
by AN-PEP (FIG. 12C).
[0034] FIG. 13 shows degradation of wheat gliadin and a 33-mer
peptide (as assessed by measuring the levels of intact 33-mer
peptide by ELISA) by GDEP-LGG in an in vitro gastrointestinal
model.
[0035] FIG. 14 shows chromatographs of HPLC analysis (panels 1 and
2) and mass spectral analysis (panel 3) of a 33-mer peptide after
treatment with digestive enzymes alone (FIG. 14A), CPY (FIG. 14B),
GDEP-M (FIG. 14C), and a combination of CPY and GDEP-M (FIG. 14D)
in an in vitro gastrointestinal model.
[0036] FIG. 15 shows degradation of wheat gliadin and a 33-mer
peptide (as assessed by measuring the levels of intact 33-mer
peptide by ELISA) by CPY and GDEP-M in an in vitro gastrointestinal
model.
[0037] FIG. 16 shows chromatographs of HPLC analysis (panels 1 and
2) and mass spectral analysis (panel 3) of a 33-mer peptide after
treatment with GDEP-LGG (FIG. 16A), CPY (FIG. 16B), and a
combination of GDEP-LGG and CPY (FIG. 16C).
[0038] FIG. 17 shows protein concentration (A280), 33-mer peptide
degradation activity, prolyl endopeptidase activity, and protease
activity of fractionated GDEP-LGG.
[0039] FIG. 18 shows residual ratio comparisons after analysis by
ELISA for gluten digestion with CPY and AN-PEP (FIG. 18A), GDEP-M.
and GDEP-LGG (FIG. 18B), Aorsin A and Aorsin B (FIG. 18C), a
Combination of GDEP-M and some peptidases (FIG. 18D), and a
Combination of GDEP-LGG and GDEP-M (FIG. 18E) all at varying
amounts of enzyme.
[0040] FIG. 19 shows degradation of 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) from a
commercial preparation of the 33-mer peptide (FIG. 19A), ravioli
(FIG. 19B), cheese macaroni (FIG. 19C), white bread (FIG. 19D) and
roll of bread (FIG. 19E) at various doses (50 mg, 100 mg, 200 mg,
and 400 mg) of GDEP-LGG.
[0041] FIG. 20 shows degradation of 33-mer peptide (as assessed by
measuring the levels of intact 33-mer peptide by ELISA) from
ravioli (FIG. 20A), macaroni (FIG. 20B), white bread (FIG. 20C),
roll of bread (FIG. 20D), lasagna (FIG. 20E) and pasta (FIG. 20F)
at various doses (50 mg, 100 mg, 200 mg, and 400 mg) of GDEP-LGG
and GDEP-M.
[0042] FIG. 21 shows a comparison of residual ratios after
degradation of a 33-mer peptide (as assessed by measuring the
levels of intact 33-mer peptide by ELISA) by three lots of GDEP-LGG
and three existing products ("Gluten Gest," "Gluten-zyme" and
"GlutenEase") as well as chromatographs after analysis by HPLC-MS
for existing products ("Gluten Gest", "Spectrumzyme", "Gluten-zyme
Plus", "Gluten-zyme" and "GlutenEase"), pepsin, a 33-mer control
and gluten degrading enzyme preparation at various times throughout
the reaction.
[0043] FIG. 22 shows chromatographs of HPLC analysis (panels 1 and
2) and mass spectral analysis (panel 3) of a 33-mer peptide after
treatment with papain.
[0044] FIG. 23 shows a chromatograph of cation-exchange analysis of
papain and mass spectral analysis of a 33-mer peptide after
treatment with the papain fractions resultant from the
cation-exchange chromatography.
[0045] FIG. 24 shows SDS-PAGE gels of papain after fractionation by
cation-exchange chromatography.
[0046] FIG. 25 shows chromatographs of HPLC analysis for Peak 2
(FIG. 25A) and Peak 3 (FIG. 25B) fractions from papain after
purification by cation-exchange chromatography.
[0047] FIG. 26 shows a chromatograph of HPLC analysis for the E9
fraction of the papain purification by cation-exchange
chromatography after further hydrophobic interaction chromatography
(FIG. 26A) and FRETS analysis (FIG. 26B).
[0048] FIG. 27 shows FRETS analysis and chromatographs of mass
spectral analysis of a 33-mer peptide after treatment with the
resulting fractions.
[0049] FIG. 28 shows chromatographs of HPLC analysis for Peaks 3-1
(FIG. 28A), 3-2 (FIG. 28B) and 3 (FIG. 28C) by reverse phase
chromatography.
[0050] FIG. 29 shows residual ratio comparisons of papain and other
enzymes after analysis by ELISA.
[0051] FIG. 30 shows the protease activity and hydrolysis activity
of papain and GDEP-LGG with a reductant (Glutathione,
dithiothreitol, L-Cysteine, or N-Acetyl-L-Cysteine).
[0052] FIG. 31 shows the relative activity of an enzyme cocktail
comprising GDEP-LGG and papain at various concentrations (FIG. 31A)
and chromatographs of HPLC analysis for each concentration (FIGS.
31B 31G)
[0053] FIG. 32 shows the results of a FRETS assay screening using a
set of 6-mers to for various compositions.
[0054] FIG. 33 shows chromatographs of HPLC analysis for the
hydrolysis activity of compositions selected from the FRETS assay
screening (FIG. 32).
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present technology generally relates to an enzyme
composition. The enzyme composition may be used to treat gluten
intolerant and/or gluten sensitive subjects, including subjects
suffering from non-Celiac gluten sensitivity ("NCGS") and/or
non-Celiac gluten intolerance ("NCGI"). 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 or prevent exposure to gluten oligopeptides, such as a
33-mer peptide derived from .alpha.-2 gliadin, to limit, reduce,
prevent, or control the development of gluten intolerance and/or
gluten insensitivity. A 33-mer peptide fragment of an
.alpha.-gliadin, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1),
is naturally formed by digestion with gastric and pancreatic
enzymes and is resistant to further degradation by digestive
enzymes.
[0056] Many food and beverages include gluten oligopeptides, such
as a 33-mer peptide derived from .alpha.-2 gliadin. The digestion
of gluten-related peptides, including the 33-mer peptide derived
from .alpha.-2 gliadin, offers physiological benefits, such as
reduced exposure to gluten and gluten-related peptides. This may be
particularly beneficial for populations or specific individuals
(including human or animal subjects) that do not effectively or
efficiently hydrolyze gluten (e.g., whose digestive systems lack
the enzyme activity needed to effectively or efficiently hydrolyze
gluten and/or gluten-related peptides) and to individuals desiring
to reduce their exposure to gluten and gluten-related peptides.
[0057] In certain embodiments, the enzyme composition of the
present technology is a nutraceutical. The term "nutraceutical" has
been used to refer to any food product or substance that is
intended to be, or reasonably expected to be, ingested and may
provide medical or health benefits, including the prevention and/or
treatment of NCGS and/or NCGI and/or related symptoms, including,
but not limited to, abdominal pain, bloating, bowel discomfort,
diarrhea, flatulence, and nutrient malabsorption. Compositions
falling under the label "nutraceutical" may range from isolated
nutrients, dietary supplements, and enzyme compositions to
genetically engineered designer foods, herbal products, and
processed foods such as cereals, soups and beverages. In a more
technical sense, the term "nutraceutical" refers to an ingestible
food product generally sold in medicinal forms and demonstrated to
have a physiological benefit by, for example, ameliorating,
treating, prevent, reducing the incidence of, or providing
protection against, an acute or chronic disease, disorder, or other
symptomology.
[0058] In certain embodiments, one or more enzyme compositions of
the present technology may be used to treat any disease amenable to
treatment by a prevention of and/or reduction in gluten exposure,
including, for example Celiac disease, thyroid diseases, such as
hypothyroidism and neurological diseases, such as autism,
cerebellar ataxia, peripheral neuropathies, and schizophrenia.
[0059] In certain embodiments, the enzyme compositions of the
present technology may comprise any enzyme or mixture of enzymes.
In certain embodiments, an enzyme of the enzyme compositions of the
present technology is a protein reducing enzyme, including, for
example, proteases and peptidases such as exoproteases,
endoproteases, and acid stable proteases. In certain embodiments,
the enzyme composition can include a protease. Generally, proteases
are enzymes that break peptide bonds between the amino acids of
proteins. A suitable subset of enzymes includes enzymes derived
from Aspergillus oryzae. For example, the enzyme composition may
comprise a gluten degrading enzyme preparation, papain, purified
papain, activated papain, chymopapain, or a combination thereof.
The enzyme composition may also include a carboxypeptidase, such as
Carboxypeptidase Y ("CPY"). The enzyme composition may also include
a semi-alkali protease. The preparation may also include a
preparation from Penicillium citrimum. Enzyme compositions of the
present technology are useful, for example, in the treatment of
gluten intolerance, and in reducing exposure to gluten
oligopeptides, including the 33-mer derived from .alpha.-2
gliadin.
[0060] As used herein, the term "gluten degrading enzyme
preparation" ("GDEP") is used to refer to an enzyme preparation
that contains at least Oryzin, Neutral protease I ("NPI") and
Neutral protease II ("NPII")
[0061] GDEP having not less than about 70 unit/g peptidase activity
at a neutral pH by the leucyl-glycyl-glycine ("LGG") method is
referred to herein as "GDEP-LGG". GDEP having not less than about
1,400 u/g peptidase activity at a neutral pH by the
leucylnaphthylamide ("LNA") method is referred to herein as
"GDEP-LNA". GDEP having not less than about 5,500 unit/g protease
activity at an acidic pH is referred to herein as "GDEP-M". GDEP
having not less than about 20,000 unit/g protease activity at a
neutral pH is referred to herein as "GDEP-2A". GDEP having not less
than about 10,000 unit/g protease activity at a neutral pH is
referred to herein as "GDEP-AH".
[0062] In one embodiment, a GDEP-LGG is a peptidase preparation
derived from Koji mold (Aspergillus oryzae). The gluten degrading
enzyme preparation production strain is derived by subjecting
Aspergillus oryzae to a course of mutigenesis, including UV
mutagenesis. For example, yellow Koji mold, which contains
Aspergillus oryzae, is subjected to UV mutagenesis to produce "A
strain," which is subjected to UV mutagenesis to produce 90-76,
which has a proteolytic activity of about 500-600 u/g. The 90-76
strain is further subjected to UV mutagenesis to produce the 90-51
strain, which has a proteolytic activity of about 800-1000 u/g.
Following UV mutagenesis, Mono Spore Isolation (M.S.I.) is
performed to obtain a mono-colony on the medium plate. This
procedure yields the gluten degrading enzyme preparation production
strain. The proteolytic activity of the gluten degrading enzyme
preparation production strain is about 2 times as much as that of
the parent strain.
[0063] An exemplary GDEP-LGG composition has not less than about 70
u/g peptidase activity at a pH of about 7.0. GDEP-LGG can be
obtained from Aspergillus oryzae by methods known in the art, and
diluted or concentrated prior to use. Preparations with GDEP-LGG
can be obtained by spray drying to avoid inactivation of
peptidases. The production process includes, for example, solid
fermentation, followed by water extraction, filtration,
ultra-filtration, and, finally, spray-drying.
[0064] In at least some embodiments of the present technology,
GDEP-LGG cuts at the carboxyl side of Gln, Ser, Thr, Met, Arg, Ala,
Lys, Phe, and Leu. As determined by assessing activity against
substrate Z-Gly-Pro-pNA, GDEP-LGG does not possess
prolyl-endopeptidase activity. Nonetheless, GDEP-LGG partially
hydrolyzes a 33-mer peptide and remains effective even at an acidic
pH. Using a 33-mer peptide as a substrate, gluten degrading enzyme
preparation digests approximately 90% of the 33-mer. In an in vitro
gastrointestinal model, 200 mg gluten degrading enzyme preparation
digests approximately 80% of the 33-mer from 10 g of wheat gluten
as compared to the 33-mer level present without added enzyme
(control).
[0065] In certain embodiments, the enzyme composition of the
present technology includes papain. A typical commercial
preparation of papain is commercially available as Papain W-40
bulk. This commercial preparation may be used at the given
concentration, or the commercial preparation may be diluted or
concentrated for use.
[0066] In certain embodiments, the enzyme composition of the
present technology includes purified papain. Purification methods
include cation-exchange chromatography and hydrophobic interaction
chromatography.
[0067] In certain embodiments, the enzyme composition of the
present technology includes chymopapain.
[0068] In certain embodiments, the enzyme composition of the
present technology includes activated papain. For example, papain
can be activated by a reductant. Suitable reductants for use in the
present technology include, but are not limited to, Glutathione,
dithiothreitol ("DTT"), L-Cysteine, and N-Acetyl-L-Cysteine.
[0069] In certain embodiments, the enzyme composition of the
present technology is a mixture of enzymes or enzyme compositions.
In certain embodiments, the components of the mixture have an
additive. In certain other embodiments, the components of the
mixture have a synergistic effect, for example, an enzyme
composition comprising a mixture of gluten degrading enzyme
preparation and papain has an unexpectedly synergistic effect on
33-mer peptide degradation.
[0070] In certain embodiments, the enzyme composition of the
present technology includes GDEP-M. GDEP-M can be obtained from
Aspergillus oryzae by methods known in the art, and diluted or
concentrated prior to use. Preparations with GDEP-M can be obtained
by spray drying or by ethanol precipitation. Spray drying produces
more peptidase activity compared to ethanol precipitation protease
activity. In certain embodiments, the GDEP-M used has a protease
activity of not less than about 5,500 u/g at a pH of about 3.0.
This preparation may be used at the given concentration, or the
preparation may be diluted or concentrated for use.
