U.S. patent application number 11/073332 was filed with the patent office on 2005-08-11 for multivalent platform molecules comprising high molecular weight polyethylene oxide.
This patent application is currently assigned to La Jolla Pharmaceutical Co.. Invention is credited to Jones, David S..
Application Number | 20050175620 11/073332 |
Document ID | / |
Family ID | 22782915 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050175620 |
Kind Code |
A1 |
Jones, David S. |
August 11, 2005 |
Multivalent platform molecules comprising high molecular weight
polyethylene oxide
Abstract
Valency platform molecules comprising high molecular weight
polyethylene oxide groups are provided, as well as conjugates
thereof with biologically active molecules, and methods for their
preparation. The high molecular weight polyethylene oxide group
has, for example, a molecular weight of greater than 22,000
Daltons, for example at least 40,000 Daltons. In one embodiment, a
composition comprising the valency platform molecules is provided,
wherein the molecules have a polydispersity less than about 1.2.
Conjugates of the valency platform molecule and a biologically
active molecule, such as a saccharide, poly(saccharide), amino
acid, poly(amino acid), nucleic acid or lipid also are provided.
Also provided are pharmaceutically acceptable compositions
comprising the conjugates disclosed herein and a pharmaceutically
acceptable carrier, as well as methods of making and using the
conjugates and compositions.
Inventors: |
Jones, David S.; (San Diego,
CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Assignee: |
La Jolla Pharmaceutical Co.
|
Family ID: |
22782915 |
Appl. No.: |
11/073332 |
Filed: |
March 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073332 |
Mar 4, 2005 |
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09877387 |
Jun 7, 2001 |
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60210439 |
Jun 8, 2000 |
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Current U.S.
Class: |
424/178.1 ;
525/54.1; 530/391.1 |
Current CPC
Class: |
A61K 47/54 20170801;
A61K 47/60 20170801; A61P 7/02 20180101 |
Class at
Publication: |
424/178.1 ;
525/054.1; 530/391.1 |
International
Class: |
A61K 039/395; C07K
016/46 |
Claims
1. A chemically defined valency platform molecule comprising at
least one high molecular weight polyethylene oxide group.
2. The valency platform molecule of claim 1, comprising at least 2
high molecular weight polyethylene oxide groups.
3. The valency platform molecule of claim 1, wherein the high
molecular weight polyethylene oxide group has a molecular weight of
greater than 22,000 Daltons.
4. The valency platform molecule of claim 1, wherein the high
molecular weight polyethylene oxide group has a molecular weight of
at least 40,000 Daltons.
5. The valency platform molecule of claim 1, wherein the high
molecular weight polyethylene oxide group has the formula:
--(CH.sub.2CH.sub.2O).su- b.n--wherein n is greater than 500.
6. The valency platform molecule of claim 5, wherein n is greater
than 600.
7. The valency platform molecule of claim 5, wherein n is greater
than 700.
8. The valency platform molecule of claim 5, wherein n is greater
than 800.
9. The valency platform molecule of claim 1, wherein the valency
platform molecule comprises a core group and at least three arms
wherein each arm comprises a terminus.
10. The valency platform molecule of claim 9, wherein the core
group comprises a high molecular weight polyethylene oxide
group.
11. The valency platform molecule of claim 9, wherein at least one
of said arms comprises a high molecular weight polyethylene oxide
group.
12. The valency platform molecule of claim 9, wherein the high
molecular weight polyethylene oxide group is attached to the core
or arm.
13. A composition comprising valency platform molecules of claim 1,
wherein the molecules have a polydispersity less than 1.2; and
wherein the average molecular weight of the high molecular weight
polyethylene oxide groups in the composition is at least
18,000.
14. The valency platform molecule of claim 1, comprising at least
three reactive conjugating groups selected from the group
consisting of hydroxyl, thiol, isocyanate, isothiocyanate, amine,
alkyl halide, alkylmercurial halide, aldehyde, ketone, carboxylic
acid halide, .alpha.-halocarbonyl, .alpha.,.beta.-unsaturated
carbonyl, haloformate ester, carboxylic acid, carboxylic ester,
carboxylic anhydride, O-acyl isourea, hydrazide, maleimide, imidate
ester, sulfonate ester, sulfonyl halide, .alpha.,.beta.-unsaturated
sulfone, aminooxy, semicarbazide, and .beta.-aminothiol.
15. The valency platform molecule of claim 1 comprising at least 3
aminooxy groups.
16. The valency platform molecule of claim 1 comprising at least 3
carbamate groups.
17. A conjugate of a valency platform molecule of claim 1 and a
biologically active molecule.
18. The conjugate of claim 17, wherein the biologically active
molecule is selected from the group consisting of
poly(saccharides), poly(amino acids), nucleic acids and lipids.
19. The conjugate of claim 17, wherein the conjugate is a B cell
toleragen.
20. The conjugate of claim 18, wherein the biologically active
molecule comprises a nucleic acid or analog thereof, which
specifically binds to an anti-double stranded DNA antibody.
21. The conjugate of claim 19, wherein the biologically active
molecule is a .beta..sub.2GPI domain 1 polypeptide or analog
thereof.
22. The conjugate of claim 21, wherein the conjugate is effective
for the treatment of antibody mediated thrombosis.
