U.S. patent application number 16/245446 was filed with the patent office on 2020-07-16 for linaclotide compositions.
The applicant listed for this patent is Forest Laboratories Holdings Limited. Invention is credited to Mohammad Mafruhul Bari, Andreas Grill, Matthew Miller, Yun Mo, Matthew Ronsheim, Ritesh Sanghvi, Joseph Stainkamp.
Application Number | 20200222541 16/245446 |
Document ID | / |
Family ID | 49914484 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200222541 |
Kind Code |
A1 |
Sanghvi; Ritesh ; et
al. |
July 16, 2020 |
LINACLOTIDE COMPOSITIONS
Abstract
The present invention is directed to stable linaclotide
compositions and methods of treating gastrointestinal disorders in
patients in need thereof by providing the stable linaclotide
compositions.
Inventors: |
Sanghvi; Ritesh; (Berkeley
Heights, NJ) ; Miller; Matthew; (Maplewood, NJ)
; Grill; Andreas; (Hauppauge, NY) ; Mo; Yun;
(Dix Hills, NY) ; Bari; Mohammad Mafruhul; (Lake
Grove, NY) ; Ronsheim; Matthew; (Port Jefferson,
NY) ; Stainkamp; Joseph; (Holtsville, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Forest Laboratories Holdings Limited |
Hamilton |
|
BM |
|
|
Family ID: |
49914484 |
Appl. No.: |
16/245446 |
Filed: |
January 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15991194 |
May 29, 2018 |
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16245446 |
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15461923 |
Mar 17, 2017 |
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15991194 |
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15068454 |
Mar 11, 2016 |
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15461923 |
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14489718 |
Sep 18, 2014 |
9283261 |
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15068454 |
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13799982 |
Mar 13, 2013 |
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14489718 |
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61670875 |
Jul 12, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/10 20130101;
A61K 47/26 20130101; A61K 9/1676 20130101; A61K 9/5015 20130101;
A61K 47/183 20130101; Y02A 50/30 20180101; A61K 47/42 20130101;
A61P 1/04 20180101; Y02A 50/414 20180101; A61K 47/02 20130101; C07K
7/08 20130101; A61K 9/0053 20130101; A61K 47/22 20130101; A61K
38/10 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 47/22 20060101
A61K047/22; C07K 7/08 20060101 C07K007/08; A61K 9/16 20060101
A61K009/16; A61K 38/10 20060101 A61K038/10; A61K 9/00 20060101
A61K009/00; A61K 47/02 20060101 A61K047/02; A61K 47/18 20060101
A61K047/18; A61K 47/26 20060101 A61K047/26; A61K 9/50 20060101
A61K009/50 |
Claims
1. A linaclotide composition comprising linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound.
2. The composition of claim 1, wherein the composition further
comprises a peptide having the structure of formula (I) or a
pharmaceutically acceptable salt thereof: ##STR00065##
3. The composition of claim 2, wherein the linaclotide composition
comprises the peptide of formula (I) in a concentration of up to 5%
by weight as compared to the weight of linaclotide.
4. The composition of claim 2, wherein the linaclotide composition
comprises the peptide of formula (I) in a concentration of up to 3%
by weight as compared to the weight of linaclotide.
5. The composition of claim 2, wherein the linaclotide composition
comprises the peptide of formula (I) in a concentration of up to 2%
by weight as compared to the weight of linaclotide.
6. The composition of claim 2, wherein the linaclotide composition
comprises the peptide of formula (I) in a concentration of up to 1%
by weight as compared to the weight of linaclotide.
7. A linaclotide composition comprising a formaldehyde scavenger
compound and an oral pharmaceutical dosage form comprising about
145 .mu.g or about 290 .mu.g of linaclotide or a pharmaceutically
acceptable salt thereof, Ca.sup.2+ or a salt thereof, leucine, and
a peptide having the structure of formula (I) or a pharmaceutically
acceptable salt thereof: ##STR00066##
8. The composition of claim 7, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 2 wt. % relative to the total weight of the
dosage form.
9. The composition of claim 7, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 1 wt. % relative to the total weight of the
dosage form.
10. The composition of claim 7, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.75 wt. % relative to the total weight of the
dosage form.
11. The composition of claim 7, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.5 wt. % relative to the total weight of the
dosage form.
12. The composition of claim 2, wherein the divalent metal cation
is Ca.sup.2+ or a salt thereof, the sterically hindered primary
amine is leucine, and wherein the linaclotide composition comprises
a molar ratio of divalent metal cation:sterically hindered primary
amine:linaclotide of about 57-63:28-32:1
13. The composition of claim 7, wherein the Ca.sup.2+ or a salt
thereof and leucine are present in the oral pharmaceutical dosage
form in a molar ratio of Ca.sup.2+:leucine:linaclotide of about
57-63:28-32:1.
14. The composition of claim 9, wherein the Ca.sup.2+ or a salt
thereof and leucine are present in the oral pharmaceutical dosage
form in a molar ratio of Ca.sup.2+:leucine:linaclotide of about
57-63:28-32:1.
15. The composition of claim 10, wherein the Ca.sup.2+ or a salt
thereof and leucine are present in the oral pharmaceutical dosage
form in a molar ratio of Ca.sup.2+:leucine:linaclotide of about
57-63:28-32:1.
16. The composition of claim 11, wherein the Ca.sup.2+ or a salt
thereof and leucine are present in the oral pharmaceutical dosage
form in a molar ratio of Ca.sup.2+:leucine:linaclotide of about
57-63:28-32:1.
17. The composition of claim 9, wherein the oral pharmaceutical
dosage form further comprises an oxidation product having a
structure of: ##STR00067## in an amount from about 0.05% to about
5% by weight relative to the total weight of the dosage form.
18. The composition of claim 17, wherein the oxidation product in
present in the dosage form in an amount of about 0.05% to about 2%
relative to the total weight of the dosage form.
19. The composition of claim 17, wherein the oxidation product in
present in the dosage form in an amount of about 0.05% to about 1%
relative to the total weight of the dosage form.
20. The composition of claim 7, wherein the Ca.sup.2+ or a salt
thereof and leucine are present in the oral pharmaceutical dosage
form in a molar ratio of Ca.sup.2+: leucine:linaclotide of about
57-63:28-32:1, wherein the formaldehyde scavenger compound is
selected from the group consisting of d-meglumine and histidine,
and wherein the peptide of formula (I) is present in the oral
pharmaceutical dosage form in an amount of about 0.001 to about 1
wt. % relative to the total weight of the dosage form.
21. The composition of claim 20, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.75 wt. % relative to the total weight of the
dosage form.
22. The composition of claim 20, wherein the peptide of formula (I)
is present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.5 wt. % relative to the total weight of the
dosage form.
23. The composition of claim 1, wherein the formaldehyde scavenger
compound is selected from the group consisting of leucine, glycine,
aspartame, d-meglumine, histidine, asparagine, proline, or a
mixture thereof.
24. The composition of claim 23, wherein the formaldehyde scavenger
compound is d-meglumine, histidine or a mixture thereof.
25. The composition of claim 23, wherein the formaldehyde scavenger
compound is d-meglumine.
26. The composition of claim 23, wherein the formaldehyde scavenger
compound is histidine.
27. The composition of claim 28, wherein the formaldehyde scavenger
compound is present in the linaclotide composition in molar ratio
of formaldehyde scavenger compound to linaclotide between 3:1 and
1:1.
Description
FIELD OF THE INVENTION
[0001] This invention relates to linaclotide compositions and
methods for treating gastrointestinal disorders.
BACKGROUND
[0002] U.S. Pat. Nos. 7,304,036 and 7,371,727 disclose peptides
that act as agonists of the guanylate cyclase C (GC-C) receptor for
the treatment of gastrointestinal disorders. One particular peptide
disclosed is linaclotide, which consists of the following amino
acid sequence (SEQ ID NO: 1):
##STR00001##
The '036 and '727 patents also disclose methods for preparing
linaclotide and related peptides. The contents of these patents are
incorporated herein by reference in their entirety.
[0003] There remains a need for improved linaclotide compositions
that have improved stability against formaldehyde, which can enter
linaclotide compositions from a variety of sources.
[0004] The present invention seeks to provide such improved
linaclotide compositions, as well as methods of treating
gastrointestinal disorders by providing the linaclotide
compositions to patients in need thereof.
SUMMARY OF THE INVENTION
[0005] The present invention relates in some embodiments to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound.
[0006] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound, and a peptide having the structure of formula
(I) or a pharmaceutically acceptable salt thereof (formula I
disclosed as SEQ ID NO: 3):
##STR00002##
[0007] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound, and a peptide having the structure of formula
(I) or a pharmaceutically acceptable salt thereof (formula VI
disclosed as SEQ ID NO: 2):
##STR00003##
[0008] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound, and a peptide having the structure of formula
(IV) or a pharmaceutically acceptable salt thereof (formula VII
disclosed as SEQ ID NO: 18):
##STR00004##
[0009] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound, and one or both of the peptide of formula (VI)
and the peptide of formula (VII).
[0010] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation, a formaldehyde
scavenger compound, a peptide having the structure of formula (I)
or a pharmaceutically acceptable salt thereof, and one or both of
the peptide of formula (VI) and the peptide of formula (VII).
[0011] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation and a peptide
having the following structure of formula (VIII) or a
pharmaceutically acceptable salt thereof (formula VIII disclosed as
SEQ ID NO: 3):
##STR00005##
[0012] In some embodiments, the present invention relates to stable
linaclotide compositions that comprises linaclotide, a sterically
hindered primary amine, a divalent metal cation, a formaldehyde
scavenger compound, a peptide having the structure of formula (I)
and one or both of the peptide of formula (III) and the peptide of
formula (IV).
[0013] In some embodiments, the present invention relates to stable
linaclotide compositions that comprise linaclotide, a sterically
hindered primary amine, a divalent metal cation and a formaldehyde
scavenger compound, a peptide having the structure of formula (I)
or a pharmaceutically acceptable salt thereof (formula I disclosed
as SEQ ID NO: 3):
##STR00006##
and a peptide having a structure of Formula (II) or a
pharmaceutically acceptable salt thereof (formula II disclosed as
SEQ ID NO: 4):
##STR00007##
[0014] In some embodiments, the present invention relates to stable
compositions that comprise linaclotide or a pharmaceutically
acceptable salt thereof, a peptide or a pharmaceutically acceptable
salt thereof that comprises the amino acid sequence Cys Cys Glu Tyr
Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 25) (wherein at
least one carboxyl group of the peptide is an alkyl ester having
the formula (-COOR) in which R is a C.sub.1-6 alkyl), and one or
more formaldehyde scavenger compounds.
[0015] In some embodiments, a stable linaclotide composition is
provided which comprises linaclotide or a pharmaceutically
acceptable salt thereof, a peptide having the structure of formula
(I) or a pharmaceutically acceptable salt thereof, and one or more
formaldehyde scavenger compounds (formula I disclosed as SEQ ID NO:
3):
##STR00008##
[0016] In some embodiments, a stable linaclotide composition is
provided which comprises linaclotide or a pharmaceutically
acceptable salt thereof, a peptide (e.g., a formaldehyde imine
product, e.g., a formaldehyde imine product) having the structure
of formula (II) or a pharmaceutically acceptable salt thereof, and
one or more formaldehyde scavenger compounds (formula II disclosed
as SEQ ID NO: 8):
##STR00009##
[0017] In some embodiments, a stable linaclotide composition is
provided which comprises linaclotide, one or more formaldehyde
scavenger compound, a first peptide having the structure of formula
(I) or a pharmaceutically acceptable salt thereof (formula I
disclosed as SEQ ID NO: 3):
##STR00010##
and a second peptide having the structure of Formula (II) or a
pharmaceutically acceptable salt thereof (formula II disclosed as
SEQ ID NO: 4):
##STR00011##
[0018] In some embodiments, a stable linaclotide composition is
provided which comprises linaclotide, one or more formaldehyde
scavenger compound, a first peptide having the structure of formula
(I) or a pharmaceutically acceptable salt thereof, and one or both
of (i) a peptide having the structure of formula (III) and (ii) a
peptide having the structure of formula (IV).