[0071] In certain embodiments, the enzyme composition of the
present technology includes GDEP-2A. GDEP-2A can be obtained from
Aspergillus oryzae by methods known in the art, and diluted or
concentrated prior to use. Preparations with GDEP-2A can be
obtained by spray drying or by ethanol precipitation. Spray drying
produces more peptidase activity compared to ethanol precipitation
protease activity. In certain embodiments, the GDEP-2A used has a
protease activity of not less than about 20,000 u/g at a pH of
about 7.0. This preparation may be used at the given concentration,
or the preparation may be diluted or concentrated for use.
[0072] In certain embodiments, the enzyme composition of the
present technology includes GDEP-AH. GDEP-AH can be obtained from
Aspergillus oryzae by methods known in the art, and diluted or
concentrated prior to use. Preparations with GDEP-AH can be
obtained by spray drying. In certain embodiments, the GDEP-AH used
has a protease activity of not less than about 10,000 u/g at a pH
of about 7.0. This preparation may be used at the given
concentration, or the preparation may be diluted or concentrated
for use.
[0073] In certain embodiments, the enzyme composition of the
present technology includes GDEP-LNA. GDEP-LNA can be obtained from
Aspergillus oryzae by methods known in the art, and diluted or
concentrated prior to use. Preparations with GDEP-LNA can be
obtained by spray drying. In certain embodiments, the GDEP-LNA has
not less than about 1,400 u/g peptidase activity at a pH of about
7.0 by leucylnaphthylamide ("LNA") method. This preparation may be
used at the given concentration, or the preparation may be diluted
or concentrated for use.
[0074] In certain embodiments, the enzyme composition of the
present technology comprises specific protease components of
GDEP-M. For example, Aorsin is a serine proteinase with
trypsin-like specificity at an acidic pH that is purified from
GDEP-M. Aorsin A and Aorsin B are characterized in Japanese Patent
Nos. JP4401555 and JP2009-232835A, each of which are incorporated
by reference in their entireties. Aorsin efficiently hydrolyzes a
33-mer peptide at an acidic pH. The nucleotide and amino acid
sequence of Aorsin was published in Lee, et al., Biochem. J. 371
(PT 2), 541-548 (2003). The nucleotide sequence for Aorsin A has
the GenBank Accession Number AB084899.1, and the corresponding
amino acid sequence has the GenBank Accession Number BAB97387. The
nucleotide sequence for Aorsin B has the GenBank. Accession Number
XM 001820783.1, and the corresponding amino acid sequence has the
GenBank Accession Number XP.sub.--001820835.1.
[0075] In certain embodiments, the enzyme composition of the
present technology comprises an enzyme having the amino acid
sequence of Aorsin or an amino acid sequence having at least about
70% identity with the amino acid sequence of Aorsin. In certain
embodiments, the amino acid may have at least about 80%, at least
about 81%, at least about 82%, at least about 83%, at least about
84%, at least about 85%, at least about 86%, at least about 87%, at
least about 88%, at least about 89%, at least about 90%, at least
about 91%, at least about 92%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, or at least about 99% identity with the amino acid
sequence of Aorsin.
[0076] In certain embodiments, the enzyme composition of the
present technology comprises an enzyme having an amino acid
sequence substantially homologous to Aorsin. Two amino acid
sequences are "substantially homologous" when at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99% of the amino
acids are identical; or at least about 80%, at least about 81%, at
least about 82%, at least about 83%, at least about 84%, at least
about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, or at least about 99% of the amino acids are similar in that
they are functionally identical. Homologous sequences may be
identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison, Wis.) pileup program, or any of sequence comparison
algorithms such as BLAST or FASTA
[0077] The present technology also includes enzyme compositions
exhibiting an enzyme activity profile similar to that of GDEP-M.
For example, in certain embodiments the enzyme composition exhibits
a protease activity of about 4,000 to about 8,000 u/g. In one
non-limiting example, such a composition has an enzyme activity
profile comprising a protease activity of about 6,500 u/g.
[0078] In certain embodiments, the enzyme composition of the
present technology includes CPY, which is also known as proteinase
C or yscY. CPY is a broad-specificity vacuolar exopeptidase that
removes amino acids from the carboxy termini of proteins and
peptides. It belongs to a family of serine carboxypeptidases that
are ubiquitous proteolytic enzymes characterized by highly
conserved and catalytically essential serine and histidine residues
around their active sites. CPY useful in the present technology may
be CPY from, for example, yeast or fungi such as saccharomyces,
schizosaccharomyces, aspergillus, candida, and pichia. For example,
CPY used in the present technology have at least about 20% sequence
identity at the amino acid level, alternatively at least about 40%
sequence identity, alternatively at least about 50% sequence
identity, alternatively at least about 60% sequence identity,
alternatively at least about 70% sequence identity, alternatively
at least about 80% sequence identity, alternatively at least about
90% sequence identity, to one of the following CPY: Saccharomyces
cerevisiae CPY, Aspergillus niger CPY, Schizosaccharomyce pombe
CPY, Aspergillus fumigatus CPY.
[0079] In certain embodiments, the enzyme composition of the
present technology comprises an enzyme having the amino acid
sequence of a CPY or an amino acid sequence having at least about
70% identity with the amino acid sequence of a CPY. In certain
embodiments, the amino acid may have at least about 80%, at least
about 81%, at least about 82%, at least about 83%, at least about
84%, at least about 85%, at least about 86%, at least about 87%, at
least about 88%, at least about 89%, at least about 90%, at least
about 91%, at least about 92%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, or at least about 99% identity with the amino acid
sequence of a CPY. In certain embodiments, the enzyme composition
comprises an enzyme having an amino acid sequence substantially
homologous to a CPY.
[0080] In another embodiment, gluten degrading activity has been
surprisingly found in an enzyme preparation from Penicillium
citrimum. In particular, the enzyme preparation from Penicillium
citrimum has gluten digesting activity in which at least 4 of the
10 6-mer substrates identified in Example 24 are digested
>1000u/g, where 1 unit is equal to 1 .mu.mol of FRET substrate
digested/min. A preparation having this activity may also be
obtained from other fungi, such as Aspergillus niger, and certain
Actinomyces, such as Streptomyces aureus. In certain embodiments,
the enzyme composition of the present technology comprises a
preparation from Penicillium citrimum that has gluten degrading
activity as well as 5'-phosphodiesterase ("5'-PDE") activity.
[0081] In another embodiment, gluten degrading activity has been
surprisingly found in an enzyme composition including a preparation
containing a semi-alkali protease. As used herein, semi-alkali
protease refers to an enzyme, which is derived from a fungal source
and is capable of breaking down proteins and their degradation
products, polypeptides and peptides, by hydrolysis, and is active
in an environment ranging from a pH of 6.0 to a pH of 11. Sources
of semi-alkali protease include certain fungi, such as Aspergillus
melleus. Semi-alkali protease may also be obtained from certain
plant germs such as barley or sorghum germ.
[0082] In certain embodiments, the enzyme composition of the
present technology may be generated by any of a number of methods.
For example, individual enzymes may be combined to achieve the
desired enzyme composition with a desired enzyme activity
profile.
[0083] Additionally or alternatively, the enzyme composition may be
obtained from a microorganism that produces enzymes naturally or
that is genetically modified to produce one or more enzymes, using
methods well known in the art. For example, the enzyme composition
of the present technology may include at least two of the
following: GDEP, GDEP-LGG, GDEP-LNA, GDEP-M, GDEP-2A, GDEP-AH,
oryzin, NPI, NPII, Aorsin A, Aorsin B, CPY, papain, activated
papain, purified papain, or chymopapain. The enzyme composition of
the present technology may further include a preparation from
Penicillium citrimum and/or a semi-alkali protease. In certain
embodiments, the enzyme composition may comprise GDEP-LGG and
papain. In another embodiment, the enzyme composition may comprise
GDEP-LGG, papain, and a preparation from Penicillium citrimum. In
another embodiment, the enzyme composition may comprise GDEP-LGG,
papain, a preparation from Penicillium citrimum and a semi-alkali
protease. In still other embodiments the enzyme composition may
comprise GDEP-LGG and activated papain. In another embodiment, the
enzyme composition may comprise GDEP-LGG, activated papain, and a
preparation from Penicillium citrimum. In another embodiment, the
enzyme composition may comprise GDEP-LGG, activated papain, a
preparation from Penicillium citrimum and a semi-alkali protease.
In still other embodiments the enzyme composition may comprise
GDEP-LGG and chymopapain. In another embodiment, the enzyme
composition may comprise GDEP-LGG, chymopapain, and a preparation
from Penicillium citrimum. In another embodiment, the enzyme
composition may comprise GDEP-LGG, chymopapain, a preparation from
Penicillium citrimum and a semi-alkali protease.
[0084] As noted above, enzymes and enzyme compositions of the
present technology may also be obtained from transformed or
transfected cells by methods well known in the art. For example, a
nucleic acid sequence encoding a desired enzyme can be inserted
into an expression vector, which can be used to transform or
transfect a host cell for production of the enzyme. Enzymes or
enzyme compositions can then be obtained from the host cell by
methods well known in the art.
[0085] The amount of a given enzyme or enzyme activity in a
composition may vary based on the desired effect of the
composition, and may be determined or measured by a variety of
methods known in the art. The amount of enzymes present in a
composition may be stated in molar amounts or molar ratios (e.g.,
nanomoles or micromoles of enzyme), weight amounts or weight ratio
(micrograms or nanograms of enzyme), or activity amounts or
activity ratios (e.g., "units" of enzyme or enzyme activity/weight
or mole of enzyme). In particular embodiments, compositions may
include protease and peptidase activities.
[0086] In certain embodiments of the present technology, the
efficacies of various protease compositions are assessed. Thus, the
present technology includes an in vitro model to assess these
protease compositions. The in vitro model may include, for example,
a gastric model, an intestinal model, or a gastrointestinal
model.
[0087] In vitro gastric model. An in vitro gastric model may
comprise, for example, a simulated gastric fluid ("SGF"). The SGF
may contain serum albumin, gastric mucosa mucin, and/or a buffer.
The buffer may be an acetate buffer. The SGF may include a
physiological salt solution comprising, for example, sodium
chloride (NaCl) and calcium chloride (CaCl.sub.2). The SGF may be
maintained at an appropriate pH to simulate gastric conditions,
such an acidic pH including pH ranging from about 1.0 to about 7.0,
and including about 2.0, about 2.1, about 2.2, about 2.3, about
2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about
3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about
3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about
4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about
4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about
5.4, about 5.5, about 5,6, about 5.7, about 5.8, about 5.9, about
6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about
6.6, about 6.7, about 6.8, or about 6.9. An acid, such as
hydrochloric acid, may be added to the SGF at periodic intervals
during the incubation phase to maintain the acidity of the SGF.
[0088] The SGF may also include one or more gastric enzymes, such
as pepsin, gelatinase, amylase, and lipase, as well as other
enzymes such as lactase and alpha-galactosidase to further simulate
gastric conditions. Commercially available digestive enzyme
products, such as EZ-GEST.RTM. (Shaklee) may be used to supplement
the simulated gastric mixture.
[0089] To assess the efficacy of a candidate enzyme composition, an
amount of the enzyme composition and a peptide-containing substance
may be added to the SGF. The peptide-containing substance may be a
homogenous peptide preparation, such as a preparation containing
natural or synthetic gluten oligopeptide, or a heterogeneous
preparation comprising, for example, any substance, including
foodstuffs, intended to be, or reasonably expected to be, ingested
by an animal. Exemplary gluten oligopeptides include the 33-mer
peptides: LQLQPEPQPQLPYPQPQLPYQPQLPYPQPQPF and
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1), as well as
peptides identified in, for example, U.S. Pat. No. 7,563,864.
[0090] The mixture comprising SGF, the enzyme composition, and the
peptide-containing substance may be incubated at about 37.degree.
C. for a period that is representative of in vivo contact with
gastric fluids; for example, about 20, about 25, about 30, about
35, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 80, about 85, about 90, about 95, about 100, about
105, about 110, about 115, or about 120 minutes. The mixture
comprising the SGF, the enzyme composition, and the
peptide-containing substance may be sampled at regular intervals to
assess digestion efficiency. Digestion efficiency may be assessed
by, for example, measuring the amount of 33-mer present in the
mixture or measuring the amount of free tyrosine production by the
Folin method.
[0091] In vitro intestinal model. An in vitro intestinal model may
comprise, for example, a simulated intestinal fluid ("SIF"). The
SIF may be maintained at an appropriate pH to simulate intestinal
conditions, such as a slightly acidic, neutral, or basic pH
including pH ranging from about 6.0 to about 14.0 and including
about 7.0, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4,
about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0,
about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6,
about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2,
about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8,
about 9.9, about 10.0, about 10.1, about 10.2, about 10.3, about
10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9,
about 11.0, about 11.1, about 11.2, about 11.3, about 11.4, about
11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12.0,
about 12.1, about 12.2, about 12.3, about 12.4, about 12.5, about
12.6, about 12.7, about 12.8, about 12.9, about 13.0, about 13.1,
about 13.2, about 13.3, about 13.4, about 13.5, about 13.6, about
13.7, about 13.8, or about 13.9. The SIF may also include
substances to neutralize acid such as sodium bicarbonate
(NaHCO.sub.3) or disodium hydrogen phosphate
(Na.sub.2HPO.sub.4).
[0092] The SIF may include one or more pancreatic enzymes such as
trypsin, chymotrypsin, and amylase, and intestinal enzymes such as
sucrase, maltase, isomaltase, and lactase. For example, the SIF may
also contain pancreatin. Pancreatin contains the pancreatic enzymes
trypsin, amylase, and lipase. Pancreatin is commercially available
as Creon.RTM. (Solvay), Nutrizym 10.RTM. (Merck), Pancrease.RTM.