23. The conjugate of claim 18, wherein the biologically active
molecule is an .alpha.Gal epitope or analog thereof that
specifically binds to an anti-.alpha.Gal antibody.
24. A pharmaceutically acceptable composition comprising the
conjugate of claim 17 and a pharmaceutically acceptable
carrier.
25. A conjugate of a chemically defined valency platform molecule
and a polypeptide comprising a .beta..sub.2GPI domain 1
polypeptide, wherein the conjugate comprises at least one high
molecular weight polyethylene oxide group.
26. The conjugate of claim 25, wherein the valency platform
molecule comprises at least 3 aminooxy groups.
27. The conjugate of claim 25, wherein the valency platform
molecule comprises at least 3 carbamate groups.
28. The conjugate of claim 25, wherein the high molecular weight
polyethylene oxide group has a molecular weight greater than 22,000
Daltons.
29. The conjugate of claim 25, wherein the valency platform
molecule comprises a core group and at least three arms, wherein
each arm comprises a terminus.
30. The conjugate of claim 25, wherein the polypeptide specifically
binds to a .beta..sub.2GPI-dependent antiphospholipid antibody.
31. The conjugate of claim 30, wherein the polypeptide lacks a T
cell epitope capable of activating T cells in an individual having
.beta..sub.2GPI dependent antiphospholipid antibodies.
32. The conjugate of claim 25, wherein the .beta..sub.2GPI domain 1
polypeptide comprises at least five contiguous amino acids of FIG.
19 (SEQ ID NO: 2).
33. The conjugate of claim 25, wherein the .beta..sub.2GPI domain 1
polypeptide comprises amino acids Nos. 2-63 of SEQ ID NO: 2.
34. The conjugate of claim 25, wherein the conjugate is selected
from the group consisting of compounds 200, 202, 203, and 205 shown
in FIG. 7 and compound 300 shown in FIG. 16, wherein D1 in said
structures is a polypeptide consisting of amino acids No. 2-63 of
SEQ ID No: 2.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. Ser. No. 60/210,439, filed Jun. 8, 2000, the
disclosure of which is incorporated herein by reference.
TECHNICAL FIELD
[0002] This application relates to valency platform molecules
comprising polyethylene oxide groups to which one or more
molecules, such as biologically active molecules, may be attached
to form a conjugate.
BACKGROUND ART
[0003] A "valency platform" is a molecule with one or more (and
typically multiple) attachment sites which can be used to
covalently attach biologically active molecules of interest to a
common scaffold. The attachment of biologically active molecules to
a common scaffold provides multivalent conjugates in which multiple
copies of the biologically active molecule are covalently linked to
the same platform. A "defined" or "chemically defined" valency
platform is a platform with defined structure, thus a defined
number of attachment points and a defined valency. A defined
valency platform conjugate is a conjugate with defined structure
and has a defined number of attached biologically active compounds.
Examples of biologically active molecules include oligonucleotides,
peptides, polypeptides, proteins, antibodies, saccharides,
polysaccharides, epitopes, mimotopes, drugs, and the like. For
example, the biologically active compounds may interact
specifically with proteinaceous receptors.
[0004] Certain classes of chemically defined valency platforms,
methods for their preparation, conjugates comprising them, and
methods for the preparation of such conjugates, have been described
in U.S. Pat. Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512;
5,726,329; 5,268,454; 5,552,391; 5,606,047; and 5,663,395. Valency
platform molecules comprising carbamate linkages are described in
U.S. Provisional Patent Application Ser. No. 60/111,641; U.S. Ser.
No. 09/457,607, filed Dec. 8, 1999; and WO 00/34231. Valency
platform molecules comprising aminooxy groups are described in U.S.
Provisional Patent Application No. 60/138,260, filed Jun. 8, 1999;
and PCT/US00/15968.
[0005] Polyethylene glycol conjugates are described, for example,
in PCT WO 99/45964, published Sep. 16, 1999; U.S. Pat. No.
5,672,662; U.S. Pat. No. 5,932,462; PCT WO 99/34833; PCT WO
95/34326; and U.S. Pat. No. 5,990,237. Polyether copolymers
including linear and dendritic blocks are described in Gitsov et
al., Angew Chem. Int. Ed. Engl., 1992, 31:1200-1203. A polyethylene
glycol multiblock copolymer as a carrier of the anticancer drug
doxorubicin is described in Pechar et al., Bioconjugate Chem.,
11:131-139 (2000). A polyethylene glycol copolymer for carrying and
releasing multiple copies of peptides is described in Huang et al.,
Bioconjugate Chem. 9:612-617 (1998). Polyethylene glycol copolymers
and their self assembly with DNA are described in Choi et al., J.
Am. Chem. Soc., 122:474-480 (2000). Polyether dendritic compounds
containing folate residues are described in Kono et al.,
Bioconjugate Chemistry, 10:1115-1121 (1999).
[0006] Direct PEGylation of polypeptides is generally done by
attaching to lysine amino groups or other side chain functionality.
This often results in a heterogeneous mixture of products and can
lead to loss of bio-activity. Thus, there is a need for improved
methods of forming multivalent conjugates of biologically active
molecules and polyethylene oxide groups.
