U.S. patent application number 14/070212 was filed with the patent office on 2014-05-08 for method and use of peptide antagonists of zonulin to prevent or delay the onset of diabetes.
This patent application is currently assigned to University of Maryland, Baltimore. The applicant listed for this patent is University of Maryland, Baltimore. Invention is credited to Alessio Fasano, Tammara L. Watts.
Application Number | 20140128321 14/070212 |
Document ID | / |
Family ID | 22761653 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140128321 |
Kind Code |
A1 |
Fasano; Alessio ; et
al. |
May 8, 2014 |
METHOD AND USE OF PEPTIDE ANTAGONISTS OF ZONULIN TO PREVENT OR
DELAY THE ONSET OF DIABETES
Abstract
A method for preventing or delaying the onset of autoimmune
diseases is disclosed.
Inventors: |
Fasano; Alessio; (West
Friendship, MD) ; Watts; Tammara L.; (Columbia,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Maryland, Baltimore |
Baltimore |
MD |
US |
|
|
Assignee: |
University of Maryland,
Baltimore
Baltimore
MD
|
Family ID: |
22761653 |
Appl. No.: |
14/070212 |
Filed: |
November 1, 2013 |
Related U.S. Patent Documents
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13461343 |
May 1, 2012 |
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14070212 |
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12536158 |
Aug 5, 2009 |
8183211 |
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13461343 |
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12165088 |
Jun 30, 2008 |
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12536158 |
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11346395 |
Feb 3, 2006 |
7531512 |
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12165088 |
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Mar 5, 2003 |
7026294 |
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Current U.S.
Class: |
514/7.3 |
Current CPC
Class: |
C07K 14/4705 20130101;
Y10S 530/844 20130101; A61P 1/14 20180101; A61P 3/10 20180101; A61K
38/00 20130101; Y10S 514/866 20130101; C07K 7/06 20130101 |
Class at
Publication: |
514/7.3 |
International
Class: |
C07K 7/06 20060101
C07K007/06 |
Goverment Interests
[0001] The development of the present invention was supported by
the University of Maryland, Baltimore, Md. The invention described
herein was supported by funding from the National Institutes of
Health (DX 48373-05). The Government has certain rights.
Claims
1. A method for prevention or delay of onset of an autoimmune
disease, comprising: administering a pharmaceutically effective
amount of a peptide antagonist of zonulin to a subject at risk of
developing the autoimmune disease, wherein the peptide antagonist
binds to zonula occludens toxin receptor but does not
physiologically modulate the opening of mammalian intestinal tight
junctions.
2. The method of claim 1, wherein the peptide antagonist comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID
NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ
ID NO: 24.
3. The method of claim 1, wherein the peptide antagonist is from
8-110 amino acids in size.
4. The method of claim 1, wherein the peptide antagonist is from
8-40 amino acids in size.
5. The method of claim 1, wherein the peptide antagonist comprises
amino acid sequence SEQ ID NO: 15.
6. (canceled)
7. The method of claim 1, wherein the peptide antagonist is
administered as an oral dosage composition for intestinal
delivery.
8. (canceled)
9. A method for prevention or delay of onset of an autoimmune
disease, comprising: administering a pharmaceutically effective
amount of an antagonist of zonulin to a subject at risk of
developing the autoimmune disease, wherein the antagonist binds to
zonula occludens toxin receptor but does not physiologically
modulate the opening of mammalian intestinal tight junctions.
10. The method of claim 9, wherein the antagonist comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID
NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ
ID NO: 24.
11. The method of claim 9, wherein the antagonist comprises a
peptide comprising 8-110 amino acids.
12. The method of claim 9, wherein the antagonist comprises a
peptide comprising 8-40 amino acids.
13. The method of claim 9, wherein the antagonist comprises amino
acid sequence SEQ ID NO: 15.
14. (canceled)
15. The method of claim 9, wherein the antagonist is administered
as an oral dosage composition for intestinal delivery.
16. (canceled)
17. A method for prevention of diabetes, comprising: administering
to a subject in need of such prevention, a pharmaceutically
effective amount of a peptide antagonist of zonulin, wherein the
peptide antagonist binds to zonula occludens toxin receptor but
does not physiologically modulate the opening of mammalian
intestinal tight junctions.
18. The method of claim 17, wherein the peptide antagonist
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, and SEQ ID NO: 24.
19. The method of claim 17, wherein the peptide antagonist is from
8-110 amino acids in size.
20. The method of claim 17, wherein the peptide antagonist is from
8-40 amino acids in size.
21. The method of claim 17, wherein the peptide antagonist
comprises amino acid sequence SEQ ID NO: 15.
22. (canceled)
23. The method of claim 17, wherein the peptide antagonist is
administered as an oral dosage composition for intestinal
delivery.
24-32. (canceled)
Description
FIELD OF THE INVENTION
[0002] The present invention relates to use of peptide antagonists
of zonulin to prevent or delay the onset of diabetes, particularly
type I diabetes. The peptide antagonists bind to the zonula
occludens toxin receptor, yet do not physiologically modulate the
opening of mammalian tight junctions.
BACKGROUND OF THE INVENTION
I. Function and Regulation of Intestinal Tight Junctions
[0003] The intestinal epithelium represents the largest interface
(more than 2,000,000 cm.sup.2) between the external environment and
the internal milieu. The maintenance of intercellular tight
junctions ("tj") competence prevents movements of potentially
harmful environmental factors, such as bacteria, viruses, toxins,
food allergens, and macromolecules across the intestinal barrier.
This competence is significantly jeopardized in a variety of
clinical conditions affecting the gastrointestinal tract, including
food allergies, enteric infections, malabsorption syndromes, and
inflammatory bowel diseases.
[0004] The tj or zonula occludens (hereinafter "ZO") are one of the
hallmarks of absorptive and secretory epithelia (Madara, J. Clin.
Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretary
Diarrhea Eds. Lebenthal et al, Chapter 11, pages 128-138 (1990)).
As a barrier between apical and basolateral compartments, they
selectively regulate the passive diffusion of ions and
water-soluble solutes through the paracellular pathway (Gumbiner,
Am. J. Physiol., 253 (Cell Physiol. 22):C749-C758 (1987)). This
barrier maintains any gradient generated by the activity of
pathways associated with the transcellular route (Diamond,
Physiologist, 20:10-18 (1977)).
[0005] Variations in transepithelial conductance can usually be
attributed to changes in the permeability of the paracellular
pathway, since the resistances of enterocyte plasma membranes are
relatively high (Madara (1989, 1990), supra). The ZO represents the
major barrier in this paracellular pathway, and the electrical
resistance of epithelial tissues seems to depend on the number of
transmembrane protein strands, and their complexity in the ZO, as
observed by freeze-fracture electron microscopy (Madara et al, J.
Cell Biol., 101:2124-2133 (1985)).
[0006] There is abundant evidence that ZO, once regarded as static
structures, are in fact dynamic and readily adapt to a variety of
developmental (Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel
et al. Cold Spring Harbor Symp. Quant. Biol., 40:443-455 (1976);
and Schneeberger et al, J. Cell Sci., 32:307-324 (1978),
physiological (Gilula et al, Dev. Biol., 50:142-168 (1976); Madara
et al, J. Membr. Biol., 100:149-164 (1987); Mazariegos et al, J.
Cell Biol., 98:1865-1877 (1984); and Sardet et al, J. Cell Biol.,
80:96-117 (1979)), and pathological (Milks et al, J. Cell Biol.,
103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537 (1988);
and Shasby et al, Am. J. Physiol., 255 (Cell Physiol.; 24:C781-C788
(1988) circumstances. The regulatory mechanisms that underlie this
adaptation are still not completely understood. However, it is
clear that, in the presence of Ca.sup.2+, assembly of the ZO is the
result of cellular interactions that trigger a complex cascade of
biochemical events that ultimately lead to the formation and
modulation of an organized network of ZO elements, the composition
of which has been only partially characterized (Diamond,
Physiologist, 20:10-18 (1977)). A candidate for the transmembrane
protein strands, occluden, has recently been identified (Furuse et
al, J. Membr. Biol., 87:141-150 (1985)).
[0007] Six proteins have been identified in a cytoplasmic
submembranous plaque underlying membrane contacts, but their
function remains to be established (Diamond, supra). ZO-1 and ZO-2
exist as a heterodimer (Gumbiner et al, Proc. Natl. Acad. Sci.,
USA, 88:3460-3464 (1991)) in a detergent-stable complex with an
uncharacterized 130 kD protein (ZO-3). Most immunoelectron
microscopic studies have localized to precisely beneath membrane
contacts (Stevenson et al, Molec. Cell Biochem., 83:129-145
(1988)). Two other proteins, cingulin (Citi et al, Nature (London),
333:272-275 (1988)) and the 7H6 antigen (Zhong et al, J. Cell
Biol., 120:477-483 (1993)) are localized further from the membrane
and have not yet been cloned. Rab 13, a small GTP binding protein
has also recently been localized to the junction region (Zahraoui
et al, J. Cell Biol., 124:101-115 (1994)). Other small GTP-binding
proteins are known to regulate the cortical cytoskeleton, i.e., rho
regulates actin-membrane attachment in focal contacts (Ridley et
al, Cell, 70:389-399 (1992)), and rac regulates growth
factor-induced membrane ruffling (Ridley et al, Cell, 70:401-410
(1992)). Based on the analogy with the known functions of plaque
proteins in the better characterized cell junctions, focal contacts
(Guan et al, Nature, 358:690-692 (1992)), and adherens junctions
(Tsukits et al, J. Cell Biol., 123:1049-1053 (1993)), it has been
hypothesize that tj-associated plaque proteins are involved in
transducing signals in both directions across the cell membrane,
and in regulating links to the cortical actin cytoskeleton.
[0008] To meet the many diverse physiological and pathological
challenges to which epithelia are subjected, the ZO must be capable
of rapid and coordinated responses that require the presence of a
complex regulatory system. The precise characterization of the
mechanisms involved in the assembly and regulation of the ZO is an
area of current active investigation.
[0009] There is now a body of evidence that tj structural and
functional linkages exist between the actin cytoskeleton and the tj
complex of absorptive cells (Gumbiner et al, supra; Madara et al,
supra; and Drenchahn et al, J. Cell Biol., 107:1037-1048 (1988)).
The actin cytoskeleton is composed of a complicated meshwork of
microfilaments whose precise geometry is regulated by a large cadre
of actin-binding proteins. An example of how the state of
phosphorylation of an actin-binding protein might regulate
cytoskeletal linking to the cell plasma membrane is the
myristoylated alanine-rich C kinase substrate (hereinafter
"MARCKS"). MARCKS is a specific protein kinase C (hereinafter
"PKC") substrate that is associated with the cytoplasmic face of
the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages 439-443
(1992)). In its non-phosphorylated form, MARCKS crosslinks to the
membrane actin. Thus, it is likely that the actin meshwork
associated with the membrane via MARCKS is relatively rigid
(Hartwig et al, Nature, 356:618-622 (1992)). Activated PKC
phosphorylates MARCKS, which is released from the membrane (Rosen
et al, J. Exp. Med., 172:1211-1215 (1990); and Thelan et al,
Nature, 351:320-322 (1991)). The actin linked to MARCKS is likely
to be spatially separated from the membrane and be more plastic.
When MARCKS is dephosphorylated, it returns to the membrane where
it once again crosslinks actin (Hartwig et al, supra; and Thelen et
al, supra). These data suggest that the F-actin network may be
rearranged by a PKC-dependent phosphorylation process that involves
actin-binding proteins (MARCKS being one of them).