(Janssen-Ortho) and Pancrex.RTM. (Paines & Byrne).
[0093] The SIF may also include one or more bile salts, such as
cholates including taurodeoxycholate ("TDCA") and deoxycholate
("DCA"), or bile acids, such as cholic acid, glyocholic acid,
taurocholic acid, deoxycholic acid, or lithocholic acid.
[0094] To assess the efficacy of a candidate enzyme composition, an
amount of the enzyme composition and a peptide-containing substance
may be added to the SIF. The peptide-containing substance may be a
homogenous peptide preparation, such as a preparation containing
natural or synthetic gluten oligopeptide, or a heterogeneous
preparation comprising, for example, include any substance,
including foodstuffs, intended to be, or reasonably expected to be,
ingested by a human or an animal. Exemplary gluten oligopeptides
include the 33-mer peptides: LQLQPEPQPQLPYPQPQLPYQPQLPYPQPQPF and
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1), as well as
peptides identified in, for example, U.S. Pat. No. 7,563,864.
[0095] The mixture comprising SIF, the enzyme composition, and the
peptide-containing substance may be incubated at about 37.degree.
C. for a period that is representative of in vivo contact with
intestinal fluids, for example, about 20, about 25, about 30, about
35, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 80, about 85, about 90, about 95, about 100, about
105, about 110, about 115, about 120, about 125, about 130, about
135, about 140, about 145, about 150, about 155, about 160, about
165, about 170, about 175, about 180, about 185, about 190, about
195, about 200, about 205, about 210, about 215, about 220, about
225, about 230, about 235, or about 240 minutes. The mixture
comprising the SIF, the enzyme composition, and the
peptide-containing substance may be sampled at regular intervals to
assess digestion efficiency. Digestion efficiency may be assessed
by, for example, measuring the amount of 33-mer present in the
mixture or measuring the amount of free tyrosine production by the
Folin method.
[0096] In vitro gastrointestinal model. An in vitro
gastrointestinal model may comprise sequential treatment in the
gastric and intestinal models described above. For example, a
candidate enzyme composition and a peptide-containing substance may
be incubated in SGF at about 37.degree. C. for a period that is
representative of in vivo contact with gastric fluids, for example,
about 20, about 25, about 30, about 35, about 40, about 45, about
50, about 55, about 60, about 65, about 70, about 80, about 85,
about 90, about 95, about 100, about 105, about 110, about 115, or
about 120 minutes. A substance to neutralize acid, such as sodium
bicarbonate (NaHCO.sub.3) or sodium phosphate (e.g.,
Na.sub.2HPO.sub.4), may be added to the mixture. The mixture may
then be incubated in SIF at about 37.degree. C. for a period that
is representative of in vivo contact with intestinal fluids, for
example, about 20, about 25, about 30, about 35, about 40, about
45, about 50, about 55, about 60, about 65, about 70, about 80,
about 85, about 90, about 95, about 100, about 105, about 110,
about 115, about 120, about 125, about 130, about 135, about 140,
about 145, about 150, about 155, about 160, about 165, about 170,
about 175, about 180, about 185, about 190, about 195, about 200,
about 205, about 210, about 215, about 220, about 225, about 230,
about 235, or about 240 minutes. Preferably, the candidate enzyme
composition and peptide-containing substance may be incubated for
about 60 minutes in SGF and about 60 minutes in SIF. Alternatively,
the candidate enzyme composition and peptide-containing substance
may be incubated for about 120 minutes in SGF and about 60 minutes
in SIF. Gastrointestinal fluids from suitable animal models and/or
simulated fluids such as Simulated Gastric and Intestinal Fluids
USP may be used.
[0097] A "pharmaceutical composition" refers to a mixture of one or
more of the enzymes or enzyme compositions of the present
technology described herein, along with other chemical components,
such as physiologically acceptable carriers and excipients as
described below. The purpose of a pharmaceutical composition is to
facilitate administration of an enzyme or enzyme composition to an
organism.
[0098] While the enzymes or enzyme compositions can be administered
in their essentially pure forms, it may be desirable to formulate
the enzymes into pharmaceutical compositions prior to
administration in order to increase, for example, enzyme
palatability, subject compliance with a treatment regimen, and/or
general ease of administration. Thus, in certain embodiments, the
enzyme compositions of the present technology comprise other
components in addition to one or more enzymes with activity against
gluten oligopeptides, such as one or more pharmaceutically
acceptable coatings, binding agents, lubricating agents, suspending
agents, sweeteners, flavoring agents, preservatives, buffers,
wetting agents, disintegrants, effervescent agents, and other
excipients. Such excipients are known in the art and can be readily
selected by the skilled artisan. The amount of additional component
present in addition to one or more enzymes may vary, ranging from
about 0.1 wt. % to about 99 wt. %, preferably from about 5 wt. % to
about 80 wt. %, more preferably from about 10 wt. % to about 70 wt.
% of the total composition. For example, the amount of additional
component present in addition to one or more enzymes may be about
15 wt. %, about 20 wt. %, about 25 wt. %, about 30 wt. %, about 35
wt. %, about 40 wt. %, about 45 wt. .degree. A, about 50 wt. %,
about 55 wt. %, about 60 wt %, or about 65 wt. %.
[0099] According to some embodiments of the present technology, a
pharmaceutical composition may include a pharmaceutically
acceptable carrier. The term "pharmaceutically acceptable carrier"
relates to one or more compatible solid or liquid fillers,
diluents, or capsule substances which are suitable for
administration to, for example, a human subject. The term "carrier"
relates to an organic or inorganic ingredient, natural or synthetic
in nature, in which the active ingredient is combined in order to
facilitate use. The ingredients of the pharmaceutical composition
are ordinarily of such a nature that no interaction which
substantially impairs the desired pharmaceutical efficacy occurs.
In certain embodiments, the carriers for use within such
compositions are biocompatible and/or biodegradable. In certain
embodiments, a carrier may be a composition comprising at least one
mucoadhesive polymer that is capable of forming a hydrogel such as
those described in US 2008/0020036.
[0100] In one embodiment, the enzyme composition is blended with at
least one pharmaceutically acceptable excipient, diluted by an
excipient or enclosed within a carrier that can be in the form of a
capsule, sachet, tablet, buccal, lozenge, oral thin film, paper, or
other container. When the excipient serves as a diluent, it may be
a solid, semi-solid, suspension, slurry, or liquid material which
acts as a vehicle, carrier, or medium for the balance of the
composition. Thus, the composition can be formulated into tablets,
pills, pastilles, powders, elixirs, suspensions, emulsions, syrups,
capsules (such as, for example, soft and hard gelatin capsules),
suppositories, lozenges, buccal dosage forms, sterile injectable
solutions, and sterile packaged powders.
[0101] In certain embodiments, a pharmaceutical composition may
comprise salts, buffer substances, preservatives, carriers and,
where appropriate, other therapeutic active ingredients. For use in
medicine, the salts should be pharmaceutically acceptable. Other
salts can, however, be used to prepare pharmaceutically acceptable
salts and are included as part of the present technology. Such
pharmacologically and pharmaceutically acceptable salts include,
for example, those prepared from the following acids: hydrobromic,
sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,
glycollic, lactic, salicylic, succinic, toluene-p-sulphonic,
tartaric, acetic, citric, formic, benzoic, malonic,
naphthalene-2-sulphonic, benzenesulphonic acids and the like.
Pharmaceutically acceptable salts can also be prepared as alkali
metal or alkaline earth metal salts such as sodium, potassium or
calcium salts.
[0102] In certain embodiments, a pharmaceutical composition may
comprise buffer substances such as acetic acid in a salt, citric
acid in a salt, boric acid in a salt and phosphoric acid in a
salt.
[0103] The pharmaceutical compositions may also include where
appropriate suitable preservatives such as parabens, including
polyparaben and methylparaben, potassium sorbate, benzoic acid and
its salts, other esters of parahydroxybenzoic acid such as
butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic
compounds such as phenol, or quarternary compounds such as
benzalkonium chloride, chlorobutanol, and thimerosal.
[0104] The pharmaceutical compositions are ordinarily supplied in a
standard dose form and can be produced in a manner known to those
of skill in the art. Pharmaceutical compositions may for example be
in the form of capsules, tablets, pastilles, suspensions, slurries,
syrups, elixirs or emulsions.
[0105] Examples of binding agents include various celluloses and
cross-linked polyvinylpyrrolidone, microcrystalline cellulose (such
as Avicel PH), silicidized microcrystalline cellulose (SMCC), and
mannitol.
[0106] Suitable lubricants, including agents that act on the
flowability of the powder formulation to be compressed, include
colloidal silicon dioxide (such as Aerosil.RTM. 200), talc, stearic
acid, magnesium stearate, calcium stearate, and silica gel.
[0107] Examples of sweeteners include natural or artificial
sweeteners, such as sucrose, xylitol, lactose, saccharin,
cyclamate, aspartame, and acesulfame and salts thereof. Examples of
flavoring agents are Magnasweet.RTM. (MAFCO), acacia syrup,
cardamom, caraway, vanilla, saccharin, glucose, glycerin,
glycyrrhiza, bubble gum flavor, peppermint, oil of wintergreen, and
fruit flavors, such as cherry or orange flavoring.
[0108] Suitable diluents include pharmaceutically acceptable inert
fillers, such as colloidal silicon dioxide, microcrystalline
cellulose (such as Avicel PH); dibasic calcium phosphate;
saccharides, including mannitol, sorbitol, lactose, sucrose, and
glucose; and/or mixtures of any of the foregoing.
[0109] Suitable disintegrants include lightly crosslinked polyvinyl
pyrrolidone, corn starch, potato starch, maize starch, and modified
starches, croscarmellose sodium, cross-povidone, sodium starch
glycolate, and mixtures thereof.
[0110] Examples of effervescent agents include effervescent
couples, such as an organic acid and a carbonate or bicarbonate.
Suitable organic acids include, for example, citric, tartaric,
malic, fumaric, adipic, succinic, and alginic acids and anhydrides
and acid salts. Suitable carbonates and bicarbonates include, for
example, sodium carbonate, sodium bicarbonate, potassium carbonate,
potassium bicarbonate, magnesium carbonate, sodium glycine
carbonate, L-lysine carbonate, and arginine carbonate.
Alternatively, only the acid component of the effervescent couple
may be present.
[0111] In certain embodiments, the pharmaceutical composition of
the present technology is an immediate release composition. In
other embodiments, the pharmaceutical composition is a controlled
release composition. The pharmaceutical composition may provide a
relatively constant level of release of one active component. In
other embodiments, however, a more rapid rate of release
immediately upon administration may be desired. In other
embodiments, release of active compounds may be event-triggered.
Events triggering the release of the active components may be
exposure to moisture, lower pH or temperature threshold. The
formulation of such compositions is well within the level of
ordinary skill in the art using known techniques. Illustrative
carriers useful in this regard include microparticles of
poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose,
dextran and the like. Other illustrative postponed-release carriers
include supramolecular biovectors, which comprise a non-liquid
hydrophilic core (e.g., a cross-linked polysaccharide or
oligosaccharide) and, optionally, an external layer comprising an
amphiphilic compound, such as phospholipids. The amount of active
compound contained in one embodiment, within a sustained release
formulation depends upon the site of administration, the rate and
expected duration of release and the nature of the condition to be
treated.
[0112] Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar, or both.
[0113] In some embodiments, the enzyme composition of the present
technology is formed into dosage units suitable for oral
administration, wherein the active compounds may be incorporated
with excipients and used in the form of ingestible tablets,
lozenges, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like.
[0114] For example, an enzyme composition can be mixed with a
solid, pulverant carrier such as, for example, sorbitol, mannitol,
starch, amylopectin, cellulose derivatives or gelatin, as well as
with an antifriction agent such as, for example, magnesium
stearate, calcium stearate, and polyethylene glycol waxes. The
mixture then can be pressed into tablets. If coated tablets are
desired, the above prepared tablets may be coated, such as with a
concentrated solution of sugar, which may contain gum arabic,
gelatin, talc, titanium dioxide, or with a lacquer dissolved in
volatile organic solvent or mixture of solvents. To this coating,
various dyes can be added in order to distinguish among tablets
with different enzymes or with different amounts of an enzyme
present.
[0115] In certain embodiments the dosage unit form is a capsule.
The capsule may contain a liquid carrier. For example, the dosage
unit form may be a soft capsule suitable for oral administration,
such as capsules which contain a mixture of the one or more enzymes
with vegetable oil or non-aqueous, water miscible materials such
as, for example, polyethylene glycol and the like. Alternatively,
the enzyme(s) can be provided in hard capsules that can contain
granules of the enzyme composition in combination with a solid,
pulverant carrier, such as, for example, sorbitol, mannitol, potato
starch, corn starch, amylopectin, cellulose derivatives, or
gelatin.
[0116] Other embodiments of the present technology include
granulated forms of the enzyme compositions described herein.
Granulated forms are useful for preparing tablets for oral use, and
are typically prepared in the following manner, although other
techniques well known in the art can be employed. The solid
substances (including the one or more enzymes) are gently ground or
sieved to a desired particle size, and the resulting mass is gently
pressed through a stainless steel sieve having a desired size. The
layers of the mixture are then dried in controlled drying units for
a determined length of time to achieve a desired particle size and
consistency. The granules of the dried mixture are gently sieved to
remove any powder. To this mixture can be added optional
disintegrating, anti-friction, and/or anti-adhesive agents.
Finally, the mixture is pressed into tablets using a machine with
the appropriate punches and dies to obtain the desired tablet size.
The operating parameters of the machine can be selected by the
skilled artisan.