DISCLOSURE OF THE INVENTION
[0007] Chemically defined valency platform molecules comprising at
least one high molecular weight polyethylene oxide group are
provided. The valency platform molecule may comprise, e.g., at
least 2, 3, 4, or more high molecular weight polyethylene oxide
groups. The high molecular weight polyethylene oxide group has, for
example, a molecular weight of greater than 18,000 Daltons; greater
than 22,000 Daltons; greater than 40,000 Daltons; greater than
50,000 Daltons; greater than 80,000 Daltons; greater than 100,000
Daltons, or at least 40,000 Daltons.
[0008] In one embodiment, in the valency platform molecule, the
high molecular weight polyethylene oxide group has the formula:
--(CH.sub.2CH.sub.2O).sub.n--
[0009] wherein n is greater than 500; n is greater than 400; n is
greater than 500; n is greater than 600; n is greater than 700; or
n is greater than 800.
[0010] In one embodiment, the valency platform molecule comprises a
core group and at least three arms wherein each arm comprises a
terminus. The core group and/or the arms may comprise a high
molecular weight polyethylene oxide group. The high molecular
weight polyethylene oxide group also may be attached to the core or
arm.
[0011] In one embodiment, a composition comprising the valency
platform molecules disclosed herein is provided, wherein the
molecules have a polydispersity less than 1.2.
[0012] In one embodiment, the valency platform molecule may
comprise at least three reactive conjugating groups such as
hydroxyl, thiol, isocyanate, isothiocyanate, amine, alkyl halide,
alkylmercurial halide, aldehyde, ketone, carboxylic acid halide,
.alpha.-halocarbonyl, .alpha.,.beta.-unsaturated carbonyl,
haloformate ester, carboxylic acid, carboxylic ester, carboxylic
anhydride, O-acyl isourea, hydrazide, maleimide, imidate ester,
sulfonate ester, sulfonyl halide, .alpha.,.beta.-unsaturated
sulfone, aminooxy, semicarbazide, or .beta.-aminothiol. In one
embodiment, the valency platform molecule comprises at least 3
aminooxy groups and/or at least 3 carbamate groups.
[0013] In one embodiment, there is provided a conjugate of a
valency platform molecule as disclosed herein and one or more, for
example 3, or more, biologically active molecules, such as a
saccharide, poly(saccharide), amino acid, poly(amino acid), nucleic
acid or lipid. In one embodiment, the conjugate is a B cell
toleragen.
[0014] In one embodiment, the biologically active molecule is a
nucleic acid or analog thereof, and the conjugate is effective for
reducing levels of anti-double stranded DNA antibodies, such as the
treatment or alleviation of lupus. In another embodiment, the
biologically active molecule is a polypeptide comprising a
.beta..sub.2GPI domain 1 polypeptide or analog thereof, and, for
example, the conjugate is effective for reduction of levels of
anti-phospholipid (aPL) antibodies and/or the treatment of diseases
associated with, for example, anti-phospholipid syndrome, such as
antibody mediated thrombosis. In one embodiment, the biologically
active molecule is an .alpha.Gal epitope or analog thereof that
specifically binds to an anti-.alpha.Gal antibody; and optionally
the conjugate is effective to induce immunological tolerance in
xenotransplantation.
[0015] In one embodiment, the biologically active molecule is an
analog of a T cell dependent immunogen wherein the analog binds
specifically to surface antibody on B cells to which the T cell
dependent immunogen binds specifically and the conjugate lacks T
cell epitopes capable of activating T cells in said individual.
[0016] Also provided are pharmaceutically acceptable compositions
comprising the conjugates disclosed herein and a pharmaceutically
acceptable carrier.
[0017] A further aspect of the invention is a method for treating
an antibody mediated disease or other condition in an individual in
need of such treatment comprising administering to the individual
an effective amount of the conjugates, wherein optionally the
conjugates specifically bind to an antibody associated with an
antibody-mediated disease.
[0018] Yet another aspect of the invention is a method of inducing
specific B cell anergy to an immunogen in an individual comprising
administering to the individual an effective amount of the
conjugates.
[0019] Another aspect of the invention is a method of treating an
individual for an antibody-mediated pathology in which undesired
antibodies are produced in response to an immunogen comprising
administering to the individual an effective amount of the
conjugates.
[0020] In one embodiment, it is preferred that the total molecular
weight of the conjugate is no greater than about 200,000 Daltons,
for example, in order for the conjugate to be effective as a
functional toleragen and not act as a T cell independent
immunogen.
[0021] Also provided are compositions, and methods for their use,
wherein the composition comprises valency platform molecules
including high molecular weight polyethylene oxide groups, wherein
the average molecular weight of the polyethylene oxide groups in
the valency platform molecules in the composition, is, for example,
greater than about 18,000, greater than about 20,000, greater than
about 22,000, greater than about 30,000, greater than about 40,000,
greater than about 50,000, or greater than about 100,000
Daltons.
[0022] Chemically defined valency platform molecules are provided
that comprise polyethylene oxide groups which in combination have a
high molecular weight. Also provided are compositions, and methods
for their use, wherein the composition comprises valency platform
molecules including polyethylene oxide groups that have a high
molecular weight in combination, and the average molecular weight
of the polyethylene oxide groups in combination on the valency
platform molecules in the composition, is, for example, greater
than about 18,000, greater than about 20,000, or, for example,
greater than about 22,000 Daltons.