[0010] A variety of intracellular mediators have been shown to
alter tj function and/or structure. Tight junctions of amphibian
gallbladder (Duffey et al, Nature, 204:451-452 (1981)), and both
goldfish (Bakker et al, Am. J. Physiol., 246:G213 -G217 (1984)) and
flounder (Krasney et al, Fed. Proc., 42:1100 (1983)) intestine,
display enhanced resistance to passive ion flow as intracellular
cAMP is elevated. Also, exposure of amphibian gallbladder to
Ca.sup.2+ ionophore appears to enhance tj resistance, and induce
alterations in tj structure (Palant et al, Am. J. Physiol.,
245:C203-C212 (1983)). Further, activation of PKC by phorbol esters
increases paracellular permeability both in kidney (Ellis et al, C.
Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol,
32):F293-F300 (1992)), and intestinal (Stenson et. al, C. Am. J.
Physiol., 265 (Gastrointest. Liver Physiol., 28):G955-G962 (1993)
epithelial cell lines.
II. Zonula Occludens Toxin
[0011] Most Vibrio cholerae vaccine candidates constructed by
deleting ctxA gene encoding cholera toxin (CT) are able to elicit
high antibody responses, but more than one-half of the vaccinees
still develop mild diarrhea (Levine et al, Infect. Immun.,
56(1):161-167 (1988)). Given the magnitude of the diarrhea induced
in the absence of CT, it was hypothesized that V. cholerae produce
other enterotoxigenic factors, which are still present in strains
deleted of the ctxA sequence (Levine et al, supra). As a result, a
second toxin, zonula occludens toxin (hereinafter "ZOT") elaborated
by V. cholerae and which contribute to the residual diarrhea, was
discovered (Fasano et al, Proc. Natl. Acad. Sci., USA, 8:5242-5246
(1991)). The zot gene is located immediately adjacent to the ctx
genes. The high percent concurrence of the zot gene with the ctx
genes among V. cholerae strains (Johnson et al, J. Clin. Microb.,
31/3:732-733 (1993), and Karasawa at al, FEBS Microbiology Letters
106:143-146 (1993)) suggests a possible synergistic role of ZOT in
the causation of acute dehydrating diarrhea typical of cholera.
Recently, the zot gene has also been identified in other enteric
pathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever
and other Salomellosis, 47(Abstr.) (1994)).
[0012] It has been previously found that, when tested on rabbit
ileal mucosa, ZOT increases the intestinal permeability by
modulating the structure of intercellular tj (Fasano et al, supra).
It has been found that as a consequence of modification of the
paracellular pathway, the intestinal mucosa becomes more permeable.
It also was found that ZOT does not affect Na.sup.+-glucose coupled
active transport, is not cytotoxic, and fails to completely abolish
the transepithelial resistance (Fasano et al, supra).
[0013] More recently, it has been found that ZOT is capable of
reversibly opening tj in the intestinal mucosa, and thus ZOT, when
co-administered with a therapeutic agent, e.g., insulin, is able to
effect intestinal delivery of the therapeutic agent, when employed
in an oral dosage composition for intestinal drug delivery, e.g.,
in the treatment of diabetes (WO 96/37196; U.S. Pat. No. 5,827,534;
U.S. Pat. No. 5,665,389 and Fasano et al, J. Clin. Invest.,
99:1158-1164 (1997) each of which is incorporated by reference
herein in their entirety). It has also been found that ZOT is
capable of reversibly opening tj in the nasal mucosa, and thus ZOT,
when co-administered with a therapeutic agent, is able to enhance
nasal absorption of a therapeutic agent (U.S. Pat. No. 5,908,825;
which is incorporated by reference herein in its entirety).
[0014] In U.S. Pat. No. 5,864,014; which is incorporated by
reference herein in its entirety, a ZOT receptor has been
identified and purified from an intestinal cell line, i.e., CaCo2
cells. Further, in U.S. Pat. No. 5,912,323; which is incorporated
by reference herein in its entirety, ZOT receptors from human
intestinal, heart and brain tissues have been identified and
purified. The ZOT receptors represent the first step of the
paracellular pathway involved in the regulation of intestinal and
nasal permeability.
III. Zonulin
[0015] In U.S. Pat. Nos. 5,945,510 and 5,948,629, which are
incorporated by reference herein in their entirety, mammalian
proteins that are immunologically and functionally related to ZOT,
and that function as the physiological modulator of mammalian tight
junctions, have been identified and purified. These mammalian
proteins, referred to as "zonulin", are useful for enhancing
absorption of therapeutic agents across tj of intestinal and nasal
mucosa, as well as across tj of the blood brain barrier.
IV. Peptide Antagonists of Zonulin
[0016] Peptide antagonists of zonulin were identified and described
for the first time in pending U.S. patent application Ser. No.
09/127,815, filed Aug. 3, 1998, which is incorporated by reference
herein is its entirety, which corresponds to WO 00/07609. Said
peptide antagonists bind to the ZOT receptor, yet do not function
to physiologically modulate the opening of mammalian tight
junctions. The peptide antagonists competitively inhibit the
binding of ZOT and zonulin to the ZOT receptor, thereby inhibiting
the ability of ZOT and zonulin to physiologically modulate the
opening of mammalian tight junctions.
V. Diabetes
[0017] The morbidity and mortality associated with diabetes is
devastating. The total number of diabetic individuals in the United
States is 15.7 million. Of these, 100% of the type I diabetic
individuals and 40% of type II diabetic individuals depend on
paternal administration of insulin. On an annual basis, thee direct
medical costs associated with diabetes exceeds 40 billion dollars.
An additional 14 billion dollars is associated with disability,
work loss, and premature mortality.
[0018] Although oral insulin drug delivery strategies have been the
focus of many research efforts, they have been largely unsuccessful
because the physiologic nature of the small intestine prevents the
absorption of macromolecules, such as insulin.
