U.S. patent application number 14/142655 was filed with the patent office on 2014-11-27 for compounds and methods for purification of serine proteases.
The applicant listed for this patent is Portola Pharmaceuticals, Inc.. Invention is credited to Anjali Pandey, Jack W. Rose.
Application Number | 20140346397 14/142655 |
Document ID | / |
Family ID | 51934750 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140346397 |
Kind Code |
A1 |
Pandey; Anjali ; et
al. |
November 27, 2014 |
COMPOUNDS AND METHODS FOR PURIFICATION OF SERINE PROTEASES
Abstract
Disclosed herein are compounds, compositions, methods and kits
for purifying a serine protease and serine proteases purified with
the compounds, compositions and methods.
Inventors: |
Pandey; Anjali; (Fremont,
CA) ; Rose; Jack W.; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Portola Pharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
51934750 |
Appl. No.: |
14/142655 |
Filed: |
December 27, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13830372 |
Mar 14, 2013 |
|
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14142655 |
|
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|
61746544 |
Dec 27, 2012 |
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Current U.S.
Class: |
252/190 ; 536/47;
546/309 |
Current CPC
Class: |
C12Y 304/21006 20130101;
C12N 9/6424 20130101; C12N 9/6432 20130101; B01J 2220/80 20130101;
C07D 213/72 20130101; B01J 20/286 20130101; B01J 20/267 20130101;
C07D 213/75 20130101 |
Class at
Publication: |
252/190 ;
546/309; 536/47 |
International
Class: |
C12N 9/64 20060101
C12N009/64 |
Claims
1-18. (canceled)
19. A compound selected from: ##STR00126## or a salt thereof.
20. (canceled)
21. An affinity solid support of Formula II: ##STR00127## or a salt
thereof, wherein: R.sup.21 is --CF.sub.3, --SO.sub.2CH.sub.3,
--X-L-Y--Z, ##STR00128## R.sup.22 is --OCH.sub.3, chloro, or
X-L-Y--Z; R.sup.23 is hydrogen or chloro; X is a covalent bond, O,
S, SO.sub.2, C(O)NH, NHC(O) or NH; L-Y--Z is -L.sup.1-Y--Z,
##STR00129## L.sup.1-Y--Z is ##STR00130## L.sup.2-Y--Z is
##STR00131## L.sup.3-Y--Z is ##STR00132## Y--Z is ##STR00133## Z is
Capto.TM. resin; q is 1, 2, 3, 4, 5, 6 or 7; r is 1, 2, 3, 4, 5, 6
or 7; s is 1, 2, 3, 4, 5, 6 or 7; t is 1, 2, 3, 4, 5, 6 or 7; v is
1, 2, 3, 4, 5, 6 or 7; u is 1, 2, 3, 4, 5, 6 or 7; w is 1, 2 or 3;
n, m, and p are either 0 or 1, with the provisos that (1) when
R.sup.21 is ##STR00134## --CF.sub.3 or --SO.sub.2CH.sub.3, and
R.sup.22 is --OCH.sub.3 or chloro, then one of n, m, and p must be
1, and the others of n, m, and p must be zero; and (2) when
R.sup.21 is other than ##STR00135## --CF.sub.3 or
--SO.sub.2CH.sub.3, or R.sup.22 is X-L-Y--Z, then all of n, m, and
p must be zero.
22. The affinity solid support of claim 21 of Formula II-A:
##STR00136## wherein the variables are as defined in claim 21.
23. The affinity solid support of claim 21 of Formula II-B:
##STR00137## wherein the variables are as defined in claim 21.
24. The affinity solid support of claim 21 of Formula II-C:
##STR00138## wherein the variables are as defined in claim 21.
25. The affinity solid support of claim 21 of Formula II-D:
##STR00139## wherein the variables are as defined in claim 21.
26. The affinity solid support of claim 21, wherein R.sup.21 is
##STR00140##
27. The affinity solid support of claim 21, wherein R.sup.22 is
--OCH.sub.3.
28. The affinity solid support of claim 21, wherein R.sup.22 is
X-L-Y--Z.
29. The affinity solid support of claim 21, wherein R.sup.23 is
hydrogen.
30. The affinity solid support of claim 21, wherein R.sup.23 is
chloro.
31. The affinity solid support of claim 21, wherein X is O.
32. The affinity solid support of claim 21, wherein X is a covalent
bond.
33. The affinity solid support of claim 21, wherein X is S or
SO.sub.2.
34. The affinity solid support of claim 21, wherein X is NH.
35. The affinity solid support of any claim 21, wherein L-Y--Z is
-L.sup.1-Y--Z or ##STR00141##
36. The affinity solid support of claim 21, wherein L-Y--Z is
selected from the group consisting of ##STR00142##
37. The affinity solid support of claim 21, wherein L.sup.1-Y--Z is
selected from the group consisting of ##STR00143##
38. The affinity solid support of claim 21, wherein each of q, r,
s, and t is at least 3.
39. The affinity solid support of claim 21, wherein --X-L-Y--Z is
selected from the group consisting of ##STR00144## ##STR00145##
##STR00146##
40. The affinity solid support of claim 21 which is ##STR00147##
##STR00148## or a salt thereof.
41. (canceled)
42. An affinity solid support of formula: ##STR00149## or a salt
thereof, wherein v is 1, 2, 3, 4, 5, 6 or 7 and Z is Capto.TM.
resin.
43-52. (canceled)
53. A kit for purifying a serine protease comprising (1) an
affinity solid support of any claim 21, and (2) an elution buffer
comprising a competitive agent.
54. (canceled)
55. A kit for purifying a serine protease comprising a compound of
Formula I-D: ##STR00150## or a salt thereof, and a resin of the
Formula ##STR00151## wherein R.sup.1 is --CF.sub.3,
--SO.sub.2CH.sub.3, --X-L-R, ##STR00152## R.sup.2 is --OCH.sub.3,
chloro, or X-L-R; R.sup.3 is hydrogen or chloro; X is a covalent
bond, O, S, SO.sub.2, C(O)NH, NHC(O) or NH; L-R is
--(CH.sub.2).sub.w--CO.sub.2H; R is CO.sub.2H; n, m, and p are
either 0 or 1, with the provisos that (1) when R.sup.1 is
##STR00153## --CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.2 is
--OCH.sub.3 or chloro, then one of n, m, and p must be 1, and the
others of n, m, and p must be zero; and (2) when R.sup.1 is other
than ##STR00154## --CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.2 is
X-L-R, then all of n, m, and p must be zero; u is 1, 2, 3, 4, 5, 6
or 7, w is 1, 2 or 3 and Z is Capto.TM. resin.
56. The kit of claim 54, further comprising an elution buffer
comprising a competitive agent.
57. The kit of claim 54, further comprising a washing buffer.
58-59. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Application Ser. No. 61/746,544, filed on Dec.
27, 2012, and is a continuation in part of U.S. application Ser.
No. 13/830,372, filed on Mar. 14, 2013, each of which is hereby
incorporated by reference in its entirety.
FIELD
[0002] This disclosure relates to compounds, compositions, methods
and kits for the purification of serine proteases, such as factor
Xa derivatives.
BACKGROUND
[0003] Anticoagulants serve a need in the marketplace in treatment
or prevention of undesired thrombosis in patients with a tendency
to form blood clots, such as, for example, those patients having
clotting disorders, confined to periods of immobility or undergoing
medical surgeries. One of the major limitations of anticoagulant
therapy, however, is the bleeding risk associated with treatment,
and limitations on the ability to rapidly reverse the anticoagulant
activity in case of overdosing or if an urgent surgical procedure
is required. Thus, specific and effective antidotes to all forms of
anticoagulant therapy are highly desirable. For safety
considerations, it is also advantageous to have an
anticoagulant-antidote pair in the development of new anticoagulant
drugs.
[0004] Previously reported modified derivatives of factor Xa (fXa),
such as those described in U.S. Pat. No. 8,153,390 and U.S. Pat.
No. 8,268,783 (which is herein incorporated by reference in its
entirety), including r-Antidote, are useful as antidotes to
anticoagulants targeting fXa. The modified derivatives of fXa bind
to and/or substantially neutralize the anticoagulant. Certain
modifications introduced to fXa, however, pose several challenges
for purification since routine methods for purification of clotting
factors cannot be used for r-Antidote.
SUMMARY
[0005] Disclosed herein are compounds, compositions, methods and
kits for purifying a serine protease. In one aspect, the serine
protease comprises a modified derivative of a fXa protein. In some
embodiments, the modified fXa protein comprises the amino acid
sequence of SEQ ID NO: 2 or a polypeptide having at least about 80%
sequence identity to SEQ ID NO: 2.
[0006] The compounds described herein have binding affinity with
the serine protease to be purified (e.g., the compounds are ligands
of the serine protease), and can be covalently attached to an
activated solid support, such as a resin. The solid support having
a small molecule compound bound thereto is referred to as an
affinity solid support. In some embodiments, the affinity solid
support is packed into a column, which is referred to as an
affinity column. A solution comprising the serine protease to be
purified is loaded to the affinity column. The serine protease to
be purified is retained in the column through binding activity with
the compound. Impurities in the solution are washed with a washing
buffer so that the proteins left on the column are mostly the
serine protease having binding affinity with the compound. The
serine protease can then be eluted by an elution buffer comprising
a competitive agent, which can disrupt the binding of the serine
protease with the compound and release the serine protease from the
affinity column, so that the purified serine protease is eluted
from the column with the elution buffer.
[0007] In some embodiments, compounds used to purify the proteins
are analogues of betrixaban or a salt thereof. Betrixaban is
described in U.S. Pat. No. 6,376,515, which is incorporated herein
by reference in its entirety, and is of the formula:
##STR00001##
[0008] Accordingly, in one aspect, provided is a compound of
Formula I:
##STR00002##
or a salt thereof,
[0009] wherein:
[0010] R.sup.1 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-R,
##STR00003##
[0011] R.sup.2 is --OCH.sub.3, chloro, or X-L-R;
[0012] R.sup.3 is hydrogen or chloro;
[0013] X is a covalent bond, O, S, SO.sub.2, C(O)NH, NHC(O) or
NH;
[0014] L-R is -L.sup.1-R,
##STR00004##
[0015] L.sup.1-R is
##STR00005##
[0016] L.sup.2-R is H,
##STR00006##
[0017] L.sup.3-R is
##STR00007##
[0018] R is NH.sub.2 or CO.sub.2H;
[0019] q is 1, 2, 3, 4, 5, 6 or 7;
[0020] r is 1, 2, 3, 4, 5, 6 or 7;
[0021] s is 1, 2, 3, 4, 5, 6 or 7;
[0022] t is 1, 2, 3, 4, 5, 6 or 7;
[0023] n, m, and p are either 0 or 1, with the provisos that
[0024] (1) when R.sup.1 is
##STR00008##
--CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.2 is --OCH.sub.3 or
chloro, then one of n, m, and p must be 1, and the others of n, m,
and p must be zero; and
[0025] (2) when R.sup.1 is other than
##STR00009##
--CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.2 is X-L-R, then all of
n, m, and p must be zero.
[0026] In some embodiments, the compound of Formula I is a compound
of Formula I-A:
##STR00010##
[0027] In some embodiments, the compound of Formula I is a compound
of Formula I-B:
##STR00011##
[0028] In another aspect, provided is an affinity solid support
comprising a compound of Formula I bound to a solid support via a
linker, which affinity solid support is of Formula II:
##STR00012##
or a salt thereof,
[0029] wherein:
[0030] R.sup.21 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-Y--Z,
##STR00013##
[0031] R.sup.22 is --OCH.sub.3, chloro, or --X-L-Y--Z;
[0032] R.sup.23 is hydrogen or chloro;
[0033] X is a covalent bond, O, S, SO.sub.2, C(O)NH, NHC(O) or
NH;
[0034] L-Y--Z is -L.sup.1-Y--Z,
##STR00014##
[0035] L.sup.1-Y--Z is
##STR00015##
[0036] L.sup.2-Y--Z is
##STR00016##
[0037] L.sup.3-Y--Z is
##STR00017##
[0038] Y--Z is
##STR00018##
[0039] Z is a solid support;
[0040] q is 1, 2, 3, 4, 5, 6 or 7;
[0041] r is 1, 2, 3, 4, 5, 6 or 7;
[0042] s is 1, 2, 3, 4, 5, 6 or 7;
[0043] t is 1, 2, 3, 4, 5, 6 or 7;
[0044] v is 1, 2, 3, 4, 5, 6 or 7;
[0045] u is 1, 2, 3, 4, 5, 6 or 7;
[0046] w is 1, 2 or 3;
[0047] n, m, and p are either 0 or 1, with the provisos that
[0048] (1) when R.sup.21 is
##STR00019##
--CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.22 is --OCH.sub.3 or
chloro, then one of n, m, and p must be 1, and the others of n, m,
and p must be zero; and
[0049] (2) when R.sup.21 is other than
##STR00020##
--CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.22 is X-L-Y--Z, then all
of n, m, and p must be zero.
[0050] In another aspect, provided is a method of preparing an
affinity solid support of Formula II comprising contacting a
compound of Formula I with an activated solid support capable of
forming a covalent bond with the compound of Formula I, wherein the
affinity solid support of Formula II, the compound of Formula I and
activated solid support are as defined herein.
[0051] In another aspect, provided is a method for purifying a
serine protease comprising [0052] (1) adding a first composition
comprising the serine protease to an affinity solid support of
Formula II to form a second composition comprising the serine
protease and the affinity solid support of Formula II, and [0053]
(2) eluting the serine protease from the second composition with an
elution buffer comprising a competitive agent,
[0054] wherein the affinity solid support of Formula II is as
defined herein.
[0055] In another aspect, provided is a purified serine protease,
which is purified by a method comprising [0056] (1) adding a first
composition comprising the serine protease to an affinity solid
support of Formula II to form a second composition comprising the
serine protease and the affinity solid support of Formula II, and
[0057] (2) eluting the serine protease from the second composition
with an elution buffer comprising a competitive agent, [0058]
wherein the affinity solid support of Formula II is as defined
herein.
[0059] In some embodiments, the competitive agent is arginine
and/or benzamidine, or a salt, such as a pharmaceutically
acceptable salt, thereof.
[0060] In still another aspect, provided is a kit for purifying a
serine protease comprising [0061] (1) an affinity solid support of
Formula II, and [0062] (2) an elution buffer comprising a
competitive agent, wherein the affinity solid support of Formula II
is as defined herein.
[0063] In still another aspect, provided is a kit for purifying a
serine protease comprising [0064] (1) a compound of Formula I and
an activated solid support capable of forming a covalent bond with
the compound of Formula I, and [0065] (2) an elution buffer
comprising a competitive agent, wherein the compound of Formula I
and the activated solid support are as defined herein.
[0066] In some embodiments, the competitive agent is arginine
and/or benzamidine, a pharmaceutically acceptable salt thereof.
[0067] A further aspect relates to a purified serine protease
produced by the methods described herein.