[0023] There are also provided compositions, and methods for their
use, comprising valency platform molecules, wherein the valency
platform molecules comprise at least one high molecular weight
polyethylene oxide group; wherein the molecules have a
polydispersity less than about 1.2; and wherein the average
molecular weight of the high molecular weight polyethylene oxide
groups in the composition is at least about 18,000.
[0024] Also provided are conjugates of a chemically defined valency
platform molecule and a polypeptide comprising a .beta..sub.2GPI
domain 1 polypeptide, wherein the conjugate comprises at least one
high molecular weight polyethylene oxide group. The high molecular
weight polyethylene oxide group has a molecular weight, for
example, greater than 22,000 Daltons. The polypeptide in one
embodiment specifically binds to a .beta..sub.2GPI-dependent
antiphospholipid antibody and optionally lacks a T cell epitope
capable of activating T cells in an individual having
.beta..sub.2GPI dependent antiphospholipid antibodies.
[0025] In the conjugate, the .beta..sub.2GPI domain 1 polypeptide,
for example, comprises at least five contiguous amino acids of FIG.
19 (SEQ ID NO: 2); or amino acids Nos. 2-63 of FIG. 19 (SEQ ID NO:
2). The conjugate is for example compound 200, 202, 203, or 205
shown in FIG. 7 or compound 300 shown in FIG. 16, wherein D1 in
said structures is for example a polypeptide consisting of amino
acids No. 2-63 of SEQ ID No: 2.
[0026] A further aspect of the invention is a method for making the
conjugates described above comprising: covalently bonding the
biologically active molecule to a chemically-defined valency
platform molecule to form a conjugate.
[0027] In one embodiment, a conjugate is provided as disclosed
herein, wherein the conjugate is suitable for inducing specific B
cell anergy to a T cell-dependent immunogen implicated in an
antibody-mediated pathology in an individual suffering from said
pathology, the conjugate comprising a preferably nonimmunogenic
valency platform molecule as described herein and at least two
analog molecules of the immunogen wherein (a) the analog molecules
bind specifically to surface antibody on B cells to which the T
cell-dependent immunogen binds specifically and (b) the conjugate
lacks T cell epitopes capable of activating T cells in said
individual. The analog molecules are for example peptides,
polypeptides, proteins, glycoproteins, lipoproteins, carbohydrates,
lipids, or lipopolysaccharides.
[0028] In another embodiment, there is provided a composition
comprising valency platform molecules comprising high molecular
weight polyethylene oxide groups, wherein the average molecular
weight of the polyethylene oxide groups is greater than about
18,000, greater than about 20,000 or greater than about 22,000.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a reaction scheme showing the synthesis of
compound 8.
[0030] FIG. 2 is a reaction scheme showing the synthesis of
compound 11.
[0031] FIG. 3 is a reaction scheme showing the synthesis of
compound 17.
[0032] FIG. 4 is a reaction scheme showing the synthesis of
compound 23.
[0033] FIG. 5 shows exemplary valency platform molecule conjugates
comprising high molecular weight polyethylene oxide groups.
[0034] FIG. 6 shows exemplary valency platform molecule conjugates
comprising high molecular weight polyethylene oxide groups.
[0035] FIG. 7 shows exemplary valency platform molecule conjugates
comprising polyethylene oxide groups.
[0036] FIG. 8 shows illustrative Formulas for valency platform
molecules comprising aminoxy groups and polyethylene oxide
groups.
[0037] FIG. 9 shows a scheme for the synthesis of multivalent
platform molecules comprising polyethylene oxide groups of varying
molecular weight.
[0038] FIG. 10 shows a scheme for the synthesis of multivalent
platform molecules comprising polyethylene oxide groups and
arms.
[0039] FIG. 11 shows a scheme for the synthesis of compound
132.
[0040] FIG. 12 shows a scheme for the synthesis of compound
136.
[0041] FIG. 13 shows a scheme for the synthesis of compound
143.
[0042] FIG. 14 shows the synthesis of an exemplary octameric bPEG
platform, where n is, for example, 112, or more, e.g., 500 or
more.
[0043] FIG. 15 shows the synthesis of a valency platform molecule
comprising two polyethylene oxide groups, wherein n is, for
example, 500 or more.
[0044] FIG. 16 shows the structure of exemplary conjugate 300.
[0045] FIG. 17 is a scheme for the synthesis of compounds 303 and
306.
[0046] FIG. 18 shows the structure of some exemplary conjugates 309
and 310.
[0047] FIG. 19 depicts the nucleotide (SEQ ID NO: 1) and amino acid
(SEQ ID NO:2) sequence of domain 1 of .beta..sub.2GPI. Numbers
below the lines indicate amino acid positions.
[0048] FIG. 20 is a scheme showing the synthesis of a transaminated
polypeptide.
MODES FOR CARRYING OUT THE INVENTION
[0049] Valency platform molecules comprising high molecular weight
polyethylene oxide groups are provided, and conjugates thereof with
biologically active molecules, as well as methods for their
synthesis and use. Also provided are pharmaceutically acceptable
compositions comprising the conjugates disclosed herein, optionally
in a pharmaceutically acceptable carrier.