[0019] In some embodiments, the linaclotide composition comprises a
linaclotide peptide comprising the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 3) which is
modified with the addition of methylene at the .alpha.-amine group
of the N-terminal Cys.sub.1 which is cross-linked to the amine
group of Cyst to form an imidazolidinone 5 membered ring at the
N-terminus of the peptide ("Cys.sub.1-IMD"), wherein
"Cys.sub.1-IMD" refers to the linaclotide imidazolidinone
derivative modified on its N-terminal amine group. In some
embodiments, the imine modification may be produced by a
formaldehyde mediated reaction in the presence of acid
catalyst.
[0020] In some embodiments, stable compositions are provided which
comprise linaclotide or a pharmaceutically acceptable salt thereof,
a peptide having the structure of formula (III) or a
pharmaceutically acceptable salt thereof, and one or more
formaldehyde scavenger compounds (formula III disclosed as SEQ ID
NO: 19):
##STR00012##
wherein R' is H or a C.sub.1-6 alkyl, and at least one R' is
C.sub.1-6 alkyl.
[0021] In some embodiments, stable compositions are provided which
comprise linaclotide or a pharmaceutically acceptable salt thereof,
a peptide having the structure of formula (IV) or a
pharmaceutically acceptable salt thereof, and one or more
formaldehyde scavenger compounds (formula IV disclosed as SEQ ID
NO: 6):
##STR00013##
wherein R is a C.sub.1-6 alkyl.
[0022] In some embodiments, stable compositions are provided which
comprise linaclotide or a pharmaceutically acceptable salt thereof,
a peptide having the structure of formula (V) or a pharmaceutically
acceptable salt thereof, and one or more formaldehyde scavenger
compounds (formula V disclosed as SEQ ID NO: 7):
##STR00014##
wherein R is C.sub.1-6 alkyl.
[0023] Another aspect of the present invention provides a method
for treating a gastrointestinal disorder, which includes providing
the linaclotide composition to a patient diagnosed with a
gastrointestinal disorder.
[0024] The details of one or more embodiments of the invention are
set forth in the accompanying description.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows the dose response of exemplary peptides of the
present invention in a T84 cell c-GMP assay.
[0026] FIG. 2 demonstrates an example of an analysis of exemplary
peptides by RP-HPLC, wherein "Cyst-IMD" refers to the linaclotide
imidazolidinone derivative modified on its N-terminal amine
group.
[0027] FIG. 3 shows specific binding of linaclotide and
Cys.sub.1-IMD to cell-surface GC-C receptors on T84 cells in a
competitive radioligand binding assay.
[0028] FIG. 4 shows the dose response of exemplary peptides of the
present invention in a T84 cell c-GMP assay.
[0029] The figures are provided by way of example and are not
intended to limit the scope of the present invention.
DETAILED DESCRIPTION
[0030] Guanylate cyclase C (GC-C) is a transmembrane receptor that
is located on the apical surface of epithelial cells in the stomach
and intestine. The receptor has an extracellular ligand-binding
domain, a single transmembrane region and a C-terminal guanylyl
cyclase domain. When a ligand binds to the extracellular domain of
GC-C, the intracellular catalytic domain catalyzes the production
of cGMP from GTP. In vivo, this increase in intracellular cGMP
initiates a cascade of events that leads to increased secretion of
chloride and bicarbonate into the intestinal lumen, increased
luminal pH, decreased luminal sodium absorption, increased fluid
secretion, and acceleration of intestinal transit. cGMP is secreted
bidirectionally from the epithelium into the mucosa and lumen. The
peptides and compositions of the present invention bind to the
intestinal GC-C receptor which is a regulator of fluid and
electrolyte balance in the intestine.
Formaldehyde Scavenger Compounds
[0031] It has been discovered that the stability of linaclotide
compositions can be increased to a surprisingly high degree by
including in the linaclotide composition a suitable/appropriate
amount of one or more formaldehyde scavenger compounds.
[0032] While not wishing to be bound by any theory, it is believed
that the formaldehyde scavenger compounds increase linaclotide
stability by reacting with formaldehyde in such a manner that the
formaldehyde has a lesser capacity/ability to react with
linaclotide. In this regard, the formaldehyde scavenger compound
can be any compound that reduces exposure of the linaclotide to
formaldehyde in the composition, such as by reacting or interacting
with at least a portion of formaldehyde that enters the linaclotide
composition or gets proximate to linaclotide. Such formaldehyde can
enter the linaclotide composition from a variety of sources and
otherwise have deleterious effects on linaclotide and drug product
stability.
[0033] In some preferred embodiments, the formaldehyde scavenger
compound comprises a nitrogen center that is reactive with
formaldehyde, such as to form one or more reversible or
irreversible bonds between the formaldehyde scavenger compound and
the formaldehyde (preferably in some embodiments one or more
irreversible covalent bonds). For example, in some preferred
embodiments, the formaldehyde scavenger compound comprises one or
more nitrogen atoms/centers that are reactive with formaldehyde to
form a schiff base imine that is capable of subsequently binding
with formaldehyde. In some preferred embodiments, the formaldehyde
scavenger compound(s) comprise one or more nitrogen centers that
are reactive with formaldehyde to form one or more 5-8 member
cyclic rings. In this regard, the formaldehyde scavenger compound
preferably comprises one or more amine or amide groups. For
example, the formaldehyde scavenger compound can be an amino acid,
an amino sugar, an alpha amine compound, or a conjugate or
derivative thereof, or a mixture thereof. In some preferred
embodiments, the formaldehyde scavenger compound comprises two or
more amines and/or amides.
[0034] It has also been surprisingly discovered in some
embodiments, that formaldehyde scavenger compounds that comprise
multiple amine binding sites achieve surprisingly high stabilizing
effects on linaclotide as is demonstrated in Example 5. For
example, some preferred formaldehyde scavenger compounds comprise
one or more (e.g., two or more) primary amines. These include, for
example, glycine, alanine, serine, threonine, cysteine, valine,
lecuine, isoleucine, methionine, phenylalanine, tyrosine, aspartic
acid, glutamic acid, arginine, lysine, ornithine, citrulline,
taurine pyrrolysine, or a conjugate or mixture thereof.
[0035] In some especially preferred embodiments, the formaldehyde
scavenger compound(s) comprises one or more secondary amines (which
have been found in some embodiments to have higher reactivity with
formaldehyde and thus higher stabilizing effects on linaclotide
drug products). Some especially preferred formaldehyde scavenger
compounds comprise two or more secondary amines. These include, for
example, megulmine, histidine, aspartame, proline, tryptophan,
citrulline, pyrrolysine, or a conjugate or mixture thereof
[0036] Other preferred formaldehyde scavenger compounds comprise
one or more (e.g., two or more) primary amines and one or more
(e.g., two or more) secondary amines. These include, for example,
arginine, citrulline, pyrrolysine, or a conjugate or mixture
thereof.
[0037] Moreover, the formaldehyde scavenger compounds can comprise
one or more amide groups. For example, the scavenger compound can
be asparagine, glutamine, citrulline, or a conjugate or mixture
thereof.
[0038] The formaldehyde scavenger compound can be introduced into
the linaclotide composition in any suitable form. For example, the
scavenger can be lyophilized. Alternatively, or in addition, the
formaldehyde scavenger compound can be applied (e.g., sprayed) onto
beads before being introduced into the linaclotide composition.
These beads can be made of any suitable material, such as for
example cellulose, glass, sugar(s) or a combination or mixture
thereof.
[0039] In some embodiments, it has been surprisingly found (see
Example 5) that formaldehyde scavenger compounds may have higher
stabilizing effects on linaclotide when the formaldehyde scavenger
compound used is in amorphous form. Therefore, in some especially
preferred embodiments, the formaldehyde scavenger compound is
amorphous.
[0040] Lastly, the formaldehyde scavenger compound can be present
in the linaclotide composition in any suitable amount. In some
preferred embodiments, the linaclotide composition comprises a
ratio of scavenger to leucine between about 10:1 and about 1:1,
preferably between about 7:1 and about 1:1, even more preferably
between about 5:1 and about 1:1 or even between about 3:1 and about
1:1.
Exemplary Peptides in the Linaclotide Composition:
[0041] The linaclotide compositions may further comprise one or
more of the peptides described herein.
[0042] In various embodiments, the linaclotide composition
comprises a peptide that is modified wherein at least one carboxyl
group of the amino acid residue of the peptide is modified to an
alkyl ester. This modification may be produced, for example, by
treating a carboxylic acid with an alcohol in the presence of a
dehydrating agent wherein the dehydrating agent can include but is
not limited to a strong acid such as sulfuric acid. Other methods
of producing alkyl esters from carboxyl groups are readily known in
those skilled in the arts and are incorporated herein.
[0043] As used herein, a carboxyl group has the formula:
(--COOH).
[0044] As used herein, the term "alkyl", refers to a saturated
linear or branched-chain monovalent hydrocarbon radical.
[0045] As used herein, a group is terminal or terminus when the
group is present at the end of the amino acid sequence.
[0046] As used herein, an amine group on a peptide has the
formula:
##STR00015##
wherein R.sup.2 is the rest of the peptide.
[0047] As used herein, an imine group on a peptide has the
formula:
##STR00016##
wherein R.sup.2 is the rest of the peptide.
[0048] In some embodiments, the carboxylic acid of the side chain
of a glutamate amino acid in a peptide sequence is modified into an
alkyl ester.
[0049] In further embodiments, the carboxylic acid on the side
chain of a glutamate amino acid a peptide sequence is modified into
an ethyl ester.
[0050] In other embodiments, the C-terminus carboxylic acid of a
tyrosine amino acid in a peptide sequence is modified into an alkyl
ester.
[0051] In further embodiments, the C-terminus carboxylic acid of a
tyrosine amino acid of a peptide sequence is modified into an ethyl
ester.
[0052] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt thereof, wherein the
peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 25), wherein at least
one carboxyl group of the peptide is an alkyl ester having the
formula (--COOR) in which R is a C.sub.1-6 alkyl.
[0053] In several embodiments, the linaclotide composition
comprises a peptide having an amino acid structure of (SEQ ID NO:
19):
##STR00017##
or a pharmaceutically acceptable salt thereof, wherein R' is H or a
C.sub.1-6 alkyl, and at least one R' is C.sub.1-6 alkyl.
[0054] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 6):
##STR00018##
wherein R is a C.sub.1-6 alkyl ("Glu3-alkyl ester").
[0055] In other embodiments, R is a C.sub.1-4 alkyl.
[0056] In further embodiments, R is methyl, ethyl, or propyl.
[0057] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of ("Glu3-ethyl ester") (SEQ
ID NO: 10):
##STR00019##
[0058] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 11):
##STR00020##
[0059] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 9):
##STR00021##
[0060] In some embodiments, the C-terminal tyrosine of the
Glu3-alkyl ester or pharmaceutically acceptable salt is absent.
[0061] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 7):
##STR00022##
[0062] wherein R is C.sub.1-6 alkyl.
[0063] In other embodiments, R is a C.sub.1-4 alkyl.
[0064] In further embodiments, R is methyl, ethyl, or propyl.
[0065] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of ("Tyr.sub.14-ethyl
ester") (SEQ ID NO: 13):
##STR00023##
[0066] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 14):
##STR00024##
[0067] In some embodiments, the linaclotide composition comprises a
peptide having an amino acid structure of (SEQ ID NO: 12):
##STR00025##
[0068] In addition, the linaclotide composition may comprise a
peptide that is modified wherein at least one amine group of the
amino acid residues of the peptide is modified into an imine. This
modification may be produced, for example, by treating an amine
group with a carbonyl, such as an aldehyde or ketone, in the
presence of acid catalyst. Other methods of producing imines from
amine groups are readily known to those skilled in the arts and are
incorporated herein.
[0069] In some embodiments, the imine modification may be produced
by a formaldehyde mediated reaction in the presence of acid
catalyst.
[0070] In further embodiments, the linaclotide composition
comprises a peptide having an imine carbon that is cross-linked to
another amine group of the peptide.
[0071] In other embodiments, the linaclotide composition comprises
a peptide that is modified into an imine at the .alpha.-amine group
of the N-terminal amino acid, wherein the imine carbon is
cross-linked with an amine group of the second amino acid residue
of the peptide forming a five membered ring.