[0068] These and other aspects are described further in the text
that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0069] FIG. 1 shows SEQ ID NO: 1, a fXa derivative (also referred
to as r-Antidote precursor) with the linker, -RKRRKR- (SEQ ID NO:
3) at amino acids 106-111;
[0070] FIG. 2 shows SEQ ID NO: 2, a fXa derivative (also referred
to as r-Antidote) with the linker removed from the r-Antidote
precursor;
[0071] FIG. 3 shows the elution profile with benzamidine as
described in Example 10;
[0072] FIG. 4 shows the elution profile with arginine as described
in Example 10;
[0073] FIGS. 5 and 6 show the elution profiles as described in
Example 11;
[0074] FIGS. 7 and 8 show the loading capacity of des-chloro
betrixaban-NHS-Sepharose affinity resin and antidote recovery as
monitored by ultraviolet (UV) spectra at 280 nm described in
Example 19;
[0075] FIG. 9 shows the antidote recovery using des-chloro
betrixaban C6 linker (A4)-Capto 5 .mu.m affinity resin as monitored
by ultraviolet (UV) spectra at three different wavelengths: 260 nm,
280 nm, and 320 nm as described in Example 19;
[0076] FIG. 10 shows the antidote recovery using des-chloro
betrixaban C6 linker (A4)-Capto 11 .mu.m affinity resin as
monitored by ultraviolet (UV) spectra at three different
wavelengths: 260 nm, 280 nm, and 320 nm as described in Example
19;
[0077] FIG. 11 shows the antidote recovery using des-chloro
betrixaban C6 linker (A4)-Capto 15 .mu.m affinity resin as
monitored by ultraviolet (UV) spectra at three different
wavelengths: 260 nm, 280 nm, and 320 nm as described in Example
19;
[0078] FIG. 12 shows the antidote recovery using des-chloro
betrixaban C6 linker (A4)-Capto 20 .mu.m affinity resin as
monitored by ultraviolet (UV) spectra at three different
wavelengths: 260 nm, 280 nm, and 320 nm as described in Example
19;
[0079] FIG. 13 shows the antidote purification using des-chloro
betrixaban C6 linker (A4) Capto 11 .mu.m affinity resin using the
SMI (small molecule inhibitor) Four Step Method of Example 21;
and
[0080] FIG. 14 shows the antidote purification using the Four Step
Method using MMC (multi-modal column) capture step of Example
21.
DETAILED DESCRIPTION
Definitions
[0081] The practice of the present disclosure will employ, unless
otherwise indicated, conventional techniques of tissue culture,
immunology, molecular biology, microbiology, cell biology and
recombinant DNA, which are within the skill of the art. See, e.g.,
Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd
edition; Ausubel et al., eds. (1987) Current Protocols In Molecular
Biology; MacPherson, B. D. Hames and G. R. Taylor eds., (1995) PCR
2: A Practical Approach; Harlow and Lane, eds. (1988) Antibodies, A
Laboratory Manual; Harlow and Lane, eds. (1999) Using Antibodies, a
Laboratory Manual; and R. I. Freshney, ed. (1987) Animal Cell
Culture.
[0082] As used herein, the term "about" generally means the stated
value plus or minus a range of 10% or 5% of that value.
[0083] As used in the specification and claims, the singular form
"a," "an" and "the" include plural references unless the context
clearly dictates otherwise.
[0084] The term "solid support" intends solid phase supports
include silica gels, resins, derivatized plastic films, glass
beads, glass slides, flasks, tissue culture flasks, cotton, plastic
beads, alumina gels, pellets, cellulose beads, pore-glass beads,
grafted co-poly beads and polyacrylamide beads. More specific
examples include polystyrene (e.g., PAM-resin obtained from Bachem
Inc., Peninsula Laboratories, etc.), POLYHIPE.TM. resin (obtained
from Aminotech, Canada), polyamide resin (obtained from Peninsula
Laboratories), polystyrene resin grafted with polyethylene glycol
(TentaGel.TM., Rapp Polymere, Tubingen, Germany) or
polydimethylacrylamide resin (obtained from Milligen/Biosearch,
California). Solid supports also include microchips and grids. The
surface of the grids may be composed of a wide variety of material
including glass, plastic, silicon, gold, gelatin or nylon. Lockhart
(2000) Nature, 405:827-836; Srinivas (2001) Clin. Chem.,
47:1901-1911. In some embodiments, the solid support is
cross-linked agarose. In some embodiments, the solid support is a
cross-linked, beaded-form of a polysaccharide polymer material
extracted from seaweed. In some embodiments, the solid support is
Sepharose.TM. resin, available from GE Healthcare. In other
embodiments, the solid support is Capto.TM. resin, available from
GE Healthcare.
[0085] The term "activated solid support" refers to a solid support
functionalized with a functional group that can form a covalent
bond with a compound of Formula I under suitable reaction
conditions. In some embodiments, the activated solid support is an
agarose bead functionalized with a N.ident.C group, e.g.,
CNBr-activated Sepharose.TM. resin. In some embodiments, the
activated solid support is an agarose bead functionalized with free
amino groups, e.g., EAH Sepharose.TM. resin. This is referred to as
"amino-functionalized agarose." In some embodiments, the activated
solid support is an agarose bead functionalized with a
(N-hydroxysuccinimide group, e.g., NHS-activated Sepharose.TM.
resin. In some embodiments, Capto.TM. resin is activated
similarly.
[0086] The term "affinity solid support" refers to a solid support
having a compound of Formula I bound thereto, wherein the compound
of Formula I exhibits binding affinity towards a serine protease to
be purified. The term "affinity resin" refers to an affinity solid
support wherein the solid support is a resin. The term "affinity
column" refers to a column comprising the affinity solid support.
In some embodiments, the compound of Formula I is covalently bound
to the solid support.
[0087] The term "protein", "peptide" and "polypeptide" are used
interchangeably and in their broadest sense to refer to a compound
of two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another embodiment, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. A protein or peptide must contain at least two
amino acids and no limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's sequence.
As used herein the term "amino acid" refers to either natural
and/or unnatural or synthetic amino acids, including glycine and
both the D and L optical isomers, amino acid analogs and
peptidomimetics.
[0088] The term "isolated" or "recombinant" as used herein with
respect to nucleic acids, such as DNA or RNA, refers to molecules
separated from other DNAs or RNAs. The term "isolated" is also used
herein to refer to polynucleotides, polypeptides and proteins that
are isolated from other cellular proteins and is meant to encompass
both purified and recombinant polypeptides. For example, an
isolated cell is a cell that is separated from tissue or cells of
dissimilar phenotype or genotype. An isolated polynucleotide is
separated from the 3' and 5' contiguous nucleotides with which it
is normally associated in its native or natural environment, e.g.,
on the chromosome. As is apparent to those of skill in the art, a
non-naturally occurring polynucleotide, peptide, polypeptide,
protein, antibody or fragment(s) thereof, does not require
"isolation" to distinguish it from its naturally occurring
counterpart.
[0089] The term "biological equivalent of" a protein, peptide or
polynucleotide refers to one that has at least about 80% homology
or identity and alternatively, at least about 85%, or alternatively
at least about 90%, or alternatively at least about 95%, or
alternatively 98% percent homology or identity, and exhibits
substantially equivalent biological activity to the reference
protein, polypeptide or nucleic acid.
[0090] A polynucleotide or polynucleotide region (or a polypeptide
or polypeptide region) having a certain percentage (for example,
80%, 85%, 90%, 95%, 97%, 98%, or 99%) of "sequence identity" to
another sequence means that, when aligned, that percentage of bases
(or amino acids) are the same in comparing the two sequences. The
alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example
those described in Current Protocols in Molecular Biology (Ausubel
et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
Preferably, default parameters are used for alignment. A preferred
alignment program is BLAST, using default parameters. In
particular, preferred programs are BLASTN and BLASTP, using the
following default parameters: Genetic code=standard; filter=none;
strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50
sequences; sort by .dbd.HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs
can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-bin/BLAST.
[0091] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares, for example, less
than 40% identity, or alternatively less than 25% identity, with
one of the sequences of the present disclosure.
[0092] The term "fraction" when used in the context of protein
isolation, refers to a collection of material separated based on a
specific property. The specific property may include, by way of
non-limiting example, size, mass, isolectric point, charge, and the
like.
[0093] "Factor Xa" or "fXa" or "fXa protein" refers to a serine
protease in the blood coagulation pathway, which is produced from
the inactive factor X (fX). Factor Xa is activated by either factor
IXa with its cofactor, factor VIIIa, in a complex known as
intrinsic Xase, or factor VIIa with its cofactor, tissue factor, in
a complex known as extrinsic Xase. fXa forms a membrane-bound
prothrombinase complex with factor Va and is the active component
in the prothrombinase complex that catalyzes the conversion of
prothrombin to thrombin. Thrombin is the enzyme that catalyzes the
conversion of fibrinogen to fibrin, which ultimately leads to blood
clot formation.
[0094] As used herein, a "fXa derivative" refers to a modified fXa
protein that does not compete with fXa in assembling into the
prothrombinase complex and has reduced or no procoagulant activity,
and yet binds and/or substantially neutralizes an anticoagulant,
such as a fXa inhibitor. An example of a fXa derivative is provided
herein as SEQ ID NO: 2 (FIG. 2) or a biological equivalent
thereof.
[0095] "r-Antidote precursor" refers to a fXa derivative
represented by SEQ ID NO: 1, which contains three mutations
relative to human fXa. The first mutation is a deletion in the
Gla-domain of the wild-type fX protein at position 6-39. The second
mutation is replacing the activation peptide sequence 143-194 amino
acids with -RKR-. This produces a -RKRRKR- (SEQ ID NO: 3) linker
connecting the light chain and the heavy chain. Upon secretion,
this linker is cleaved in CHO resulting in a cleaved two-chain
polypeptide. The term "cleaved two-chain polypeptide" refers to a
polypeptide of SEQ ID NO: 2, or a polypeptide having 80% identity
to SEQ ID NO: 2, having two-chains and being linked together by a
disulfide bond. The N-terminal chain consist of amino acids 1-105
of SEQ ID NO: 2 and the C-terminal chain consists of amino acids
106-359 of SEQ ID NO: 2. Optionally, the LC chain may contain 1, 2,
3, 4, 5 or 6 amino acid residues of the linker. Such additional
residues result from the incomplete removal of the linker
polypeptide. The third mutation is mutation of active site residue
S379 to an Ala residue (based on secreted human fX sequence). This
amino acid substitution corresponds to amino acid 296 and 290 of
SEQ ID NOS: 1 and 2, respectively.
[0096] The term "r-Antidote" may refer to the polypeptide (SEQ ID
NO: 1) before removal of the linker (SEQ ID NO: 3) or after removal
of the linker (SEQ ID NO: 3). The r-Antidote with the linker
removed has two forms: alpha form (SEQ ID NO: 2), and beta form
(SEQ ID NO. 4), which lacks the beta-peptide (GLPKAKSHAPEVITSSPLK,
SEQ ID NO. 5) at the c-terminus of the heavy chain. These
polypeptides are described in Tables 1-3 below.
TABLE-US-00001 TABLE 1 SEQ ID NO. 1-Polypeptide sequence of the
r-antidote precursor, prior to removal of the-RKRRKR-(SEQ ID NO. 3)
linker Light Chain 1 ANSFL F WNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC
TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121
GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181 IVGGQE
CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241 AKRFKVRVGD
RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301
ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ
361 NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG
IYTKVTAFLK 421 WIDRSMKTRG LPKAKSHAPE VITSSPLK
TABLE-US-00002 TABLE 2 Sequence ID NO. 2-Polypeptide sequence of
the r-antidote, the alpha form Light Chain 1 ANSFL F WNKYKDGDQC
ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC
HEEQNSVVCS CARGYTLADN 121 GKACIPTGPY PCGKQTLER Heavy Chain 181
IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241 AKRFKVRVGD
RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301
ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ
361 NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG
IYTKVTAFLK 421 WIDRSMKTRG LPKAKSHAPE VITSSPLK
TABLE-US-00003 TABLE 3 SEQ ID NO. 4-Polypeptide sequence of the
r-antidote, the beta form, which lacks the beta-peptide
(GLPKAKSHAPEVITSSPLK, SEQ ID NO. 5) at the c-terminus of the heavy
chain Light Chain 1 ANSFL F WNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC
TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121
GKACIPTGPY PCGKQTLER Heavy Chain 181 IVGGQE CKDGECPWQA LLINEENEGF
CGGTILSEFY ILTAAHCLYQ 241 AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK
HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301 ACLPERDWAE STLMTQKTGI
VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361 NMFCAGYDTK
QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421
WIDRSMKTR
[0097] The term "competitive agent" is a molecule that can aid in
the elution of the serine protease from the affinity solid support
of Formula II either by disruption of a charge-charge interaction
between the affinity solid support of Formula II and the serine
protease or by competing with the affinity solid support of Formula
II for binding to the serine protease. Non-limiting examples of
competitive agents include arginine and benzamidine.
[0098] The term "chaotropic agent" intends a substance which
disrupts the structure of, and denatures, macromolecules such as
proteins and nucleic acids. Chaotropic agents include, for example,
butanol, ethanol, guanidinium chloride, lithium perchlorate,
magnesium chloride, phenol, propanol, sodium dodecyl sulfate,
thiourea, and urea.
[0099] The term "salt" refers to an ionic compounds that result
from the neutralization reaction of an acid and a base, and is
composed of at least one cations (positively charged ion) and at
least one anion (negative ion). In some embodiments, a salt is
electrically neutral (without a net charge). The term
"pharmaceutically acceptable salts" is meant to include salts of
the compounds which are prepared with relatively nontoxic acids or
bases, depending on the particular substituents found on the
compounds described herein. Pharmaceutically acceptable salts
derived from inorganic bases include aluminum, ammonium, calcium,
copper, ferric, ferrous, lithium, magnesium, manganic, manganous,
potassium, sodium, and zinc salts, and the like. Pharmaceutically
acceptable salts derived from organic bases include salts of
primary, secondary and tertiary amines, including substituted
amines, cyclic amines, naturally-occurring amines and the like,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine and the like. Acids that can form
pharmaceutically acceptable salts include inorganic acids such as
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
and relatively nontoxic organic acids such as acetic, propionic,
isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic,
phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, e.g.,
Berge, S. M., et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19).
Compounds
[0100] In one aspect, provided is a compound of Formula I:
##STR00021##
or a salt thereof, wherein:
[0101] R.sup.1 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-R,
##STR00022##
[0102] R.sup.2 is --OCH.sub.3, chloro, or X-L-R;
[0103] R.sup.3 is hydrogen or chloro;
[0104] X is a covalent bond, O, S, SO.sub.2, C(O)NH, NHC(O) or
NH;
[0105] L-R is -L.sup.1-R,
##STR00023##
[0106] L.sup.1-R is
##STR00024##
[0107] L.sup.2-R is H,
##STR00025##
[0108] L.sup.3-R is
##STR00026##
[0109] R is NH.sub.2 or CO.sub.2H;
[0110] q is 1, 2, 3, 4, 5, 6 or 7;
[0111] r is 1, 2, 3, 4, 5, 6 or 7;
[0112] s is 1, 2, 3, 4, 5, 6 or 7;
[0113] t is 1, 2, 3, 4, 5, 6 or 7;
[0114] n, m, and p are either 0 or 1, with the provisos that
[0115] (1) when R.sup.1 is
##STR00027##
--CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.2 is --OCH.sub.3 or
chloro, then one of n, m, and p must be 1, and the others of n, m,
and p must be zero; and
[0116] (2) when R.sup.1 is other than
##STR00028##
--CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.2 is X-L-R, then all of
n, m, and p must be zero.
[0117] In some embodiments, the compound of Formula I is a compound
of Formula I-A:
##STR00029##
i.e., wherein R is NH.sub.2.
[0118] In some embodiments, the compound of Formula I is a compound
of Formula I-B:
##STR00030##
i.e., wherein R is CO.sub.2H.