[0050] In the complexes of valency platform molecules comprising
high molecular weight polyethylene oxide groups with biologically
active molecules, advantageously, in a preferred embodiment, the
polyethylene oxide groups are not attached directly to the
biologically active molecule, such as a polypeptide.
Advantageously, the polyethylene oxide group is attached directly
to the platform, rather than the protein or other agent, thus
potentially reducing the potential of the interference with
binding. The attachment of a polyethylene oxide of a selected
molecular weight or molecular weight range to the platform is well
defined, thus conjugates of biologically active molecules and
valency platform molecules comprising high molecular weight
polyethylene oxide groups also are homogeneous and well
defined.
[0051] The valency platform molecules disclosed herein include one
or more high molecular weight polyalkylene oxide groups. The
presence of the polyalkylene oxide groups on the valency platform
molecules also can advantageously improve serum half life and
improve activity of biologically active molecules conjugated
thereto. In the embodiments described herein, where the term
polyethylene oxide is used, also included within the scope of this
invention are other polyalkylene oxides such as polypropylene
oxide.
[0052] Pharmaceutically Acceptable Compositions
[0053] Pharmaceutically acceptable valency platform molecules
comprising high molecular weight polyethylene oxide groups are
provided, and conjugates thereof with biologically active
molecules, as well as methods for their synthesis and use. Also
provided are pharmaceutically acceptable compositions comprising
the molecules and conjugates disclosed herein, optionally in a
pharmaceutically acceptable carrier.
[0054] Carriers for different routes of administration, including
oral, intravenous, and aerosol administration are described in the
art, for example, in "Remington: The Science and Practice of
Pharmacy," Mack Publishing Company, Pennsylvania, 1995, the
disclosure of which is incorporated herein by reference. Carriers
can include, for example, water, saccharides, polysaccharides,
buffers, excipients, and biodegradable polymers such as polyesters,
polyanhydrides, polyamino acids and liposomes.
[0055] Pharmaceutically acceptable compositions are compositions in
a form suitable for administration to an individual, for example,
systemic or localized administration to individuals in unit dosage
forms, sterile parenteral solutions or suspensions, sterile
non-parenteral solutions or oral solutions or suspensions, oil in
water or water in oil emulsions and the like.
[0056] Polyethylene Oxide
[0057] In one embodiment, the valency platform molecules include
one or more polyethylene oxide groups with a molecular weight, for
example, greater than about 5,000; greater than about 10,000;
greater than about 15,000; or greater than about 20,000. The
polyethylene oxide group can have a molecular weight, for example,
of about 5,000 to 10,000; about 8,000 to 20,000; about 10,000 to
20,000; or about 15,000 to 20,000.
[0058] In a composition comprising valency platform molecules, the
molecular weight of the polyethylene oxide groups in the valency
platform molecule may be a molecular weight that is an average
molecular weight of the polyethylene oxide groups, since there may
be a molecular weight distribution. Preferably, the molecular
weight distribution is narrow. The molecular weights disclosed
herein in one embodiment refer to the average molecular weight of a
composition comprising the valency platform molecules.
[0059] In a preferred embodiment, the valency platform molecule
comprises at least one high molecular weight polyethylene oxide
group. The valency platform molecule also can include plural high
molecular weight polyethylene oxide groups, for example, 2, 3 or
more. Alternatively, the high molecular weight polyethylene oxide
may be present as a plurality of different polyethylene oxide
groups in different locations on the platform which have a high
molecular weight in combination. The high molecular weight in
combination may be, for example, greater than about 18,000, greater
than about 20,000, or for example, greater than about 22,000.
[0060] As used herein, the term "high molecular weight polyethylene
oxide" group refers to a polyethylene oxide group having a
molecular weight greater than about 18,000, greater than about
20,000, or, for example, greater than about 22,000 Daltons. The
molecular weight may be, for example, about 20,000-22,000 or about
18,000-22,000.
[0061] For example, the high molecular weight polyethylene oxide
may be a group having the formula:
--(CH.sub.2CH.sub.2O).sub.n--,
[0062] wherein n is, for example, greater than about 400, greater
than about 450, greater than about 500, or greater than about 550.
For example, n is about 400-550, 520 to 600, 550 to 700,600 to 800,
600 to 900, or 600 to 1000, or more. In another embodiment, n is at
least about 600, at least about 700, at least about 800, at least
about 900 or at least about 1000.
[0063] The high molecular weight polyethylene oxide group can, for
example, have a molecular weight greater than about 25,000; greater
than about 30,000; or greater than about 40,000 daltons. The high
molecular weight polyethylene oxide group further may have, for
example, a molecular weight greater than about 50,000, for example
about 50,000 to 100,000 or more, for example about 50,000 to
100,000.
[0064] In some embodiments, the valency platform molecules include
core groups and a plurality of arms extending from the core,
wherein said arms comprise a terminus. See, for example, FIGS.