[0072] In other embodiments, the linaclotide composition comprises
a peptide having the amino acid sequence Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 3) wherein the peptide
may be modified with the addition of methylene at the .alpha.-amine
group of the N-terminal Cys.sub.1 which is cross-linked to the
amine group of Cys.sub.2 to form an imidazolidinone 5 membered ring
at the N-terminus of the peptide ("Cys.sub.1-IMD").
[0073] In several embodiments, the linaclotide composition
comprises a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide comprising the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 8), wherein
at least one amine group of the peptide is an imine having the
formula
##STR00026##
wherein R.sup.2 is the rest of the peptide.
[0074] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt comprises a peptide
wherein the N-terminal amine group of the peptide is an imine
having the formula
##STR00027##
wherein R.sup.2 is the rest of the peptide.
[0075] In further embodiments, the linaclotide composition
comprises a peptide or pharmaceutically acceptable salt thereof
comprising an amino acid structure of (SEQ ID NO: 8):
##STR00028##
[0076] In several embodiments, the linaclotide composition
comprises a peptide or pharmaceutically acceptable salt thereof
comprising an amino acid structure of (SEQ ID NO: 3):
##STR00029##
[0077] In some embodiments, the C-terminal tyrosine of the
Cys.sub.1-IMD peptide or pharmaceutically acceptable salt thereof
is absent. In some embodiments, the Cys.sub.1-IMD peptide or
pharmaceutically acceptable salt thereof further comprises one or
more peptide modifications, wherein at least one carboxyl group of
the peptide is an alkyl ester having the formula (--COOR) in which
R is a C.sub.1-6 alkyl.
[0078] In several embodiments, the linaclotide composition
comprises a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide consists of the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 25),
wherein at least one carboxyl group of the peptide is an alkyl
ester having the formula (--COOR) in which R is a C.sub.1-6
alkyl.
[0079] In some embodiments, the linaclotide composition comprises
linaclotide, a sterically hindered primary amine, a divalent metal
cation and a formaldehyde scavenger compound, and a peptide having
the structure of formula (I) or a pharmaceutically acceptable salt
thereof (formula VI disclosed as SEQ ID NO: 2):
##STR00030##
[0080] In some embodiments, the linaclotide composition comprises
linaclotide, a sterically hindered primary amine, a divalent metal
cation and a formaldehyde scavenger compound, and a peptide having
the structure of formula (IV) or a pharmaceutically acceptable salt
thereof (formula VII disclosed as SEQ ID NO: 18):
##STR00031##
[0081] In some embodiments, the linaclotide composition comprises
linaclotide, a sterically hindered primary amine, a divalent metal
cation and a formaldehyde scavenger compound, and one or both of
the peptide of formula (VI) and the peptide of formula (VII).
[0082] In some embodiments, the linaclotide composition comprises
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, a peptide having the
structure of formula (I) or a pharmaceutically acceptable salt
thereof, and one or both of the peptide of formula (VI) and the
peptide of formula (VII).
[0083] In some embodiments, the linaclotide composition comprises
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, a peptide having the
following structure of formula (VIII) or a pharmaceutically
acceptable salt thereof (formula VIII disclosed as SEQ ID NO:
3):
##STR00032##
[0084] In several embodiments, the linaclotide composition
comprises a peptide that consists of an amino acid structure of
(SEQ ID NO: 19):
##STR00033##
[0085] or a pharmaceutically acceptable salt thereof, wherein R' is
H or a C.sub.1-6 alkyl, and at least one R' is C.sub.1-6 alkyl.
[0086] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of an amino acid structure of (SEQ ID NO: 6):
##STR00034##
wherein R is a C.sub.1-6 alkyl. In further embodiments, the
C-terminal tyrosine is absent.
[0087] In other embodiments, R is a C.sub.1-4 alkyl.
[0088] In further embodiments, R is methyl, ethyl, or propyl.
[0089] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of a peptide having an amino acid structure of (SEQ ID NO: 10):
##STR00035##
[0090] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of a peptide having an amino acid structure of (SEQ ID NO: 11):
##STR00036##
[0091] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of a peptide having an amino acid structure of (SEQ ID NO: 9):
##STR00037##
[0092] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof wherein the
peptide has an amino acid structure of (SEQ ID NO: 7):
##STR00038##
wherein R is C.sub.1-6 alkyl.
[0093] In other embodiments, R is a C.sub.1-4 alkyl.
[0094] In further embodiments, R is methyl, ethyl, or propyl.
[0095] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of an amino acid structure of (SEQ ID NO: 13):
##STR00039##
[0096] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of an amino acid structure of (SEQ ID NO: 14):
##STR00040##
[0097] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of an amino acid structure of (SEQ ID NO: 12):
##STR00041##
[0098] In several embodiments, the linaclotide composition
comprises a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide consists of the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 8), wherein
at least one amine group of the peptide is an imine having the
formula
##STR00042##
wherein R.sup.2 is the rest of the peptide.
[0099] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt thereof having the
structure of formula (III) (SEQ ID NO: 2):
##STR00043##
[0100] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt thereof having the
structure of formula (IV) (SEQ ID NO: 18):
##STR00044##
[0101] In some embodiments, the linaclotide composition comprises
one or both of a peptide or a pharmaceutically acceptable salt
thereof having the structure of formula (III) and a peptide having
the structure of formula (IV).
[0102] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt thereof having the
structure of formula (III) and a peptide or a pharmaceutically
acceptable salt thereof having the structure of formula (IV).
[0103] In some embodiments, the linaclotide composition comprises a
peptide or a pharmaceutically acceptable salt that consists of a
peptide wherein the N-terminal amine group of the peptide is an
imine having the formula
##STR00045##
wherein R.sup.2 is the rest of the peptide.
[0104] In further embodiments, the linaclotide composition
comprises a peptide or pharmaceutically acceptable salt thereof
that consists of an amino acid structure of (SEQ ID NO: 8):
##STR00046##
[0105] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt thereof that consists
of an amino acid structure of (SEQ ID NO: 3):
##STR00047##
[0106] In some embodiments, the linaclotide composition comprises
the Cys.sub.1-IMD peptide or pharmaceutically acceptable salt
wherein the C-terminal tyrosine of the Cys.sub.1-IMD peptide or
pharmaceutically acceptable salt thereof is absent. In some
embodiments, the Cys.sub.1IMD peptide or pharmaceutically
acceptable salt thereof further comprises one or more peptide
modifications, e.g., wherein at least one carboxyl group of the
peptide is an alkyl ester having the formula (--COOR), wherein R is
a C.sub.1-6 alkyl.
Production of Peptides
[0107] The peptides or precursor peptides described herein can be
produced in any suitable manner such as recombinantly in any known
protein expression system, including, without limitation, bacteria
(e.g., E. coli or Bacillus subtilis), insect cell systems (e.g.,
Drosophila Sf9 cell systems), yeast cell systems (e.g., S.
cerevisiae, S. saccharomyces) or filamentous fungal expression
systems, or animal cell expression systems (e.g., mammalian cell
expression systems). Peptides or precursor peptides of the
invention may also be chemically synthesized.
[0108] If the peptide or variant peptide is to be produced
recombinantly, e.g., E. coli, the nucleic acid molecule encoding
the peptide may also encode a leader sequence that permits the
secretion of the mature peptide from the cell. Thus, the sequence
encoding the peptide can include the pre sequence and the pro
sequence of, for example, a naturally-occurring bacterial ST
peptide. The secreted, mature peptide can be purified from the
culture medium.
[0109] The sequence encoding a peptide described herein can be
inserted into a vector capable of delivering and maintaining the
nucleic acid molecule in a bacterial cell. The DNA molecule may be
inserted into an autonomously replicating vector (suitable vectors
include, for example, pGEM3Z and pcDNA3, and derivatives thereof).
The vector nucleic acid may be a bacterial or bacteriophage DNA
such as bacteriophage lambda or M13 and derivatives thereof.
Construction of a vector containing a nucleic acid described herein
can be followed by transformation of a host cell such as a
bacterium. Suitable bacterial hosts include but are not limited to,
E. coli, B. subtilis, Pseudomonas and Salmonella. The genetic
construct also includes, in addition to the encoding nucleic acid
molecule, elements that allow expression, such as a promoter and
regulatory sequences. The expression vectors may contain
transcriptional control sequences that control transcriptional
initiation, such as promoter, enhancer, operator, and repressor
sequences. A variety of transcriptional control sequences are well
known to those in the art. The expression vector can also include a
translation regulatory sequence (e.g., an untranslated 5' sequence,
an untranslated 3' sequence, or an internal ribosome entry site).
The vector can be capable of autonomous replication or it can
integrate into host DNA to ensure stability during peptide
production.
[0110] The protein coding sequence that includes a peptide
described herein can also be fused to a nucleic acid encoding a
peptide affinity tag, e.g., glutathione S-transferase (GST),
maltose E binding protein, protein A, FLAG tag, hexa-histidine (SEQ
ID NO: 26), myc tag or the influenza HA tag, in order to facilitate
purification. The affinity tag or reporter fusion joins the reading
frame of the peptide of interest to the reading frame of the gene
encoding the affinity tag such that a translational fusion is
generated. Expression of the fusion gene results in translation of
a single peptide that includes both the peptide of interest and the
affinity tag. In some instances where affinity tags are utilized,
DNA sequence encoding a protease recognition site will be fused
between the reading frames for the affinity tag and the peptide of
interest.
[0111] Genetic constructs and methods suitable for production of
immature and mature forms of the peptides and variants described
herein in protein expression systems other than bacteria, and well
known to those skilled in the art, can also be used to produce
peptides in a biological system.
[0112] In other embodiments, peptides containing amino acids not
normally incorporated by the translation machinery and described
above (e.g. --.beta.-carboxylated Asp, .gamma.-carboxylated Glu,
Asu, Aad and Apm) may be recombinantly produced by tRNA
modification methods. Methods for modifying tRNA including, but not
limited to, modifying the anti-codon, the amino acid attachment
site, and/or the accepter stem to allow incorporation of unnatural
and/or arbitrary amino acids are known in the art (Biochem.
Biophys. Res. Comm. (2008) 372: 480-485; Chem. Biol. (2009)
16:323-36; Nat. Methods (2007) 4:239-44; Nat. Rev. Mol. Cell Biol.
(2006) 7:775-82; Methods (2005) 36:227-238; Methods (2005)
36:270-278; Annu. Rev. Biochem. (2004) 73:147-176; Nuc. Acids Res.
(2004) 32:6200-6211; Proc. Natl. Acad. Sci. USA (2003)
100:6353-6357; Royal Soc. Chem. (2004) 33:422-430).
[0113] In some embodiments, peptides may be chemically produced.
Peptides can be synthesized by a number of different methods
including solution and solid phase synthesis using traditional BOC
or FMOC protection. For example, the peptide can be synthesized on
2-Chlorotrityl or Wang resin using consecutive amino acid
couplings. The following protecting groups can be used:
Fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl (alpha-amino
groups, N-terminus); trityl or tert-butyl (thiol groups of Cys);
tert-butyl (.gamma.-carboxyl of glutamic acid and the hydroxyl
group of threonine, if present); and trityl (.beta.-amid function
of the asparagine side chain and the phenolic group of tyrosine, if
present). Coupling can be effected with DIC and HOBt in the
presence of a tertiary amine, and the peptide can be deprotected
and cleaved from the solid support in using cocktail K
(trifluoroacetic acid 81%, phenol 5%, thioanisole 5%,
1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium
iodide 1.5% w/w). After removal of trifluoroacetic acid and other
volatiles the peptide can be precipitated using an organic solvent
. Disulfide bonds between Cys residues can be formed using dimethyl
sulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62) or
using an air oxidation strategy. The resulting peptide can be
purified by reverse-phase chromatography and lyophilized.
[0114] These peptides can be made, isolated or used either in form
of the base or as pharmaceutically acceptable salts thereof.
Examples of salts include, without limitation, acetate, chloride,
sulfate and phosphate salts of the peptide.