[0117] Other embodiments of the present technology include powders
for inhalation.
[0118] The one or more enzymes or enzyme compositions of the
present technology also can be formulated in compositions that
release the enzyme or enzyme compositions over an extended period
of time, such as, for example, between about two to about sixteen
hours. In some embodiments, the enzyme or enzyme compositions are
released over a time of between about 3 to about 12 hours. For
example, the enzyme or enzyme compositions may be released over
about 4, about 5, about 6, about 7, about 8, about 9, about 10, or
about 11 hours. In yet other embodiments, the enzyme or enzyme
compositions are released over a time of between about four to
about eight hours. Those who are skilled in the art can prepare
such sustained release formulation by methods that are known in the
art.
[0119] In certain embodiments, the enzyme composition of the
present technology is stabilized to resist digestion in acidic
stomach conditions. In certain embodiments, the enzyme composition
may be contained in an enteric coating that allows delivery of the
active agent(s) to the intestine. In certain embodiments, the
enzyme composition may be present in a core surrounded by one or
more layers including, for example, an enteric coating layer with
or without a protective sub-coating as known to the ordinarily
skilled artisan relative to pharmaceutical formulations. If no
sub-coating is employed, then the enteric coating can be selected
such that it does not degrade the active ingredient in the
core.
[0120] The enteric layer typically comprises a polymer with enteric
properties. Exemplary enteric polymers include, but are not limited
to, methacrylic acid copolymer, hydroxypropyl methylcellulose
phtalate and hydroxypropyl methylcellulose acetate succinate.
Different types of methacrylic acid copolymers can be used, such
as, for example, methacrylic acid copolymer type A (Eudragit.RTM.
L-100), methacrylic acid copolymer type B (Eudragit.RTM. S 100),
methacrylic acid copolymer type C (Eudragit.RTM. L 100-55),
methacrylic acid copolymer dispersion (Eudragit.RTM. L 30 D-55), a
copolymer of methacrylic acid methyl methacrylate and methyl
methacrylate (Eudragit.RTM. FS) and mixtures thereof, for instance,
a mixture of Eudragit.RTM. L 100-55 and Eudragit.RTM. S 100 at a
weight ratio of about 3:1 to about 2:1, or a mixture of
Eudragit.RTM. L 30 D-55 and Eudragit.RTM. FS at a weight ratio of
about 3:1 to about 5:1.
[0121] The enteric layer may further comprise other agents such as
cellulose acetate phthalate, polyvinyl acetate phthalate, cellulose
acetate trimellitate, shellac and/or zein. Optionally, the enteric
layer further comprises anti-tackiness agents such as talc or
glyceryl monostearate; plasticizers such as triethylcitrate or
polyethylene glycol; and/or pigments such as titanium dioxide or
ferric oxides. The enteric layer may further comprise one or more
plasticizers including, but not limited to, acetyl triethyl
citrate, acetyltributyl citrate, acetylated monoglycerides,
glycerin, triacetin, propylene glycol, phthalate esters (e.g.,
diethyl phthalate, dibutyl phthalate), castor oil, sorbitol and
dibutyl seccate. Certain embodiments provide an enterically coated
formulation where the enteric layer comprises hydroxypropyl
methylcellulose phthalate, triacetin, silica and stearic acid.
[0122] Dosage units for rectal administration also are
contemplated. These can be prepared in the form of suppositories
which contain the enzyme(s) and a neutral fat base. Alternatively,
they can be prepared in the form of gelatin-rectal capsules which
contain the enzyme(s) in a mixture with a vegetable oil or paraffin
oil.
[0123] Compositions suitable for parenteral administration include
ordinarily a sterile aqueous or nonaqueous preparation of the
active ingredient, which is preferably isotonic with the
recipient's blood. Suitable carriers and solvents are for example
Ringer's solution and isotonic sodium chloride solution. Ordinarily
employed additionally as dissolving or suspending medium are
sterile, fixed oils.
[0124] Methods and compositions are provided herein for the
administration of one or more enzyme compositions to a gluten
intolerant subject or a subject desiring to reduce his or her
gluten exposure. In certain embodiments, these methods and
compositions will allow a subject' to ingest gluten-containing food
without serious health consequences, much the same as individuals
that do not suffer from gluten intolerance. In certain embodiments,
a subject not suffering from Celiac disease may wish to reduce or
limit his or her exposure to gluten without strict adherence to a
gluten-free diet. As further examples, the enzyme composition may
also be administered to subjects to treat any disease amenable to
treatment by a reduction in gluten exposure, including, for example
thyroid diseases, such as hypothyroidism and neurological diseases,
such as autism, cerebellar ataxia, peripheral neuropathies, and
schizophrenia.
[0125] In certain embodiments, enzymes or enzyme compositions can
be administered alone or in combination with each other, or with
other active or inactive agents. When combinations of enzymes or
enzyme compositions are used, simultaneous or sequential
administration of at least two different enzymes or enzyme
compositions is contemplated.
[0126] In certain embodiments simultaneous administration is
contemplated and the combination of enzymes or enzyme compositions
is present in a dosage unit form suitable for oral administration,
wherein the active compounds may be incorporated with excipients
and used in the form of ingestible tablets, lozenges, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers
and the like. The combination of enzymes or enzyme compositions may
include at least two of GDEP-LGG, GDEP-LNA, GDEP-M, GDEP-2A,
GDEP-AH, oryzin, NPI, NPII, Aorsin A, Aorsin B, CPY, papain,
activated papain, purified papain, or chymopapain. The enzyme
composition of the present technology may further include a
preparation from Penicillium citrimum.
[0127] As used herein, the phrase a "therapeutically effective
amount" connotes an amount effective to treat gluten intolerance,
including NCGS or NCGI. In certain embodiments, the phrase a
"therapeutically effective amount" may also be used to refer to an
amount effective to reduce a subject's exposure to gluten
oligopeptides, including the 33-mer derived from .alpha.-2 gliadin,
or to reduce the antigenicity of gluten oligopeptides, including
the 33-mer derived from .alpha.-2 gliadin. This amount may vary
from subject to subject, and may generally range from about 20 to
about 1000 mg per dose for a human subject. In another embodiment,
the amounts range from about 100 to about 500 mg per dose for a
human subject. For example, the amount may be about 50, about 100,
about 150, about 200, about 250, about 300, about 350, about 400,
or about 450 mg per dose for a human subject. An exemplary dosage
is about 200 mg for a human subject. A therapeutically effective
amount of an enzyme composition of the present technology can be
administered once daily. Alternatively, a therapeutically effective
amount can be administered in divided amounts multiple times per
day. An illustrative dosing regimen is about 200 mg of the enzyme
composition given twice per day. Another illustrative dosing
regimen for a human subject is about 200 mg of the enzyme
composition given whenever gluten-containing food is ingested.
Alternate suitable dosing regimens can be determined by the skilled
artisan.
[0128] One skilled in the art will recognize that a therapeutically
effective amount of active ingredient will vary depending upon a
variety of factors including, for example, the activity of the
specific compound employed; the age, body weight, general health,
sex, and diet of a particular patient or patient population; the
time of administration, rate of absorption, and rate of excretion;
the potential interactions with other drugs taken separately or in
combination; and the severity of the particular disease. condition,
or disorder for which a therapeutic effect is desired. The size of
the dose will also be determined by the existence, nature, and
extent of any adverse side effects that might accompany the
administration of a particular compound. Other factors, which
affect the specific dosage, include, for example, bioavailability,
metabolic profile, and the pharmacodynamics associated with the
particular compound to be administered in a particular subject. For
example, a therapeutically effective amount can include the amount
or quantity of an enzyme composition, which is sufficient to elicit
the required or desired therapeutic response, e.g., an amount,
which is sufficient to elicit a biological or therapeutic response
when administered to a subject.
[0129] In certain embodiments, the term "administering" refers to
bringing a subject in contact with the compositions provided
herein. For example, in certain embodiments, the compositions
provided herein are suitable for oral administration, whereby
bringing the subject in contact with the composition comprises
ingesting the compositions. In other embodiments, a method of
administration may include genetic modification of cells, such as
enterocytes, to express increased levels of particular enzymes or
the introduction of micro-organisms expressing enzyme compositions
to the subject's intestinal tract. Such cells (which include cells
that are not derived from the patient but that are not
immunologically rejected when administered to the patient) and
microorganisms are, in some embodiments, formulated in a
pharmaceutically acceptable excipient, or introduced in foods. In
still other embodiments, food may be pretreated or combined with an
enzyme composition to reduce or to remove the toxic oligopeptides
of gluten.
[0130] A person skilled in the art would readily recognize that the
methods of bringing the subject in contact with the compositions
provided herein, will depend on many variables such as, without any
intention to limit the modes of administration; age, pre-existing
conditions, other agents administered to the subject, the severity
of symptoms, subject weight or propensity to gain weight,
refraction to other medication and the like. In one embodiment,
provided herein are embodiments of methods for administering the
compounds of the present technology to a subject, through any
appropriate route, as will be appreciated by one skilled in the
art.
[0131] As used herein, the term "treating" refers to abrogating;
preventing; substantially inhibiting, slowing or reversing the
progression of; substantially ameliorating clinical and/or
non-clinical symptoms of; or substantially preventing or delaying
the appearance of clinical and/or non-clinical symptoms of a
disease, disorder or condition.
[0132] In one embodiment, the term "subject" refers to a mammal
including a human in need of therapy for, or susceptible to, a
condition or its sequelae. The subject may include dogs, cats,
pigs, cows, sheep, goats, horses, rats, and mice and humans. The
term "subject" does not exclude an individual that is normal in all
respects.
[0133] In the preceding paragraphs, use of the singular may include
the plural except where specifically indicated. As used herein, the
words "a," "an," and "the" mean "one or more," unless otherwise
specified. In addition, where aspects of the present technology are
described with reference to lists of alternatives, the technology
includes any individual member or subgroup of the list of
alternatives and any combinations of one or more thereof.
[0134] The disclosures of all patents, publications, including
published patent applications, depository accession numbers, and
database accession numbers are hereby incorporated by reference to
the same extent as if each patent, publication, depository
accession number, and database accession number were specifically
and individually incorporated by reference.
[0135] It is to be understood that the scope of the present
technology is not to be limited to the specific embodiments
described above. The present technology may be practiced other than
as particularly described and still be within the scope of the
accompanying claims.
[0136] Likewise, the following examples are presented in order to
more fully illustrate the present technology. They should in no way
be construed, however, as limiting the broad scope of the
technology disclosed herein.
EXAMPLES
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 .mu.L reaction volume comprised 10 .mu.L 33-mer
peptide, 10 .mu.L enzyme solution, and 10 .mu.L 3.times. SIF
buffer. The reactions were carried out for about 15, 30, 60, 120,
and 240 minutes at about 37.degree. C. and, then, 5 min at
90.degree. 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.apprxeq.6.8). A 30 .mu.L reaction volume comprised
10 .mu.l 33-mer peptide, 10 .mu.L enzyme solution, and 10 .mu.L
3.times. SIF buffer. The reactions were carried out for about 20,
60, 120, and 240 minutes at about 37.degree. C. and, then, 5 min at
90.degree. 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 33-mer peptide. Oryzin alone did
not degrade the 33-mer peptide. NP I and NP II each exhibited a
limited ability to degrade the 33-mer peptide. Re-construction of
GDEP-LGG using the three purified enzymes (Oryzin, NP I, and NP II)
resulted in about 50% degradation of the 33-mer peptide at 120 and
240 minutes.
[0142] Further combinations of Oryzin, NP I, and/or NP II were
tested for their ability to degrade the 33-mer peptide. The 33-mer
peptide (1 mg/ml) was incubated with various concentrations of
enzyme solution (0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml, or 0.8 mg/ml)
comprising gluten degrading enzyme preparation; Oryzin; NP I;
and/or NP II in the presence of simulated intestinal fluid (SIF; pH
6.8). A 30 .mu.L reaction volume comprised 10 .sub.RL 33-mer
peptide, 10 .mu.L enzyme solution, and 10 .mu.L 3.times. SIF
buffer. The reactions were carried out for about 60 minutes at
about 37.degree. C. and, then, 5 min at 90.degree. C. to stop the
enzyme reaction.
[0143] 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 and various combinations thereof is shown in FIG.
3. NP I alone degraded about 40% of the 33-mer peptide (FIG. 3B).
NP II alone degraded about 20% to about 30% of the 33-mer peptide
(FIG. 3B). Oryzin, at the lowest concentrations, did not degrade
the 33-mer peptide (FIG. 3B). However, at higher concentrations
(0.4 mg/ml and 0.8 mg/ml), Oryzin degraded about 10% to about 20%
of the 33-mer peptide (FIG. 3B). Re-construction of GDEP-LGG using
the three purified enzymes (Oryzin, NP I, and NP II) resulted in
degradation of about 30% to about 50% of the 33-mer peptide.
However, the combination did not achieve the same percentage of
degradation of the 33-mer as GDEP-LGG (FIG. 3B). Thus, another
protease or peptidase in GDEP-LGG might be involved in 33-mer
digestion. Neither the combination of Oryzin and NP I nor the
combination of NP I and NP II appear to result in a synergy effect
(FIGS. 3C and 3E). However, the combination of Oryzin and NP II may
achieve a synergy effect (FIG. 3D).
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.apprxeq.6.8) or
simulated gastric fluid (SGF; pH.apprxeq.2.0). A 40 .mu.L reaction
volume comprised 10 .mu.L 33-mer peptide, 10 .mu.L enzyme solution,
and 20 .mu.L 2.times. SIF or SGF buffer. The reactions were carried
out for about 15 hours at 37.degree. C. and, then, 5 min at
90.degree. 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.times.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.fwdarw.10 min, 25% B.fwdarw.40 min, 40% B.fwdarw.45 min, 100%
B.fwdarw.50 min, 100% B). Flow rate was set at 0.2 mL/min and the
injection volume was 5 .mu.L. Detection was performed with a diode
array detector (210, 280) and a mass spectrometer.