[0019] An oral dosage composition comprising ZOT for targeting
delivery insulin to the paracellular pathway for the treatment of
diabetes has been described in U.S. Pat. Nos. 5,827,534 and
5,665,389. By physiologically modulating the paracellular pathway
using ZOT, it is now possible to introduce a wide variety of
therapeutic agents into the systemic circulation. This drug
delivery system adds targeting specificity, which has long hampered
the design of many oral pharmaceutical agents. The utility of this
system is not limited to insulin delivery, and may represent a new
way of designing orally administered pharmaceutical agents.
[0020] While offering an innovative treatment strategy for a
disease as debilitating as diabetes is promising, preventing or
delaying the onset of disease has widespread implications.
Understanding the pathogenesis of any disease process is a daunting
task. Heretofore, there has been no prior evidence of a
pharmaceutical agent with the capability of preventing or delaying
the onset of diabetes. In the present invention new light has been
shed on the pathogenesis, prevention and delaying of onset of
diabetes by demonstrating that a critical and early step in disease
progression resides in alterations in paracellular permeability. In
the present invention, it has been demonstrated that an increase in
paracellular permeability is necessary for the progression toward
diabetes. Peptide antagonists of zonulin, which block this
endogenous pathway, have been found in the present invention to
prevent the progression to diabetes. Thus, the present invention is
believed to be useful to prevent long-term complications of
diabetes. Further, the permeability changes associated with
autoimmune diseases are long standing, and early intervention per
the present invention is believed to have untold benefits to the
diabetic patient.
SUMMARY OF THE INVENTION
[0021] An object of the present invention is to provide a method
for the prevention or delay the onset of diabetes.
[0022] This and other objects of the present invention, which will
be apparent from the detailed description of the invention provided
hereinafter, have been met, in one embodiment, by a method for
preventing or delay the onset of diabetes (particularly, type I
diabetes) comprising administering to a subject in need of such
prevention or delay of onset, a pharmaceutically effective amount
of a peptide antagonist of zonulin, wherein said peptide antagonist
binds to ZOT receptor, yet does not physiologically modulate the
opening of mammalian tight junctions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a comparison of the N-terminal sequences of
zonulin purified from various human tissues and IgM heavy chain
with the N-terminal sequence of the biologically active fragment
(amino acids 288-399) of ZOT.
[0024] FIG. 2 shows the effect of ZOT, zonulin.sub.i,
zonulin.sub.h, either alone (closed bars), or in combination with
the peptide antagonist FZI/0 (open bars) or in combination with
FZI/1 (shaded bars), as compared to the negative control, on the
tissue resistance (Rt) of rabbit ileum mounted in Ussing chambers.
N equals 3-5; and * equals p<0.01.
[0025] FIG. 3 shows the concentrations (ng/ml) of intraluminal
zonulin in both diabetic-prone and diabetic-resistant rats, which
was determined using a sandwich ELISA assay. Samples were obtained
by intestinal lavage in normal saline. The first bar in each case
represents diabetic-resistant rate (DR). The second bar represents
diabetic-prone animals (DP), and the third bar represents rats with
chronic diabetes (CD). <9% of the diabetic-prone rats do not
become diabetic, and <9% of the diabetic-resistant rats develop
diabetes.
[0026] FIG. 4 shows the percentage of rats used in the study that
progressed to diabetes.
[0027] FIG. 5 shows the concentrations (ng/ml) of intraluminal
zonulin in diabetic rats, which was determined using a sandwich
ELISA assay.
[0028] FIG. 6 shows ex vivo intestinal permeability in diabetic
resistant (DR) rats, untreated diabetic-prone rats (DP-untreated;
second bar) determined in Ussing chambers, diabetic-prone rats
treated with the peptide antagonist of zonulin (DP-treated;
third-bar). * equals p<0.05; ** equals p<0.05, and
p<0.0001 compared to DP-treated.
[0029] FIG. 7 shows ex vivo intestinal permeability in the small
intestines of untreated diabetes-prone rats that either developed
or did not develop diabetes. * equals p<0.04.
DETAILED DESCRIPTION OF THE INVENTION
[0030] As discussed above, in one embodiment, the above-described
object of the present invention have been met by a method for
preventing or delaying the onset of diabetes (particularly, type I
diabetes) comprising administering to a subject in need of such
prevention or delay of onset, a pharmaceutically effective amount
of a peptide antagonist of zonulin, wherein said peptide antagonist
binds to ZOT receptor, yet does not physiologically modulate the
opening of mammalian tight junctions
[0031] The particular peptide antagonist of zonulin employed in the
present invention is not critical thereto. Examples of said peptide
antagonists include peptides which comprise an amino acid sequence
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11; SEQ ID NO:12,
SEQ ID:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ. ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, and SEQ ID NO:24.
[0032] The size of the peptide antagonist is not critical to the
present invention. Generally, the size of the peptide antagonist
will range from 8 to 110, amino acids, preferably from 8 to 40
amino acids, more preferably will be 8 amino acids.
[0033] The peptide antagonists can be chemically synthesized and
purified using well-known techniques, such as described in High
Performance Liquid Chromatography of Peptides and Proteins:
Separation Analysis and Conformation, Eds. Mant et. al, C.R.C.
Press (1991), and a peptide synthesizer; such as Symphony (Protein
Technologies, Inc); or by using recombinant DNA techniques, i.e.,
where the nucleotide sequence encoding the peptide is inserted in
an appropriate expression vector, e.g., an E. coli or yeast
expression vector, expressed in the respective host cell, and
purified therefrom using well-know techniques.