[0115] In some embodiments, the linaclotide composition comprises
linaclotide or a pharmaceutically acceptable salt and a two or more
peptides selected from:
i. a peptide or a pharmaceutically acceptable salt thereof, wherein
the peptide comprises the amino acid structure of (SEQ ID NO:
2):
##STR00048##
ii. a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide comprises an amino acid structure of (SEQ ID
NO: 1):
##STR00049##
and iii. a peptide or a pharmaceutically acceptable salt thereof,
wherein the peptide comprises the amino acid sequence Cys Cys Glu
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 25),
wherein at least one carboxyl group of the peptide is an alkyl
ester having the formula (--COOR) in which R is a C.sub.1-6
alkyl.
[0116] In other embodiments, the linaclotide composition comprises
linaclotide or a pharmaceutically acceptable salt thereof, one or
more formaldehyde scavenger compounds, and a peptide or a
pharmaceutically acceptable salt thereof, wherein the peptide
comprises an amino acid structure of formula (I) (SEQ ID NO:
3):
##STR00050##
wherein the linaclotide composition contains up to 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, or 90% by weight of the peptide of formula
(I) as compared to the weight of linaclotide. In some preferred
embodiments, the linaclotide composition contains up to 8% by
weight of the peptide of formula (I) as compared to the weight of
linaclotide. In some preferred embodiments, the linaclotide
composition contains up to 7% by weight of the peptide of formula
(I) as compared to the weight of linaclotide. In some preferred
embodiments, the linaclotide composition contains up to 6% by
weight of the peptide of formula (I) as compared to the weight of
linaclotide. In some preferred embodiments, the linaclotide
composition contains up to 5% by weight of the peptide of formula
(I)as compared to the weight of linaclotide. In some preferred
embodiments, the linaclotide composition contains up to 4% by
weight of the peptide of formula (I) as compared to the weight of
linaclotide. In some preferred embodiments, the linaclotide
composition contains up to 3% by weight of the peptide of formula
(I) as compared to the weight of linaclotide. In some preferred
embodiments, the linaclotide composition contains up to 2% by
weight of the peptide of formula (I) as compared to the weight of
linaclotide. In some preferred embodiments, the linaclotide
composition contains up to 1% by weight of the peptide of formula
(I) as compared to the weight of linaclotide. In some preferred
embodiments, the linaclotide composition contains up to 0.75% by
weight of the peptide of formula (I) as compared to the weight of
linaclotide. In some preferred embodiments, the linaclotide
composition contains up to 0.5% by weight of the peptide of formula
(I) as compared to the weight of linaclotide.
[0117] In some preferred embodiments, the linaclotide composition
contains one or more formaldehyde scavenger compounds and an oral
pharmaceutical dosage form that comprises linaclotide and the
peptide of formula (I) wherein the peptide of formula (I) is
present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 2 wt. % relative to the total weight of the
dosage form.
[0118] In some preferred embodiments, the linaclotide composition
contains one or more formaldehyde scavenger compounds and an oral
pharmaceutical dosage form that comprises linaclotide and the
peptide of formula (I) wherein the peptide of formula (I) is
present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 1 wt. % relative to the total weight of the
dosage form.
[0119] In some preferred embodiments, the linaclotide composition
contains one or more formaldehyde scavenger compounds and an oral
pharmaceutical dosage form that comprises linaclotide and the
peptide of formula (I) wherein the peptide of formula (I) is
present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.75 wt. % relative to the total weight of the
dosage form.
[0120] In some preferred embodiments, the linaclotide composition
contains one or more formaldehyde scavenger compounds and an oral
pharmaceutical dosage form that comprises linaclotide and the
peptide of formula (I) wherein the peptide of formula (I) is
present in the oral pharmaceutical dosage form in an amount of
about 0.001 to about 0.5 wt. % relative to the total weight of the
dosage form.
[0121] In some embodiments, the imidazolidinone derivative of
linaclotide comprises up to about 15% by weight of the linaclotide
composition, up to about 10% by weight of the linaclotide
composition, up to about 7% by weight of the linaclotide
composition or up to about 5% by weight of the linaclotide
composition. In other exemplary embodiments, the imidazolidinone
derivative of linaclotide comprises from about 0.01% to about 15%
by weight of the linaclotide composition, about 0.05% to about 10%
by weight of the linaclotide composition, about 0.05% to about 7%
by weight of the linaclotide composition or about 0.05% to about 5%
by weight of the linaclotide composition.
[0122] In other embodiments, the linaclotide composition comprises
linaclotide and a peptide or a pharmaceutically acceptable salt
thereof, wherein the peptide comprises an amino acid structure of
(SEQ ID NO: 13):
##STR00051##
wherein the peptide or pharmaceutically acceptable salt thereof
comprises up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by
weight compared to the weight of linaclotide.
[0123] In further embodiments, the linaclotide composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of (SEQ ID NO: 13):
##STR00052##
wherein the peptide or pharmaceutically acceptable salt thereof
comprises up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight
compared to the weight of linaclotide.
[0124] In some embodiments, the Tyr.sub.14-ethyl ester peptide
comprises up to about 15% by weight of the linaclotide composition,
up to about 10% by weight of the linaclotide composition, up to
about 7% by weight of the linaclotide composition or up to about 5%
by weight of the linaclotide composition. In other exemplary
embodiments, the Tyr.sub.14-ethyl ester comprises from about 0.01%
to about 15% by weight of the linaclotide composition, about 0.05%
to about 10% by weight of the linaclotide composition, about 0.05%
to about 7% by weight of the linaclotide composition or about 0.05%
to about 5% by weight of the linaclotide composition.
[0125] In other embodiments, the linaclotide composition comprising
linaclotide and a peptide or a pharmaceutically acceptable salt
thereof, wherein the peptide comprises an amino acid structure of
(SEQ ID NO: 10):
##STR00053##
wherein the peptide or pharmaceutically acceptable salt thereof
comprises up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by
weight compared to the weight of linaclotide.
[0126] In further embodiments, the linaclotide composition
comprises linaclotide and a peptide or a pharmaceutically
acceptable salt thereof, wherein the peptide comprises an amino
acid structure of (SEQ ID NO: 10):
##STR00054##
wherein the peptide or pharmaceutically acceptable salt thereof
comprises up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight
compared to the weight of linaclotide.
[0127] In some embodiments, the Glu3-ethyl ester peptide comprises
up to about 15% by weight of the linaclotide composition, up to
about 10% by weight of the linaclotide composition, up to about 7%
by weight of the linaclotide composition or up to about 5% by
weight of the linaclotide composition. In other exemplary
embodiments, the Glu3-ethyl ester comprises from about 0.01% to
about 15% by weight of the linaclotide composition, about 0.05% to
about 10% by weight of the linaclotide composition, about 0.05% to
about 7% by weight of the linaclotide composition or about 0.05% to
about 5% by weight of the linaclotide composition.
[0128] In some embodiments, the linaclotide composition comprises a
peptide or pharmaceutically acceptable salt, wherein the peptide
consists of the amino acid structure of (SEQ ID NO: 3):
##STR00055##
[0129] In other embodiments, the linaclotide composition consists
essentially of a peptide or pharmaceutically acceptable salt
thereof, wherein the peptide comprises the amino acid structure of
(SEQ ID NO: 3):
##STR00056##
[0130] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and one or more of the
peptides having the structures of formula (VI) and (VII) or
pharmaceutically acceptable salt(s) thereof, wherein the
linaclotide composition contains a total combined amount of the
peptides of formula (VI) and (VII) up to 3%, 2%, 1%, 0.75%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to 0.01% by
weight as compared to the weight of linaclotide.
[0131] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (IX) or a pharmaceutically acceptable salt
thereof (formula IX disclosed as SEQ ID NO: 20):
##STR00057##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (IX) as compared to the
weight of linaclotide.
[0132] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (X) or a pharmaceutically acceptable salt
thereof (formula X disclosed as SEQ ID NO: 21):
##STR00058##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (X) as compared to the
weight of linaclotide.
[0133] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (XI) or a pharmaceutically acceptable salt
thereof (formula XI disclosed as SEQ ID NO: 22):
##STR00059##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (XI) as compared to the
weight of linaclotide.
[0134] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (XII) or a pharmaceutically acceptable salt
thereof (formula XII disclosed as SEQ ID NO: 23):
##STR00060##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (XII) as compared to the
weight of linaclotide.
[0135] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (XIII) or a pharmaceutically acceptable salt
thereof (formula XIII disclosed as SEQ ID NO: 24):
##STR00061##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (XIII) as compared to the
weight of linaclotide.
[0136] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (XIV) or a pharmaceutically acceptable salt
thereof (formula XIV disclosed as SEQ ID NO: 10):
##STR00062##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (XIV) as compared to the
weight of linaclotide.
[0137] In other embodiments, the linaclotide composition consists
linaclotide, a sterically hindered primary amine, a divalent metal
cation, a formaldehyde scavenger compound, and a peptide having the
structure of formula (XV) or a pharmaceutically acceptable salt
thereof (formula XV disclosed as SEQ ID NO: 13):
##STR00063##
wherein the linaclotide composition contains up to 3%, 2%, 1%,
0.75%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05%, or even up to
0.01% by weight of the peptide of formula (XV) as compared to the
weight of linaclotide.
Methods of Treatment
[0138] In various embodiments, a patient diagnosed with a
gastrointestinal disorder is provided with the linaclotide
composition described herein, wherein the gastrointestinal disorder
is selected from the group consisting of irritable bowel syndrome
(IBS) (such as constipation-predominant IBS), constipation (such as
chronic idiopathic constipation), a functional gastrointestinal
disorder, gastroesophageal reflux disease, functional heartburn,
dyspepsia, visceral pain, gastroparesis, chronic intestinal
pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease,
ulcerative colitis, and inflammatory bowel disease.
[0139] In a further embodiment, the gastrointestinal disorder is
constipation. The constipation can be chronic idiopathic
constipation, idiopathic constipation, due to post-operative ileus,
or caused by opiate use. Clinically accepted criteria that define
constipation include the frequency of bowel movements, the
consistency of feces and the ease of bowel movement. One common
definition of constipation is less than three bowel movements per
week. Other definitions include abnormally hard stools or
defecation that requires excessive straining (Schiller 2001,
Aliment Pharmacol Ther 15:749-763). Constipation may be idiopathic
(functional constipation or slow transit constipation) or secondary
to other causes including neurologic, metabolic or endocrine
disorders. These disorders include diabetes mellitus,
hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis,
Parkinson's disease, spinal cord lesions, Neurofibromatosis,
autonomic neuropathy, Chagas disease, Hirschsprung's disease and
Cystic fibrosis. Constipation may also be the result of surgery
(postoperative ileus) or due to the use of drugs such as analgesics
(like opioids), antihypertensives, anticonvulsants,
antidepressants, antispasmodics and antipsychotics.
[0140] In other embodiments, the gastrointestinal disorder is
irritable bowel syndrome (IBS). The irritable bowel syndrome can be
constipation-predominant irritable bowel syndrome (c-IBS),
diarrhea-predominant irritable bowel syndrome (d-IBS) or
alternating between the two irritable bowel syndromes (a-IBS).
[0141] In other embodiments, the gastrointestinal disorder is
dyspepsia.
[0142] In other embodiments, the gastrointestinal disorder is
gastroparesis. The gastroparesis can be selected from idiopathic,
diabetic or post-surgical gastroparesis.
[0143] In still other embodiments, the gastrointestinal disorder is
chronic intestinal pseudo obstruction.
[0144] In other embodiments, the gastrointestinal disorder is
Crohn's disease.
[0145] In some embodiments, the gastrointestinal disorder is
ulcerative colitis.
[0146] In some embodiments, the gastrointestinal disorder is
inflammatory bowel disease.
[0147] In still another embodiment, the invention features a method
for treating a gastrointestinal disorder comprising providing a
patient diagnosed with IBS-C or CC with the linaclotide composition
described herein.
[0148] It has been discovered in some embodiments, that linaclotide
is only to be administered to patients without known or suspected
mechanical gastrointestinal obstruction. In this regard, prior to
administering or providing the linaclotide composition to patients
diagnosied with a GI disorder, the patient is evaluated for
symptoms suggestive of mechanical gastrointestinal obstruction
prior to initiating treatment.