[0146] The mass spectrometer was operated in API-ES positive mode
using the following conditions: drying gas flow 13.0 L/min; drying
gas temperature 280.degree. C.; nebulizer pressure 60 psig; and
capillary voltage 2000 V.
[0147] At gastric conditions (pH.apprxeq.2.0), pepsin did not
hydrolyze most of the 33-mer peptide (FIG. 4A). Similarly, at
intestinal conditions (pH.apprxeq.6.8), proteinase K did not
hydrolyze most of the 33-mer peptide (FIG. 4B). At intestinal
conditions, GDEP-LGG hydrolyzed the 33-mer peptide, but did not
eliminate all peptide fragments (FIG. 4C).
Example 4
Comparison of Gluten Degrading Enzyme Preparation and Other
Aspergillus-Derived Proteases
[0148] The 33-mer peptide (1 mg/ml) was incubated with gluten
GDEP-LGG, GDEP-2A, GDEP-LNA, GDEP-M, or Protease P, in the presence
of SIF (pH.apprxeq.6.8). A 30 .mu.L reaction volume comprised 10
.mu.L 33-mer peptide, 10 .mu.L enzyme solution (0.1 mg/ml), and 10
.mu.L 3.times. SIF buffer. The reactions were carried out for about
4 hours at 37.degree. C. and, then, 5 min at 90.degree. C. Analysis
by HPLC-MS was as described in Example 3.
[0149] Protease P is a semi-alkali protease and a proteolytic
enzyme preparation. Protease P is manufactured by a unique
fermentation process with a selected strain of Aspergillus, which
is cultured on the wheat bran. Protease P enzyme is extracted with
water and purified by fractionation with ethanol. Protease P has
high proteolytic activity.
[0150] PZH(SD) is a semi-alkali protease from Aspergillus melleus.
This is also referred to as Protease P, Seaprose or Protease DS
depending on the purification and application of the enzyme.
Protease P is less purified; Seaprose (semi alkaline protease) has
been used as a drug in Japan for lung related diseases; Protease DS
is an enzyme preparation produced by Aspergillus melleus.
[0151] When incubated with the 33-mer peptide for 4 hours at
intestinal conditions (pH.apprxeq.6.8), GDEP-LGG hydrolyzed the
33-mer (FIG. 5A). However, hydrolysis of the 33-mer by GDEP-LGG was
less efficient when the reaction time was reduced to 4 hours as
compared to 15 hours (see Example 3). The other Aspergillus
proteases show similar hydrolysis of the 33-mer peptide as gluten
degrading enzyme preparation. (FIG. 5B-5E).
Example 5
33-mer Hydrolysis Activity of GDEP-M and GDEP-LGG at Various pH
[0152] The 33-mer peptide (1 mg/ml) was incubated with GDEP-LGG or
GDEP-M at various pH. A 30 .mu.L reaction volume comprised 10 .mu.L
33-mer peptide, 10 .mu.L enzyme solution (0.1 mg/ml), and 10 .mu.L
50 mM citrate buffer. The pH of the buffer for GDEP-M ranged from
3.0 to 6.0 (FIG. 6A-6D). The pH of the buffer for gluten degrading
enzyme preparation ranged from 4.0 to 6.0 (FIG. 6E-6G). A half
amount of GDEP-M was also tested at pH 4.0 (FIG. 6H). The reactions
were carried out for about 4 hours at 37.degree. C. and, then, 5
min at 90.degree. C. Analysis by HPLC-MS was as described in
Example 3.
[0153] Both GDEP-LGG and GDEP-M, individually, hydrolyzed the
33-mer peptide at high efficiency at acidic pH. GDEP-M exhibited
more efficient hydrolysis than GDEP-LGG at acidic pH.
Example 6
Protease Inhibitor Analysis
[0154] The preceding examples show that GDEP-M and GDEP-LGG
hydrolyze a 33-mer peptide, such as that derived from .alpha.-2
gliadin, efficiently at an acidic pH. Various protease inhibitors
were used to evaluate the component of GDEP-M and GDEP-LGG
effective for the degradation of the 33-mer.
[0155] The 33-mer peptide (1 mg/ml) was incubated with 0.1 mg/ml
enzyme solution (either GDEP-LGG or GDEP-M). A 33 .mu.L reaction
volume comprised 10 .mu.L 33-mer peptide, 10 .mu.L enzyme solution
(0.1 mg/ml), 10 .mu.L 50 mM citrate buffer, and 3 .mu.L protease
inhibitor--PMSF, Pepstatin, or EDTA. The pH of the buffer was about
4.0. The reactions were carried out for about 4 hours at 37.degree.
C. and, then, 5 min at 90.degree. C. Analysis by HPLC-MS was as
described in Example 3.
[0156] When incubated with the 33-mer peptide for 4 hours in acidic
conditions (pH.apprxeq.4.0), GDEP-LGG hydrolyzed the 33-mer (FIG.
7A). However, hydrolysis of the 33-mer by GDEP-LGG was less
efficient when in the presence of PMSF (FIG. 7B). Neither pepstatin
nor EDTA inhibited 33-mer peptide hydrolysis by GDEP-LGG (FIGS. 7C
and 7D).
[0157] When incubated with the 33-mer peptide for 4 hours in acidic
conditions (p.apprxeq.4.0), GDEP-M hydrolyzed the 33-mer (FIG. 7E).
However, hydrolysis of the 33-mer by GDEP-M was less efficient when
in the presence of PMSF (FIG. 7F). Neither pepstatin nor EDTA
inhibited 33-mer peptide hydrolysis by GDEP-M (FIGS. 7G and
7H).
[0158] Neither pepstatin nor EDTA inhibited 33-mer peptide
hydrolysis by GDEP-LGG or GDEP-M. PMSF inhibited 33-mer peptide
hydrolysis by gluten degrading enzyme preparation or GDEP-M. Thus,
an acidic serine protease is the main component of 33-mer peptide
hydrolysis by gluten degrading enzyme preparation or GDEP-M.
Example 7
33-mer Peptide Degradation by Aorsin, a Component of GDEP-M
[0159] The experiments presented above indicate that an acidic
serine protease contributes to the hydrolytic activity of GDEP-M
against the 33-mer. Aorsin is an acidic serine protease present in
GDEP-M.
[0160] Individual components of GDEP-M, including Aorsin A and
Aorsin B, were tested for enzymatic activity against the 33-mer.
The 33-mer peptide (1 mg/ml) was incubated with 1.0 mg./rill enzyme
solution comprising Aorsin A or Aorsin B in the presence of citrate
buffer (pH.apprxeq.4.0). A 30 .mu.L reaction volume comprised 10
.mu.L 33-mer peptide, 10 .mu.L Aorsin solution, and 10 .mu.L 50 mM
citrate buffer. The reactions were carried out for about 15, 30,
60, and 240 minutes at about 37.degree. C. and, then, 5 min at
90.degree. C. to stop the enzyme reaction.
[0161] Analysis by HPLC-MS was as described in Example 3. When
incubated with the 33-mer peptide in acidic conditions
(pH.apprxeq.4.0), Aorsin A hydrolyzed the 33-mer. (FIGS. 8A-8D).
Similarly, when incubated with the 33-mer peptide in acidic
conditions (pH.apprxeq.4.0), Aorsin B hydrolyzed the 33-mer. (FIGS.
8E-8H).
[0162] Samples of 33-mer treated with Aorsin for about 240 minutes
were also analyzed as in Example 1. Degradation of the 33-mer by
Aorsin is shown in FIG. 9. Incubation with either Aorsin A or B for
about 240 minutes resulted in complete degradation of the 33-mer
peptide.
Example 8
33-mer Hydrolysis Activity of Carboxypeptidase Y and Myxococcus
xanthus Prolyl Endopeptidase at Various pH
[0163] The 33-mer peptide (1 mg/ml) was incubated with a CPY
derived from Saccharomyces cerevisiae or Myxococcus xanthus prolyl
endopeptidase ("MX-PEP") at various pH. A 30 .mu.L reaction volume
comprised 10 .mu.L 33-mer peptide, 10 .mu.L enzyme solution (0.1
mg/ml), and 10 .mu.L buffer. The pH of the buffer ranged from 4.0
to 7.0 (FIG. 10A-10D for CPY and 10E-10H for MX-PEP). The reactions
were carried out for about 4 hours at 37.degree. C. and, then, 5
min at 90.degree. C. Analysis by HPLC-MS was as described in
Example 3.
[0164] MX-PEP did not hydrolyze the 33-mer peptide at an acidic pH.
CPY completely hydrolyzed the 33-mer peptide at acidic pH. This
result is surprising because CPY is known as an exo-peptidase
having a neutral pH optimum. However, the data presented here show
that CPY has an acidic pH optimum against the 33-mer peptide.
Example 9
Comparison of Carboxypeptidase Y and Prolyl Endopeptidases
[0165] The 33-mer peptide (1 mg/ml) was incubated with enzyme
solution (containing CPY, MX-PEP, or Aspergillus niger prolyl
endopeptidase ("AN-PEP")). AN-PEP is commercially available as, for
example, Brewers Clarex.TM. (DSM). CPY and AN-PEP were incubated at
an acidic pH (pH.apprxeq.5.0). MX-PEP was incubated in the presence
of SIF (pH.apprxeq.6.8). A 30 .mu.L reaction volume comprised 10
.mu.L 33-mer peptide, 10 .mu.L enzyme solution, and 10 .mu.L
buffer.
[0166] Each enzyme was tested at 4 concentrations: 0.1 mg/ml, 0.05
mg/ml; 0.025 mg/ml, and 0.0125 mg/ml. The reactions were carried
out for about 4 hours at about 37.degree. C. and, then, 5 min at
90.degree. C. to stop the enzyme reaction. The results of the dose
response for CPY, MX-PEP, and AN-PEP are presented in FIGS. 11A-11D
(CPY), 11E-11H (MX-PEP), and 11I-11L (AN-PEP).
[0167] Each enzyme (at a concentration of 0.1 mg/ml) was also
tested at 4 time points: about 15 min, 30 min, 60 min, and 240 min.
Analysis by HPLC-MS was as described in Example 3 and the results
of this analysis for CPY, MX-PEP, and AN-PEP are presented in FIGS.
11M-11P (CPY), 11Q-11T (MX-PEP), and 11U-11V (AN-PEP). Analysis by
ELISA was also performed as described in Example 1 and the results
of this analysis are presented in FIG. 12.
[0168] CPY, MX-PEP, and AN-PEP hydrolyze the 33-mer peptide. AN-PEP
appears to have the strongest activity against the 33-mer peptide,
followed by CPY and then MX-PEP.
Example 10
In Vitro Gastrointestinal Model
[0169] In vitro gastrointestinal models were used to assess
digestion efficiency of various enzymes, including GDEP-LGG,
GDEP-M, and CPY.
[0170] To examine the digestion efficiency of GDEP-LGG against
wheat gluten and commercially available foodstuffs, intended to be,
or reasonably expected to be, ingested by an animal, the following
model was employed.
[0171] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2
g+CaCl.sub.22H2o 0.22 g/100ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of 1.47%
EZ-GEST.RTM. (Shaklee) were added to the solution. At t=0 min a
peptide-containing substance (e.g., wheat gluten or a commercially
available food) was added to the solution and incubated for about
120 min. During the incubation phase, 4 ml of 0.2N HCl was added to
the mixture to maintain the acidity of the mixture (at t=5 min,
t=20 min, t=35 min, t=50 min, t=65 min).
[0172] At t=120 min, 3.0 g sodium bicarbonate (NaHCO.sub.3), 0.31
g. TDCA, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then be incubated
at about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min.
[0173] The digestion efficiency of GDEP-LGG against the 33-mer
peptide using various commercially available foods as a source of
the 33-mer peptide are presented in Tables 1 and 2. The data in
Table 1 reflect incubation of the indicated source of a 33-mer with
200 mg GDEP-LGG for 180 min. The data presented in Table 2 reflect
incubation of the indicated source of a 33-mer with the indicated
amount of GDEP-LGG for 180 min.
TABLE-US-00001 TABLE 1 Amount of 33-mer peptide digested by 200
GDEP-LGG. Source of 33-mer (Food) Serving Size Digestion Efficiency
Wheat Gluten 10 g 68% Lasagna 252 g 89% Easy Mac 58 g 51% Cheez-It
.RTM. Crackers 30 g 81% Ramen Noodles 43 g 49% Sesame Chicken 283 g
49% Frozen Burrito 142 g 64%
TABLE-US-00002 TABLE 2 Amount of 33-mer peptide digested by
GDEP-LGG. Source of 33-mer Digestion (Food) Serving Size GDEP-LGG
Efficiency Chicken & Cheddar 127 g 7 .mu.g 37% Hot Pockets
Turkey Pot Pie 284 g 10 .mu.g 83% Chow Mein 65 g 10 .mu.g 62%
Oyster Sauce 16 g 5 .mu.g 39% Onion Soup 8 g 6 .mu.g 39%
[0174] In addition, various doses of GDEP-LGG, ranging from 0 mg to
200 mg, were tested against 10 g wheat gluten in the
gastrointestinal model described above. FIG. 13 shows digestion of
wheat gliadin and the 33-mer peptide by GDEP-LGG. A dOse of about
200 mg GDEP-LGG digested approximately 80% of gliadin and
approximately 80% of the 33-mer peptide.
[0175] An alternative in vitro gastrointestinal model was employed
to examine the digestion efficiency of CPY and GDEP-M against the
33-mer peptide.