[0034] The peptide antagonists can be administered as oral dosage
compositions for small intestinal delivery. Such oral dosage
compositions for small intestinal delivery are well-known in the
art, and generally comprise gastroresistent tablets or capsules
(Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol, Mack
Publishing Co., Chapter 89 (1980); Digenis et al, J. Pham. Sci.,
83:915-921 (1994); Vantini et al, Clinica Terapeutica, 145:445-451
(1993); Yoshitomi et al, Chem. Pharm. Bull., 40:1902-1905 (1992);
Thoma et al, Pharmazie, 46:331-336 (1991); Morishita et al, Drug
Design and Delivery, 7:309-319 (1991); and Lin et al,
Pharmaceutical Res., 8:919-924 (1991)); each of which is
incorporated by reference herein in its entirety).
[0035] Tablets are made gastroresistant by the addition of, e.g.,
either cellulose acetate phthalate or cellulose acetate
terephthalate.
[0036] Capsules are solid dosage forms in which the peptide
antagonist(s) is enclosed in either a hard or soft, soluble
container or shell of gelatin. The gelatin used in the manufacture
of capsules is obtained from collagenous material by hydrolysis.
There are two types of gelatin. Type A, derived from pork skins by
acid processing, and Type B, obtained from bones and animal skins
by alkaline processing. The use of hard gelatin capsules permit a
choice in prescribing a single peptide antagonist or a combination
thereof at the exact dosage level considered best for the
individual subject. The hard gelatin capsule consists of two
sections, one slipping over the other, thus completely surrounding
the peptide antagonist. These capsules are filled by introducing
the peptide antagonist, or gastroresistent beads containing the
peptide antagonist, into the longer end of the capsule, and then
slipping on the cap. Hard gelatin capsules are made largely from
gelatin, FD&C colorants, and sometimes an opacifying agent,
such as titanium dioxide. The USP permits the gelatin for this
purpose to contain 0.15% (w/v) sulfur dioxide to prevent
decomposition during manufacture.
[0037] In the context of the present invention, oral dosage
compositions for small intestinal delivery also include liquid
compositions which contain aqueous buffering agents that prevent
the peptide antagonist from being significantly inactivated by
gastric fluids in the stomach, thereby allowing the peptide
antagonist to reach the small intestines in an active form.
Examples of such aqueous buffering agents which can be employed in
the present invention include bicarbonate buffer (pH 5.5 to 8.7,
preferably about pH 7.4).
[0038] When the oral dosage composition is a liquid composition, it
is preferable that the composition be prepared just prior to
administration so as to minimize stability problems. In this case,
the liquid composition can be prepared by dissolving lyophilized
peptide antagonist in the aqueous buffering agent.
[0039] The pharmaceutically effective amount of peptide antagonist
employed is not critical to the present invention and will vary
depending upon the age, weight and sex of the subject being
treated. Generally, the amount of peptide antagonist employed in
the present invention to prevent or delay the onset of diabetes, is
in the range of about 7.5.times.10.sup.-6 M to 7.5.times.10.sup.-3
M, preferably about 7.5.times.10.sup.-6 M to 7.5.times.10.sup.-4 M.
To achieve such a final concentration in, e.g., the intestines or
blood, the amount of peptide antagonist in a single oral dosage
composition of the present invention will generally be about 1.0
.mu.g to 1000 .mu.g, preferably about 1.0 .mu.g to 100 .mu.g.
[0040] The following examples are provided for illustrative
purposes only, and are in no way intended to limit the scope of the
present invention.
EXAMPLE 1
Peptide Antagonists of Zonulin
[0041] Given that ZOT, human intestinal zonulin (zonulin.sub.i) and
human heart zonulin (zonulin.sub.h) all act on intestinal (Fasano
et al, Gastroenterology, 112:839. (1997); Fasano et al, J. Clin.
Invest., 96:710 (1995)) and endothelial tj and that all three have
a similar regional effect (Fasano et al. (1997), supra) that
coincides with the ZOT receptor distribution within the intestine
(Fasano et al (1997), supra; and Fasano et al (1995), supra), it
was postulated in U.S. patent application Ser. No. 09/127,815,
filed Aug. 3, 1998; that these three molecules interact with the
same receptor binding site. A comparison of the primary amino acid
structure of ZOT and the human zonulins was thus carried out
therein to provide insights as to the absolute structural
requirements of the receptor-ligand interaction involved in the
regulation of intestinal tj. The analysis of the N-termini of these
molecules revealed the following common motif (amino acid residues
8-15 boxed in FIG. 1): non-polar (Gly for intestine, Val for
brain), variable, non-polar, variable, non-polar, polar, variable,
polar (Gly). Gly in position 8, Val in position 12 and Gln in
position 13, all are highly conserved in ZOT, zonulin.sub.i and
zonulin.sub.h (see FIG. 1), which is believed to be critical for
receptor binding function within the intestine. To verify the same,
the synthetic octapeptide Gly Gly Val Leu Val Gln Pro Gly (SEQ ID
NO:15) (named FZI/0, and corresponding to amino acid residues 8-15
of human fetal zonulin.sub.i) was chemically synthesized.
[0042] Next, rabbit ileum mounted in Ussing chambers as described
above, were exposed to 100 .mu.g of FZI/0 (SEQ ID NO:15), 100 .mu.g
of FZI/1 (SEQ ID NO:29), 1.0 .mu.g of 6.times.His-ZOT (obtained as
described in Example 1 of U.S. patent application Ser. No.
09/127,815, filed Aug. 3, 1998), 1.0 .mu.g of zonulin.sub.i
(obtained as described in Example 3 of U.S. patent application Ser.
No. 09/127,815, filed Aug. 3, 1998), or 1.0 .mu.g of zonulin.sub.i,
(obtained as described in Example 3 of U.S. patent application Ser.
No. 09/127,815, filed Aug. 3, 1998), alone; or pre-exposed for 20
min to 100 .mu.g of FZI/0 or FZI/1, at which time 1.0 .mu.g of
6.times.His-ZOT, 1.0 .mu.g of zonulin.sub.i, or 1.0 .mu.g of
zonulin.sub.h, was added. .DELTA.Rt was then calculated as
described above. The results are shown in FIG. 2.