[0149] Moreover, in some embodiments, the linaclotide composition
is provided to patients diagnosed with a GI disorder and not
currently or frequently experiencing diarrhea.
[0150] In some embodiments, the method comprises after
administering or providing the linaclotide composition to a
patient, monitoring the patient for diarrhea, flatulence, abdominal
pain, abdominal distension, Defecation urgency, dyspepsia,
gastroesophageal reflux disease, upper abdominal pain, vomiting;
viral gastroenteritis; muscle strain; dizziness; sinus congestion,
fecal incontinence, dehydration and/or headache, and optionally
decreasing the linaclotide dose administered to patients
experiencing one or more of these adverse reactions to the initial
dosage.
[0151] In some embodiments, the method comprises after
administering or providing the linaclotide composition to a
patient, periodically assess the need for continued treatment with
linaclotide.
[0152] In some embodiments, the method comprises after
administering or providing the linaclotide composition to a
patient, determining that the patient receiving the linaclotide
composition is experiencing severe or intolerable diarrhea, and
reducing the linaclotide dose or suspending treatment.
[0153] In some embodiments, the method comprises diagnosing the
patient with IBS-C or CC, administering or providing the
linaclotide composition to the patient and counseling the patient
that improvement of bowel symptoms should occur within the first
week of treatment, but improvement of abdominal symptoms may take
longer.
[0154] In some embodiments, the method comprises periodically
assessing the need for continued treatment with the linaclotide
composition.
[0155] In this regard, the efficacy of oral dosage forms comprising
290 mg for the management of IBS-C was established in two
double-blind, placebo-controlled, randomized, multicenter studies
in adult patients. A total of 800 patients in Study 1 and 804
patients in Study 2 (overall mean age of 43.9 years [range 18-87
years with 5.3%.gtoreq.65 years of age], 90.1% female, 77.4% white,
18.8% black, and 12.0% Hispanic) received treatment with LINZESS
290 mcg or placebo once daily and were evaluated for efficacy. All
patients met Rome II criteria for IBS and were required to report a
mean abdominal pain score of .gtoreq.3 on a 0-to-10-point numeric
rating scale, <3 complete spontaneous bowel movements (CSBMs; a
CSBM is a spontaneous bowel movement [SBM] that is associated with
a sense of complete evacuation; an SBM is a bowel movement
occurring in the absence of laxative use), and .ltoreq.5 SBMs per
week during a 2-week baseline period. The study designs were
identical through the first 12 weeks, and thereafter differed only
in that Study 1 included a 4-week randomized withdrawal (RW)
period, and Study 2 continued for 14 additional weeks (total of 26
weeks) of double-blinded treatment .
[0156] Efficacy of the oral dosage form of linaclotide was assessed
using responder and change-from-baseline endpoints. Results for
endpoints were based on information provided daily by patients. An
abdominal pain responder was a patient who had .gtoreq.30%
reduction in mean abdominal pain from baseline in a given week for
.gtoreq.6 out of 12 weeks of the treatment period. A CSBM responder
was a patient who had an increase of .gtoreq.1 CSBM from baseline
in a given week for .gtoreq.6 out of 12 weeks of the treatment
period. To be a combined responder, a patient had to meet both
abdominal pain and CSBM weekly responder criteria in the same week
for .gtoreq.6 out of 12 weeks of the treatment period. The efficacy
results are shown in Table 2. In both studies, the proportion of
patients who were responders to the oral dosage form of 290 mcg
linaclotide was statistically significantly higher than with
placebo.
[0157] For change-from-baseline endpoints, patients who received
the oral dosage form of 290 mcg linaclotide across the 2 studies
had statistically significantly (p<0.0001) greater improvements
compared with patients receiving placebo in abdominal symptoms,
including abdominal pain, abdominal discomfort, and bloating; and
bowel function, including stool frequency (CSBM and SBM) and
consistency (i.e., hardness of stool), as well as straining.
Sixty-seven percent of the patients had an SBM within 24 hours of
taking their first dose versus 42% of placebo patients
(p<0.0001).
[0158] The proportions of patients who met response criteria of
increasing levels of symptom improvement compared to baseline
(i.e., decreases of .gtoreq.0%, .gtoreq.10%, .gtoreq.20%,
.gtoreq.30%, .gtoreq.40%, .gtoreq.50%, and .gtoreq.60% in abdominal
pain and increases of .gtoreq.0, .gtoreq.1, .gtoreq.2, .gtoreq.3,
.gtoreq.4, .gtoreq.5, and .gtoreq.6 CSBMs per week) over 12 weeks
of treatment were analyzed. At each level, a statistically
significantly greater proportion of patients treated with the oral
dosage form of 290 mcg linaclotide met the response criterion
compared to placebo patients. Moreover, the oral dosage form of 290
mcg linaclotide demonstrated a statistically significant separation
from placebo that was present at the first week and sustained
across the 26 weeks of the treatment period (p<0.001 at all time
points during the treatment period). Similar results for
improvement in CSBM frequency were demonstrated throughout the
26-week treatment period. Maximum effect on CSBM frequency occurred
by Week 1, but the effect on abdominal pain continued to increase
over the first 6 to 8 weeks. During the 4-week RW period in Study 2
when treatment with the oral dosage form of 290 mcg linaclotide was
discontinued, bowel symptoms returned toward baseline within the
first week with no evidence of rebound worsening compared to
baseline; abdominal symptoms also returned toward baseline with no
evidence of rebound.
[0159] The efficacy of the oral dosage form of 290 mcg linaclotide
for the management of CC was established in two double-blind,
placebo-controlled, randomized, multicenter studies in adult
patients. A total of 642 patients in Study 3 and 630 patients in
Study 4 (overall mean age of 47.8 years [range 18-85 years with
12.1%.gtoreq.65 years of age], 88.9% female, 76.2% white, 21.5%
black, Hispanic 10.0%) received treatment with the oral dosage form
of 145 or 290 mcg linaclotide, or placebo once daily and were
evaluated for efficacy. All patients met Rome II criteria for CC
and were required to report <3 CSBMs and .ltoreq.6 SBMs per week
during a 2-week baseline period. Patients were excluded if they met
criteria for IBS. The study designs differed only in that Study 3
had a 4-week RW period following the 12-week treatment period.
[0160] Efficacy of the oral dosage form of 145 or 290 mcg
linaclotide was assessed using responder and change-from-baseline
endpoints. Results for endpoints were based on information provided
daily by patients. A CSBM responder was defined differently in the
CC studies than it was in the IBS-C studies. A CSBM responder in
the CC studies was a patient who had .gtoreq.3 CSBMs and an
increase of .gtoreq.1 CSBM from baseline in a given week for
.gtoreq.9 out 12 weeks of the treatment period. both studies, the
proportion of patients who were CSBM responders was statistically
significantly greater with each dose of LINZESS (145 and 290 mcg)
than with placebo.
[0161] For change-from-baseline endpoints, patients who received
either dose of the oral dosage form of linaclotide across the 2
studies had statistically significantly (p<0.0001) greater
improvements compared with patients receiving placebo in abdominal
discomfort, bloating, stool frequency (CSBM and SBM), stool
consistency (i.e., hardness of stool), and straining. Sixty-seven
percent and 57% of linaclotide 145 and 290 mcg patients,
respectively, had an SBM within 24 hours of taking their first dose
versus 39% of placebo patients (p<0.0001 for both doses versus
placebo).
[0162] The proportions of patients who met response criteria of
increasing levels of stool frequency compared to baseline (i.e.,
increases of >0, .gtoreq.1, .gtoreq.2, .gtoreq.3, .gtoreq.4,
.gtoreq.5, and .gtoreq.6 CSBMs per week) over 12 weeks of treatment
were analyzed. At each level, a statistically significantly greater
proportion of patients treated with either dose of the oral dosage
form of linaclotide met the response criterion compared with
placebo patients.
[0163] For CSBM and SBM frequency, each dose of LINZESS (145 and
290 mcg) demonstrated a statistically significant separation from
placebo that was present at the first week and sustained across the
12 weeks of the treatment period (p<0.001 for each dose vs.
placebo at all time points). During the 4-week RW period in Study 3
when LINZESS treatment was discontinued, bowel function, including
CSBMs and SBMs, returned toward baseline within the first week with
no evidence of rebound worsening (see FIG. 3).
[0164] In some preferred embodiments, the linaclotide composition
is stored up to 25.degree. C. (77.degree. F.); excursions permitted
between 15.degree. C. and 30.degree. C. (59.degree. F. and
86.degree. F.) [see USP Controlled Room Temperature]. In some
embodiments, the linaclotide composition is stored is a low
moisture environment.
[0165] As used herein, unless otherwise indicated, the phrase
"consisting of" when used in reference to the linaclotide
composition or a single component of the composition means that the
linaclotide composition or single component defined by the phrase
contains no other components than those specified but may contain
additional components that are unrelated to the invention and/or
impurities ordinarily associated with the recited steps or
components.
Dosage and Excipients
[0166] The linaclotide composition may any suitable oral
pharmaceutical dosage form (e.g., capsules) containing any suitable
therapeutic dosage of linaclotide. In some embodiments, the
linaclotide composition comprises capsules/tablets that comprise
290 mcg which are to be taken orally once daily on an empty
stomach, such as for treating IBS-c (MS with constipation. In some
embodiments, the linaclotide composition comprises capsules/tablets
that comprise 145 or 290 mcg which are to be taken orally once
daily on an empty stomach for treating chronic idiopathic
constipation.
[0167] The linaclotide composition can include additional
ingredients or excipient. In certain embodiments, one or more
therapeutic agents of the dosage unit may exist in an extended or
control release formulation and additional therapeutic agents may
not exist in extended release formulation. For example, a peptide
or agonist described herein may exist in a controlled release
formulation or extended release formulation in the same dosage unit
with another agent that may or may not be in either a controlled
release or extended release formulation. Thus, in certain
embodiments, it may be desirable to provide for the immediate
release of one or more of the agents described herein, and the
controlled release of one or more other agents.
[0168] The linaclotide composition can comprise any
pharmaceutically tolerable carrier or medium, e.g. solvents,
dispersants, coatings, absorption promoting agents, controlled
release agents, and one or more inert excipients (which include
starches, polyols, granulating agents, microcrystalline cellulose
(e.g. celphere, Celphere beads.RTM.), diluents, lubricants,
binders, disintegrating agents, and the like), etc. If desired,
tablet dosages of the disclosed compositions may be coated by
standard aqueous or nonaqueous techniques.
[0169] Examples of excipients for use as the pharmaceutically
acceptable carriers and the pharmaceutically acceptable inert
carriers and the aforementioned additional ingredients include, but
are not limited to binders, fillers, disintegrants, lubricants,
anti-microbial agents, and coating agents.
[0170] As used herein, the term "binder" refers to any
pharmaceutically acceptable binder that may be used in the practice
of the invention. Examples of pharmaceutically acceptable binders
include, without limitation, a starch (e.g., corn starch, potato
starch and pre-gelatinized starch (e.g., STARCH 1500.RTM. and
STARCH 1500 LM.RTM., sold by Colorcon, Ltd.) and other starches),
maltodextrin, gelatin, natural and synthetic gums such as acacia,
powdered tragacanth, guar gum, cellulose and its derivatives (e.g.,
methylcellulose, hydroxyethyl cellulose, hydroxyethyl
methylcellulose, hydroxypropyl cellulose and hydroxypropyl
methylcellulose (hypromellose), ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose,
carboxymethylcellulose, microcrystalline cellulose (e.g. AVICEL.TM.
such as, AVICEL-PH-101.TM., -103.TM. and 105.TM., sold by FMC
Corporation, Marcus Hook, PA, USA)), polyvinyl alcohol, polyvinyl
pyrrolidone (e.g., polyvinyl pyrrolidone K30), and mixtures
thereof.