[0176] The 33-mer (300 .mu.g) and pepsin (30 .mu.g) were added to
100 .mu.L of 20 mM acetate buffer (pH 4.5). CPY alone, GDEP-M
alone, or a combination of CPY and GDEP-M were also added. The
solution was incubated under these gastric conditions at 37.degree.
C. for about 60 min. The pH was then adjusted to 6.8 by the
addition of 500 mM Na.sub.2HPO.sub.4. Intestinal enzymes, trypsin
(20 .mu.g) and chymotrypsin (20 .mu.g), were also added at this
time. The solution was further incubated under intestinal
conditions at 37.degree. C. for about 60 min and then, 5 min at
90.degree. C. to stop the enzyme reaction. Analysis by HPLC-MS was
as described in Example 3.
[0177] Analysis by HPLC-MS was as described in Example 3. The
digestive enzymes only (pepsin, trypsin, and chymotrypsin) failed
to digest the 33-mer peptide (FIG. 14A). GDEP-M, CPY, and the
combination of GDEP-M and CPY efficiently digested the 33-mer
peptide in the artificial gastrointestinal condition (FIGS.
14B-14D). Analysis was also carried out by ELISA as described in
Example 1. GDEP-M alone digested about 87% of the 33-mer peptide,
CPY alone digested about 95% of the 33-mer peptide, and the
combination of GDEP-M and CPY digested about 99-100% of the 33-mer
peptide (FIG. 15).
Example 11
Hydrolysis of 33-mer Peptide by CPY and GDEP-LGG
[0178] The 33-mer peptide (1 mg/ml) was incubated with GDEP-LGG,
CPY or a combination of GDEP-LGG and CPY in the presence of SIF
(pH.apprxeq.6.8). A 30 .mu.L reaction volume comprised 10 .mu.L
33-mer peptide, 10 .mu.L enzyme solution (0.1 mg/ml), and 10 .mu.L
3.times. SIF buffer. The reactions were carried out for about 4
hours at 37.degree. C. and, then, 5 min at 90.degree. C. Analysis
by HPLC-MS was as described in Example 3.
[0179] GDEP-LGG hydrolyzed the 33-mer peptide and produced residual
peptides such as 1-30, 6-33, and 4-33 (FIG. 16A). CPY efficiently
hydrolyzed the 33-mer peptide and residual peptides were
approximately 20 amino acids in length (FIG. 16B). Treatment with
GDEP-LGG and CPY hydrolyzed the 33-mer peptide; residual peptides
were far fewer than with either enzyme composition alone (FIG.
16C).
Example 12
GDEP-LGG Protease Activity
[0180] GDEP-LGG was fractionated with DEAE chromatography. Each
fraction was analyzed for protein concentration, 33-mer peptide
degradation activity, prolyl endopeptidase activity, and protease
activity. Protein concentration was assessed at 280 nm. Prolyl
endopeptidase activity was assessed using the substrate
Z-Gly-Pro-pNA. Protease activity was analyzed using azocasein and
the commercially available Fluorescence Resonance Energy Transfer
Substrates-25Xaa ("FRETS-25Xaa") Series.
[0181] FRETS-25Xaa products have the general structure:
##STR00001##
[0182] Each substrate (Code 3701-v--Code 3719-v) in the FRETS-25Xaa
series contains a highly fluorescent 2-(N-methylamino)benzoyl (Nma)
group linked to the side chain of the amino-terminal D-2,3-diamino
propionic acid (D-A2pr) residue, which is efficiently quenched by a
2,4-dinitrophenyl (Dnp) group linked to the function of Lys. Xaa
represents a fixed position of each of the 19 natural amino acids
excluding Cys (noted in product name Code 3701-v--Code 3719-v). A
mixture of 5 amino acid residues (P, Y, K, I, and D) is at the Yaa
position, along with a mixture of 5 amino acid residues (F, A, V,
E, and R) at the Zaa position for each fixed Xaa. This provides a
peptide mixture of 25 combinations of each Xaa series resulting in
a combinatorial library totaling 475 peptide substrates. Both Nma
and Dnp groups are linked to the side chain of the individual
residues, allowing for the determination of the cleavage site by a
specific enzyme through mass spectrometric analysis and Edman
degradation as well. When an enzyme of interest cleaves any peptide
bond between D-A2pr(Nma) and Lys(Dnp) in the substrate, the
fluorescence at .lamda.ex=340 nm and .lamda.em=440 nm increases in
proportion to the release of the Nma fluorophore form the internal
Dnp quencher.
[0183] GDEP-LGG has significant protease activity as assessed by
degradation of azocasein and FRETS-25Pro (FIG. 17A). Protease
activity was divided in two major fractions, DEAE flow through
fraction and DEAE absorbed fraction (FIG. 17A). GDEP-LGG has
negligible prolyl endopeptidase activity as measured the substrate
Z-Gly-Pro-pNA (FIG. 17B). Nonetheless, GDEP-LGG surprisingly has
significant activity against the 33-mer peptide, which was divided
in two major fractions, DEAE flow through fraction and DEAE
absorbed fraction (FIG. 17B).
Example 13
In Vitro Gastrointestinal Model (Enzymes at Doses of 12.5 mg, 25
mg, 37.5 mg, 50 mg, 100 mg, 200 mg and 400 mg)
[0184] In vitro gastrointestinal models were used to assess
digestion efficiency of various enzymes at doses of 12.5 mg, 25 mg,
37.5 mg, 50 mg, 100 mg, 200 mg and 400 mg.
[0185] To examine the digestion efficiency of various enzymes
against gluten, intended to be, or reasonably expected to be,
ingested by an animal, the following model was employed.
[0186] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2
g+CaCl.sub.22H2o 0.22 g/100 ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of various doses
of enzyme (12.5 mg, 25 mg, 37.5 mg, 50 mg, 100 mg, 200 mg, or 400
mg) were added to the solution. At t=0 min gluten was added to the
solution and incubated for about 120 min. During the incubation
phase, 4 ml of 0.2N HCl aliquots were added to the mixture to
maintain the acidity of the mixture (at t=5 min, t=20 min, t=35
min, t=50 min, t=65 min).
[0187] At t=120 min, 3.0 g sodium bicarbonate (NaHCO3), 0.81 g
deoxycholate, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then incubated at
about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min.
[0188] The residual ratio of enzyme against the 33-mer peptide
using gluten as a source of the 33-mer peptide is presented in
FIGS. 18A-18E. The figures reflect incubation of gluten with 12.5
mg, 25 mg, 37.5 mg, 50 mg, 100 mg, 200 mg, or 400 mg of the
indicated enzyme or enzymes for 180 min. Analysis by ELISA was as
described in Example 1.
[0189] GDEP-LGG was effective to greatly reduce or eliminate the
33-mer from the gluten source of 33-mer peptide (FIGS. 18B,
18E).
Example 14
In Vitro Gastrointestinal Model (Gluten Degrading Enzyme
Preparation at Doses of 50 mg, 100 mg, 200 mg and 400 mg)
[0190] In vitro gastrointestinal models were used to assess
digestion efficiency of GDEP-LGG at doses of 50 mg, 100 mg, 200 mg
and 400 mg.
[0191] To examine the digestion efficiency of gluten degrading
enzyme preparation against commercially available foodstuffs,
intended to be, or reasonably expected to be, ingested by an
animal, the following model was employed.
[0192] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2 g
+CaCl.sub.22H2o 0.22g/100ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of various doses
of GDEP-LGG (50, 100, 200 or 400 mg) were added to the solution. At
t=0 min a peptide-containing substance (e.g., a commercially
available food) was added to the solution and incubated for about
120 min. During the incubation phase, 4 ml of 0.2N HCl aliquots
were added to the mixture to maintain the acidity of the mixture
(at t=5 min, t=20 min, t=35 min, t=50 min, t=65 min).
[0193] At t=120 min, 3.0 g sodium bicarbonate (NaHCO.sub.3), 0.81 g
deoxycholate, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then incubated at
about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min
[0194] The residual ratio of the 33-mer peptide from commercially
available foods as a source of the 33-mer peptide following
treatment with GDEP-LGG is presented in FIGS. 19A-19E and
summarized in Table 3. The data in Table 3 reflect incubation of
the indicated source of the 33-mer with 50 mg, 100 mg, 200 mg or
400 mg GDEP-LGG for 180 min. Analysis by ELISA was as described in
Example 1.
TABLE-US-00003 TABLE 3 Amount of 33-mer peptide digested by
GDEP-LGG Source of 33-mer (Food) GDEP-LGG Dose Residual Ratio
Commercial 33-mer peptide 50 mg 10% 100 mg -1% 200 mg 1% 400 mg 1%
Ravioli (Chef Boyardee 50 mg 11.7% Mini Beef Ravioli) 100 mg 7.8%
200 mg 7.6% 400 mg 4.9% Cheese Macaroni (Kraft 50 mg 6.7% Easy Mac)
100 mg 1.4% 200 mg 3.7% 400 mg 2.3% White Bread 50 mg 8.6% 100 mg
7.2% 200 mg 7.8% 400 mg 0.8% Roll of Bread 50 mg -4.0% 100 mg -1.9%
200 mg -5.2% 400 mg -1.2%
[0195] GDEP-LGG was effective to greatly reduce or eliminate the
33-mer from various commercial sources of 33-mer peptide. The
residual ratio of 33-mer generally decreased with higher doses of
GDEP-LGG across all sources (FIG. 19A-19E).
Example 15
In Vitro Gastrointestinal Model: Comparison of GDEP-LGG and GDEP-M
at Doses of 50 mg, 100 mg, 200 mg and 400 mg
[0196] In vitro gastrointestinal models were used to compare
digestion efficiency of each of GDEP-LGG and GDEP-M at doses of 50
mg, 100 mg, 200 mg and 400 mg.
[0197] To examine the digestion efficiency of GDEP-LGG and GDEP-M
against commercially available foodstuffs, intended to be, or
reasonably expected to be, ingested by an animal, the following
model was employed.
[0198] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2
g+CaCl.sub.22H2o 0.22 g/100 ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of various doses
of GDEP-LGG or GDEP-M (50, 100, 200 or 400 mg) were added to the
solution. At t=0 min a peptide-containing substance (e.g., a
commercially available food) was added to the solution and
incubated for about 120 min. During the incubation phase, 4 ml of
0.2N HCl aliquots were added to the mixture to maintain the acidity
of the mixture (at t=5 min, t=20 min, t=35 min, t=50 min, t=65
min).
[0199] At t=120 min, 3.0 g sodium bicarbonate (NaHCO.sub.3), 0.81 g
deoxycholate, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then incubated at
about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min.
[0200] The residual ratio of the 33-mer peptide from commercially
available foods as a source of the 33-mer peptide following
treatment with GDEP-LGG or GDEP-M is presented in FIGS. 20A-20H and
summarized in Table 4. The data in Table 4 reflect incubation of
the indicated source of a 33-mer with 50 mg, 100 mg, 200 mg or 400
mg GDEP-LGG or GDEP-M for 180 min. Analysis by ELISA was as
described in Example 1.
TABLE-US-00004 TABLE 4 Amount of 33-mer peptide digested by
GDEP-LGG or GDEP-M Source of 33-mer GDEP-LGG or Residual Ratio
Residual Ratio (Food) GDEP-M Dose GDEP-LGG GDEP-M Ravioli (Chef 50
mg 11.7% 38.6% Boyardee Mini 100 mg 7.8% 20.8% Beef Ravioli) 200 mg
7.6% 8.9% 400 mg 4.9% 7.2% Cheese Macaroni 50 mg 6.7% 18.3% (Kraft
Easy Mac) 100 mg 1.4% 10.6% 200 mg 3.7% 3.6% 400 mg 2.3% 4.0% White
Bread 50 mg 8.6% 19.8% 100 mg 7.2% 15.2% 200 mg 7.8% 2.7% 400 mg
0.8% -1.1% Roll of Bread 50 mg -4.0% 8.0% 100 mg -1.9% 0.9% 200 mg
-5.2% -1.9% 400 mg -1.2% -4.5% Lasagna 50 mg 20.6% 44.2% 100 mg
13.3% 40.6% 200 mg 3.0% 29.7% 400 mg -0.6% 10.3% Pasta 50 mg 20.0%
49.3% 100 mg 8.3% 27.8% 200 mg 4.5% 10.3% 400 mg 2.5% 5.3%
[0201] GDEP-LGG was effective to greatly reduce or eliminate the
33-mer from various commercial sources of 33-mer peptide and was
more effective than GDEP-M at reducing or eliminating the 33-mer
from various commercial sources of 33-mer peptide. The residual
ratio of the 33-mer generally decreased with higher doses of
GDEP-LGG or GDEP-M across all sources (FIG. 20A-20H).
Example 16
Comparison of GDEP-LCC with Existing Products
[0202] In order to assess the digestion efficiency of GDEP-LGG as
compared to existing products, the following in vitro model was
employed.
[0203] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2
g+CaCl.sub.22H2o 0.22 g/100ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of an enzyme
composition (100 mg GDEP-LGG; 2 capsules of "Gluten Gest,"
available from Allergy Research Group; 2 capsules of "Gluten-zyme,"
available from BioCare; or 1 capsule of "GlutenEase," available
from Enzymedica; 2 capsules of "Gluten-zyme Plus" available from
BioCare; or 2 capsules of "Spectrumzyme" available from BioCare)
were added to the solution. At t=0 min a commercially available
33-mer peptide was added to the solution and incubated for about
120 min. During the incubation phase, 4 ml of 0.2N HCl aliquots
were added to the mixture to maintain the acidity of the mixture
(at t=5 min, t=20 min, t=35 min, t=50 min, t=65 min).