[0043] As shown in FIG. 2, FZI/0 did not induce any significant
change in Rt (0.5% as compared to the negative control) (see closed
bar). On the contrary, pre-treatment for 20 min with FZI/0
decreased the effect of ZOT, zonulin.sub.i, and zonulin.sub.h on Rt
by 75%, 97%, and 100%, respectively (see open bar). Also as shown
in FIG. 2, this inhibitory effect was completely ablated when a
second synthetic peptide (FZI/1, SEQ ID NO:29) was chemically
synthesized by changing the Gly in position 8, the Val in position
12, and the Gln in position 13 (as referred to zonulin.sub.i) with
the correspondent amino acid residues of zonulin.sub.h (Val, Gly,
and Arg, respectively, see SEQ ID NO:30) was used (see shaded
bar).
[0044] The above results demonstrate that there is a region
spinning between residue 8 and 15 of the N-terminal end of ZOT and
the zonulin family that is crucial for the binding to the target
receptor, and that the amino acid residues in position 8, 12, and
13 determine the tissue specificity of this binding.
EXAMPLE 2
Diabetic Rat Model
[0045] Alterations in intestinal permeability have been shown to be
one of the preceding physiologic changes associated with the onset
of diabetes (Meddings, Am. J. Physiol., 276:G951-957 (1999)).
Paracellular transport and intestinal permeability is regulated by
intracellular tj via mechanisms which have not been completely
elucidated.
[0046] Zonulin and its prokaryotic analog, ZOT, both alter
intestinal permeability by modulating tj. In this example, it has
been demonstrated for the first time that zonulin-related
impairment of tj is involved in the pathogenesis of diabetes, and
that diabetes can be prevented, or the onset delayed, by
administration of a peptide antagonist of zonulin.
[0047] Initially, two genetic breeds, i.e., BB/Wor diabetic-prone
(DP) and diabetic-resistant (DR) rats (Haber et al, J. Clin.
Invest., 95:832-837 (1993)), were evaluated to determine whether
they exhibited significant changes in intraluminal secretion of
zonulin and intestinal permeability.
[0048] More specifically, age-matched DP and DR rats (20, 50, 75,
and >100 days of age) were sacrificed. After the rats were
sacrificed, a 25G needle was placed within the lumen of the ileum,
and intestinal lavage with Ringer's solution was performed to
determine the presence of intraluminal zonulin. Zonulin
concentration was evaluated using a sandwich enzyme linked
immunosorbent assay (ELISA) as follows:
[0049] Plastic microtiter plates (Costar, Cambridge, Mass.) were
coated with polyclonal rabbit anti-ZOT antibodies (obtained as
described in Example 2 of U.S. application Ser. No. 09/127,815;
filed Aug. 3, 1998) (dilution 1:100) overnight at 4.degree. C.,
washed three times with PBS containing 0.05% (v/v) Tween 20, then
blocked by incubation with 300 .mu.l of PBS containing 0.1% (v/v)
Tween 20, for 15 min at room temperature. Next, purified human
intestine zonulin (obtained as described in Example 3 of U.S.
application Ser. No. 09/127,815 filed Aug. 3, 1998) was coated on
the plates.
[0050] A standard curve was obtained by diluting zonulin in PBS
containing 0.05% (v/v) Tween 20 at different concentration: 0.78
ng/ml, 1.56 ng/ml, 3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml
and 50 ng/ml.
[0051] 100 .mu.l of each standard concentration or 100 .mu.l of
intestinal lavage sample were pipetted into the wells, and
incubated for 1 hr at room temperature, using a plate shaker.
Unbound zonulin was washed-out using PBS, and the wells were
incubated with 100 .mu.l of anti-ZOT antibodies conjugated with
alkaline phosphate for 1 hr at room temperature with shaking.
Unbound conjugate was washed-out with PBS, and a color reaction was
developed by first adding 100 .mu.l of Extra-Avidin (SIGMA, St.
Louis, Mo.) diluted 1/20000 in 0.1 M Tris-HCl (pH 7.3), 1.0 mM
MgCl.sub.2, 1.0% (w/v) BSA for 15 min, and then incubating each
well for 30 min at 37.degree. C. with 100 .mu.l of a solution
containing 1.0 mg/ml of p-nitrophenyl-phosphate substrate (SIGMA,
St. Louis, Mo.), Adsorbance was read on an enzyme immunoassay
reader at 405 nm.
[0052] In order to evaluate the intra- and inter-assay precision of
the ELISA-sandwich method, the coefficient variation (CV) was
calculated using three replicates from two samples with different
concentrations of zonulin, on three consecutive days. The
inter-assay test of the ELISA-sandwich method produced CV values of
9.8%. The CV of the intra-assay test was 4.2% at day 1, 3.3% at day
2 and 2.9% at day 3.
[0053] Zonulin concentration was expressed as ng/mg protein
detected in the intestinal lavages and normalized by exposed
surface area (in mm.sup.2). The results are shown in FIG. 3.
[0054] As shown in FIG. 3, a 4-fold increase in intraluminal
zonulin was first observed in diabetic-prone rats (age 50 days)
(second bar). This increase in intraluminal zonulin was found to
correlate with an increase in intestinal permeability. The increase
in intraluminal zonulin remains high in these diabetic-prone rats,
and found to correlate with the progression toward full-blown
diabetes. Of note, the diabetic-prone rat (age >100 days) did
not have an increase in intraluminal zonulin. This is remarkable,
as this rat did not progress to diabetes. Blood glucose for this
rat was normal. Thus, zonulin is responsible for the permeability
changes associated with the pathogenesis of type I diabetes. The
increase in zonulin secretion is age-related, and proceeds the
onset of diabetes.