[0119] In some embodiments, the compound of Formula I is a compound
of Formula I-C:
##STR00031##
[0120] In some embodiments, R.sup.1 is
##STR00032##
[0121] In some embodiments, R.sup.3 is hydrogen. In some
embodiments, R.sup.3 is chloro.
[0122] In some embodiments, R.sup.2 is --OCH.sub.3. In some
embodiments, R.sup.2 is X-L-R.
[0123] In some embodiments, X is O. In some embodiments, X is S. In
some embodiments,
[0124] X is SO.sub.2. In some embodiments, X is NH. In some
embodiments, X is C(O)NH. In some embodiments, X is NHC(O). In some
embodiments, X is a covalent bond.
[0125] In some embodiments, L-R is -L.sup.1-R. In some embodiments,
L-R is
##STR00033##
In some embodiments, L.sup.1-R is
##STR00034##
[0126] In some embodiments, L-R is
##STR00035##
[0127] In some embodiments, L-R is
##STR00036##
[0128] In some embodiments, --X-L-R is
##STR00037##
##STR00038##
[0129] In some embodiments, --X-L-R is
##STR00039##
In some embodiments, --X-L-R is
##STR00040##
[0130] In some embodiments, q is at least 3. In some embodiments, r
is at least 3. In some embodiments, s is at least 3. In some
embodiments, t is at least 3.
[0131] In some embodiments, the compound of Formula I inhibits fXa
with an IC.sub.50 of from about 100 nM to about 1 .mu.M, from about
150 nM to about 700 nM, or from about 200 nM to about 500 nM.
[0132] In some embodiments, the compound of Formula I is selected
from
##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045##
or a salt thereof.
Affinity Solid Supports
[0133] In another aspect, provided is an affinity solid support of
Formula II:
##STR00046##
or a salt thereof, wherein:
[0134] R.sup.21 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-Y--Z,
##STR00047##
[0135] R.sup.22 is --OCH.sub.3, chloro, or X-L-Y--Z;
[0136] R.sup.23 is hydrogen or chloro;
[0137] X is a covalent bond, O, S, SO.sub.2, C(O)NH, NHC(O) or
NH;
[0138] L-Y--Z is -L.sup.1-Y--Z,
##STR00048##
[0139] L.sup.1-Y--Z is
##STR00049##
[0140] L.sup.2-Y--Z is
##STR00050##
[0141] L.sup.3-Y--Z is
##STR00051##
[0142] Y--Z is
##STR00052##
[0143] Z is a solid support;
[0144] q is 1, 2, 3, 4, 5, 6 or 7;
[0145] r is 1, 2, 3, 4, 5, 6 or 7;
[0146] s is 1, 2, 3, 4, 5, 6 or 7;
[0147] t is 1, 2, 3, 4, 5, 6 or 7;
[0148] v is 1, 2, 3, 4, 5, 6 or 7;
[0149] u is 1, 2, 3, 4, 5, 6 or 7;
[0150] w is 1, 2 or 3;
[0151] n, m, and p are either 0 or 1, with the provisos that
[0152] (1) when R.sup.21 is
##STR00053##
--CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.22 is --OCH.sub.3 or
chloro, then one of n, m, and p must be 1, and the others of n, m,
and p must be zero; and
[0153] (2) when R.sup.21 is other than
##STR00054##
--CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.22 is X-L-Y--Z, then all
of n, m, and p must be zero.
[0154] In some embodiments, the affinity solid support of Formula
II is an affinity solid support of Formula II-A
##STR00055##
i.e., wherein Y--Z is
##STR00056##
[0155] In some embodiments, the affinity solid support of Formula
II is of Formula II-B
##STR00057##
i.e., wherein Y--Z is
##STR00058##
[0156] In some embodiments, the affinity solid support of Formula
II is of Formula II-C
##STR00059##
i.e., wherein L-Y--Z is
##STR00060##
u is 1, 2, 3, 4, 5, 6 or 7, and w is 1, 2 or 3.
[0157] In some embodiments, u is 4 or 5, and w is 1.
[0158] In some embodiments, the affinity solid support is of
Formula II-D:
##STR00061##
[0159] In some embodiments, R.sup.21 is
##STR00062##
[0160] In some embodiments, R.sup.22 is --OCH.sub.3. In some
embodiments, R.sup.22 is X-L-Y--Z.
[0161] In some embodiments, R.sup.23 is hydrogen. In some
embodiments, R.sup.23 is chloro.
[0162] In some embodiments, X is O. In some embodiments, X is S. In
some embodiments, X is SO.sub.2. In some embodiments, X is NH. In
some embodiments, X is C(O)NH. In some embodiments, X is NHC(O). In
some embodiments, X is covalent bond.
[0163] In some embodiments, L-Y--Z is -L.sup.1-Y--Z. In some
embodiments, L-Y--Z is
##STR00063##
[0164] In some embodiments, L.sup.1-Y--Z is
##STR00064##
[0165] In some embodiments. L-Y--Z is
##STR00065##
[0166] In some embodiments, L-Y--Z or L.sup.1-Y--Z is
##STR00066##
[0167] In some embodiments, L-Y--Z or L.sup.1-Y--Z is
##STR00067##
[0168] In some embodiments, L-Y--Z or L.sup.1-Y--Z is
##STR00068##
[0169] In some embodiments, --X-L-Y--Z is
##STR00069##
[0170] In some embodiments, --X-L-Y--Z is
##STR00070##
[0171] In some embodiments, --X-L-Y--Z is
##STR00071##
[0172] In some embodiments, q is at least 3. In some embodiments, r
is at least 3. In some embodiments, s is at least 3. In some
embodiments, t is at least 3.
[0173] In some embodiments, Z is selected from the group consisting
of polystyrene, polystyrene resin grafted with polyethylene glycol,
polyamide resin, polyacrylamide resin, polydimethylacrylamide
resin, silica, dextran and polysaccharide resin. In some
embodiments, Z is cross-linked agarose. In some embodiments, Z is a
resin comprising a crosslinked, polysaccharide polymer, which can
be extracted from seaweed. In some embodiments, Z is Sepharose.TM.
resin. In other embodiments, Z is Capto.TM. resin.
[0174] In some embodiments, the affinity solid support comprises
betrixaban covalently bound to the solid support through a linker.
Such an affinity solid support can be referred to as
betrixaban-solid support, such as betrixaban--Sepharose.TM. when
the solid support is Sepharose.TM. or betrixaban--Capto.TM. when
the solid support is Capto.
[0175] In some embodiments, the affinity solid support comprises
des-chloro betrixaban covalently bound to the solid support through
a linker. Des-chloro betrixaban is of the formula:
##STR00072##
Such an affinity solid support can be referred to as des-chloro
betrixaban-solid support, such as des-chloro
betrixaban--Sepharose.TM. when the solid support is Sepharose.TM.
or des-chloro betrixaban--Capto.TM. when the solid support is
Capto.TM.
[0176] In some embodiments, the affinity solid support of Formula
II is
##STR00073## ##STR00074##
or a salt thereof, wherein Z is a solid support.
[0177] In some embodiments, Z is Sepharose.TM.. In other
embodiments, Z is Capto.TM..
[0178] In some embodiments, the salt is a pharmaceutically
acceptable salt.
Preparation Methods
[0179] The compounds and affinity solid supports of this invention
can be prepared from readily available starting materials according
to the general methods and procedures, and procedures in examples
provided herein. It will be appreciated that where typical or
preferred process conditions (i.e., reaction temperatures, times,
mole ratios of reactants, solvents, pressures) are given, other
process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0180] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions.
Suitable protecting groups for various functional groups as well as
suitable conditions for protecting and deprotecting particular
functional groups are known in the art. For example, numerous
protecting groups are described in T. W. Greene and P. G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and references cited therein.
[0181] Furthermore, the compounds of this invention may contain one
or more chiral centers. Accordingly, if desired, such compounds can
be prepared or isolated as pure stereoisomers, i.e., as individual
enantiomers or diastereomers, or as stereoisomer-enriched mixtures.
All such stereoisomers (and enriched mixtures) are included within
the scope of this invention, unless otherwise indicated. Pure
stereoisomers (or enriched mixtures) may be prepared using, for
example, optically active starting materials or stereoselective
reagents well-known in the art. Alternatively, racemic mixtures of
such compounds can be separated using, for example, chiral column
chromatography, chiral resolving agents, and the like.
[0182] The starting materials for preparing the compounds or
affinity solid supports are generally known compounds or can be
prepared by known procedures or obvious modifications thereof. For
example, many of the starting materials are available from
commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.,
USA), Bachem (Torrance, Calif., USA), Emka-Chemce or Sigma (St.
Louis, Mo., USA). Others may be prepared by procedures, or obvious
modifications thereof, described in standard reference texts such
as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15
(John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds,
Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989),
Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991),
March's Advanced Organic Chemistry, (John Wiley and Sons, 4.sup.th
Edition), and Larock's Comprehensive Organic Transformations (VCH
Publishers Inc., 1989).
[0183] The various starting materials, intermediates, and compounds
of the invention may be isolated and purified where appropriate
using conventional techniques such as precipitation, filtration,
crystallization, evaporation, distillation, and chromatography.
Characterization of these compounds may be performed using
conventional methods such as by melting point, mass spectrum,
nuclear magnetic resonance, and various other spectroscopic
analyses.
[0184] In one aspect, provided is a method of preparing an affinity
solid support of Formula II or a salt thereof comprising contacting
a compound of Formula I or a salt thereof with a solid support
capable of forming a covalent bond with the compound of Formula I,
wherein the affinity solid support of Formula II, the compound of
Formula I and the solid support are as defined herein.
[0185] In some embodiments, provided is a method of preparing an
affinity solid support of Formula II-A or a salt thereof comprising
contacting a compound of Formula I-A, or a salt thereof, with a
solid support of the formula NC--O--Z, wherein the affinity solid
support of Formula II-A, the compound of Formula I-A and Z are as
defined herein. The method is illustrated in Scheme 1.
##STR00075##
[0186] The variables in the Scheme 1 are as defined herein. The
compound of Formula I-A can be coupled to commercially available
CNBr-activated Sepharose.TM. using standard coupling techniques.
For example, CNBr-activated Sepharose.TM. 4-FF or CNBr-activated
Sepharose.TM. 4B (Amersham) resin can be hydrated and optionally
washed with a low pH aqueous solution (e.g., about 1 mM aqueous HCl
solution). A solution comprising a compound of Formula I-A in a
suitable solvent, such as a mixture of a water miscible organic
solvent (e.g., DMSO) and a suitable buffer (e.g., pH at about 8-9,
e.g., about 8.3) can be added to the resin. The mixture is kept at
room temperature for a sufficient period of time (e.g., about
several hours) while adjusting the pH to about 8-9, e.g., about
8.3, to allow coupling reaction between the compound of Formula I-A
and the CNBr group of the resin. The reaction can be monitored by
conventional analytical methods, such as HPLC or UPLC. Upon
completion of the coupling reaction, unreacted CNBr can be
optionally capped with a suitable buffer, such as 0.1 M Tris-HCl
buffer at pH 8.0. The coupled resin can be optionally washed with a
suitable buffer, such as an acetate buffer (e.g., 0.1 M at a pH of
3 to 4) and/or a Tris-HCl buffer (e.g., 0.1 M at a pH of 8 to 9).
The buffers can optionally comprising a suitable amount of NaCl
(e.g., 0.5 M). The wash can be repeated.
[0187] Similarly, Capto.TM. resin can be used instead of
Sepharose.TM..
[0188] General methods of preparing the solid support for the
reaction and reaction conditions for the solid support are
described in more detail in Instructions 71-5000-15 AF, 2011, by
General Electric Company, which is incorporated by reference in its
entirety.
[0189] In some embodiments, provided is a method of preparing an
affinity solid support of Formula II-B or a salt thereof comprising
contacting a compound of Formula I-A, or a salt thereof, with a
resin of the formula
##STR00076##
wherein the affinity solid support of Formula II-B, the compound of
Formula I-A and Z are as defined herein. The method is illustrated
in Scheme 2 wherein all variables are as defined herein.
##STR00077##
[0190] In some embodiments, the reaction is conducted at a pH of
about 6 to 9, such as in a buffer of 0.2 M NaHCO.sub.3, 0.5 M NaCl,
pH 8.3.
[0191] In some embodiments, the solid support is
##STR00078##
such as NHS-activated Sepharose.TM. 4 Fast Flow, available from GE
Healthcare. General methods of preparing the solid support for the
reaction and reaction conditions for the solid support are
described in more detail in Instructions 71-5000-14 AD, 2011, by
General Electric Company, which is incorporated by reference in its
entirety.
[0192] In another aspect, provided is a method of preparing an
affinity solid support of Formula II-C or a salt thereof comprising
contacting a compound of Formula I-D:
##STR00079##
or a salt thereof, with a resin of the formula
##STR00080##
wherein
[0193] R.sup.1 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-R,
##STR00081##
[0194] R.sup.2 is --OCH.sub.3, chloro, or X-L-R;
[0195] R.sup.3 is hydrogen or chloro;
[0196] X is a covalent bond, O, S, SO.sub.2, C(O)NH, NHC(O) or
NH;
[0197] L-R is --(CH.sub.2).sub.w--CO.sub.2H;
[0198] R is CO.sub.2H;
[0199] n, m, and p are either 0 or 1, with the provisos that
[0200] (1) when R.sup.1 is
##STR00082##
--CF.sub.3 or --SO.sub.2CH.sub.3, and R.sup.2 is --OCH.sub.3 or
chloro, then one of n, m, and p must be 1, and the others of n, m,
and p must be zero; and
[0201] (2) when R.sup.1 is other than
##STR00083##
--CF.sub.3 or --SO.sub.2CH.sub.3, or R.sup.2 is X-L-R, then all of
n, m, and p must be zero;
[0202] the affinity solid support of Formula II-C, u, w and Z are
as defined herein.
[0203] The method is illustrated in Scheme 3 wherein all variables
are as defined herein.
##STR00084##
[0204] In Scheme 3, Compound I-D can be coupled to the resin under
conditions comprising an amide coupling reagent. Amide coupling
reagent refers to a reagent that may be used to form an amide bond
between an amino group and a carboxy group. Examples of coupling
reagents include, but are not limited to, carbodiimides such as
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIPCDI), and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide
(EDCI); aminium compounds such as
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-meth-
ylmethanaminium hexafluorophosphate N-oxide (HATU),
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HBTU),
N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan-
aminium hexafluorophosphate N-oxide (HCTU),
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
tetrafluoroborate N-oxide (TBTU), and
N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan-
aminium tetrafluoroborate N-oxide (TCTU); and phosphonium compounds
such as 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP) and
benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP). In some embodiments, the coupling
reagent is a carbodiimide. In some embodiments, the coupling
reagent is EDC. In some embodiments, the coupling is conducted at a
pH of about 4.5-6. In some embodiments, the concentration of the
coupling reagent is about 10-100 times of the concentration of the
functional group on the solid support. In some embodiments, the
coupling reagent is in a solution comprising water and optionally a
water soluble organic solvent such as dioxane or ethylene
glycol.
[0205] In some embodiments, the solid support is
##STR00085##
such as EAH Sepharose.TM. 4B, available from GE Healthcare. General
methods of preparing the solid support for the reaction and
reaction conditions for the solid support are described in more
detail in Instructions 71-7097-00 AE, 2009, by General Electric
Company, which is incorporated by reference in its entirety.