[0204] At t=120 min, 3.0 g sodium bicarbonate (NaHCO.sub.3), 0.81 g
deoxycholate, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then be incubated
at about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min.
[0205] Analysis was performed by ELISA as described in Example 1
and HPLC-MS as described in Example 3. The results are presented in
FIG. 21A and summarized in Table 4.
TABLE-US-00005 TABLE 5 Comparison of gluten degrading enzyme
preparation with existing products Product Residual Ratio 100 mg
Lot1 GDEP-LGG 7.4% 100 mg Lot2 GDEP-LGG 13.6% 100 mg Lot3 GDEP-LGG
6.2% 2 Capsules of "Gluten Gest" 84.6% 2 Capsules of "Gluten- 66.7%
zyme" 1 Capsule of "GlutenEase" 56.8%
[0206] All 3 lots of GDEP-LGG greatly reduced or eliminated the
33-mer peptide. All 3 lots of gluten degrading enzyme preparation
had a significantly lower residual ratio than the three tested
existing products, with Lot3 presenting the lowest residual ratio
at 6.2%. Individual chromatographs for each commercial product are
presented in FIGS. 21B-21O.
Example 17
33-mer Peptide Degradation by Papain
[0207] The 33-mer peptide (1 mg/ml) was incubated with papain at a
pH of about 4.5. A 30 .mu.L reaction volume comprised 10 .mu.L
33-mer peptide, 10 .mu.L enzyme solution (0.1 mg/ml) and 10 .mu.L
buffer. The pH of the buffer was about 4.5. The reaction was
carried out for about 1 hour at 37.degree. C. and, then, 5 min at
90.degree. C. to stop the enzyme reaction. Analysis by HPLC-MS was
as described in Example 3.
[0208] The results of the analysis are presented in FIG. 22. Papain
completely hydrolyzed the 33-mer peptide at a pH of about 4.5.
Example 18
Papain Purification by Cation-Exchange Chromatography
[0209] Papain was purified by cation-exchange chromatography using
an AKTA purifier from GE Health Care. A 20% enzyme solution of
papain W-40 bulk in a 50 mM Acetate buffer at a pH of about 5.0 was
desalted using a PD10 column with 50 mM Acetate buffer at a pH of
about 5.0. The sample was diluted about 1.4 times. Samples were
loaded onto a 40 ml SP Sepharose FF column. The mobile phase
comprised of (A) 50 mM Bis-Tris HCl buffer at a pH of about 5.0 and
(B) 50 mM Bis-Tris HCl buffer at a pH of about 5.0 with 1M NaCl was
used to elute targets in gradient mode (40 mL application; 80 mL
flow through; 600 mL elution 1 with 0.fwdarw.600 mM NaCl; 120 mL
elution 2 with 600 mM NaCl; 120 mL wash with 1M NaCl). Flow rate
was set at 5.0 mL/min, the injection volume was 10 mL and the
fraction size was 10 mL. Detection was performed at an optical
density of 280 nm.
[0210] The results of the purification are presented in FIG. 23.
Each fraction was incubated with the 33-mer peptide at a pH of
about 4.5. A 30 .mu.L reaction volume comprised 10 .mu.L 33-mer
peptide, 10 .mu.L enzyme solution (0.1 mg/ml) and 10 .mu.L buffer.
The pH of the buffer was about 4.5. The reaction was carried out
for about 1 hour at 37.degree. C. and, then, 5 min at 90.degree. C.
to stop the enzyme reaction. Analysis by HPLC-MS was as described
in Example 3.
[0211] The results of the analysis are presented in FIG. 23. The
fractions from peak 3 completely hydrolyzed the 33-mer peptide at a
pH of about 4.5.
[0212] SDS-PAGE gels were prepared for each fraction of the
cation-exchange chromatography analysis. The results of the
SDS-PAGE gels are presented in FIG. 24. Each peak (protein) shows
consistent molecular weight.
[0213] Peaks 2 and 3 from the cation-exchange chromatography
purification results were further purified by reverse phase
chromatography and identification was performed by N-Terminal
Sequence analysis. The results of the analysis are presented in
FIGS. 25A and 25B.
Example 19
Papain Purification by Hydrophobic Interaction Chromatography
[0214] The E9 fraction of the cation-exchange chromatography
analysis performed in Example 18 was further purified by
hydrophobic interaction chromatography using an AKTA purifier from
GE health care. The enzyme solution of the E9 fraction was added to
the same volume of 2.4M ammonium sulfate in 50 mM Pipes buffer at a
pH of about 6.5 to form a final sample of 1.2M ammonium sulfate in
25 mM Pipes buffer at a pH of about 6.5. Samples were loaded onto a
5 mL HiTrap Butyl FF column. The mobile phase comprised of (A) 1.2M
ammonium sulfate in 25 mM Pipes buffer at a pH of about 6.5 and (B)
25 mM Pipes buffer at a pH of about 6.5 was used to elute targets
in gradient mode (20 mL application; 5 mL flow through; 100 mL
elution 1 with 1.2M.fwdarw.0 mM ammonium sulfate; 25 mL elution 2
with 0 mM ammonium sulfate). Flow rate was set at 4.0mL/min, the
injection volume was 10 mL and the fraction size was 2.5 mL.
Detection was performed at an optical density of 280 nm.
[0215] The results of the purification are presented in FIG. 26.
Unlike the results of the cation-exchange chromatography of Example
18, Peak 3 presents as two separate peaks. The peaks are labeled
Peak 3-1 and Peak 3-2 in Figure (FIG. 26A-26B).
[0216] The fractions were hydrolyzed using the FRETS-25Xaa method
as described in Example 18. 10 .mu.L of enzyme solution was added
to 100 .mu.L of 10 .mu.M substrate in 50 mM acetate buffer and
incubated at 37.degree. C. for about 1 hour and, then, 5 min at
90.degree. C. to stop the enzyme reaction.
[0217] The results of the FRETS-25Xaa analysis are presented in
FIG. 27. The fractions represented by Peak 3-1 completely
hydrolyzed the 33-mer peptide.
Example 20
Papain Purification by Reverse Phase Chromatography
[0218] The enzyme solutions presented as Peaks 3-1 and 3-2 in
Example 19 were further purified using reverse phase
chromatography.
[0219] The results of the reverse phase chromatography analysis are
presented in FIG. 28.
Example 21
Comparison of Papain and Other Enzymes
[0220] In vitro gastrointestinal models were used to assess
digestion efficiency of papain and other enzymes at varying
doses.
[0221] A 135 ml solution was prepared using 100 ml of 2.7% bovine
serum albumin; 5 ml of 3% gastric mucosa mucin; 10 ml NaCl 13.2 g
+CaCl.sub.22H2o 0.22 g/100 ml; and 20 ml of pH 4.0 acetate buffer.
The solution was incubated at 37.degree. C. for about 20 min. After
this pre-incubation, 5 ml of 0.2% pepsin and 10 ml of various doses
of enzyme (5, 10, 40, 50, 100 and 400 mg) were added to the
solution. At t=0 min a commercially available 33-mer peptide was
added to the solution and incubated for about 120 min. During the
incubation phase, 4 ml of 0.2N HCl aliquots were added to the
mixture to maintain the acidity of the mixture (at t=5 min, t=20
min, t=35 min, t=50 min, t=65 min).
[0222] At t=120 min, 3.0 g sodium bicarbonate (NaHCO.sub.3), 0.81 g
deoxycholate, and 150 mg pancreatin were added to the mixture to
simulate intestinal conditions. The mixture was then incubated at
about 37.degree. C. for about 60 minutes (until t=180 min). The
mixture was sampled by removing 1 ml at t=0 min, t=30 min, t=60
min, t=90 min, t=120 min, t=130 min, t=140 min, t=150 min, and
t=180 min.
[0223] The residual ratio of the enzyme against the 33-mer peptide
is presented in FIG. 29. Analysis by ELISA was as described in
Example 1.
[0224] All forms of papain were effective to greatly reduce or
eliminate the 33-mer from. The residual ratio of 33-mer generally
decreased with higher doses of enzyme (FIG. 29).
Example 22
Activation of Papain by Reducing Agent
[0225] In order to test the activity of papain in the presence of a
reductant, the following test method was employed.
[0226] Papain and GDEP-LGG with and without the presence of a
reductant (Glutathione, dithiothreitol ("DTT"), L-Cysteine, or
N-Acetyl-L-Cysteine) were analyzed for protease activity using the
FRETS-25Xaa method as described in Example 16. 10 .mu.L of enzyme
solution was added to 100 .mu.L of 10 .mu.M
Nma-YPQPQLPYPK(Dnp)-DArg-DArg-NH2 peptide in 50 mM Acetate buffer
at pH 4.5 or 50 mM Acetate buffer with 1 mM reductant. The solution
was incubated for about 1 hour at 37.degree. C. and, then, 5 min at
90.degree. C. to stop the enzyme reaction.
[0227] The DTT and Glutathione reductants activated the FRETS
activity of papain but did not significantly activate the FRETS
activity of GDEP-LGG (FIG. 30A). All reductants activated papain
(FIG. 30B).
[0228] The 33-mer hydrolysis activity of the enzyme solutions with
reductants was further tested. A 30 .mu.L reaction volume comprised
of 10 .mu.L of 1 mg/mL 33-mer peptide, 10 .mu.L of enzyme solution
(papain or GDEP-LGG) and 10 .mu.L of 50 mM acetate buffer at pH 4.5
or 50 mM Acetate buffer with 1 mM reductant was incubated for about
1 hour at 37.degree. C. and, then, 5 min at 90.degree. C. to stop
the enzyme reaction. Analysis by HPLC-MS was as described in
Example 3. All reductants activated the 33-mer hydrolysis activity
of papain (FIG. 30C).
Example 23
Comparison of GDEP-LGG, Various Papain GDEP-LGG Combinations, and
Papain
[0229] The FRETS activity of various concentrations of enzyme
solution (100% GDEP-LGG, 80% GDEP-LGG with 20% papain, 60% GDEP-LGG
with 40% papain, 40% GDEP-LGG with 60% papain, 20% GDEP-LGG with
80% papain, and 100% papain) was measured using the FRETS-25Xaa
method as described in Example 16. GDEP-LGG concentration was 0.057
unit/mL and papain concentration was 0.060 unit/mL.
[0230] 10 .mu.L of enzyme solution was added to 100 .mu.L of 10
.mu.M Nma-YPQPQLPYPK(Dnp)-DArg-DArg-NH2 peptide in 50 mM Acetate
buffer at pH 4.5. The solution was incubated for 1 hour at
37.degree. C. and, then, 5 min at 90.degree. C. to stop the enzyme
reaction.
[0231] Relative activity (%) of GDEP-LGG and papain at various
concentrations is shown in FIG. 31A. GDEP-LGG and papain alone
showed a relative activity of about 100%. The combination of
GDEP-LGG and papain resulted in a synergistic effect (FIG. 31A)
with the combination of GDEP-LGG and papain having activity in
excess of 100%. The sample with 60% GDEP-LGG and 40% papain
resulted in the highest relative activity at about 150% to about
170%.
[0232] The 33-mer hydrolysis activity of the enzyme solutions was
further tested. A 60 .mu.L reaction volume comprised of 20 .mu.L of
1 mg/mL 33-mer peptide, 20 .mu.L of enzyme solution (100% GDEP-LGG,
80% GDEP-LGG with 20% papain, 60% GDEP-LGG with 40% papain, 40%
GDEP-LGG with 60% papain, 20% GDEP-LGG with 80% papain, and 100%
papain) and 20 .mu.L of 150 mM acetate buffer at pH 4.5 was
incubated for 1 hour at 37.degree. C. and, then, 5 min at
90.degree. C. to stop the enzyme reaction. Analysis by HPLC-MS was
as described in Example 3. The results of the hydrolysis experiment
are presented in FIG. 31B-31G. The sample with 60% GDEP-LGG and 40%
papain resulted in the highest 33-mer hydrolysis activity.
Example 24
Comparison of Various Enzyme Preparations
[0233] The 33-mer hydrolysis activity of various enzyme
preparations was tested according to the method described in
Example 23. A set of 6-mers were used in a FRETS assay to screen
various compositions for activity against the 33-mer. FIG. 32
summarizes the results of the screening. Enzymes with the best
activity were selected. It was surprisingly found that certain
preparations from Penicillium citrimum had activity against the
33-mer. Table 6 summarizes the reaction blends and maximum residual
peptides found.
TABLE-US-00006 TABLE 6 Comparison of various gluten degrading
enzyme preparation containing enzyme blends Enzyme Blend Dose/serve
Maximum Residual Peptide GDEP-LGG 100 mg >10 mer GDEP-LGG and
papain 100 mg 9 mer GDEP-LGG, papain and 150 mg 8 mer Protease P
GDEP-LGG, papain, 200 mg 4 mer Protease P and a preparation from
Penicillium citrinum
Chromatographs showing the results of the hydrolysis experiment are
presented in FIG. 33. Analysis by HPLC/MC was as described in
Example 3. The products of the hydrolysis experiment are smallest
for the blend comprising GDEP-LGG, papain, Protease P and a
preparation from Penicillium citrimum.
Specific Embodiments
[0234] An enzyme cocktail described herein can be illustrated by
the following embodiments enumerated in the numbered sentences that
follow:
[0235] 1. An enzyme cocktail comprising:
[0236] (a) GDEP-LGG; and
[0237] (b) papain;
[0238] wherein the enzyme cocktail is capable of cleaving a gluten
oligopeptide.
[0239] 2. The enzyme cocktail of sentence 1, further comprising a
semi-alkali protease and/or a preparation from Penicillium
citrimum.
[0240] 3. The enzyme cocktail of any one of the preceding
sentences, wherein the papain is activated papain or
chymopapain.