14-16. Optionally, the arms also may branch, increasing the number
of termini. The high molecular weight polyethylene groups may be
present in the core or in one or more of the arms or may be
attached to the valency platform molecule. For example, the valency
platform molecule may have the formula:
Rc[G.sub.1AG.sub.2].sub.y Formula 20
[0065] wherein Rc represents the core and A represents the two or
more arms, wherein Rc and A are independently an organic moiety,
and at least one of Rc and A comprises a high molecular weight
polyethylene oxide; and
[0066] G.sub.1 if present is an organic moiety;
[0067] G.sub.2 if present is an organic moiety, for example
comprising a reactive conjugating group; and
[0068] y is two or more, for example, 3, 4, 5, 6, 7, 8, 16 or
more.
[0069] The core group or one of the arms of the valency platform
molecule thus can comprise the polyethylene oxide groups, or the
polyethylene oxide group may be optionally attached to a selected
location on the valency platform molecule such as on the core or
one or more arms. Preferably the arms include a terminus that
comprises a reactive group for the attachment of biologically
active molecules. The valency platform molecule also may include
branching groups that increase the number of arms, and therefore
termini of the platform molecule.
[0070] Polyethylene oxide groups of molecular weight of for
example, about 10,000 to 40,000 Daltons may be useful for promoting
suitably long plasma half lives.
[0071] Valency Platforms
[0072] Any of a variety of valency platform molecules known in the
art can be synthesized in a manner to include high molecular weight
polyethylene oxide groups as disclosed herein. Methods for making
valency platform molecules are described, for example, in U.S. Pat.
Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512; 5,726,329;
5,268,454; 5,552,391; 5,606,047; 5,663,395 and 5,874,409, as well
as in U.S. Ser. No. 60/111,641 and PCT US97/10075. In general,
these platforms contain core groups or branched core groups which
can terminate in, for example, hydroxyl groups, thiols, carboxyl
groups, amino groups, aldehydes, ketones, alkyl halides, or
aminooxy groups, which optionally can be further modified to
provide a preselected reactive conjugating group to permit further
attachment of selected molecules thereto. Preferably, the valency
platform molecules include at least three reactive conjugating
groups. Examples of reactive conjugating groups include hydroxyl,
thiol, isocyanate, isothiocyanate, amine, alkyl halide,
alkylmercurial halide, aldehyde, ketone, carboxylic acid halide,
.alpha.-halocarbonyl, .alpha.,.beta.-unsaturated carbonyl,
haloformate ester, carboxylic acid, carboxylic ester, carboxylic
anhydride, O-acyl isourea, hydrazide, maleimide, imidate ester,
sulfonate ester, sulfonyl halide, .alpha.,.beta.-unsaturated
sulfone, aminooxy, semicarbazide, and .beta.-aminothiol.
[0073] Valency platforms are prepared from core groups which
contain the desired valence, or the valence of a core group can be
increased by derivatizing the terminal functionality with branching
moieties.
[0074] Methods known the art for making valency platform molecules,
include, for example, a propagation method, or segmental approach.
Such methods can be modified, using the appropriate reagents, to
provide the desired valency.
[0075] In one aspect, valency platform molecules are provided that
are substantially monodisperse. The valency platform molecules
advantageously have a narrow molecular weight distribution. A
measure of the breadth of distribution of molecular weight of a
sample of a valency platform molecule is the polydispersity of the
sample. Polydispersity is used as a measure of the molecular weight
homogeneity or nonhomogeneity of a polymer sample. Polydispersity
is calculated by dividing the weight average molecular weight (Mw)
by the number average molecular weight (Mn). The value of Mw/Mn is
unity for a perfectly monodisperse polymer. Polydispersity (Mw/Mn)
is measured by methods available in the art, such as gel permeation
chromatography. The polydispersity (Mw/Mn) of a sample of a valency
platform molecule is preferably less than 2, more preferably, less
than 1.5, or less than 1.2, less than 1.07, less than 1.02, or,
e.g., about 1.05 to 1.5 or about 1.05 to 1.2. Typical polymers
generally have a polydispersity of 2-5, or in some cases, 20 or
more. Advantages of the low polydispersity property of the valency
platform molecules include improved biocompatibility and
bioavailability since the molecules are substantially homogeneous
in size, and variations in biological activity due to a wide
variations in molecular weight are minimized. The low
polydispersity molecules thus are pharmaceutically optimally
formulated and easy to analyze. Further there is controlled valency
of the population of molecules in the sample.
[0076] In a composition comprising valency platform molecules
including high molecular weight polyethylene oxide groups, the
average molecular weight of the polyethylene oxide groups on the
valency platform molecules in the composition, is, for example,
greater than about 18,000, greater than about 20,000, or, for
example, greater than about 22,000 Daltons.
[0077] In a composition comprising valency platform molecules
including polyethylene oxide groups that have a high molecular
weight in combination, the average molecular weight of the
polyethylene oxide groups in combination on the valency platform
molecules in the composition, is, for example, greater than about
18,000, greater than about 20,000, or, for example, greater than
about 22,000 Daltons.
[0078] In some embodiments, the valency platform molecule may be
described as "dendritic," owing to the presence of successive
branch points. Dendritic valency platform molecules possess
multiple termini, typically 4 or more termini, e.g., 8 termini, or
16 termini.