[0206] Compounds of Formula I can be prepared by the following
exemplifying synthetic schemes.
##STR00086##
[0207] In Scheme 4, Lv is a leaving group, Pr.sup.1 is an amino
protecting group, and Pr.sup.2 is an acid protecting group,
R.sup.41 is --CF.sub.3, --SO.sub.2CH.sub.3, --OCH.sub.3, or
##STR00087##
R.sup.42 is --OCH.sub.3 or chloro, R.sup.31 is --CF.sub.3,
--SO.sub.2CH.sub.3, --OH, R.sup.32 is --OH, --OCH.sub.3 or chloro,
R.sup.1 is --CF.sub.3, --SO.sub.2CH.sub.3, --X-L-R or
##STR00088##
X is O, R.sup.2, R.sup.3, L, R, m, n, and p are as defined herein
unless otherwise stated. Compound 1 can be prepared according to
methods described in U.S. Pat. No. 6,376,515. Compound 2 can also
be prepared according to methods described in U.S. Pat. No.
6,376,515, or can be prepared from Compound 1 by demethylation of
the methoxy group with under appropriate conditions, such as using
BBr.sub.3 in a suitable organic solvent, such as methylene
chloride. Compound 2 can then be coupled to a compound of
Lv-L-NH--Pr.sup.1 or Lv-L-C(O)O--Pr.sup.2, followed by deprotection
to provide a compound of Formula I wherein X is O, R.sup.1 is
--CF.sub.3, --SO.sub.2CH.sub.3,
##STR00089##
or --X-L-R.
[0208] Leaving groups, amino protecting groups and acid protecting
groups and methods of deprotection are generally known in the
field, and many are described in T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and references cited therein, which are incorporated by
reference in their entirety. Non-limiting examples of leaving
groups include chloro, bromo, iodo, tosylate, triflate, etc.
Non-limiting examples of amino protecting groups include
N-tert-butoxycarbonyl (t-Boc), 9-fluorenylmethoxycarbonyl (Fmoc),
carboxybenzyl (Cbz), acetyl (Ac), benzoyl (Bz), p-methoxybenzyl
carbonyl (Moz or MeOZ), benzyl (Bn), p-methoxybenzyl (PMB),
3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), etc.
Non-limiting examples of carboxy protecting groups include esters
of C.sub.1-C.sub.6 alkyl, such as methyl or ethyl, which can be
deprotected by hydrolysis with a base (e.g., sodium hydroxide or
potassium carbonate), t-butyl (t-Bu) which can be deprotected by
acid hydrolysis (e.g., hydrochloric acid (HCl) or trifluoroacetic
acid (TFA)), or benzyl which can be deprotected by hydrogenation
with hydrogen in the presence of a catalyst, such as palladium.
[0209] Compound 2 in Scheme 1 can be replaced with Compound 3,
wherein R.sup.51 is --CF.sub.3, --SO.sub.2CH.sub.3,
##STR00090##
or NH.sub.2, R.sup.52 is --OCH.sub.3, NH.sub.2 or chloro, which can
react with Lv-L-NH--Pr.sup.1 or Lv-L-C(O)O--Pr.sup.2, followed by
deprotection to give compound of Formula I where X is NH, R.sup.1
is --CF.sub.3, --SO.sub.2CH.sub.3,
##STR00091##
or --X-L-R. Compound 3 can also be prepared by methods described in
U.S. Pat. No. 6,376,515.
##STR00092##
##STR00093##
[0210] Scheme 5 shows an exemplifying procedure for preparing a
compound of Formula I wherein X is S or SO.sub.2. In Scheme 2, Lv
is a leaving group, Pr.sup.1 an amino protecting group and Pr.sup.2
is an acid protecting group, R.sup.61 is --CF.sub.3,
--SO.sub.2CH.sub.3,
##STR00094##
or SH, R.sup.62 is --OCH.sub.3, SH or chloro, R.sup.1 is
--CF.sub.3, --SO.sub.2CH.sub.3,
##STR00095##
or --X-L-R, R.sup.2, R.sup.3, L, R, m, n, and p are as defined
herein unless otherwise stated. Compound 4, which can also be
prepared according to methods described in U.S. Pat. No. 6,376,515,
reacts with Lv-L-NH--Pr.sup.1 or Lv-L-C(O)O--Pr.sup.2, followed by
deprotection to give Compound 5, i.e., a compound of Formula I
where X is S. Compound 5 can be oxidized by a suitable oxidation
reagent, such as hydrogen peroxide, m-chloroperbenzoic acid and
manganese dioxide, to provide Compound 6, i.e., a compound of
Formula I where X is SO.sub.2.
Purification Methods and Serine Proteases
[0211] In certain embodiments, the serine protease that is purified
by the methods described herein is a fXa derivative. Certain fXa
derivatives are described in U.S. Pat. No. 8,153,590, which is
herein incorporated by reference in its entirety. For example, the
serine protease is a polypeptide comprising the amino acid sequence
of SEQ ID NO: 1, 2 or 4 or a polypeptide having at least about 80%
sequence identity to SEQ ID NO: 1, 2 or 4. The fXa derivative
represented by SEQ ID NO: 1 contains three mutations relative to
wild-type fXa. The first mutation is the deletion in the Gla-domain
of FX at position 6-39 in the wild-type protein. The second
mutation replaces the activation peptide sequence 143-194 aa with
-RKR-. This produced a -RKRRKR- (SEQ ID NO: 3) linker connecting
the light chain and the heavy chain. Upon secretion, this linker is
cleaved in CHO resulting in a two-chain fXa molecule (SEQ ID NO:
2). The third mutation is mutation of active site residue S379 to
an Ala residue (based on secreted human fX amino acid sequence).
This amino acid substitution corresponds to amino acid at position
296 and position 290 of SEQ ID NOS: 1 and 2, respectively. The fXa
derivative does not compete with fXa in assembling into the
prothrombinase complex, but instead bind and/or substantially
neutralize the anticoagulants, such as fXa inhibitors. The
derivatives useful as antidotes are modified to reduce or remove
intrinsic procoagulant and anticoagulant activities, while
retaining the ability to bind to the inhibitors. Structurally, in
one embodiment, the derivatives are modified to provide either no
procoagulant activity or reduced procoagulant activity.
"Procoagulant activity" is referred to herein as an agent's ability
to cause blood coagulation or clot formation. Reduced procoagulant
activity means that the procoagulant activity has been reduced by
at least about 50%, or more than about 90%, or more than about 95%
as compared to wild-type fXa. In a related embodiment, the amino
acid sequence having at least 80% sequence identity to SEQ ID NO: 2
has reduced procoagulant activity compared to wild-type factor Xa.
In a further embodiment, the amino acid sequence having at least
80% sequence identity to SEQ ID NO: 2 does not assemble into a
prothrombinase complex. The serine protease purified herein
includes salts of the serine protease.
[0212] A further aspect disclosed herein relates to a purified
serine protease comprising the amino acid sequence of SEQ ID NO: 2
or a polypeptide having at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 97%, at least
about 98%, or at least about 99% sequence identity to SEQ ID NO: 2
wherein the polypeptide is produced by the methods described
herein. U.S. Pat. Nos. 8,153,590 and 8,268,783 describe serine
protease proteins, modifications, and methods of preparing the
proteins, and are incorporated by reference in their entirety. In
some embodiments, the purified serine protease comprises at least
85% of the amino acid sequence of SEQ ID NO: 2 (the alpha form of
r-Antidote) and no more than 10% of the amino acid sequence of SEQ
ID NO: 4 (the beta form of r-Antidote). In some embodiments, the
purified serine protease comprises no more than 8% of the amino
acid sequence of SEQ ID NO: 4 (the beta form of r-Antidote).
[0213] The serine protease can be recombinantly produced as
previously described, e.g., in U.S. Patent Publication No. US
2013-0230901, or by other methods of recombinant protein production
known in the art. For example, proteins may be cloned into a DNA
construct (i.e. plasmids, viral vectors, cosmids, expression
vectors, phagemids, fosmids, and artificial chromosomes such as
bacterial artificial chromosomes, yeast artificial chromosomes, and
human artificial chromosomes) and introduced into a suitable host
cell by gene transfer techniques such chemical-based transfection,
such as calcium phosphate transfection and polyfection, and non
chemical-based transfection such as electroporation, optical
transfection, and gene electrotransfer. Suitable host cells include
prokaryotic and eukaryotic cells, which include, but are not
limited to bacterial cells, yeast cells, insect cells, animal
cells, mammalian cells, murine cells, rat cells, sheep cells,
simian cells and human cells. Cells can then be lysed by physical
techniques such as sonication or freeze-thaw or by the use of
detergents or lysis buffers such as RIPA Buffer
(Radi-Immunoprecipitation Assay) containing 150 mM NaCl, 1.0%
IGEPAL.TM. CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM
Tris, pH 8.0, or by physical separation, such as centrifugation or
filtration, to obtain the clarified harvested culture fluid from
mammalian cell cultures. The resulting soluble protein extract may
be then used in the purification methods described herein.
[0214] U.S. Patent Publication No. US 2013-0230901, which is herein
incorporated by reference in its entirety, describes methods and
cells for the improved or enhanced processing of the one-chain
r-Antidote precursor to the cleaved two-chain r-Antidote protein
that acts as an antidote to fXa inhibitors. WO 2013/188587
describes methods for purifying serine proteases (e.g., r-Antidote)
in active form from a composition containing the serine proteases a
STI based affinity resin. STI affinity resin having a protein
usually is reusable for a limited number of times and is expensive
to manufacture. The purification methods described herein employ
small molecule compounds that can be readily prepared and attached
to a solid support, and are reusable. The methods are suitable for
large scale purification, and can provide higher binding capacity
and improved purity. The small molecule compounds can provide
different levels of binding affinities with different serine
proteases so that selectivity and specificity with a particular
serine protease can be obtained. The methods are contemplated to
provide increased yield.
[0215] In one aspect, the method comprises
[0216] (1) adding a first composition comprising the serine
protease to an affinity solid support of Formula II or a salt
thereof to form a second composition comprising the serine protease
and the affinity solid support of Formula II, and
[0217] (2) eluting the serine protease from the second composition
with an elution buffer comprising a competitive agent,
[0218] wherein the affinity solid support of Formula II is as
defined herein.
[0219] In another aspect, provided is a purified serine protease,
which is purified by a method comprising [0220] (1) adding a first
composition comprising the serine protease to an affinity solid
support of Formula II or a salt thereof to form a second
composition comprising the serine protease and the affinity solid
support of Formula II, and [0221] (2) eluting the serine protease
from the second composition with an elution buffer comprising a
competitive agent, [0222] wherein the affinity solid support of
Formula II is as defined herein.
[0223] As described herein, the affinity solid support of Formula
II comprises a compound covalently bound to the solid support which
compound has binding affinity towards the serine protease. The
second composition comprising the serine protease and the affinity
solid support of Formula II is formed through non-covalent binding
between the serine protease and the compound on the affinity solid
support of Formula II. Such non-covalent binding includes one or
more binding interactions, such as hydrogen bonds, ionic bonds, van
der Waals forces, and hydrophobic interactions, etc., between the
compound on the affinity solid support of Formula II and one or
more amino acid residues of the serine protease.
[0224] In some embodiments, the amount of the serine protease bound
to the affinity solid support is at least 50%, at least 60%, at
least 70% or at least 80% of the binding capacity of the affinity
solid support.
[0225] In some embodiments, the amount of the serine protease bound
to the affinity solid support is at least 150%, 200%, 250% or 300%
of that bound to a STI affinity solid support per unit volume of
the solid support.
[0226] In some embodiments, at least 50%, at least 60%, at least
70% or at least 80% of the seine protease is recovered after
purification.
[0227] In some embodiments, the method further comprises washing
the second composition with a washing buffer after step (1) and
prior to step (2).
[0228] In one embodiment, the affinity solid support of Formula II
is contained in a column. The serine protease may be added to the
column under conditions that allow for the absorption of the serine
protease on to the column, and the column may be washed with a
washing buffer that allows for the continued absorption of the
serine protease to the column and the elution of contaminating
proteins or molecules in the flow-through.
[0229] The serine protease may then be eluted with an elution
buffer comprising a competitive agent, a salt, a detergent, or a
chaotropic agent. The competitive agent may be benzamidine and/or
arginine, or a pharmaceutically acceptable salt thereof.
[0230] In one embodiment, the competitive agent is arginine.
Elution with arginine is advantageous because it is a GRAS
(Generally Recognized As Safe) excipient and does not need to be
removed from the purified protein. An additional benefit of
arginine is that it actually improves the solubility of a serine
protease (e.g., r-Antidote) and can be used as an excipient in the
final formulation.
[0231] The concentration of arginine or the competitive agent
employed in the elution buffer may be from about 250 mM to about
1000 mM. In one embodiment, the concentration of arginine or the
competitive agent in the elution buffer is about 500 mM. In further
embodiments, the concentration is about 250 mM, or about 300 mM, or
about 350 mM, or about 400 mM, or about 450 mM, or about 550 mM, or
about 600 mM, or about 650 mM, or about 700 mM, or about 750 mM, or
about 800 mM, or about 850 mM, or about 900 mM, or about 1 M. The
elution buffer optionally further comprises a salt, a detergent, or
a chaotropic agent. Salts useful in the elution buffer of the
methods and kits disclosed herein include sodium chloride, ammonium
chloride, sodium citrate, potassium citrate, potassium chloride,
magnesium chloride, calcium chloride. sodium phosphate. calcium
phosphate, ammonium phosphate, magnesium phosphate, potassium
phosphate, sodium sulfate, ammonium sulfate, potassium sulfate,
magnesium sulfate, calcium sulfate, etc. Detergents useful in the
elution buffer of the methods and kits disclosed herein include,
for example, polysorbate 80, urea, guanidine, etc.
[0232] The pH of the elution buffer is one that allows for the
effective elution of a serine protease protein absorbed on the
resin without causing inactivation and/or precipitation of the
serine protease. Certain fXa derivatives such as r-Antidote are
inactivated or precipitate at low pH. In certain embodiments, the
pH of the elution buffer is from about 4.5 to about 10.5. In
another embodiment, the pH of the elution buffer is about pH 5.0.
In another embodiment, the pH of the elution buffer is about pH
7.4. Alternatively, the pH of the elution buffer is at least about
4.5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8.0,
about 8.5, about 9, about 9.5, or at least about 10. In another
embodiment, the pH of the elution buffer is not higher than about
5.5, about 6, about 6.5, about 7, about 7.5, about 8.0, about 8.5,
about 9, about 9.5, about 10, or not higher than about 10.5. In one
embodiment, the pH of the elution buffer is about 7.4 when
benzamidine is used as the competitive agent. In one embodiment,
the pH of the elution buffer is about 5.0 when arginine is used as
the competitive agent.
[0233] In one embodiment, the washing buffer may comprise a salt
and be at a neutral pH. The term "neutral pH" is intended to mean a
pH from about 6 to about 8. In certain embodiments, the washing
buffer comprises from about 200 to about 500 mM NaCl at a neutral
pH. In another embodiment, the buffer further comprises about 10 to
50 mM, for example, about 20 mM Tris. In other embodiments, the pH
is about 6, or about 7, or about 8.