[0171] As used herein, the term "filler" refers to any
pharmaceutically acceptable filler that may be used in the practice
of the invention. Examples of pharmaceutically acceptable fillers
include, without limitation, talc, calcium carbonate (e.g.,
granules or powder), dibasic calcium phosphate, tribasic calcium
phosphate, calcium sulfate (e.g., granules or powder),
microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305),
powdered cellulose, dextrates, kaolin, mannitol, silicic acid,
sorbitol, starch (e.g., Starch 1500), pre-gelatinized starch,
lactose, glucose, fructose, galactose, trehalose, sucrose, maltose,
isomalt, raffinose, maltitol, melezitose, stachyose, lactitol,
palatinite, xylitol, myoinositol, and mixtures thereof
[0172] Examples of pharmaceutically acceptable fillers that may be
particularly used for coating the peptides include, without
limitation, talc, microcrystalline cellulose (e.g., Avicel PH101 or
Celphere CP-305), powdered cellulose, dextrates, kaolin, mannitol,
silicic acid, sorbitol, starch, pre-gelatinized starch, lactose,
glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt,
dibasic calcium phosphate, raffinose, maltitol, melezitose,
stachyose, lactitol, palatinite, xylitol, mannitol, myoinositol,
and mixtures thereof.
[0173] As used herein, the term "additives" refers to any
pharmaceutically acceptable additive. Pharmaceutically acceptable
additives include, without limitation, disintegrants, dispersing
additives, lubricants, glidants, antioxidants, coating additives,
diluents, surfactants, flavoring additives, humectants, absorption
promoting additives, controlled release additives, anti-caking
additives, anti-microbial agents (e.g., preservatives), colorants,
desiccants, plasticizers and dyes. As used herein, an "excipient"
is any pharmaceutically acceptable additive, filler, binder or
agent.
[0174] The linaclotide composition may also optionally include
other therapeutic ingredients, anti-caking agents, preservatives,
sweetening agents, colorants, flavors, desiccants, plasticizers,
dyes, glidants, anti-adherents, anti-static agents, surfactants
(wetting agents), anti-oxidants, film-coating agents, and the like.
Any such optional ingredient must be compatible with the compound
described herein to insure the stability of the formulation. The
composition may contain other additives as needed, including for
example lactose, glucose, fructose, galactose, trehalose, sucrose,
maltose, raffinose, maltitol, melezitose, stachyose, lactitol,
palatinite, starch, xylitol, mannitol, myoinositol, and the like,
and hydrates thereof, and amino acids, for example alanine, glycine
and betaine, and peptides and proteins, for example albumen.
[0175] The linaclotide composition can include, for example,
various additional solvents, dispersants, coatings, absorption
promoting additives, controlled release additives, and one or more
inert additives (which include, for example, starches, polyols,
granulating additives, microcrystalline cellulose, diluents,
lubricants, binders, disintegrating additives, and the like), etc.
If desired, tablet dosages of the disclosed compositions may be
coated by standard aqueous or non-aqueous techniques. Compositions
can also include, for example, anti-caking additives,
preservatives, sweetening additives, colorants, flavors,
desiccants, plasticizers, dyes, and the like.
[0176] Suitable disintegrants include, for example, agar-agar,
calcium carbonate, microcrystalline cellulose, croscarmellose
sodium, crospovidone, povidone, polacrilin potassium, sodium starch
glycolate, potato or tapioca starch, other starches,
pre-gelatinized starch, clays, other algins, other celluloses,
gums, and mixtures thereof.
[0177] Suitable lubricants include, for example, calcium stearate,
magnesium stearate, mineral oil, light mineral oil, glycerin,
sorbitol, mannitol, polyethylene glycol, other glycols, stearic
acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil
(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive
oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl
laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co.,
Baltimore, Md. USA), a coagulated aerosol of synthetic silica
(Evonik Degussa Co., Plano, Tex. USA), a pyrogenic silicon dioxide
(CAB-O-SIL, Cabot Co., Boston, Mass. USA), and mixtures
thereof.
[0178] Suitable glidants include, for example, leucine, colloidal
silicon dioxide, magnesium trisilicate, powdered cellulose, starch,
talc, and tribasic calcium phosphate.
[0179] Suitable anti-caking additives include, for example, calcium
silicate, magnesium silicate, silicon dioxide, colloidal silicon
dioxide, talc, and mixtures thereof.
[0180] Suitable anti-microbial additives that may be used, e.g., as
a preservative for the peptides compositions, include, for example,
benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl
alcohol, butyl paraben, cetylpyridinium chloride, cresol,
chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben,
phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric
acetate, phenylmercuric nitrate, potassium sorbate, propylparaben,
sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic
acid, thimersol, thymo, and mixtures thereof.
[0181] Suitable antioxidants include, for example, BHA (butylated
hydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propyl
gallate, ascorbic acid and salts or esters thereof, tocopherol and
esters thereof, alpha-lipoic acid and beta-carotene.
[0182] Suitable coating additives include, for example, sodium
carboxymethyl cellulose, cellulose acetate phthalate,
ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl
cellulose phthalate, methylcellulose, polyethylene glycol,
polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,
carnauba wax, microcrystalline wax, and mixtures thereof. Suitable
protective coatings include Aquacoat (e.g. Aquacoat Ethylcellulose
Aquaeous Dispersion, 15% w/w, FMC Biopolymer, ECD-30), Eudragit
(e.g. Eudragit E PO PE-EL, Roehm Pharma Polymers) and Opadry (e.g
Opadry AMB dispersion, 20% w/w, Colorcon).
[0183] In certain embodiments, suitable additives for the peptides
composition include one or more of sucrose, talc, magnesium
stearate, crospovidone or BHA.
[0184] The compositions of the present invention can also include
other excipients, agents, and categories thereof including but not
limited to L-histidine, Pluronic.RTM., Poloxamers (such as
Lutrol.RTM. and Poloxamer 188), ascorbic acid, glutathione,
permeability enhancers (e.g. lipids, sodium cholate, acylcarnitine,
salicylates, mixed bile salts, fatty acid micelles, chelators,
fatty acid, surfactants, medium chain glycerides), protease
inhibitors (e.g. soybean trypsin inhibitor, organic acids), pH
lowering agents and absorption enhancers effective to promote
bioavailability (including but not limited to those described in
U.S. Pat. Nos. 6,086,918 and 5,912,014), materials for chewable
tablets (like dextrose, fructose, lactose monohydrate, lactose and
aspartame, lactose and cellulose, maltodextrin, maltose, mannitol,
microcrystalline cellulose and guar gum, sorbitol crystalline);
parenterals (like mannitol and povidone); plasticizers (like
dibutyl sebacate, plasticizers for coatings, polyvinylacetate
phthalate); powder lubricants (like glyceryl behenate); soft
gelatin capsules (like sorbitol special solution); spheres for
coating (like sugar spheres); spheronization agents (like glyceryl
behenate and microcrystalline cellulose); suspending/gelling agents
(like carrageenan, gellan gum, mannitol, microcrystalline
cellulose, povidone, sodium starch glycolate, xanthan gum);
sweeteners (like aspartame, aspartame and lactose, dextrose,
fructose, honey, maltodextrin, maltose, mannitol, molasses,
sorbitol crystalline, sorbitol special solution, sucrose); wet
granulation agents (like calcium carbonate, lactose anhydrous,
lactose monohydrate, maltodextrin, mannitol, microcrystalline
cellulose, povidone, starch), caramel, carboxymethylcellulose
sodium, cherry cream flavor and cherry flavor, citric acid
anhydrous, citric acid, confectioner's sugar, D&C Red No. 33,
D&C Yellow #10 Aluminum Lake, disodium edetate, ethyl alcohol
15%, FD& C Yellow No. 6 aluminum lake, FD&C Blue #1
Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminum
lake, FD&C Green No.3, FD&C Red No. 40, FD&C Yellow No.
6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No.10,
glycerol palmitostearate, glyceryl monostearate, indigo carmine,
lecithin, manitol, methyl and propyl parabens, mono ammonium
glycyrrhizinate, natural and artificial orange flavor,
pharmaceutical glaze, poloxamer 188, Polydextrose, polysorbate 20,
polysorbate 80, polyvidone, pregelatinized corn starch,
pregelatinized starch, red iron oxide, saccharin sodium, sodium
carboxymethyl ether, sodium chloride, sodium citrate, sodium
phosphate, strawberry flavor, synthetic black iron oxide, synthetic
red iron oxide, titanium dioxide, and white wax.
[0185] In some embodiments, there is provided a pharmaceutical
composition comprising a peptide described herein and one or more
agents selected from Mg.sup.2+, Ca.sup.2+, Zn.sup.2+, Mn.sup.2+,
Na.sup.+ or Al.sup.3+, a combination thereof, and/or a sterically
hindered primary amine. In further embodiments, the agent is
Mg.sup.2+, Ca.sup.2+ or Zn.sup.2+ or a combination thereof. In some
embodiments, the divalent metal cation is provided, without
limitation, as magnesium acetate, magnesium chloride, magnesium
phosphate, magnesium sulfate, calcium acetate, calcium chloride,
calcium phosphate, calcium sulfate, zinc acetate, zinc chloride,
zinc phosphate, zinc sulfate, manganese acetate, manganese
chloride, manganese phosphate, manganese sulfate, potassium
acetate, potassium chloride, potassium phosphate, potassium
sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium
sulfate, aluminum acetate, aluminum chloride, aluminum phosphate or
aluminum sulfate. In further embodiments, the cation is provided as
magnesium chloride, calcium chloride, calcium phosphate, calcium
sulfate, zinc acetate, manganese chloride, potassium chloride,
sodium chloride or aluminum chloride. In other embodiments, the
cation is provided as calcium chloride, magnesium chloride or zinc
acetate.
[0186] In another embodiment, the agent is a sterically hindered
primary amine. In a further embodiment, the sterically hindered
primary amine is an amino acid. In yet a further embodiment, the
amino acid is a naturally-occurring amino acid. In a still further
embodiment, the naturally-occurring amino acid is selected from the
group consisting of: histidine, phenylalanine, alanine, glutamic
acid, aspartic acid, glutamine, leucine, methionine, asparagine,
tyrosine, threonine, isoleucine, tryptophan, glycine and valine;
yet further, the naturally-occurring amino acid is leucine,
isoleucine, alanine or methionine. In a still further embodiment,
the naturally-occurring amino acid is leucine. In another
embodiment, the sterically hindered primary amine is a
non-naturally occurring amino acid (e.g., 1-aminocyclohexane
carboxylic acid). In a further embodiment, the sterically hindered
primary amine is cyclohexylamine, 2-methylbutylamine or chitosan.
In another embodiment, one or more sterically hindered primary
amines may be used in a composition.
##STR00064##
[0187] In some cases, the sterically hindered primary amine has the
formula:, wherein R.sub.1, R.sub.2 and R.sub.3 are independently
selected from: H, C(O)OH, C1-C6 alkyl, C1-C6 alkylether, C1-C6
alkylthioether, C1-C6 alkyl carboxylic acid, C1-C6 alkyl
carboxylamide and alkylaryl, wherein any group can be singly or
multiply substituted with: halogen or amino, and provided that no
more than two of R.sub.1, R.sub.2 and R.sub.3 are H. In another
embodiment, no more than one of R.sub.1, R.sub.2 and R.sub.3 is
H.
[0188] In other embodiments, there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier,
peptide, a cation selected from Mg.sup.2+, Ca.sup.2+, Zn.sup.2+,
Mn.sup.2+, Na.sup.+ or Al.sup.3+, or a mixture thereof, and a
sterically hindered primary amine. In one embodiment, the cation is
Mg.sup.2+, Ca.sup.2+ or Zn.sup.2+ or a mixture thereof. In a
further embodiment, the linaclotide composition further comprises a
pharmaceutically acceptable binder and/or a pharmaceutically
acceptable glidant, lubricant or additive that acts as both a
glidant and lubricant and/or an antioxidant. In some embodiments,
the linaclotide composition is applied to a carrier. In some
embodiments, the carrier is a filler.
[0189] In some cases the molar ratio of cation:sterically hindered
primary amine: peptide in the aqueous solution applied to the
carrier is 5-100:5-50:1. In some cases, the molar ratio of
cation:sterically hindered primary amine may be equal to or greater
than 2:1 (e.g., between 5:1 and 2:1). Thus, in some cases the molar
ratio of cation:sterically hindered primary amine: peptide applied
to the carrier is 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1,
60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1,
10:5:1 or 5:10:1. In some preferred embodiments, the molar ratio of
divalent metal cation:sterically hindered primary amine:linaclotide
is about 57-63:28-32:1. When binder, e.g., methylcellulose, is
present in the GC-C agonist peptide solution applied to the carrier
it can be present at 0.5%-2.5% by weight (e.g., 0.7%-1.7% or 0.7% -
1% or 1.5% or 0.7%).