[0241] 4. The enzyme cocktail of sentence 3, wherein the activated
papain has been activated by a reductant.
[0242] 5. The enzyme cocktail of any one of the preceding
sentences, further comprising a reductant.
[0243] 6. A pharmaceutical composition comprising the enzyme
cocktail of any one of the preceding sentences.
[0244] 7. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises
GDEP-M.
[0245] 8. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises an enzyme
having an amino acid sequence at least about 80% homologous to CPY,
or a fragment thereof.
[0246] 9. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises an enzyme
having an amino acid sequence at least about 90% homologous to CPY,
or a fragment thereof.
[0247] 10. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises CPY.
[0248] 11. The enzyme cocktail of sentence 10, wherein the CPY is
selected from the group consisting essentially of Saccharomyces
cerevisiae CPY, Aspergillus niger CPY, Schizosaccharomyces pombe
CPY, Aspergillus fumigatus CPY.
[0249] 12. The enzyme cocktail of any one of the preceding
sentences, wherein the gluten oligopeptide is a 33-mer peptide
fragment of .alpha.-gliadin.
[0250] 13. The enzyme cocktail of sentence 12, wherein the 33-mer
peptide fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO.1).
[0251] 14. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is stable in acid
conditions.
[0252] 15. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is formulated in a
pharmaceutically acceptable excipient.
[0253] 16. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is formulated for oral
delivery.
[0254] 17. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is contained in a
formulation that contains an enteric coating.
[0255] 18. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is contained in a
formulation that includes a pharmaceutically acceptable
carrier.
[0256] 19. The enzyme cocktail of sentence 18, wherein the
pharmaceutically acceptable carrier is a solid.
[0257] 20. The enzyme cocktail of sentence 18, wherein the
pharmaceutically acceptable carrier is a capsule.
[0258] 21. The enzyme cocktail of sentence 18, wherein the
pharmaceutically acceptable carrier is a liquid.
[0259] An enzyme cocktail described herein can be illustrated by
the following embodiments enumerated in the numbered sentences that
follow:
[0260] 1. An enzyme cocktail comprising:
[0261] (a) GDEP-LGG; and
[0262] (b) an acidic serine protease polypeptide having an amino
acid sequence at least about 80% homologous to Aorsin, or a
fragment thereof;
[0263] wherein the enzyme cocktail is capable of cleaving a gluten
oligopeptide.
[0264] The enzyme cocktail of sentence I, wherein the acidic serine
protease polypeptide has an amino acid sequence at least about 90%
homologous to Aorsin, or a fragment thereof.
[0265] 3. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail comprises GDEP-M.
[0266] 4. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises an enzyme
having an amino acid sequence at least about 80% homologous to CPY,
or a fragment thereof.
[0267] 5. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises an enzyme
having an amino acid sequence at least about 90% homologous to CPY,
or a fragment thereof.
[0268] 6. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail further comprises CPY.
[0269] 7. The enzyme cocktail of sentence 6, wherein the CPY is
selected from the group consisting essentially of Saccharomyces
cerevisiae CPY, Aspergillus niger CPY, Schizosaccharomyces pombe
CPY, Aspergillus fumigatus CPY.
[0270] 8. The enzyme cocktail of any one of the preceding
sentences, wherein the gluten oligopeptide is a 33-mer peptide
fragment of .alpha.-gliadin.
[0271] 9. The enzyme cocktail of sentence 8, wherein the 33-mer
peptide fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1)
[0272] 10. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is stable in acid
conditions.
[0273] 11. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is formulated in a
pharmaceutically acceptable excipient.
[0274] 12. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is formulated for oral
delivery.
[0275] 13. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is contained in a
formulation that contains an enteric coating.
[0276] 14. The enzyme cocktail of any one of the preceding
sentences, wherein the enzyme cocktail is contained in a
formulation that includes a pharmaceutically acceptable
carrier.
[0277] 15. The enzyme cocktail of sentence 14, wherein herein the
pharmaceutically acceptable carrier is a solid.
[0278] 16. The enzyme cocktail of sentence 14, wherein herein the
pharmaceutically acceptable carrier is a capsule
[0279] 17. The enzyme cocktail of sentence 14, wherein herein the
pharmaceutically acceptable carrier is a liquid.
[0280] A formulation described herein can be illustrated by the
following embodiments enumerated in the numbered sentences that
follow:
[0281] 1. A formulation for use in reducing gluten exposure or
treating gluten intolerance comprising:
[0282] an enzyme composition selected from the group consisting
essentially of GDEP-LGG, papain, activated papain, purified papain,
chymopapain, GDEP-M, Aorsin, CPY, a semi-alkali protease, and a
preparation from Penicillium citrimum;
[0283] wherein the enzyme composition is capable of cleaving an
immunogenic gluten oligopeptide into non-toxic fragments in
vitro.
[0284] 2. The formulation of sentence 1, wherein the enzyme
composition capable of cleaving at least about 70% of an
immunogenic gluten oligopeptide into non-toxic fragments in
vitro.
[0285] 3. The formulation of any one of the preceding sentences,
wherein the enzyme composition capable of cleaving at least about
80% of an immunogenic gluten oligopeptide into non-toxic fragments
in vitro.
[0286] 4. The formulation of any one of the preceding sentences,
wherein the enzyme composition capable of cleaving at least about
90% of an immunogenic gluten oligopeptide into non-toxic fragments
in vitro.
[0287] 5. The formulation of any one of the preceding sentences,
wherein the formulation is suitable for oral administration.
[0288] 6. The formulation of any one of the preceding sentences,
wherein the formulation includes a pharmaceutically acceptable
carrier.
[0289] 7. The formulation of sentence 6, wherein the
pharmaceutically acceptable carrier is a solid.
[0290] 8. The formulation of sentence 6, wherein the
pharmaceutically acceptable carrier is a capsule.
[0291] 9. The formulation of sentence 6, wherein the
pharmaceutically acceptable carrier is a liquid.
[0292] A formulation described herein can be illustrated by the
following embodiments enumerated in the numbered sentences that
follow:
[0293] 1. A formulation for use in reducing gluten exposure or
treating gluten intolerance comprising:
[0294] at least two enzyme compositions selected from the group
consisting essentially of GDEP; GDEP-LGG; GDEP-M; GDEP-LNA;
GDEP-2A; GDEP-AH; papain; Aorsin; an acidic serine protease
polypeptide having an amino acid sequence at least about 80%
homologous to Aorsin, or a fragment thereof; CPY; an enzyme having
an amino acid sequence at least about 80% homologous to CPY; a
semi-alkali protease; and a preparation from Penicillium
citrimum;
[0295] wherein the at least two enzyme compositions are capable of
digesting a gluten oligopeptide in an in vitro gastrointestinal
model.
[0296] 3. The formulation of sentence I, wherein the at least two
enzyme compositions are GDEP-LGG and papain.
[0297] 3. The formulation of sentence 1, wherein the at least two
enzyme compositions are Carboxypeptidase Y and Aorsin.
[0298] 4. The formulation of any one of the preceding sentences,
wherein the model comprises incubating the at least two enzyme
compositions with the gluten oligopeptide in simulated gastric
fluid at about 37.degree. C. for a period representative of in vivo
contact with gastric fluids.
[0299] 5. The formulation of sentence 4, wherein the period
representative of in vivo contact with gastric fluids is about 120
minutes.
[0300] 6. The formulation of any one of sentences 4 or 5, wherein
the simulated gastric fluid comprises gastric mucosa mucin, pepsin,
gelatinase, amylase, and lipase.
[0301] 7. The formulation of any one of the preceding sentences,
wherein the model comprises incubating the at least two enzyme
compositions with the gluten oligopeptide in simulated intestinal
fluid at about 37.degree. C. for a period representative of in vivo
contact with intestinal fluids.
[0302] 8. The formulation of sentence 7, wherein the period
representative of in vivo contact with intestinal fluids is about
60 minutes.
[0303] 9. The formulation of any one of the preceding sentences,
wherein the simulated intestinal fluid comprises one or more
pancreatic enzymes and a bile salt.
[0304] 10. The formulation of sentence .9, wherein the one or more
pancreatic enzymes are selected from the group consisting
essentially of trypsin, chymotrypsin, amylase, and lipase.
[0305] 11. The formulation of any one of the preceding sentences,
wherein the model comprises:
[0306] incubating a mixture comprising the at least two enzyme
compositions, the gluten oligopeptide, gastric mucosa mucin,
pepsin, gelatinase, amylase, and lipase in acidic conditions at
about 37.degree. C. for about 120 minutes;
[0307] adding an acid neutralizing substance to the mixture;
and
[0308] incubating the mixture with trypsin, amylase, and lipase,
and a bile salt in neutral conditions at about 37.degree. C. for
about 60 minutes.
[0309] 12. The formulation of any one of the preceding sentences,
wherein the model comprises:
[0310] incubating a mixture comprising the at least two enzyme
compositions, the gluten oligopeptide, and pepsin in acidic
conditions at about 37.degree. C. for about 60 minutes;
[0311] adding an acid neutralizing substance to the mixture;
and
[0312] incubating the mixture with trypsin and chymotrypsin in
neutral conditions at about 37.degree. C. for about 60 minutes.
[0313] 13. The formulation of any one of, the preceding sentences,
wherein the gluten oligopeptide is a 33-mer peptide fragment of
.alpha.-gliadin.
[0314] 14. The formulation of sentence 13, wherein the 33-mer
peptide fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1)
[0315] A method described herein can be illustrated by the
following embodiments enumerated in the numbered sentences that
follow:
[0316] 1. A method of treating gluten intolerance or reducing
gluten exposure in a human subject comprising:
[0317] providing the subject with a therapeutically effective
amount of an enzyme cocktail,
[0318] wherein the enzyme cocktail is capable of cleaving a gluten
oligopeptide at acidic conditions.
[0319] 2. The method of sentence 1, wherein the enzyme cocktail
comprises a composition selected from the group consisting
essentially of at least two enzyme compositions selected from the
group consisting essentially of GDEP; GDEP-LGG; GDEP-M; GDEP-LNA;
GDEP-2A; GDEP-AH; papain; Aorsin; an acidic serine protease
polypeptide having an amino acid sequence at least about 80%
homologous to Aorsin, or a fragment thereof; CPY; an enzyme having
an amino acid sequence at least about 80% homologous to CPY, or a
fragment thereof; a semi-alkali protease; and a preparation from
Penicillium citrimum.
[0320] 3. The method of any one of the preceding sentences, wherein
the enzyme cocktail includes a composition derived from Aspergillus
oryzae.
[0321] 4. The method of any one of the preceding sentences, wherein
the enzyme cocktail is formulated in a pharmaceutically acceptable
excipient.
[0322] 5. The method of any one of the preceding sentences, wherein
the enzyme cocktail is formulated for oral delivery.
[0323] 6. The method of any one of the preceding sentences, wherein
the enzyme cocktail is contained in a formulation that contains an
enteric coating.
[0324] 7. The method of any one of the preceding sentences, wherein
enzyme cocktail is contained in a formulation that includes a
pharmaceutically acceptable carrier.
[0325] The method of sentence 7, wherein the pharmaceutically
acceptable carrier is a solid.
[0326] 9. The method of sentence 7, wherein the pharmaceutically
acceptable carrier is a capsule.
[0327] 10. The method of sentence 7, wherein the pharmaceutically
acceptable carrier is a liquid.
[0328] A method described herein can be illustrated by the
following embodiments enumerated in the numbered sentences that
follow:
[0329] 1. A method of assessing the efficacy of an enzyme
composition, comprising the steps of:
[0330] (i) incubating a mixture comprising the enzyme composition
and a gluten oligopeptide in simulated gastric fluid at about
37.degree. C. for a period representative of in vivo contact with
gastric fluids;
[0331] (ii) adding an acid neutralizing substance to the
mixture;
[0332] (iii) incubating the mixture in simulated intestinal fluid
at about 37.degree. C. for a period representative of in vivo
contact with intestinal fluids; and
[0333] (iv) determining the amount of intact gluten oligopeptide in
the mixture.
[0334] 2. The method of sentence 1, wherein the period
representative of in vivo contact with gastric fluids is about 120
minutes.
[0335] 3. The method of any one of the preceding sentences, wherein
the simulated gastric fluid comprises gastric mucosa mucin, pepsin,
gelatinase, amylase, and lipase.
[0336] 4. The method of any one of the preceding sentences, wherein
the period representative of in vivo contact with intestinal fluids
is about 60 minutes.
[0337] 5. The method of any one of the preceding sentences, wherein
the simulated intestinal fluid comprises one or more pancreatic
enzymes and a bile salt.
[0338] 6. The method of sentence 5, wherein the one or more
pancreatic enzymes are selected from the group consisting
essentially of trypsin, chymotrypsin, amylase, and lipase.
[0339] 7. The method of any one of the preceding sentences, wherein
the gluten oligopeptide is a 33-mer peptide fragment of
.alpha.-gliadin.
[0340] 8. The method of sentence 7, wherein the 33-mer peptide
fragment has the amino acid sequence
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1).
[0341] The presently described technology is now described in such
full, clear, concise and exact terms as to enable any person
skilled in the art to which it pertains, to practice the same. It
is to be understood that the foregoing describes preferred
embodiments of the technology and that modifications may be made
therein without departing from the spirit or scope of the invention
as set forth in the appended claims.
Sequence CWU 1
1
1133PRTTriticum aestivum 1Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln
Leu Pro Tyr Pro Gln Pro1 5 10 15Gln Leu Pro Tyr Pro Gln Pro Gln Leu
Pro Tyr Pro Gln Pro Gln Pro 20 25 30Phe
* * * * *