[0234] The methods disclosed herein may further comprise other
purification and chromatographic steps such as, for example, gel
electrophoresis such as polyacrylamide gel electrophoresis,
ion-exchange chromatography, reverse phase chromatography,
mixed-mode resins, exclusion chromatography, affinity
chromatography, or other chromatography techniques, isoelectric
focusing, precipitation with ammonium sulfate, PEG (polyethylene
glycol), antibodies and the like or by heat denaturation, followed
by centrifugation; filtration such as gel filtration,
hydroxylapatite; or combinations of such and other techniques. In
one embodiment, the method further comprises applying the solution
containing the polypeptide to an ion-exchange column.
[0235] Suitable cation-exchange resins include a wide variety of
materials known in the art, including those capable of binding
polypeptides over a wide pH range. For example, carboxymethylated,
sulfonated, agarose-based, or polymeric polystyrene/divinyl benzene
cation-exchange matrices are particularly preferred. Other useful
matrix materials include, but are not limited to, cellulose
matrices, such as fibrous, microgranular, and beaded matrices;
dextran, polyacrylate, polyvinyl, polystyrene, silica, and
polyether matrices; and composites. Other suitable materials for
use in cation exchange chromatography are within the knowledge of
those skilled in the art.
[0236] Anion-exchange chromatography is carried out using media
appropriate therefor, as are known in the art. Suitable media
include, e.g., polymeric polystyrene/divinyl benzene resins and
agarose-based resins, as well as agarose beads, dextran beads,
polystyrene beads, media that comprise an insoluble, particulate
support derivatized with tertiary or quaternary amino groups., and
supports derivatized with trimethylaminoethyl groups. Examples of
suitable such media include DE92 (diethylaminoethyl cellulose,
Whatman); DEAE CELLULOSE (Sigma), BAKERBOND ABX 40 mu (J. T. Baker,
Inc.); DEAE resins such as FRACTOGEL EMD DEAE-650 (EM Separations),
FRACTOGEL EMD TMAE-650 (S).TM. (EM Science, Gibbstown, N.J.), TSK
gel DEAE-SPW (Tosohaas), DEAE-SEPHAROSE CL-6BT'' and chelating
SEPHAROSE (Amersham Pharmacia Biotech AB), DEAE MERE SEP. IOOO.TM.
(Millipore), and DEAE SPHERODEX (Sepracor); RESOURCE Q.TM. and Q
SEPHAROSE (QSFF) (Amersham Pharmacia Biotech AB); MACRO-PEP Q.TM.
(Bio-Rad Laboratories, Hercules, Calif.); Q-HYPERD (BioSepra, Inc.,
Marlborough, Mass.); and the like. Other suitable anion-exchange
chromatography materials, as well as the selection and use of these
materials for the present application, are conventional in the
art.
[0237] The ion-exchange chromatography, filtration, nanofiltration,
or additional purification step may be prior to or after the
affinity chromatography described herein. Additional steps may also
include viral inactivation steps by, for example, solvent and
detergent treatment of the protein extract or through
nanofiltration.
[0238] Multi-modal or mixed-mode chromatography (MMC) methods are
also used for purification of proteins and other biologics.
Examples of commercial multi-modal chromatography resins include
ceramic hydroxyapatite (CHT), Capto-MMC, Capto-Adhere, Capto-Q,
Capto-S, Capto-Octyl, Capto-CHT, and the like.
[0239] Generally, "purified" will refer to a protein or peptide
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. A substantially purified
protein or peptide in a composition forms the major component of
the composition, such as constituting at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95% or more of the proteins in the
composition.
[0240] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
Kits
[0241] Also provided herein is a kit for purifying a serine
protease.
[0242] In one aspect, provided is kit for purifying a serine
protease comprising [0243] (1) an affinity solid support of Formula
II or a salt thereof, and [0244] (2) an elution buffer comprising a
competitive agent, [0245] wherein the affinity solid support of
Formula II is as defined herein.
[0246] In another aspect, provided is a kit for purifying a serine
protease comprising a compound of Formula I or a salt thereof and
an activated solid support capable of forming a covalent bond with
the compound of Formula I, wherein the compound of Formula I and
the activated solid support are as defined herein.
[0247] In some embodiments, the kit further comprises an elution
buffer comprising a competitive agent.
[0248] In some embodiments, the competitive agent is arginine
and/or benzamidine, or a salt, such as a pharmaceutically
acceptable salt thereof.
[0249] In one embodiment, the kit further comprises a washing
buffer. In a related embodiment, the washing buffer comprises about
250 mM NaCl at a neutral pH. In another embodiments, the buffer
further comprises about 10 to 50 mM, for example, about 20 mM
Tris.
EXAMPLES
[0250] The examples below as well as throughout the application,
the following abbreviations have the following meanings. If not
defined, the terms have their generally accepted meanings. [0251]
atm=atmosphere [0252] Boc=tert-butoxycarbonyl [0253]
BOP=(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate [0254] DCM=dichloromethane [0255]
DIEA=N,N-diisopropylethylamine [0256] DMAP=dimethylaminopyridine
[0257] DMF=dimethylformamide [0258] DMSO=dimethylsulfoxide [0259]
eq.=equivalent [0260] ESMS=electrospray mass spectrometry [0261]
EtOAc=ethyl acetate [0262] EtOH=ethanol [0263]
HPLC=high-performance liquid chromatography [0264] g=gram [0265]
MeOH=methanol [0266] mg=milligram [0267] min=minute [0268]
mL=milliliter [0269] mM=millimolar [0270] mmol=millimole [0271]
MS=mass spectrometry [0272] N (when used as concentration
unit)=normal [0273] nm=nanometer [0274] nM=nanomolar [0275]
pM=picomolar [0276] TEA=triethylamine [0277] TFA=trifluoroacetic
acid [0278] UPLC=ultra performance liquid chromatography [0279]
UV=ultraviolet spectrum [0280] .mu.L=microliter [0281]
.mu.M=micromolar [0282] .lamda.=wavelength
Example 1
Preparation of
5-(2-((6-amidohexyl)amino)-2-oxoethyoxy)-N-(5-chloropyridin-2-yl)-2-(4-(N-
,N-dimethylcarbamimidoyl)benzamido)benzamide (Compound A3)
1. Preparation of tert-butyl(6-(2-chloroacetamido)hexyl)carbamate,
Compound B2
##STR00096##
[0284] To a solution of N-Boc-1,6-hexanediamine, Compound B1 (376
mg, 1.74 mmol) in CH.sub.2Cl.sub.2 (8 mL) was added DIEA (0.500 mL,
2.87 mmol) at room temperature. To this was added dropwise
chloroacetyl chloride (0.138 mL, 1.73 mmol). The mixture was
stirred at room temperature for 4 hour, diluted with EtOAc, washed
with 1N HCl and 5% NaHCO.sub.3. The organic layer was dried,
filtered and concentrated in vacuum to give semi-solid compound B2
(434 mg).
2. Preparation of Compound A3
##STR00097##
[0286] To a mixture of betrixaban, Compound A1 (1.00 g, 2.21 mmol)
in dichloromethane (15 mL) was added BBr.sub.3 (1.5 mL, 15.70
mmol). The reaction mixture was stirred at room temperature
overnight. To the reaction mixture was added water, the solid
precipitated was collected by filtration, dried under vacuum to
afford compound A2 (1.10 g).
[0287] A mixture of Compound A2 (240 mg, 0.548 mmol), Compound B2
(210 mg, 0.718 mmol) and NaH (60%, 65 mg, 1.62 mmol) in DMF (4 mL)
was stirred at room temperature for 6 hours. To the mixture was
added water and the sticky solid precipitated was taken to the next
step as such.
[0288] The solid from the above reaction was treated with neat TFA
at room temperature for 1 hour. The mixture was concentrated and
subjected to reverse phase preparative HPLC to provide the title
Compound A3 (150 mg). MS found for C.sub.30H.sub.36ClN.sub.7O.sub.4
as (M+H).sup.+ 594.6. UV: .lamda.=202, 287.8 nm.
Example 2
Preparation of
5-(2-((6-amidohexyl)amino)-2-oxoethyoxy)-2-(4-(N,N-dimethylcarbamimidoyl)-
benzamido)-N-(pyridine-2-yl)benzamide (Compound A4)
Route 1:
##STR00098##
[0290] A mixture of Compound A3 (102 mg, 0.171 mmol) and Pd/C (10%,
49 mg) in MeOH was hydrogenated under balloon H.sub.2 for 4 hours.
The reaction mixture was filtered through celite plug, concentrated
in vacuo and purified by reverse phase preparative HPLC to isolate
the title Compound A4 (31 mg). MS found for
C.sub.30H.sub.37N.sub.7O.sub.4 as (M+H).sup.+ 560.43. UV:
.lamda.=204, 231.5, 294 nm.
Route 2:
[0291] Compound A4 can be prepared according to:
##STR00099##
Route 3:
##STR00100## ##STR00101##
[0293] To a dichloromethane solution of
5-methoxy-2-nitro-N-(5-chloro-pyridine-2-yl)benzamide (C1, 2 g, 6.5
mmol) was added BBr.sub.3 (1.5 mL, 15.6 mmol). The reaction mixture
was stirred at room temperature overnight. To the reaction mixture
was added water, the solid precipitated was collected by
filtration, dried under vacuum to afford compound C2 (1.8 g).
[0294] Compound C2 and methyl 2-bromoacetate (1 g, 3.4 mmol) was
dissolved in DMF (20 mL) followed by addition of K.sub.2CO.sub.3
(94 mg, 6.8 mmol) and the reaction mixture was heated at 40.degree.
C. After 1 hour stirring, the reaction was complete. Water was
added and the mixture was extracted with ethyl acetate. The ethyl
acetate layer was dried, filtered and evaporated to afford compound
C3 (1.59 g). The crude residue was purified by RP-HPLC. ESCI-MS:
366.1 & 368.1 (chlorine pattern).
[0295] To a THF solution of compound C3 (1.5 g, 4.3 mmol) was added
LiOH (1 M aqueous, 17.2 mL, 17.2 mmol) and the reaction was stirred
at room temperature for 1 hour. The progress of the reaction was
monitored by HPLC and after 1 hour, the reaction showed complete
disappearance of compound C3. The reaction mixture was
concentrated, added water and acidified with 1N HCl. The solid
precipitated was collected to give compound C4 (480 mg). ESMS:
M+H=352.1, 354.1 (Cl pattern).
[0296] Compound C4 (480 mg, 1.7 mmol), BOP (757 mg, 1.7 mmol) and
triethylamine (392 .mu.L, 1.7 mmol) were dissolved in 10 mL of DMF
and stirred at room temperature for 30 mins. To the mixture added
Boc protected C.sub.6 linked diamine (380 .mu.L, 1.7 mmol) and the
reaction mixture was stirred for 30 mins. The product compound C5
was isolated with ethyl acetate after aqueous work-up. ESMS: 550
& 552 (Cl pattern) and M-Boc=450.2.
[0297] Compound C5 was suspended in ethyl acetate and added 10%
Pd/C. The mixture was hydrogenated with H.sub.2 balloon overnight.
The reaction mixture was filtered through celite and the filtrate
was evaporated to afford the desired compound C6. ESMS confirmed
M+H=486.4 (No Cl pattern).
[0298] Compound C6, BOP (1.1 eq.) and DMAP (1.2 eq.) were dissolved
in DMF and stirred for 1 hour at room temperature. To the mixture
was added 4-(N,N-dimethylcarbamimidoyl)benzoic acid (1.05 eq.) and
reaction mixture was stirred overnight. To the reaction mixture was
added water and the crude product was extracted with ethyl acetate.
The residue was dissolved in ethanol followed by addition of 6 N
HCl. The product crashed out. The solid was filtered to give the
title compound. ESMS confirms M+H=561.
Route 4:
[0299] Compound A4 was also prepared according to:
##STR00102##
[0300] To a 250 mL flask was charged 5-methoxy-2-nitrobenzoic acid
(100 g, 507 mmol), 2-aminopyridine (71.6 g, 761 mmol, 1.5 eq.),
acetonitrile (550 mL), then pyridine (120 g, 1520 mmol, 3.0 eq.)
and the mixture was stirred and cooled to 0-5.degree. C. under
nitrogen. Then POCl.sub.3 (93 g, 609 mmol, 1.2 eq.) was added
drop-wise over about 60 minutes, keeping the temperature below
5.degree. C. Upon reaction completion (about 1 hour, based on HPLC)
the reaction mixture was quenched by slow addition of water (750
mL). The resulting solids went into solution in a few minutes, but
precipitated out upon addition of KOH. After stirring overnight the
organics were removed from the sticky solid by distillation, water
(1000 mL) was added and the mixture was stirred at 0-5.degree. C.
for 30 minutes, then collected by filtration. The product was dried
under vacuum to provide 121 g of D1 that was 95.4% pure by
HPLC.
[0301] To 2.235 kg of Compound D1 (8.18 mole) in DMAc (19.0 kg;
20.3 L) was added LiBr (6.64 kg, 76.5 mole) and the mixture was
stirred at 147.degree. C. for about 20 hours. The product was
isolated by filtration, dried first under nitrogen then finished in
a vacuum drying oven to provide a total of 1425 g of Compound D2
that was 98.1% pure (with 1.9% D1 as only measurable impurity).
[0302] To 1.810 kg (6.98 mole) of Compound D2 and 1.348 kg (4.60
mole) of Compound B2 in DMAc (20 L) were added 2.431 kg of
K.sub.2PO.sub.4. After 45 hours at 84-86.degree. C., the mixture
was cooled, quenched into water and subjected to an aqueous work up
followed by crystallization from EtOAc/heptane. After drying under
vacuum a total of 1481 g of Compound D3 was obtained with HPLC
purity of 98.7%.
[0303] 250 g Compound D3 was reduced with H.sub.2 in EtOH/EtOAc
with Pt/V on C at 35 psi and 38.degree. C. to provide an
intermediate that is not isolated but carried directly on to the
coupling with NNDC.HCl (150.0 g, 1.35 eq.) using EDAC (138 g, 1.5
eq) as the coupling reagent in DMAc. Compound D4 was isolated by
quenching the reaction mixture into aqueous
Na.sub.2CO.sub.3/NaHCO.sub.3 with a small amount of MTBE present to
prevent Compound D4 from becoming a sticky mass. After filtration,
washing with water and MTBE, and vacuum drying a total of 291 g of
Compound D4 was obtained as a bright yellow solid with HPLC purity
of 96.7%.
[0304] A total of 2068 g (1491 g) of crude Compound D4 was charged
to a 50 L reactor and heated with 30 L of EtOAc to 70.degree. C.
The slurry was then filtered into a clean reactor, heptane was
added slowly (6 L), and the mixture slowly cooled to ambient
temperature for an overnight stir period. The product was isolated
by filtration and dried under nitrogen to provide 1113.6 g of
purified Compound D4 was obtained with HPLC purity of 98.6%
(AUC).
[0305] A total of 1.179 kg of purified Compound D4 was dissolved in
27.8 L 1,4-dioxane and treated with 2.89 kg of a solution of 4M HCl
in 1,4-dioxane. After stirring at 13.degree. C. for 20 hours, an
additional 0.23 kg of 4M HCl in dioxane was charged and after an
additional overnight stir period the reaction was complete. The
product was filtered and dried under a nitrogen purge for 6 days
under vacuum oven to finish 1,4-dioxane removal. A total of 1195 g
of Compound A4 (3HCl salt) was obtained after vacuum drying to
remove 1,4-dioxane.