[0055] Next, in order to demonstrate that diabetes can be prevented
by administration of a peptide antagonist of zonulin, BB/Wor rats
(ages 21-26 days), were obtained from Biomedical Research Models,
Inc. (Rutland, Mass.), and were randomized into two groups (n=5 per
group), i.e., a treated group and a control group. Both groups were
maintained on a standard diet of rat chow (Harlan Teklab Diet
#7012). All food and water were previously autoclaved. Each day,
daily water intake was measured and 100 ml of fresh water was
given. The treated group received 10 .mu.g/ml of the zonulin
peptide antagonist (SEQ ID NO:15) supplemented in the drinking
water. The rats were housed in hepa-filter cages.
[0056] Diabetes in the rats was diagnosed as follows: The rats were
weighed twice a week. Blood glucose was determined weekly using the
OneTouch.RTM. glucose monitoring system (Johnson & Johnson).
Each week, reagent strips for urinalysis were used to monitor
glucose (Diastix.RTM.) and ketones (Ketositx.RTM.) (Bayer). Rats
with a blood glucose >250 mg/dl were fasted overnight, and blood
glucose levels >200 mg/dl were considered diabetic. These
guidelines are in accordance with the data supplied by Biomedical
Research Models, Inc. The results are shown in FIG. 4.
[0057] As shown in FIG. 4, 80% of the control rats (4/5) and 40% of
the rats treated with the peptide antagonist of zonulin (2/5)
developed diabetes by age 80 days. Alterations in zonulin secretion
paralleled the onset of diabetes.
[0058] Following clinical presentation of diabetes, the rats were
sacrificed as follows: the rats were anesthesized using ketamine
anesthesia and a midline incision was made allowing access to the
heart. An 18G needle was placed into the heart and death occurred
by exsanguinations. Then, zonulin assays were conducted as
described above. For those rats that did not present with diabetes,
the endpoint of the study was age 80 days. According to Biomedical
Research Models, Inc., 80% of diabetes prone rats present with
diabetes by age 80 days. The results of the zonulin assays are
shown in FIG. 5.
[0059] As shown in FIG. 5, the diabetic rats that were not treated
with the peptide antagonist of zonulin were observed to have an
increase in intraluminal zonulin, which was consistent with the
results shown in FIG. 3. Further, intraluminal zonulin was
increased 2 to 4-fold. In diabetic rats (DR), as compared to both
diabetic-prone rats that did not develop diabetes (DP-treated) and
control rate (DP-untreated). Non-diabetic control rats that did not
develop diabetes had negligible levels of zonulin, consistent with
the levels of zonulin shown in FIG. 3. Moreover, two diabetic-prone
rats that developed diabetes despite treatment with the peptide
antagonist of zonulin showed intraluminal zonulin levels that were
significantly higher than the successfully treated rats, and the
untreated control rats. The levels of zonulin were sufficient to
initiate the permeability changes necessary to progress to
diabetes, but the ZOT/zonulin receptors were effectively blocked by
the peptide antagonist.
[0060] Also, following clinical presentation of diabetes, the
intestinal tissues of the sacrificed rats were mounted in Ussing
chamber to assess for changes in ex vivo permeability.
[0061] More specifically, sections of jejunum and ileum were
isolated from the sacrificed rats, and rinsed free of intestinal
contents. Six sections of each intestinal segment was prepared and
mounted in Lucite Ussing chambers (0.33 cm.sup.2 opening),
connected to a voltage clamp apparatus (EVC 4000; World Precision
Instruments, Saratosa, Fla.), and bathed with freshly prepared
buffer comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM Na.sub.2SO.sub.4,
30.5 mM mannitol, 1.69 mM Na.sub.2PO.sub.4, 0.3 mM NaHPO.sub.4,
1.25 mM CaCl.sub.2, 1.1 mM MgCl.sub.2, and 25 mN NaHCO.sub.3 (pH
7.4). The bathing solution was maintained at 37.degree. C. with
water-jacketed reservoirs connected to a constant temperature
circulating pump and gassed with 95% O.sub.2 and 5% CO.sub.2.
Potential difference was measured and short-circuit current and
tissue resistance was calculated as described by Fasano et al,
Proc. Natl. Acad. Sci. USA, 88:5242-5246 (1991). The results are
shown in FIGS. 6-7.
[0062] As demonstrated in the ex vivo Ussing chamber permeability
studies, and shown in FIG. 6, all of the rats that progressed to
diabetes had an increase in their intestinal permeability. Diabetic
resistant (DR) rats had no appreciable alterations in paracellular
permeability (first bar). Untreated diabetic-prone rats
(DP-untreated; second bar) had a significant increase in
paracellular permeability of the jejunum and ileum. More
importantly, diabetic-prone rats treated with the peptide
antagonist of zonulin (DP-treated; third bar) had a significant
increase in paracellular permeability of the small intestine
restricted to the jejunum. However, as shown in FIG. 6,
pre-treatment with the zonulin peptide antagonist prevented these
changes in the distal ileum. Consequently alterations in
paracellular permeability associated with the pathogenesis are
restricted to the ileum. Also, as shown in FIG. 6, there are no
significant changes in permeability of the colon, which coincides,
with the regional distribution of the zonulin receptor
distribution.
[0063] These results were further validated by a comparison of ex
vivo intestinal permeability in the small intestines of untreated
diabetes-prone rats that either developed (DP-D) or did not
developed (DP-N) diabetes (FIG. 7). While no significant changes in
jejunal Rt were observed between DP-D and DP-N rats, a significant
lower Rt of the ileal mucosa of DP-D rats was observed as compared
to DP-N rats (FIG. 7).