[0079] Note that the Formulas disclosed herein are intended to
encompass both symmetric and non-symmetric valency platforms. See,
for example, the symmetric molecule M in FIG. 14 and the
non-symmetric molecule 300 in FIG. 16.
[0080] Using methods described in the detailed description below
and the Examples, high molecular weight polyethylene oxide groups
can be readily incorporated into the valency platform using the
appropriate starting materials and reagents such that the
polyethylene oxide units are within the molecule, for example in
the core, or in one or more of the arms extending from the core, or
are attached to the valency platform molecule via reactive groups
present on the molecule.
[0081] Exemplary Chemically Defined Valency Platform Molecules
[0082] Valency platform molecules can be used that are defined with
respect to their chemical structure, valency, homogeneity and a
defined chemistry which is amenable to effective conjugation with
the appropriate biological and/or chemical molecules.
[0083] Chemically-defined, non-polymeric valency platform molecules
suitable for use within the present invention include, but are not
limited to, biologically compatible and nonimmunogenic carbon-based
compounds of the following formulae: 1 G [ 1 ] { T [ 1 ] } n [ 1 ]
Formula 1 a G [ 2 ] { L [ 2 ] - J [ 2 ] - Z [ 2 ] ( T [ 2 ] ) p [ 2
] } n [ 2 ] Formula 2 a
[0084] wherein
[0085] each of G.sup.[1] and G.sup.[2], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000, or 10,000 or more chain atoms selected from the
group C, N, O, Si, P and S;
[0086] more preferably, G.sup.[2], if present, is a radical derived
from a polyalcohol, a polyamine, or a polyglycol; for example,
G.sup.[2] is selected from the group --(CH.sub.2).sub.q-- wherein
q=0 to 20, --CH.sub.2(CH.sub.2OCH.sub.2).sub.rCH.sub.2--, wherein
r=0 to 300, and C(CH.sub.2OCH.sub.2CH.sub.2--).sub.s(OH).sub.4-s
wherein s=1 to 4, more preferably s=3 to 4;
[0087] each of the n.sup.[1] moieties shown as T.sup.[1] and each
of the p.sup.[2] x n.sup.[2] moieties shown as T.sup.[2] is
independently chosen from the group NHR.sup.SUB (amine),
C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB (hydrazine),
C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER (activated
ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride), C(.dbd.O)X (acid
halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.- SUB)NHR.sup.SUB(carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
sulfone);
[0088] in one embodiment each of the n.sup.[1] moieties shown as
T.sup.[1] and each of the p.sup.[2] x n.sup.[2] moieties shown as
T.sup.[2] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX (alkyl
halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CHCHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2(.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2(.alpha.,.beta.-unsaturated
sulfone);
[0089] for example each of the n.sup.[1] moieties shown as
T.sup.[1] and each of the p.sup.[2] x n.sup.[2] moieties shown as
T.sup.[2] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)CH.sub.2X (haloacetyl), NR.sup.1R.sup.2 wherein
R.sup.1R.sup.2 is --C(.dbd.O)CHCHC(.dbd.O)-- (maleimide), and
C(.dbd.O)CR.sup.B.dbd.CR.s- up.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl);
[0090] in one embodiment, all of the n.sup.[1] moieties shown as
T.sup.[1] and all of the p.sup.[2] x n.sup.[2] moieties shown as
T.sup.[2] are identical;
[0091] wherein
[0092] each X is independently a halogen of atomic number greater
than 16 and less than 54 or other good leaving group (i.e., weak
bases such as alkyl or alkyl-substituted sulfonates or sulfates and
the like, aryl or aryl-substituted sulfonates or sulfates and the
like that act similarly to a halogen in this setting);
[0093] each R.sup.ALK is independently a linear, branched, or
cyclic alkyl (1-20C) group;
[0094] each R.sup.SUB is independently H, linear, branched, or
cyclic alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C);
[0095] each R.sup.ESTER is independently N-succinimidyl,
p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl,
pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl,
cyanomethyl and the like, or other activating group such as
5-chloro,8-quinolone, 1-piperidine, N-benzotriazole and the
like;
[0096] each R.sup.B is independently a radical comprising 1-50
atoms selected from the group C, H, N, O, Si, P and S;
[0097] each of the n.sup.[2] moieties shown as L.sup.[2], if
present, is independently chosen from the group O, NR.sup.SUB and
S;
[0098] each of the n.sup.[2] moieties shown as J.sup.[2], if
present, is independently chosen from the group C(.dbd.O) and
C(.dbd.S);
[0099] n.sup.[1]=1 to 32, more preferably n.sup.[1]=2 to 16, even
more preferably n.sup.[1]=2 to 8, most preferably n.sup.[1]=2 to
4;