Example 3
Preparation of
5-(2-((6-aminopentyl)amino)-2-oxoethyoxy)-N-(5-chloropyridin-2-yl)-2-(4-(-
N,N-dimethylcarbamimidoyl)benzamido)benzamide
##STR00103##
[0307] The title compound was prepared according to a procedure
similar to that illustrated in Example 1 using
tert-butyl(5-(2-chloroacetamido)pentyl)carbamate. MS found for
C.sub.29H.sub.34ClN.sub.7O.sub.4 as (M+H).sup.+ 584.6. UV:
.lamda.=202, 287.8 nm.
Example 4
Preparation of
5-(2-((6-aminopropyl)-2-oxoethyoxy)-N-(5-chloropyridin-2-yl)-2-(4-(N,N-di-
methylcarbamimidoyl)benzamido)benzamide
##STR00104##
[0309] The title compound was prepared according to a procedure
similar to that illustrated in Example 1 using
tert-butyl(5-(2-chloropropyl)carbamate. MS found for
C.sub.27H.sub.30ClN.sub.7O.sub.4 as (M+H).sup.+ 552.21. UV:
.lamda.=202, 287.8 nm.
Example 5
Preparation of
5-(2-((4-aminobutyl)amino)-2-oxoethyoxy)-N-(5-chloropyridin-2-yl)-2-(4-(N-
,N-dimethylcarbamimidoyl)benzamido)benzamide
##STR00105##
[0311] The title compound was prepared according to a procedure
similar to that illustrated in Example 1 using
tert-butyl(5-(2-chlorobutyl)carbamate. MS found for
C.sub.28H.sub.32ClN.sub.7O.sub.4 as (M+H).sup.+ 566.22. UV:
.lamda.=202, 287.8 nm.
Example 6
Preparation of
5-(4-aminobutoxy)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-N-(pyridin-2-
-yl)benzamide
##STR00106##
[0313] The title compound was prepared according to a procedure
similar to that illustrated in Example 2, route 4, starting with
Compound D2. MS found for C.sub.26H.sub.30N.sub.6O.sub.3 as
(M+H).sup.+ 475.3.
Example 7
5-((6-aminohexyl)oxy)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-N-(pyridi-
n-2-yl)benzamide
##STR00107##
[0315] The title compound was prepared according to a procedure
similar to that illustrated in Example 2, route 4, starting with
Compound D2. MS found for C.sub.28H.sub.34N.sub.6O.sub.3 as
(M+H).sup.+ 503.3.
Example 8
5-(2-((3-aminopropyl)amino)-2-oxoethoxy)-2-(4-(N,N-dimethylcarbamimidoyl)b-
enzamido)-N-(pyridin-2-yl)benzamide
##STR00108##
[0317] The title compound was prepared according to a procedure
similar to that illustrated in Example 2, route 4, starting with
Compound D2. MS found C.sub.27H.sub.31N.sub.7O.sub.4 as (M+H).sup.+
518.3.
Example 9
N.sup.1-(4-aminobutyl)-N.sup.4-(4-methoxy-2-(pyridin-2-ylcarbamoyl)phenyl)-
terephthalamide
##STR00109##
[0319] The title compound was prepared according to the following
procedure starting with Compound D1 in Example 2, route 4.
##STR00110##
[0320] Compound D1 (2.73 g, 10 mmol) was charged in a pressure
bottle. Ethanol (15 mL) and ethyl acetate (7 mL) were added, and
the resulting slurry was degassed and purged nitrogen. Then
platinum/vanadium on carbon (0.27 g) was added. The reaction
mixture was degassed and purged hydrogen (40 psi) and was heated at
40.degree. C. After stirring at 40.degree. C. under hydrogen
atmosphere (40 psi) for 3 h, HPLC and TLC indicated the reaction
completion. Upon the completion, the catalyst was removed by
filtration through a Celite pad and washed with ethyl acetate. The
filtrate was concentrated under reduced pressure. The residual oil
was dried under vacuum to isolate 2.56 g of crude Compound D5 as a
yellow thick oil, which solidified over the time
(quantitative).
[0321] Compound D5 (0.5 g, 2.06 mmol) was dissolved in DMAc (6 mL)
and put under reduced pressure at 45.degree. C. (water bath) to
remove any residual solvent from the previous step. The solution
was placed in a flask. Compound D6 (0.47 g, 2.61 mmol) was added,
followed by the addition of conc HCl (11 .mu.L, 0.13 mmol). The
resulting solution was cooled down to 15.degree. C. EDCI (0.55 g,
2.88 mmol) was divided into four portions and was added every 20
min at 15.+-.2.degree. C. HPLC confirmed the reaction completion in
30 min after the last addition of EDCI and the reaction was
quenched by pouring into a solution of sodium carbonate (0.3 g) and
sodium bicarbonate (0.17 g) in water (6.5 mL) and MTBE (1.5 mL).
The quench was slightly exothermic and the internal temperature was
up to 30.degree. C. During the quench, a yellow precipitate was
formed. The resulting yellow slurry was stirred at 0.degree. C. for
1 h and the precipitate was isolated by filtration, washed with
water and MTBE, and dried in the vacuum oven at 35-40.degree. C.
overnight to afford 1.5 g of crude Compound D7 as a pale yellow
solid with the purity of 90%. Thus, the further purification was
performed before the hydrolysis step. The solid was suspended in
MTBE (25 mL) and stirred at ambient temperature for 1 h to remove
some of the impurities. The purity was improved to 96%. To achieve
higher purity, a recrystallization from isopropanol was carried
out. First, 100 mg of the solid was recrystallized from isopropanol
(5 mL) and 83 mg of Compound D7 was isolated in 98.5% purity (83%
recovery). The rest of the solid was recrystallized from
isopropanol (45 mL) to isolate 455 mg of Compound D7 (97.8% purity)
(total 0.54 g, 64% yield, 98% purity).
[0322] A solution of lithium hydroxide (36 mg, 0.75 mmol) in water
(0.6 mL) was added to a solution of Compound D7 (100 mg, 0.25 mmol)
in THF (3.6 mL) at ambient temperature. The reaction mixture was
stirred at ambient temperature for 4 h. The reaction was monitored
by HPLC which showed 96% of Compound D8 and 4% of Compound D7. The
reaction was quenched with water and the desired compound D8 was
extracted in ethyl acetate.
[0323] Compound D8 (0.5 g, 1.278 mmol) was suspended in DMF (10 mL)
and was put under reduced pressure to remove any residual solvent
from the previous step. Then the slurry was diluted with DMF (40
mL) and HOBt (0.26 g, 1.92 mmol) was added. The mixture was stirred
at ambient temperature and was added EDCI (0.29 g, 1.534 mmol) at
once. The resulting pale brown slurry was stirred at ambient
temperature. After overnight the reaction mixture became pale brown
slightly unclear solution. A solution of N-Boc-1,4-butanediamine
(0.36 g, 1.916 mmol) in DMF (2.5 mL) was added drop-wise at ambient
temperature. DMF (2.5 mL) was used to rinse. After 4 h, the
reaction mixture was diluted with ethyl acetate (200 mL) and
hexanes (5 mL). The solution was washed with water (30 mL.times.3).
The aqueous layers were combined and extracted with ethyl
acetate/hexanes (20/1, 100 mL). The organic layers were combined,
washed with 1N HCl solution (30 mL), water (30 mL), saturated
sodium bicarbonate solution (30 mL), saturated sodium chloride
solution (30 mL), and dried over sodium sulfate. The solid was
removed by filtration and washed with ethyl acetate. The filtrate
was concentrated under reduced pressure. The crude solid (1.34 g)
was purified by recrystallization from ethyl acetate (.about.50 mL)
and heptanes (1015 mL). The obtained slurry was cooled down to
0.degree. C. by an ice-water bath and stirred for 2 h and the
precipitate was isolated by filtration, washed with ethyl
acetate/heptanes (3/1, .about.150 mL total), and dried in the
vacuum oven at 40-45.degree. C. for 2-3 h. 451 mg of Compound D9 as
a white solid (63%, >99% purity).
[0324] Compound D9 (300 mg, 0.534 mmol) was dissolved in
1,4-dioxane (60 mL), along with methanol (20 ml) and 4M HCl
solution in 1,4-dioxane (4 mL, 16 mmol) was added drop-wise at
ambient temperature. The reaction mixture was stirred overnight.
The reaction mixture was monitored by HPLC, and the reaction
completion was observed after 24 h at ambient temperature. The
precipitate was isolated by filtration, washed with MTBE several
times, and dried in the vacuum oven at 40-45.degree. C. for 3 h to
isolate 241 mg of title compound as a yellow solid (84.4%, >99%
purity). MS found for C.sub.25H.sub.27N.sub.5O.sub.4 as (M+H).sup.+
462.2.
Example 10
N.sup.1-(3-aminopropyl)-N.sup.4-(4-methoxy-2-(pyridin-2-ylcarbamoyl)phenyl-
)terephthalamide
##STR00111##
[0326] The title compound was prepared according to a procedure
similar to that illustrated in Example 9 and isolated as yellow
solid (HPLC >99% purity). MS found for
C.sub.24H.sub.26N.sub.5O.sub.4 as (M+H).sup.+ 448.2.
Example 11
N.sup.1-(6-aminohexyl)-N.sup.4-(4-methoxy-2-(pyridin-2-ylcarbamoyl)phenyl)-
terephthalamide
##STR00112##
[0328] The title compound was prepared according to a procedure
similar to that illustrated in Example 9. MS found for
C.sub.27H.sub.31N.sub.5O.sub.4 as (M+H).sup.+ 490.2.
Example 12
2-(4-(4-aminobutoxy)benzamido)-5-methoxy-N-(pyridin-2-yl)benzamide
##STR00113##
[0330] The title compound was prepared according to the following
procedure starting with methyl 4-hydroxy benzoate.
##STR00114##
[0331] Methyl 4-hydroxybenzoate (76 mg, 0.5 mmol),
4-(Boc-amino)-1-butanol (114 mg, 0.6 mmol), and triphenylphosphine
(197 mg, 0.75 mmol) were dissolved in THF (anhyd, 2 mL) and cooled
down to .about.5.degree. C. DIAD (0.15 mL, 0.75 mmol) was added
drop-wise. The addition was exothermic and the internal temperature
was up to .about.10.degree. C. After the addition, the reaction
mixture was warmed up to ambient temperature and stirred for 1 h.
The reaction mixture was diluted with hexanes/ethyl acetate (2/1,
v/v), the organic layer was washed with 1N NaOH solution,
water.times.2 (neutral), saturated sodium chloride solution, and
dried over sodium sulfate. The solid was removed by filtration
through a short pad of silica gel and concentrated under reduced
pressure to provide 0.32 g of D10 as a yellow oil.
[0332] To the crude D10 were added 1N NaOH solution and ethanol and
the reaction mixture was stirred at ambient temperature overnight,
and then heated at 50.degree. C. The reaction mixture was washed
with ethyl acetate. The aqueous layer was acidified with 10%
KHSO.sub.4 solution to pH.about.2 and extracted with ethyl acetate.
The organic layer was separated, washed with water (2.times.),
saturated sodium chloride solution, and dried over sodium sulfate.
The solid was removed and the filtrate was concentrated under
reduced pressure to yield 152 mg of Compound D11 as a white solid
(98% over two steps).
##STR00115##
[0333] Amine D13 (0.25 g, 1.03 mmol) and acid D11 (0.41 g, 1.31
mmol) were dissolved in DMAc (5 mL) and put under reduced pressure
to remove any residual solvent from the previous step (at
.about.50.degree. C., .about.10 mmHg, for 30 min). The solution was
diluted with DMAc (10 mL), and was added conc HCl (6 .mu.L, 0.065
mmol). The solution was cooled down to .about.5.degree. C. and was
added 1/4 of EDCI (0.28 g, 1.44 mmol) in every 15 min. After the
last addition, the reaction mixture was warmed up to ambient
temperature. Upon completion, the reaction mixture was poured into
a solution of sodium carbonate (0.17 g) and sodium bicarbonate (0.1
g) in water (5 mL). The precipitate was generated, but became gummy
oil by the addition of excess water. Thus, the aqueous layer was
extracted with ethyl acetate. The organic layer was separated,
washed with water until neutral, saturated sodium chloride
solution, and dried over sodium sulfate. The solid was removed and
the filtrate was concentrated under reduced pressure to isolate
0.97 g of pale brown crude oil which was solidified in a mixture of
ethyl acetate (2.5 mL) and hexanes (5 ml). After drying under
vacuum, 0.5 g of compound D13 was obtained (91%).
[0334] Compound D13 (0.25 g, 0.47 mmol) was dissolved in
1,4-dioxane (25 mL) by slight heating (.about.45.degree. C.). The
solution was cooled down to ambient temperature, and was added 4M
HCl solution in 1,4-dioxane (1.75 mL, 7.01 mmol) drop-wise. After 1
h, methanol (3 mL) was added. After 17 h, an additional amount of
4M HCl solution was added (0.5 mL, 2 mmol) and the reaction mixture
was stirred for another 3 h. The reaction mixture was diluted with
MTBE and the solid was isolated, washed with MTBE, and dried in the
vacuum oven at 40-45.degree. C. for overnight to isolate 172 mg of
the title compound as a yellow-orange solid in 71% (99% purity). MS
found for C.sub.24H.sub.26N.sub.4O.sub.4 as (M+H).sup.+ 435.2.
Example 13
FXa Inhibitory Activity of Compounds
[0335] This example illustrates methods for evaluating the
compounds, along with results obtained for such assays. As
mentioned above, the compound may be selected based on its factor
Xa inhibitory activity. The in vitro factor Xa activities of the
compounds can be determined by various procedures known in the art.
The potent affinities for factor Xa inhibition exhibited by the
compounds can be measured by an IC.sub.50 value (in nM). The
IC.sub.50 value is the concentration (in nM) of the compound
required to provide 50% inhibition of factor Xa proteolytic
activity. The smaller the IC.sub.50 value, the more active (potent)
is a compound for inhibiting factor Xa activity.
[0336] An in vitro assay for detecting and measuring inhibition
activity against factor Xa is as follows:
[0337] a. IC.sub.50 and Ki Determinations
Substrate:
[0338] The substrate S-2765 (Z-D-Arg-Gly-Arg-pNA.HCl) can be
obtained from Diapharma (West Chester, Ohio).
Enzyme:
[0339] The human plasma protein factor Xa can be purchased from
Haematologic Technologies (Essex Junction, Vt.).
Methods:
[0340] IC.sub.50 Determinations
[0341] All assays, which are performed in 96-well microtiter
plates, measure proteolytic activity of the enzyme (factor Xa) by
following cleavage of a paranitroanilide peptide substrate. The
assay buffer used for proteolytic assays was Tris buffered saline
(20 mM Tris, 150 mM NaCl, 5 mM CaCl.sub.2, 0.1% Bovine serum
albumin (BSA), 5% dimethyl sulfoxide (DMSO) pH 7.4). In a 96-well
microtiter plate, inhibitor was serially diluted to give a range of
final concentrations from 0.01 nM to 10 .mu.M. Duplicate sets of
wells were assayed and control wells without inhibitor were
included. Enzyme was added to each well, (factor Xa concentration=1
nM), the plate was shaken for 5 seconds and then incubated for 5
minutes at room temperature. S2765 was added (100 .mu.M final) and
the plate was shaken for 5 seconds (final volume in each well was
200 .mu.L). The degree of substrate hydrolysis was measured at 405
nm on a Thermomax plate reader (Molecular Devices, Sunnyvale,
Calif.) for 2 minutes. The initial velocities of substrate cleavage
(mOD/min), for each range of inhibitor concentrations, were fitted
to a four parameter equation using Softmax data analysis software.