[0064] Thus, the following conclusions can be made: (1) the peptide
antagonist was able to effectively block the permeability changes
required for the development of diabetes; and (2) in those rats
treated with the peptide antagonist, the levels of intraluminal
zonulin are 3-fold higher than the treated rats that did not
develop diabetes. In this population of treated rats that developed
diabetes, the amount of peptide antagonist may not have been enough
to block a sufficient number of ZOT/zonulin receptors necessary to
prevent diabetes.
[0065] 60% of the treated rats did not develop diabetes. In this
population of rats, the peptide antagonist of zonulin effectively
prevented the increase in intestinal permeability necessary for the
onset of diabetes. As shown in FIG. 5, the treated rats had levels
of intraluminal zonulin comparable with the untreated controls, but
due to the presence of the peptide antagonist of zonulin, the
overall permeability the small intestine was not altered enough to
initiate the pathophysiologic changes necessary for the progression
to diabetes. Interestingly, as shown in FIG. 5, the one control
animal that did not develop diabetes had negligible levels of
zonulin, further supporting the role of zonulin in the pathogenesis
of diabetes.
[0066] Accordingly, an early event in the pathogenesis of diabetes
in BB/Wor rats involves changes in zonulin-mediated intestinal
paracellular permeability. Furthermore, inhibition of the zonulin
signaling system with the use of peptide antagonists of zonulin
prevents, or at least delays, the onset of diabetes.
[0067] While the invention has been described in detail, and with
reference to specific embodiments thereof; it will be apparent to
one of ordinary skill in the art that various changes and
modifications can be made therein without departing from the spirit
and scope thereof.
Sequence CWU 1
1
3318PRTArtificial Sequencetight junction antagonist peptide 1Gly
Arg Val Cys Val Gln Pro Gly1 528PRTArtificial Sequencetight
junction antagonist peptide 2Gly Arg Val Cys Val Gln Asp Gly1
538PRTArtificial Sequencetight junction antagonist peptide 3Gly Arg
Val Leu Val Gln Pro Gly1 548PRTArtificial Sequencetight junction
antagonist peptide 4Gly Arg Val Leu Val Gln Asp Gly1
558PRTArtificial Sequencetight junction antagonist peptide 5Gly Arg
Leu Cys Val Gln Pro Gly1 568PRTArtificial Sequencetight junction
antagonist peptide 6Gly Arg Leu Cys Val Gln Asp Gly1
578PRTArtificial Sequencetight junction antagonist peptide 7Gly Arg
Leu Leu Val Gln Pro Gly1 588PRTArtificial Sequencetight junction
antagonist peptide 8Gly Arg Leu Leu Val Gln Asp Gly1
598PRTArtificial Sequencetight junction antagonist peptide 9Gly Arg
Gly Cys Val Gln Pro Gly1 5108PRTArtificial Sequencetight junction
antagonist peptide 10Gly Arg Gly Cys Val Gln Asp Gly1
5118PRTArtificial Sequencetight junction antagonist peptide 11Gly
Arg Gly Leu Val Gln Pro Gly1 5128PRTArtificial Sequencetight
junction antagonist peptide 12Gly Arg Gly Leu Val Gln Asp Gly1
5138PRTArtificial Sequencetight junction antagonist peptide 13Gly
Gly Val Cys Val Gln Pro Gly1 5148PRTArtificial Sequencetight
junction antagonist peptide 14Gly Gly Val Cys Val Gln Asp Gly1
5158PRTArtificial Sequencetight junction antagonist peptide 15Gly
Gly Val Leu Val Gln Pro Gly1 5168PRTArtificial Sequencetight
junction antagonist peptide 16Gly Gly Val Leu Val Gln Asp Gly1
5178PRTArtificial Sequencetight junction antagonist peptide 17Gly
Gly Leu Cys Val Gln Pro Gly1 5188PRTArtificial Sequencetight
junction antagonist peptide 18Gly Gly Leu Cys Val Gln Asp Gly1
5198PRTArtificial Sequencetight junction antagonist peptide 19Gly
Gly Leu Leu Val Gln Pro Gly1 5208PRTArtificial Sequencetight
junction antagonist peptide 20Gly Gly Leu Leu Val Gln Asp Gly1
5218PRTArtificial Sequencetight junction antagonist peptide 21Gly
Gly Gly Cys Val Gln Pro Gly1 5228PRTArtificial Sequencetight
junction antagonist peptide 22Gly Gly Gly Cys Val Gln Asp Gly1
5238PRTArtificial Sequencetight junction antagonist peptide 23Gly
Gly Gly Leu Val Gln Pro Gly1 5248PRTArtificial Sequencetight
junction antagonist peptide 24Gly Gly Gly Leu Val Gln Asp Gly1
52520PRTHomo sapiens 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu 20269PRTHomo sapiens
26Val Thr Phe Tyr Thr Asp Ala Val Ser1 52720PRTHomo
sapiensmisc_feature(16)..(16)Xaa can be any naturally occurring
amino acid 27Met Leu Gln Leu Ala Glu Ser Gly Gly Val Leu Val Gln
Pro Gly Xaa1 5 10 15Ser Asp Arg Leu 202811PRTHomo
sapiensmisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 28Glu Val Gln Leu Val Glu Ser Gly Gly Xaa Leu1 5
10298PRTArtificial Sequencetight junction antagonist peptide 29Val
Gly Val Leu Gly Arg Pro Gly1 5308PRTArtificial Sequencetight
junction antagonist peptide 30Val Asp Gly Phe Gly Arg Ile Gly1
53122PRTHomo sapiensmisc_feature(1)..(1)Xaa can be any naturally
occurring amino acid 31Xaa Gly Leu Val Leu Val Gly Val Asn Gly Phe
Gly Arg Ile Gly Arg1 5 10 15Ile Gly Arg Leu Val Ile 203220PRTHomo
sapiens 32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu 203314PRTVibrio cholerae 33Phe Cys
Ile Gly Arg Leu Cys Val Gln Asp Gly Phe Val Thr1 5 10
* * * * *