[0190] In a further embodiment, the linaclotide composition further
comprises a pharmaceutically acceptable binder or additive, and/or
a pharmaceutically acceptable glidant, lubricant or additive that
acts as both a glidant and lubricant and/or an antioxidant.
[0191] Suitable pharmaceutical compositions in accordance with the
invention will generally include an amount of the active
compound(s) with an acceptable pharmaceutical diluent or excipient,
such as a sterile aqueous solution, to give a range of final
concentrations, depending on the intended use. The techniques of
preparation are generally well known in the art, as exemplified by
Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing
Company, 1995).
[0192] As used herein, unless otherwise indicated, the phrase
"consisting essentially of," when used in reference to the
linaclotide composition or a single component of the composition
means that the composition or single component defined by the
phrase contains no active pharmaceutical ingredients other than
those specified but that it may contain any additional inactive
components or excipients or formaldehyde scavenger compound(s).
[0193] The term "linaclotide composition" is used herein, unless
otherwise indicated, to mean a composition that comprises
linaclotide, excipients necessary for stabilizing, storing and
delivering linaclotide as described herein (e.g., a divalent metal
cation and an amino acid, for example, in a molar ratio of divalent
metal cation:amino acid:linaclotide of about 57-63:28-32:1) and
optionally one or more formaldehyde scavenger compounds in any
suitable configuration or conformation. For example, in some
embodiments, the linaclotide composition comprises an oral
pharmaceutical dosage form (e.g., capsules or tablets) that
comprises linaclotide (in any desired dosage, such as 145 .mu.g or
290 .mu.g), excipients necessary for stabilizing, storing and
delivering linaclotide as described herein (e.g., a divalent metal
cation and an amino acid for example in a molar ratio of divalent
metal cation:amino acid:linaclotide of about 57-63:28-32:1) and one
or more formaldehyde scavenger compounds. In other preferred
embodiments, the linaclotide composition comprises an oral
pharmaceutical dosage form (e.g., capsules or tablets) that
comprises linaclotide (in any desired dosage, such as 145 .mu.g or
290 .mu.g) and excipients necessary for stabilizing, storing and
delivering linaclotide such as described herein (e.g., a divalent
metal cation and an amino acid in a molar ratio of divalent metal
cation:amino acid:linaclotide of about 57-63:28-32:1) and one or
more separate formaldehyde scavenger compounds that are outside of
the oral dosage form (e.g., but in the same bottle, cannister, or
container).
[0194] The present invention has been described with reference to
certain exemplary embodiments thereof. However, it will be readily
apparent to those skilled in the art that it is possible to embody
the invention in specific forms other than those of the exemplary
embodiments described above. This may be done without departing
from the spirit of the invention. The exemplary embodiments are
merely illustrative and should not be considered restrictive in any
way. The scope of the invention is defined by the appended claims
and their equivalents, rather than by the preceding
description.
EXAMPLES
[0195] The GC-C agonist peptides or pharmaceutically acceptable
salts thereof as described herein were prepared by solid phase
chemical synthesis and natural folding (air oxidation) by American
Peptide Company (Sunnyvale, Calif.). In some cases, the peptides
were modified after synthesis as described herein.
[0196] The Cys.sub.1-IMD peptide was synthesized by mixing 4.6 g
(3.0 mmol) of linaclotide in 200 ml of EtOH. Formaldehyde at 37%
(1.12 ml/5eq) was added to this mixture. The reaction mixture was
incubated in a water bath (45.quadrature.C) for overnight. The
following day the solvent was removed by rota-evaporation. The
peptide was further purified through reverse-phase
chromatography.
[0197] The Glu3-ethyl ester peptide was synthesized on a 20 mmol
Fmoc-Tyr(tBu)-Wang resin. Protecting groups used for amino acids
are: t-Butyl group for Tyr and Thr, Trt group for Asn and Cys. The
peptide chain was assembled on the resin by repetitive removal of
the Fmoc protecting group and coupling of protected amino acid. DIC
and HOBt were used as coupling reagents and NMM was used as the
base for this reaction. 20% piperidine in DMF was used as
de-Fmoc-reagent. After removal of last Fmoc protecting group, resin
was treated with cocktail K for 3 hours to cleave the peptide from
resin and removal of the side chain protecting groups. The eluted
peptide was precipitated in cold ether and dried. The dried peptide
was dissolved in a mixture of TFA/TIS/water (95:3:2v/v) in a ratio
of 1 to 10 (g/v). This mixture was stirred at room temperature for
1 hour. The isolated peptide was also precipitated in cold ether,
collected by filtration and dried under high vacuum.
[0198] The Tyr.sub.14-ethyl ester peptide was synthesized by a
fragment condensation method. Fragment A
(Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro--
Ala-Cys(Trt)-Thr(tBu)-Gly-OH (SEQ ID NO: 27)) was prepared on
15mmol CTC resin using Fmoc chemistry. This peptide chain was also
assembled on the resin by repetitive removal of the Fmoc protecting
group and coupling of protected amino acid. DIC and HOBt were used
as coupling reagents and NMM was used as the base. 20% piperidine
in DMF was used as de-Fmoc-reagent. After removal of last Fmoc
protecting group, Boc was coupled to protect the N-terminal amino
group. The peptide resin was washed, dried, and treated with 1%
TFA/DCM to cleave peptide from resin. Fragment B (Cys(Trt)-Tyr-OEt)
was prepared from coupling of Fmoc-Cys(Trt)-OH and Tyr-OEt. HCl.
The Fmoc group was removed by treating this di-peptide with 20%
piperidine in DMF.
[0199] The Tyr.sub.14-ethyl ester peptide was finally synthesized
by coupling the two fragments in DMF. HBTU/HOBt/NMM was used as the
coupling reagent for this reaction. The protecting groups were
removed by treating the peptide with cocktail K for 2 hours. This
peptide was precipitated in cold ether and dried. The dried peptide
was dissolved in a mixture of TFA/TIS/water (95:3:2v/v) in a ratio
of 1 to 10 (g/v). This mixture was stirred at room temperature for
1 hour. The isolated peptide was again precipitated in cold ether,
collected by filtration and dried under high vacuum.
Example 1: cGMP Accumulation in T84 Cells for Analysis of GC-C
Activity
[0200] For the cGMP assay, 4.5.times.10.sup.5 cells/mL of T84 cells
were grown overnight in 24 well tissue culture plates. On next day,
the T84 cells were washed twice with 1 mL of DMEM (pH 7). After the
second wash, the cells were incubated with 450 .mu.L of 1 mM
isobutylmethylxanthine (IBMX) in pH 7 buffer for 10 minutes at
37.degree. C. to inhibit any phosphodiesterase activity. The
peptides were then diluted in DMEM buffer (pH 7) to a 10.times.
concentration. The peptide solution of 50 .mu.L was diluted to a
final volume of 500 .mu.L with the T84 cells, bringing each peptide
concentration to lx. The peptides were tested in duplicate at 100
nM.
[0201] There was no peptide control used to determine endogenous
levels of cGMP. Peptides were incubated for 30 minutes at
37.degree. C. After 30 minutes, the supernatants were removed and
the cells were lysed with 0.1 M HCl. The cells were lysed for 30
minutes on ice. After 30 minutes, lysates were pipetted off and
placed into a 96 well HPLC plate and spun at 10,000.times.G for 10
minutes to remove any cell debris. Supernatants from the previous
spin were removed and placed into a fresh 96 well HPLC plate.
[0202] cGMP concentrations were determined from each sample using
the LC/MS conditions (Table 1 below) and calculated standard curve.
EC.sub.50 values were calculated from concentration-response curves
generated with GraphPad Prism Software.
TABLE-US-00001 TABLE 1 LC/MS conditions MS: Thermo Quantum Ion
Mode: ESI.sup.+ Scan Type: MRM Dwell Collision Retention Time
Energy Tube Time Compound: Transition (msec) (V) Lens (min) cGMP
346 > 152 100 28 139 1.0 HPLC: Agilent Technologies 1200 Series
with CTC Analytics HTS PAL Column: Thermo Hypersil Gold 2.1 .times.
50 mm 5 micron particle size Flow Rate: 400 uL/min Column RT
Temperature: Autosampler 6.degree. C. Temperature: Injection 20 uL
Volume: Mobile A = 98:2 Water:Acetonitrile + 0.1% Formic Acid
Phases: B = 2:98 Water:Acetonitrile + 0.1% Formic Acid Time
Gradient: (min) % A % B 0 100 0 0.3 30 70 2.00 30 70 2.01 100 0 4
100 0
Example 2: Relative Binding Affinity of Exemplary Peptides to the
GC-C Receptor of T84 Cells
[0203] The relative binding affinities of linaclotide and
Cys.sub.1-IMD to the guanylate cyclase-C receptor (GC-C) were
determined using a competitive-binding assay in which the peptides
competed with a known GC-C agonist, porcine-derived heat-stable
enterotoxin (pSTa), for binding sites on cell-surface GC-C
receptors on human colonic epithelial (T84) cells. The pSTa was
radiolabeled with .sup.125I to enable measurement of its receptor
binding. The competitive-binding assay was performed by adding
various concentrations of each peptide (0.1 to 3,000 nM) to 0.20mL
reaction mixtures containing Dulbecco's modified Eagle's medium
(DMEM), 0.5% bovine serum albumin (BSA), 2.0.times.10.sup.5 T84
cells, and 170 pM [.sup.125I]STa (200,000 cpm). The data were used
to construct competitive radioligand-binding curves and determine
the relative binding affinities of linaclotide and Cys.sub.1-IMD,
as measured by their IC.sub.50 and K.sub.i values.
[0204] Both linaclotide and Cys.sub.1-IMD competitively inhibited
the specific binding of [.sup.125I]-pSTa to cell-surface GC-C
receptors on T84 cells. Their relative binding affinities, as
measured by their inhibition constants (K), were as follows:
Linaclotide K=3.9.+-.1.6 nM and Cys.sub.1-IMD K=1.4.+-.0.5 nM (FIG.
3).
Example 3: cGMP Response in T84 Cells Induced by Exemplary
Peptides
[0205] Linaclotide and Cys.sub.1-IMD were tested for guanylate
cyclase-C (GC-C) agonist activity in T84 cells as follows. In each
well of a 96-well plate, approximately 200,000 T84 cells/well was
first incubated with 1 mM 3-isobutyl-1-methylxanthine (IBMX) in
0.18 mL of Dulbecco's modified Eagle's medium (DMEM) for 10 minutes
at 37.degree. C. Each peptide was diluted to final concentrations
ranging from 0.1 to 10,000 nM, and 0.02 mL of each dilution was
added in duplicate to a 96-well plate containing the T84 cells, for
a final volume of 0.20 mL per well. The peptide reactions were
incubated for 30 min at 37.degree. C. Following the incubation, the
supernatants were removed and discarded and the cells were lysed
with cold 0.1 M hydrochloric acid (HCl) for 30 min on ice. The cell
debris was removed by centrifugation and the concentration of
guanosine 3',5'-cyclic monophosphate (cyclic GMP) in each lysate
was determined using liquid chromatography with tandem mass
spectrometry. The data were used to construct dose-response curves
and calculate half-maximal effective concentration (EC.sub.50)
values for each test article.
[0206] Linaclotide and Cys.sub.1-IMD showed GC-C agonist activity
in T84 cells, as measured by the increase in intracellular cGMP
(FIG. 4). The EC.sub.50 values for linaclotide and Cys.sub.1-IMD
were 315.+-.105 nM and 172.+-.32 nM, respectively.