The parameter C, derived from the resulting curve-fit, corresponded
to the concentration for half maximal inhibition (IC.sub.50).
[0342] K.sub.i Determination
[0343] The assay buffer for this series of assays was Hepes
buffered saline (20 mM Hepes, 150 mM NaCl, 5 mM CaCl.sub.2, 0.1%
PEG-8000, pH 7.4). In a 96-well microtiter plate, inhibitor was
serially diluted in a duplicate set of wells to give a range of
final concentrations from 5 pM to 3 .mu.M. Controls without
inhibitor (8 wells) were included. The enzyme, factor Xa (final
concentration=1 nM) was added to the wells. The substrate S-2765
(final concentration=200 .mu.M) was added and the degree of
substrate hydrolysis was measured at 405 nm on a Thermomax plate
reader for 5 minutes, using Softmax software. Initial velocities
(mOD/min) were analyzed by non-linear least squares regression in
the Plate K.sub.i software (BioKin Ltd, Pullman, Wash.) [Kusmic, et
al., Analytical Biochemistry 281: 62-67, 2000]. The model used for
fitting the inhibitor dose-response curves was the Morrison
equation. An apparent K.sub.i(Ki*) was determined. The overall
K.sub.i was calculated using the following equation:
Ki = Ki * 1 + [ S ] Km ##EQU00001##
where [S] is substrate concentration (200 .mu.M) and K.sub.m, the
Michaelis constant for S2765.
[0344] Table 4 shows the fXa inhibitory activity of selected
compounds.
TABLE-US-00004 TABLE 4 Compound fXa IC.sub.50 (nM) ##STR00116## 227
##STR00117## 24 ##STR00118## 615 ##STR00119## 158 ##STR00120##
320
Example 14
Preparation of Affinity Resin with Betrixaban Ligand A3 for the
Purification of r-Antidote
1. Coupling of Compound A3 to CNBr-Activated Sepharose 4 Fast Flow
Matrix
##STR00121##
[0346] CNBr-activated Sepharose 4-FF matrix was hydrated with 1 mM
HCl. The resin was washed 10 times with 2 mL volumes of 1 mM HCl.
After this step, 20 mL of resin was obtained. The coupling solution
was prepared by dissolving Compound A3 (150 mg) in 2.5 mL of DMSO.
This solution was diluted to 5.0 mL with a buffer containing 0.1M
NaHCO.sub.3 and 0.5 M NaCl at pH 8.3. The coupling solution was
added to 10 mL of resin and reacted for room temperature for 3
hours while adjusting the pH to about 8.3. The reaction was
monitored by UPLC after completion of the coupling, unreacted CNBr
was capped with 0.1 M Tris-HCl buffer at pH 8.0. The coupled resin
was washed three times with 0.1 M acetate buffer pH 3 to 4
containing 0.5 M NaCl, and then with 0.1M Tris-HCl buffer pH 8 to 9
containing 0.5 M NaCl. The above wash cycle was repeated five
times. The coupled ligand affinity resin is available for
r-antidote purification.
[0347] CNBr-activated Sepharose 4 Fast Flow is a pre-activated
matrix that combines the advantages of CNBr coupling with the high
flow stability characteristics of Sepharose 4 Fast Flow.
2. Coupling of Compound A4 to CNBr-Activated Sepharose 4 Fast Flow
Matrix
[0348] A similar procedure was utilized to couple Compound A4 to
CNBr-activated Sepharose 4-FF resin to prepare affinity resin with
des-chloro betrixaban Compound A4.
Example 15
Coupling of Betrixaban with NHS Activated Resin
##STR00122##
[0350] NHS-activated Sepharose 4-FF matrix was hydrated with 1 mM
HCl. The resin was washed 10 times with 2 mL volumes of 1 mM HCl.
After this step, 20 mL of resin was obtained. The coupling solution
was prepared by dissolving Compound A3 (15 mg) in 0.5 mL of DMSO.
This solution was diluted to 3.0 mL with buffer containing 0.1 M
NaHCO.sub.3, 0.5 M NaCl at pH 8.3. This coupling solution was added
to the NHS-activated Sepharose matrix and reacted at room
temperature for 3 hours while adjusting the pH at about 8.3. The
reaction was monitored by UPLC. After completion of the coupling,
unreacted resin was blocked with 0.1 M Tris-HCl buffer at pH 8.0.
The coupled betrixaban NHS-Sepharose resin was washed with 3 times
with 0.1M acetate buffer pH 3 to 4 containing 0.5 M NaCl and 0.1 M
Tris-HCl buffer pH 8 to 9 containing 0.5 M NaCl. 5 mL of
des-chloro-betrixaban NHS-Sepharose resin was obtained using a
similar procedure. The substitution is 3 mg of compound per mL of
resin or 5 .mu.mol compound per mL of resin. The coupled ligand
affinity resin is available for r-antidote purification.
[0351] Other ligand affinity NHS resins can be prepared similarly,
for example:
##STR00123##
Example 16
Coupling of Betrixaban with EAH Sepharose.TM. Resin
##STR00124##
[0353] Coupling of betrixaban with EAH Sepharose.TM. resin can be
conducted according to methods described in Instructions 71-7097-00
AE, 2009, by General Electric Company.
Example 17
Coupling of Des Chloro-C6 Betrixaban Linker A4 with NHS Activated
Capto Resin
##STR00125##
[0355] NHS-activated Capto matrix was hydrated with 1 mM HCl. The
resin was washed 10 times with 2 mL volumes of 1 mM HCl. After this
step, 20 mL of resin was obtained. The coupling solution was
prepared by dissolving Compound A4 in buffer. This solution was
diluted to 3.0 mL with buffer containing 0.1 M NaHCO.sub.3, 0.5 M
NaCl at pH 8.3. This coupling solution was added to the
NHS-activated Capto matrix and reacted at room temperature for 3
hours while adjusting the pH at about 8.3. After completion of the
coupling, unreacted resin was blocked with 0.1 M Tris-HCl buffer at
pH 8.0. The coupled betrixaban NHS-Capto resin was washed 3 times
with 0.1M acetate buffer pH 3 to 4 containing 0.5 M NaCl and 0.1 M
Tris-HCl buffer pH 8 to 9 containing 0.5 M NaCl. Using this
process, resins with 5, 11, 15 and 20 .mu.m binding capacity were
synthesized.
[0356] Similarly NHS activated sepharose resin 5 and 11 .mu.m
binding capacity were synthesized with A4.
Example 18
Purification of r-Antidote
[0357] A 1.0 mL column was packed with the betrixaban-affinity or
des-chloro betrixaban-affinity resin. The cell culture BSR7
conditioned media (.about.1 mg r-Antidote) was loaded through pump
at 0.2 mL/min. After the r-Antidote sample is loaded, the column
was washed to baseline with equilibration buffer (20 mM Tris/250 mM
NaCl/pH 7.4). The r-Antidote was stepwise eluted either with 0.5 M
arginine in 25 mM Na-acetate buffer pH 5, or with 0.5 M benzamidine
buffers (20 mM acetate, pH 5.0).
[0358] 1 N sodium hydroxide wash and treatment with equilibration
buffer 20 mM Tris/HCl, 250 mM NaCl, pH 7.4 regenerates the resin
for reuse.
[0359] The eluent was analyzed using gel electrophoresis according
to the following procedure: [0360] SDS-PAGE (Sodium dodecyl
sulfate-Polyacrylamide Gel Electrophoresis) [0361] 1. Sample
Preparation-Reducing Conditions: 15 .mu.L of sample is mixed with 5
.mu.L of NuPAGE.RTM. LDS Sample Buffer and 2 .mu.L of NuPAGE.RTM.
Reducing Agent. Samples are then heated to 70.degree. C. for 10
minutes [0362] 2. Noyes 10% Gel 1.0 mm, 12 well gel preparation.
The cassette is removed from its packaging, rinsed with water, and
then inserted into XCell SureLock.TM. Mini-Cell. The upper and
lower chambers are filled with 1.times. NuPAGE.RTM. SDS Running
Buffer; the wells are then rinsed twice with running buffer. [0363]
3. Sample Loading and Gel Running 20 .mu.L of sample is loaded into
a well. The gel is then run at 135 Volts, 400 mA for 65 minutes.
[0364] 4. The cassette is then removed from the Mini-Cell; the gel
is then removed from the cassette and fixed with 10% Acetic
Acid/50% Methanol in water (v/v) for fifteen minutes. The gel is
then stained over night using Gelcode Blue Stain Reagent. The gel
is then destained with water.
[0365] FIG. 3 shows that r-Antidote is eluted with benzamidine.
FIG. 4 shows that r-Antidote is eluted with arginine. The gel
electrophoresis in FIG. 3 shows that benzamidine was effective in
eluting r-Antidote from both betrixaban-affinity and des-chloro
betrixaban-affinity columns, as compared to NaCl (compare lanes 4
and 5 from right to lanes 2 and 3 from right). The ability of
arginine to elute r-Antidote, to some degree, depends on the column
type. From betrixaban-affinity column, arginine was able to elute
r-Antidote, but not as effective as benzamidine (compare lanes 2
and 3 in FIG. 4). From des-chloro betrixaban-affinity columns,
however, arginine appeared even more effective than benzamidine
(FIG. 4, lanes 4 and 5).
Example 19
Process for the Packing of the Column Followed by Purification
Sepharose Affinity Resin
[0366] A 1.0 mL column was packed with the betrixaban-NHS-Sepharose
affinity or des-chloro betrixaban-NHS-Sepharose affinity resin
prepared according to Example 8. Cell culture EB2 conditioned media
(.about.1 mg r-Antidote) was loaded through pump at 0.2 mL/min.
After the antidote sample is loaded the column was washed to
baseline with equilibration buffer (20 mM Tris/250 mM NaCl/pH 7.4).
The antidote was then stepwise eluted either with 0.5 M arginine in
25 mM Na-acetate, pH 5 or 0.5 M benzamidine buffers.
[0367] The SDS-PAGE of purified r-Antidote are shown in FIGS. 5 and
6. FIG. 5 shows complete elution of the antidote with 500 mM
arginine from des-chloro betrixaban-NHS-Sepharose affinity resin.
Lane 1 shows the SDS-PAGE of the equilibration buffer wash. Lane 2
shows the SDS-PAGE of the antidote purified with STI affinity
resin. Lane 3 shows the unpurified antidote. Lane 4 shows antidote
eluted with arginine buffer. Lanes 5 and 6 show elution with
benzamidine buffer and wash with NaOH, respectively, after elution
by arginine buffer, indicating that substantially all antidote was
eluted by arginine buffer.
[0368] FIG. 6 shows partial elution of the antidote with 500 mM
arginine and complete elution with 500 mM of benzamidine from
betrixaban-NHS-Sepharose affinity resin. Lane 2 shows the SDS-PAGE
of the unpurified antidote. Lanes 3 and 4 show the SDS-PAGE of
antidote purified with STI affinity resin. Lane 6 shows antidote
eluted with arginine buffer. Lane 7 shows that a significant amount
of antidote was eluted with benzamidine buffer after elution by
arginine buffer, indicating partial elution by arginine buffer.
[0369] 12 mg of r-Antidote was loaded to a des-chloro
betrixaban-NHS-Sepharose affinity resin at 15 mg antidote per mL of
resin (80% capacity, FIG. 7). 10 mg of antidote was recovered after
elution (83% of recovery rate, FIG. 8).
Capto Resin
[0370] SMI Capto resins were prepared using a 2.0 mL column packed
with des chloro-C6 Betrixaban linker A4 with NHS Activated Capto
Resin prepared according to Example 17. Stage 31 Format A Culture
Fluid was loaded through pump on 5, 11, 15 and 20 .mu.M/mL SMI
Capto column. After the antidote sample was loaded (1% Triton, 0.3%
TnBP), the column was washed to baseline with equilibration buffer
(20 mM Tris/250 mM NaCl/pH 7.4). The antidote was then stepwise
eluted with 1 M arginine in 20 mM Tris/HCl, at pH 7.4.
[0371] 90 mg of r-Antidote was loaded to a des-chloro betrixaban
compound A4-5 .mu.m SMI Capto Prototype resin and 42.9 mg (47.7%
yield) mg of antidote was recovered after elution (FIG. 9).
[0372] 90 mg of r-Antidote was loaded to a des-chloro betrixaban
compound A4-11 .mu.m SMI Capto Prototype resin and 56.6 mg (62.9%)
of antidote was recovered after elution (FIG. 10).
[0373] 90 mg of r-Antidote was loaded to a des-chloro betrixaban
compound A4-15 .mu.m SMI Capto Prototype resin and 64.5 mg (71.7%)
of antidote was recovered after elution (FIG. 11).
[0374] 90 mg of r-Antidote was loaded to a des-chloro betrixaban
compound A4-20 .mu.m SMI Capto Prototype resin and 63.2 mg (70.2%)
of antidote was recovered after elution (FIG. 12).
[0375] The plots for FIG. 9-12 are in triplicates as they are run
at three different wavelengths: 260 nm, 280 nm, and 320 nm.
Example 20
Comparison of Small Molecule Inhibitor Affinity Sepharose Resin
with Soyabean Trypsin Inhibitor (STI) Affinity Resin
[0376] Antidote purification using small molecule inhibitor
(des-chloro betrixaban) affinity Sepharose resin was compared with
STI affinity resin. A minimum 3-fold higher binding capacity for
small molecule inhibitor affinity Sepharose resin was obtained
compared to STI affinity resin.
[0377] Approximately 400 .mu.g of r-Antidote eluted from small
molecule inhibitor or STI affinity column was concentrated to 3-5
mg/mL and buffer exchanged 25 fold into lyo formulation buffer
using Amicon 10 kDa Ultracel centrifugal filter (Millipore
UFC501096, 0.5 mL).
[0378] Table 5 shows the purification results using small molecule
inhibitor affinity Sepharose resin or STI affinity resin. The small
molecule inhibitor affinity resin consistently gave higher
percentage of the alpha form (which is preferred) and lower
percentage of the beta form than the STI affinity resin. It is
preferred that the purified antidote contains no more than 10% of
the beta form.
[0379] The yield of the antidote purified using small molecule
inhibitor affinity resin is higher than the antidote purified by a
9-10 step GMP process with similar impurity profiles. The 9-10 step
GMP process comprises four chromatography steps including a
multi-modal cation exchange (Capto MMC, GE Healthcare) column, a
multi-modal anion exchange (Capto adhere, GE Healthcare) column, a
ceramic hydroxyapatite column (CHT, Bio-Rad) and a hydrophobic
interaction column with a yield of about 55% for the four
steps.
TABLE-US-00005 TABLE 5 Beta Form Peak Area Resin Alpha Form Peak
Area (%) (%) GMP 89.45 8.06 Small Molecule 89.16 7.41 Inhibitor
Affinity 89.41 7.48 Sepharose Resin 88.97 7.33 STI Affinity Resin
82.38 15.22 84.50 12.98
Example 21
Purification with Small Molecule Inhibitor Affinity Capto Resin SMI
Four Step Method
[0380] Antidote purification was carried out using small molecule
inhibitor (Des chloro-C6 Betrixaban linker A4) affinity 11 um Capto
resin as below:
[0381] Sample Preparation
[0382] 100 mL of frozen CCF (clarified culture fluid) was thawed at
room temperature. The CCF was centrifuged to remove precipitate and
then filtered thru a 0.22 um filter. The filtrate was treated with
10% Triton for a final concentration of 1% and made 0.3% with
N-Tributyl Phosphate. The resulting solution was then stirred at
room temp for 30 minutes for viral inactivation.
[0383] SMI Capture Step (1)
[0384] The treated CCF was applied to a 2.0 mL (5.times.100 mm) 11
um SMI Capto column equilibrated with 20 mM Tris/HCl, 200 mM NaCl,
pH 7.4 with a flow rate of 200 cm/hr. The chromatography was
monitored at UV wavelengths 280, 260, and 320 nm; conductivity and
pH were also monitored. 5 CV fractions were collected for the
sample application. After the sample finished applying, the column
was washed with 10 CV of 20 mM Tris/HCl, 200 mM NaCl, pH 7.4 and
then washed with 10 CV 20 mM Tris/HCl, pH 7.4.
[0385] The bound Antidote was eluted with a 20 CV Linear Gradient
of 0.fwdarw.1 M Arginine in 20 mM Tris/HCl, pH 7.4. Fractionation
was started at 50 mAU and ended at 100 mAU; a single fraction was
collected (SMI Eluate).
[0386] Adhere Step (2)
[0387] The SMI Eluate was diluted 1:15 with 25 mM Tris/HCl, pH 8.0
and applied to a 4.7 mL Capto Adhere GE-HiScreen column was
equilibrated with 25 mM Tris/HCl, 50 mM NaCl, pH 8.0 with a flow
rate of 200 cm/hr. The chromatography was monitored at UV
wavelengths 280, 260, and 320 nm; conductivity and pH were also
monitored. After the sample finished applying, the column was
washed with 10 CV of 25 mM Tris/HCl, 50 mM NaCl, pH 8.0.
[0388] The bound Antidote was step eluted with 335 mM Arginine, 50
mM HEPES, pH 7.0 Fractionation was started at 50 mAU and ended at
100 mAU; a single fraction was collected (Adhere Eluate).
[0389] CHT Step (3)
[0390] The Adhere Eluate was diluted 1:5 with 50 mM MES, 5 mM
Sodium Phosphate, pH 7.0 and applied to a 5 mL Bio-Scale Mini CHT,
Type 1 column equilibrated with 50 mM MES, 5 mM Sodium Phosphate,
pH 7.0 with flow rate of 200 cm/hr. The chromatography was
monitored at UV wavelengths 280, 260, and 320 nm; conductivity and
pH were also monitored. After the sample finished applying, the
column was washed with 5 CV of 50 mM MES, 5 mM Sodium Phosphate, pH
7.0.
[0391] The bound Antidote was eluted with a 15 CV Linear Gradient
of O.sub.2 M NaCl in 50 mM MES, 5 mM Sodium Phosphate, pH 7.0.
Fractionation was started at 200 mAU and ended at 100 mAU; a single
fraction was collected (CHT Eluate).
[0392] Octyl Step (4)
[0393] The CHT Eluate was applied to a 1 mL Octyl Sepharose FF
HiTrap Column equilibrated with 50 mM MES, 5 mM Sodium Phosphate, 1
M NaCl, pH 7.0 with a flow rate of 200 cm/hr. The chromatography
was monitored at UV wavelengths 280, 260, and 320 nm; conductivity
and pH were also monitored. After the sample finished applying, the
column was washed with 50 mM MES, 5 mM Sodium Phosphate, 1 M NaCl,
pH 7.0.
[0394] This is a Pass-Thru Collection; fractionation was started at
50 mAU and ended at 50 mAU; a single fraction was collected (Octyl
Pool). The data is as shown in the table below and also in FIG. 13.
The
TABLE-US-00006 Process Conc Step/ % Pre- % Main % Beta % Post-
mg/mL (by RT min Sample Peaks Peak Peak Peaks TPA) Main Peak SMI
1.7 87.3 9.5 1.5 1.0 20.5 Adhere 1.1 88.1 10.8 0.0 2.6 20.4 CHT 0.7
89.4 9.9 0.0 1.1 20.4 Octyl 0.7 89.4 10.0 0.0 0.9 20.4
Four Step Method Using MMC Capture Step (Non-Affinity and
Non-Specific)
[0395] Alternatively, antidote purification was carried out using
MMC capture step (1), Adhere Step (2), CHT Step (3), and Octyl Step
(4). The data is shown in Table below and in FIG. 14. FIG. 14 as
compared to FIG. 13 indicates that the SMI four step method works
significantly better than the four step method using MMC capture
step that does not use the SMI.
TABLE-US-00007 Process Conc Step/ % Pre- % Main % Beta % Post-
mg/mL (by RT min Sample Peaks Peak Peak Peaks TPA) Main Peak MMC
5.3 68.2 10.1 16.5 4.6 20.4 Adhere 4.8 79.8 11.8 3.6 2.4 20.5 CHT
2.6 85.8 10.1 1.5 1.0 20.5 Octyl 2.4 87.0 10.1 0.4 0.8 20.4
Alternate Methods
SMI Three Step Method
[0396] Alternative purification method was developed to exploit the
power of the SMI affinity capture step and remove the CHT and Octyl
columns from the process. The CHT column is difficult to run, under
loading results in poor recovery and over loading does not clear
impurities. Octyl column is not necessary for Host Cell Protein
(HCP) removal as SMI, 1.sup.st capture step cleans HCP
significantly. Thus, alternatively, antidote purification was
carried out using small molecule inhibitor (Des chloro-C6
Betrixaban linker A4) affinity 20 um Capto resin using SMI Capture
Step (1) and Adhere Step (2) as listed above followed by the
following MMC ImpRes step (3).
[0397] MMC ImpRes
[0398] The Adhere Eluate was diluted 1:15 with 50 mM HEPES, 50 mM
NaCl, pH 7.0 and applied to a 4.7 mL MMC ImpRes Column equilibrated
with 50 mM HEPES, 50 mM NaCl, pH 7.0 with a flow rate of 200 cm/hr.
The chromatography was monitored at UV wavelengths 280, 260, and
320 nm; conductivity and pH were also monitored. After the sample
finished applying, the column was washed with 10 CV of 50 mM HEPES,
50 mM NaCl, pH 7.0.
[0399] The bound Antidote was Step Eluted with 350 mM Arginine, 50
mM Tris/HCl, pH 8.0 Fractionation was started at 50 mAU and ended
at 100 mAU; a single fraction was collected (MMC ImpRes
Eluate).
[0400] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other physical and electronic documents.
[0401] The disclosure has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
disclosure. This includes the generic description of the disclosure
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0402] Other embodiments are within the following claims. In
addition, where features or aspects of the disclosure are described
in terms of Markush groups, those skilled in the art will recognize
that the disclosure is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
51365PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Ala Asn Ser Phe Leu Phe Trp Asn Lys Tyr Lys
Asp Gly Asp Gln Cys 1 5 10 15 Glu Thr Ser Pro Cys Gln Asn Gln Gly
Lys Cys Lys Asp Gly Leu Gly 20 25 30 Glu Tyr Thr Cys Thr Cys Leu
Glu Gly Phe Glu Gly Lys Asn Cys Glu 35 40 45 Leu Phe Thr Arg Lys
Leu Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln 50 55 60 Phe Cys His
Glu Glu Gln Asn Ser Val Val Cys Ser Cys Ala Arg Gly 65 70 75 80 Tyr
Thr Leu Ala Asp Asn Gly Lys Ala Cys Ile Pro Thr Gly Pro Tyr 85 90
95 Pro Cys Gly Lys Gln Thr Leu Glu Arg Arg Lys Arg Arg Lys Arg Ile
100 105 110 Val Gly Gly Gln Glu Cys Lys Asp Gly Glu Cys Pro Trp Gln
Ala Leu 115 120 125 Leu Ile Asn Glu Glu Asn Glu Gly Phe Cys Gly Gly
Thr Ile Leu Ser 130 135 140 Glu Phe Tyr Ile Leu Thr Ala Ala His Cys
Leu Tyr Gln Ala Lys Arg 145 150 155 160 Phe Lys Val Arg Val Gly Asp
Arg Asn Thr Glu Gln Glu Glu Gly Gly 165 170 175 Glu Ala Val His Glu
Val Glu Val Val Ile Lys His Asn Arg Phe Thr 180 185 190 Lys Glu Thr
Tyr Asp Phe Asp Ile Ala Val Leu Arg Leu Lys Thr Pro 195 200 205 Ile
Thr Phe Arg Met Asn Val Ala Pro Ala Cys Leu Pro Glu Arg Asp 210 215
220 Trp Ala Glu Ser Thr Leu Met Thr Gln Lys Thr Gly Ile Val Ser Gly
225 230 235 240 Phe Gly Arg Thr His Glu Lys Gly Arg Gln Ser Thr Arg
Leu Lys Met 245 250 255 Leu Glu Val Pro Tyr Val Asp Arg Asn Ser Cys
Lys Leu Ser Ser Ser 260 265 270 Phe Ile Ile Thr Gln Asn Met Phe Cys
Ala Gly Tyr Asp Thr Lys Gln 275 280 285 Glu Asp Ala Cys Gln Gly Asp
Ala Gly Gly Pro His Val Thr Arg Phe 290 295 300 Lys Asp Thr Tyr Phe
Val Thr Gly Ile Val Ser Trp Gly Glu Gly Cys 305 310 315 320 Ala Arg
Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Thr Ala Phe Leu 325 330 335
Lys Trp Ile Asp Arg Ser Met Lys Thr Arg Gly Leu Pro Lys Ala Lys 340
345 350 Ser His Ala Pro Glu Val Ile Thr Ser Ser Pro Leu Lys 355 360
365 2359PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Ala Asn Ser Phe Leu Phe Trp Asn Lys Tyr Lys
Asp Gly Asp Gln Cys 1 5 10 15 Glu Thr Ser Pro Cys Gln Asn Gln Gly
Lys Cys Lys Asp Gly Leu Gly 20 25 30 Glu Tyr Thr Cys Thr Cys Leu
Glu Gly Phe Glu Gly Lys Asn Cys Glu 35 40 45 Leu Phe Thr Arg Lys
Leu Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln 50 55 60 Phe Cys His
Glu Glu Gln Asn Ser Val Val Cys Ser Cys Ala Arg Gly 65 70 75 80 Tyr
Thr Leu Ala Asp Asn Gly Lys Ala Cys Ile Pro Thr Gly Pro Tyr 85 90
95 Pro Cys Gly Lys Gln Thr Leu Glu Arg Ile Val Gly Gly Gln Glu Cys
100 105 110 Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu Ile Asn Glu
Glu Asn 115 120 125 Glu Gly Phe Cys Gly Gly Thr Ile Leu Ser Glu Phe
Tyr Ile Leu Thr 130 135 140 Ala Ala His Cys Leu Tyr Gln Ala Lys Arg
Phe Lys Val Arg Val Gly 145 150 155 160 Asp Arg Asn Thr Glu Gln Glu
Glu Gly Gly Glu Ala Val His Glu Val 165 170 175 Glu Val Val Ile Lys
His Asn Arg Phe Thr Lys Glu Thr Tyr Asp Phe 180 185 190 Asp Ile Ala
Val Leu Arg Leu Lys Thr Pro Ile Thr Phe Arg Met Asn 195 200 205 Val
Ala Pro Ala Cys Leu Pro Glu Arg Asp Trp Ala Glu Ser Thr Leu 210 215
220 Met Thr Gln Lys Thr Gly Ile Val Ser Gly Phe Gly Arg Thr His Glu
225 230 235 240 Lys Gly Arg Gln Ser Thr Arg Leu Lys Met Leu Glu Val
Pro Tyr Val 245 250 255 Asp Arg Asn Ser Cys Lys Leu Ser Ser Ser Phe
Ile Ile Thr Gln Asn 260 265 270 Met Phe Cys Ala Gly Tyr Asp Thr Lys
Gln Glu Asp Ala Cys Gln Gly 275 280 285 Asp Ala Gly Gly Pro His Val
Thr Arg Phe Lys Asp Thr Tyr Phe Val 290 295 300 Thr Gly Ile Val Ser
Trp Gly Glu Gly Cys Ala Arg Lys Gly Lys Tyr 305 310 315 320 Gly Ile
Tyr Thr Lys Val Thr Ala Phe Leu Lys Trp Ile Asp Arg Ser 325 330 335
Met Lys Thr Arg Gly Leu Pro Lys Ala Lys Ser His Ala Pro Glu Val 340
345 350 Ile Thr Ser Ser Pro Leu Lys 355 36PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Arg
Lys Arg Arg Lys Arg 1 5 4340PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Ala Asn Ser Phe Leu Phe
Trp Asn Lys Tyr Lys Asp Gly Asp Gln Cys 1 5 10 15 Glu Thr Ser Pro
Cys Gln Asn Gln Gly Lys Cys Lys Asp Gly Leu Gly 20 25 30 Glu Tyr
Thr Cys Thr Cys Leu Glu Gly Phe Glu Gly Lys Asn Cys Glu 35 40 45
Leu Phe Thr Arg Lys Leu Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln 50
55 60 Phe Cys His Glu Glu Gln Asn Ser Val Val Cys Ser Cys Ala Arg
Gly 65 70 75 80 Tyr Thr Leu Ala Asp Asn Gly Lys Ala Cys Ile Pro Thr
Gly Pro Tyr 85 90 95 Pro Cys Gly Lys Gln Thr Leu Glu Arg Ile Val
Gly Gly Gln Glu Cys 100 105 110 Lys Asp Gly Glu Cys Pro Trp Gln Ala
Leu Leu Ile Asn Glu Glu Asn 115 120 125 Glu Gly Phe Cys Gly Gly Thr
Ile Leu Ser Glu Phe Tyr Ile Leu Thr 130 135 140 Ala Ala His Cys Leu
Tyr Gln Ala Lys Arg Phe Lys Val Arg Val Gly 145 150 155 160 Asp Arg
Asn Thr Glu Gln Glu Glu Gly Gly Glu Ala Val His Glu Val 165 170 175
Glu Val Val Ile Lys His Asn Arg Phe Thr Lys Glu Thr Tyr Asp Phe 180
185 190 Asp Ile Ala Val Leu Arg Leu Lys Thr Pro Ile Thr Phe Arg Met
Asn 195 200 205 Val Ala Pro Ala Cys Leu Pro Glu Arg Asp Trp Ala Glu
Ser Thr Leu 210 215 220 Met Thr Gln Lys Thr Gly Ile Val Ser Gly Phe
Gly Arg Thr His Glu 225 230 235 240 Lys Gly Arg Gln Ser Thr Arg Leu
Lys Met Leu Glu Val Pro Tyr Val 245 250 255 Asp Arg Asn Ser Cys Lys
Leu Ser Ser Ser Phe Ile Ile Thr Gln Asn 260 265 270 Met Phe Cys Ala
Gly Tyr Asp Thr Lys Gln Glu Asp Ala Cys Gln Gly 275 280 285 Asp Ala
Gly Gly Pro His Val Thr Arg Phe Lys Asp Thr Tyr Phe Val 290 295 300
Thr Gly Ile Val Ser Trp Gly Glu Gly Cys Ala Arg Lys Gly Lys Tyr 305
310 315 320 Gly Ile Tyr Thr Lys Val Thr Ala Phe Leu Lys Trp Ile Asp
Arg Ser 325 330 335 Met Lys Thr Arg 340 519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Leu Pro Lys Ala Lys Ser His Ala Pro Glu Val Ile Thr Ser Ser 1 5 10
15 Pro Leu Lys
* * * * *