Example 4: Measurement of Content and Purity of Exemplary
Peptides
[0207] Content and purity of the peptides of the present invention
may be determined by reverse phase gradient liquid chromatography
using an Agilent Series 1100 LC System with Chemstation Rev A.09.03
software or equivalent. A YMC Pro.TM. C18 column (dimensions:
3.0.times.150 mm, 3.5 um, 120 .ANG.; Waters Corp., Milford, Mass.)
or equivalent is used and is maintained at 40.degree. C. Mobile
phase A (MPA) consists of water with 0.1% trifluoroacetic acid
while mobile phase B (MPB) consists of 95% acetonitrile:5% water
with 0.1% trifluoroacetic acid. Elution of the peptides is
accomplished with a gradient from 0% to 47% MPB in 28 minutes
followed by a ramp to 100% MPB in 4 minutes with a 5 minute hold at
100% M wash the column. Re-equilibration of the column is performed
by returning to 0% MPB in 1 minute followed by a 10 minute hold at
100% MPA. The flow rate is 0.6 mL/min and detection is accomplished
by UV at 220 nm.
[0208] Samples for analysis are prepared by addition of the
contents of capsules of exemplary peptides to 0.1 N HC1 to obtain a
target concentration of 20 .mu.g peptide/mL. 100 .mu.L of this
solution is injected onto the column.
Cys.sub.1-IMD Peptide
[0209] The Cys.sub.1-IMD peptide was purified using a 2-inch Waters
C18 column with 0.1% TFA buffer with a linear gradient of 15-45% in
60 minutes of buffer B at flow rate of 100 mL/min. The pooled
fractions with purity around 95% were loaded onto C18 column. After
equilibrating the column with TEAP buffer and AA buffer, the
peptide was purified and eluted out with HAC buffer with a linear
gradient of 15-75% of buffer B in 60 minutes. Pooled fractions with
purified peptide were lyophilized to dryness. An example of an
analysis of linaclotide and Cys.sub.1-IMD product by RP-HPLC is
shown in FIG. 2.
Glu3-ethyl Ester Peptide
[0210] The Glu3-ethyl ester peptide (6.0 g) was dissolved in 12 L
of 0.05M ammonium bicarbonate in water, and the oxidation process
was monitored by Ellman's test, MS and analytical HPLC. The
oxidation process took approximately 48 hours for completion.
[0211] The above solution was filtered and loaded onto a 2-inch C18
column, and purified by using 0.05M ammonium acetate buffer with a
linear gradient of 10-40% of buffer B in 60 minutes at flow rate of
100 mL/min.
[0212] The pooled fractions with purity of >95% were lyophilized
to dryness. After the peptides were dried, the peptide was
re-dissolved in acetonitrile-water and acidified to pH around 4-5
by addition of acetic acid and re-lyophilized to dryness.
Tyr.sub.14-ethyl Ester Peptide
[0213] The Tyr.sub.14-ethyl ester peptide was purified by
dissolving 2.5 g of the isolated peptide in 5 L of 0.05M ammonium
bicarbonate in water, and the oxidation process was monitored by
Ellman's test, MS and analytical HPLC. This oxidation process took
approximately 16 hours for completion.
The above solution was filtered and loaded onto a 2-inch Polymer
column, and purified by using 0.05M ammonium bicarbonate buffer
with a linear gradient of 15-45% of buffer B in 60 minutes at flow
rate of 100 mL/min. The pooled fractions with the peptide were
lyophilized to dryness. After the peptide was dried, the peptide
was re-dissolved in acetonitrile-water and acidified to pH 4-5 by
addition of acetic acid and re-lyophilized to dryness.
[0214] The contents of the purified peptides were measured by
determining the peptide concentration in the prepared sample
against a similarly prepared external peptide standard.
Example 5
[0215] The effect of including various formaldehyde scavenger
compounds on the stability of the linaclotide composition was next
assessed.
[0216] 60 cc square HPDE bottle (with 33 mm caps with induction
seals) were loaded with (i) capsules containing beads coated with
145 .mu.g or 290 .mu.g of linaclotide, a binder, calcium chloride
and leucine with an approximate molar ratio of CaCl.sub.2: leucine:
linaclotide of 60:30:1, (ii) either no formaldehyde scavenger
compound (negative control) or a canister comprising one of the
formaldehyde scavenger compounds disclosed in Table 3, and (iii)
optionally a canister containing 3 grams of a silica gel
desiccant.
[0217] In addition, some of the linaclotide compositions contained
a formaldehyde stressor (in particular, an adhesive label on the
desiccant canister that was applied by the manufacturer of the
desiccant canister (Sud Chemie) (its presence in some linaclotide
compositions is indicated in the table as "labeled canister").
Regarding the formaldehyde stressor canister, it was observed by
applicants that the canister emits about 27.5 ug of formaldehyde in
24 hours at 60.degree. C.
[0218] The linaclotide compositions were stored in a 40.degree. C.,
75% relative humidity stability chamber. Next, the quantity of
Cys.sub.1-IMD in each linaclotide composition was determined
following 4, 8 and 13 week storage via Reverse Phase-HPLC using a
gradient elution with UV detection at 220 nm. Quantitation was
based on percent of chromatogram area.
[0219] Test samples of linaclotide capsules are prepared by
emptying the bead contents into flasks, adding diluent and shaking
for 30 minutes. The analysis used the bead contents of a composite
of 6 or 8 capsules (for 290 .mu.g and 145 .mu.g, respectively).
Operating conditions for the analytical test method by HPLC are
outlined in Table 2. The results of the stability assay are set
forth in Table 3.
TABLE-US-00002 TABLE 2 HPLC Purity and Degradation Conditions
Mobile Phase A 98:2:0.1 Water:Acetonitrile:FA Mobile Phase B
95:5:0.1 Acetonitrile:Water:TFA Diluent 0.1N Hydrochloric Acid
Gradient Time Profile (min) % A % B Comments 0 100 0 Initial
Conditions 4 100 0 4-minute hold 9 90 10 5-minute linear gradient
43 77 23 34-minute linear gradient 49 66 34 6-minute linear
gradient 59 20 80 10-minute linear gradient 60 100 0 Return to
initial conditions 67 100 0 Re-equilibration UV Detection 220 nm
Injection 50 uL Volume Sample 200 ug/mL Concentration Column YMC
Pro C18, 150 mm .times. 3.0 mm ID, 3 um or equivalent Column
40.degree. C. Temperature Flow Rate 0.6 mL/min
TABLE-US-00003 TABLE 3 Effect of formaldehyde scavenger compounds
on the Linaclotide Composition Stability % decrease in
Cys.sub.1-IMD as compared % Cys.sub.1-IMD after to negative storage
control Formaldehyde Scavenger 4 8 13 (after Compound Weeks Weeks
Weeks 13 weeks) negative control 1.21 1.87 3.53 -- (labeled
canister with no scavenger) d-meglumine 0.59 0.81 0.84 -76% (coated
on beads in labeled canister) histidine 0.60 0.68 1.00 -72% (coated
on beads in labeled canister) lyophilized leucine 0.52 0.78 1.36
-61% (in labeled canister) asparagine 0.56 1.11 1.45 -59% (coated
on beads in labeled canister) glycine-leucine 0.61 1.03 1.51 -57%
(coated on beads in labeled canister) lysine 0.87 1.27 1.87 -47%
(coated on beads in labeled canister) glycine-glycine 0.89 1.40
1.98 -44% (coated on beads in labeled canister) leucine 0.84 1.26
2.44 -31% (coated on beads in labeled canister) aspartame 0.82 1.54
2.58 -27% (coated on beads in labeled canister)
[0220] As is demonstrated in Table 3, linaclotide compositions that
contained formaldehyde scavenger compounds exhibited surprisingly
and unexpectedly higher stability that those not containing a
formaldehyde scavenger compound (negative control).
[0221] All publications and patents referred to in this disclosure
are incorporated herein by reference to the same extent as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Should the
meaning of the terms in any of the patents or publications
incorporated by reference conflict with the meaning of the terms
used in this disclosure, the meaning of the terms in this
disclosure are intended to be controlling. Furthermore, the
foregoing discussion discloses and describes merely exemplary
embodiments of the present invention. One skilled in the art will
readily recognize from such discussion and from the accompanying
drawings and claims, that various changes, modifications and
variations can be made therein without departing from the spirit
and scope of the invention as defined in the following claims.
Sequence CWU 1
1
27114PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 1Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10214PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated peptideMOD_RES(1)..(1)Alpha-amine of the
Cys is deaminatedDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13)
2Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10314PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated peptideMOD_RES(1)..(1)Cysteine modified
into an
imidazolidinoneDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13)
3Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10414PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated peptideMOD_RES(1)..(1)Cys1 sulfur atom is
oxidizedDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 4Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10514PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3 residue
is esterified with C1-C6 alkylDISULFID(5)..(13)C-terminus is
esterified with C1-C6 alkyl 5Cys Cys Glu Tyr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10614PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3 is
esterified with C1-C6 alkylDISULFID(5)..(13) 6Cys Cys Glu Tyr Cys
Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5 10714PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13)C-terminus
is esterified with C1-C6 alkyl 7Cys Cys Glu Tyr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 10814PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideMOD_RES(1)..(1)Cysteine with an N-terminal
imineDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 8Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10914PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains a methyl esterDISULFID(5)..(13) 9Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 101014PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains an ethyl esterDISULFID(5)..(13) 10Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 101114PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(5)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains a propyl esterDISULFID(5)..(13) 11Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 101214PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(5)DISULFID(2)..(10)DISULFID(5)..(13)C-terminus
contains a methyl ester 12Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
Thr Gly Cys Tyr1 5 101314PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13)C-terminus
contains an ethyl ester 13Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
Thr Gly Cys Tyr1 5 101414PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13)C-terminus
contains a propyl ester 14Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
Thr Gly Cys Tyr1 5 101514PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains a methyl esterDISULFID(5)..(13)C-terminus contains a
methyl ester 15Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
Tyr1 5 101614PRTArtificial SequenceDescription of Artificial
Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains an ethyl esterDISULFID(5)..(13)C-terminus contains an
ethyl ester 16Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
Tyr1 5 101714PRTArtificial SequenceDescription of Artificial
Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(3)..(3)Glu3
contains a propyl esterDISULFID(5)..(13)C-terminus contains a
propyl ester 17Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
Tyr1 5 101814PRTArtificial SequenceDescription of Artificial
Sequence Synthetically generated peptideMOD_RES(1)..(1)Cysteine is
deaminated and the alpha ketone is in geminal diol
formDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 18Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
101914PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MISC_FEATURE(3)..(3)Either
or both of Glu3 and Tyr14 are alkylated with C1-C6
alkylDISULFID(5)..(13)MISC_FEATURE(14)..(14)Either or both of Glu3
and Tyr14 are alkylated with C1-C6 alkyl 19Cys Cys Glu Tyr Cys Cys
Asn Pro Ala Cys Thr Gly Cys Tyr1 5 102013PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 20Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys1 5 102114PRTArtificial
SequenceDescription of Artificial Sequence Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)DISULFID(5)..(13) 21Cys Cys
Glu Tyr Cys Cys Asp Pro Ala Cys Thr Gly Cys Tyr1 5
102214PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MOD_RES(5)..(5)D-CysDISULFID(5)..-
(13) 22Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
102314PRTArtificial SequenceDescription of Artificial Sequence
Synthetically generated
peptideDISULFID(1)..(6)DISULFID(2)..(10)MISC_FEATURE(5)..(13)Cys5-Cys13
contains a trisulfide bond 23Cys Cys Glu Tyr Cys Cys Asn Pro Ala
Cys Thr Gly Cys Tyr1 5 102414PRTArtificial SequenceDescription of
Artificial Sequence Synthetically generated
peptideMOD_RES(1)..(1)ACETYLATIONDISULFID(1)..(6)DISULFID(2)..(10)DISULFI-
D(5)..(13) 24Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
Tyr1 5 102514PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideSee specification as filed for detailed
description of substitutions and preferred embodiments 25Cys Cys
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr1 5
10266PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 26His His His His His His1 52712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptideN-term
BocMOD_RES(1)..(1)Cys(Trt)MOD_RES(2)..(2)Cys(Trt)MOD_RES(3)..(3)Glu(OtBu)-
MOD_RES(4)..(4)Tyr(tBu)MOD_RES(5)..(5)Cys(Trt)MOD_RES(6)..(6)Cys(Trt)MOD_R-
ES(7)..(7)Asn(Trt)MOD_RES(10)..(10)Cys(Trt)MOD_RES(11)..(11)Thr(tBu)
27Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly1 5 10
* * * * *