[0100] n.sup.[2]=1 to 32, more preferably n.sup.[2]=1 to 16, even
more preferably n.sup.[2]=1 to 8, yet more preferably n.sup.[2]=1
to 4, most preferably n.sup.[2]=1 to 2;
[0101] p.sup.[2]=1 to 8, more preferably p.sup.[2]=1 to 4, most
preferably p.sup.[2]=1 to 2;
[0102] with the proviso that the product n.sup.[2] x p.sup.[2] be
greater than 1 and less than 33;
[0103] each of the n.sup.[2] moieties shown as Z.sup.[2] is
independently a radical comprising 1-200 atoms selected from the
group C, H, N, O, Si, P and S, containing attachment sites for at
least p.sup.[2] functional groups on alkyl, alkenyl, or aromatic
carbon atoms;
[0104] in one embodiment, all of the n.sup.[2] moieties shown as
Z.sup.[2] are identical;
[0105] in one embodiment, each of the n.sup.[2] moieties shown as
Z.sup.[2] is independently described by a formula chosen from the
group:
Z.sup.[2] is W.sup.[3]-Y.sup.[3] (attachment site).sub.p[2] Formula
3a 2 Z [ 2 ] is W [ 4 ] --N { Y [ 4 ] ( attachment site ) p [ 2 ] /
2 } 2 Formula 4 a Z [ 2 ] is W [ 5 ] --CH { Y [ 5 ] ( attachment
site ) p [ 2 ] / 2 } 2 Formula 5 a
[0106] wherein
[0107] each of the n.sup.[2] moieties shown as W.sup.[3],
W.sup.[4], or W.sup.[5] if present, is independently a radical
comprising 1-100 atoms selected from the group C, H, N, O, Si, P
and S;
[0108] each of the n.sup.[2] moieties shown as Y.sup.[3], each of
the 2 x n.sup.[2] moieties shown as Y.sup.[4], and each of the 2 x
n.sup.[2] moieties shown as Y.sup.[5] is independently a radical
comprising 1-100 atoms selected from the group C, H, N, O, Si, P
and S, containing attachment sites for at least p.sup.[2] (for
Y.sup.[3]) or p.sup.[2]/2 (for Y.sup.[4] and Y.sup.[5], where
p.sup.[2]/2 is an integer) functional groups on alkyl, alkenyl, or
aromatic carbon atoms;
[0109] in one embodiment, each of the n.sup.[2] moieties shown as
W.sup.[3], if present, is independently chosen from the group
(CH.sub.2).sub.r, (CH.sub.2CH.sub.2O).sub.r,
NR.sup.SUB(CH.sub.2CH.sub.2O- ).sub.rCH.sub.2CH.sub.2, and
NR.sup.SUB(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)- , wherein r=1 to
10;
[0110] in one embodiment, each of the n.sup.[2] moieties shown as
Y.sup.[3] is independently linear, branched, or cyclic alkyl
(1-20C), aryl (6-20C), or alkaryl (7-30C); most preferably, each of
the n.sup.[2] moieties shown as Y.sup.[3] is independently chosen
from the group C.sub.6H.sub.4 (phenyl-1,4-diradical),
C.sub.6H.sub.3 (phenyl-1,3,5-triradical), and (CH.sub.2).sub.r
wherein r=1 to 10;
[0111] for example, each of the n.sup.[2] moieties shown as
W.sup.[4], if present, is independently chosen from the group
(CH.sub.2).sub.rC(.dbd.O) and (CH.sub.2).sub.rNR.sup.SUBC(.dbd.O),
wherein r=1 to 10;
[0112] for example, each of the 2 x n.sup.[2] moieties shown as
Y.sup.[4], is independently chosen from the group (CH.sub.2).sub.r,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2).sub.q,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O)(CH-
.sub.2).sub.r,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUBC(CH.sub.2).sub.qNR.sup.-
SUBC(.dbd.O) (CH.sub.2).sub.r,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.-
2CH.sub.2O).sub.qCH.sub.2CH.sub.2, and
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB-
(CH.sub.2CH.sub.2O).sub.qCH.sub.2CH.sub.2, wherein r=1 to 10, more
preferably r=2 to 6, and q=1 to 10, more preferably q=1 to 3;
[0113] in one embodiment, each of the n.sup.[2] moieties shown as
W.sup.[5], if present, is independently chosen from the group
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB and
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.- O)NR.sup.SUB, wherein r=1 to
10;
[0114] in one embodiment, each of the 2 x n.sup.[2] moieties shown
as Y.sup.[5], is independently chosen from the group
(CH.sub.2).sub.r and
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q, wherein r=1 to
10 and q=1 to 10.
[0115] In a further embodiment, a conjugate comprises a
chemically-defined, non-polymeric valency platform molecule and a
biologically active molecule is provided. The biologically active
molecule may be coupled to a linker molecule before being coupled
to a valency platform molecule.
[0116] Exemplary of suitable linker molecules are 6 carbon thiols
such as HAD, a thio-6 carbon chain phosphate, and HAD.sub.pS, a
thio-6 carbon chain phosphorothioate. Chemically-defined valency
platform molecules are formed, for example, by reacting amino
modified-PEG with 3,5-bis-(iodoacetamido) benzoyl chloride
(hereinafter .differential.DABA");
3-carboxypropionamide-N,N-bis-[(6'-N'-carbobenzylox-
yanino-hexyl)acetamide]4"-nitrophenyl ester (hereinafter "BAHA");
3-carboxypropionamide-N,N-bis-[(8'-N'-carbobenzyloxyamino-3',6'-dioxaocty-
l)acetamide]4"-nitrophenyl ester (hereinafter "BAHA.sup.ox"); or by
reacting PEG-bis-chloroformate with
N,N-di(2-[6'-N'-carbobenzyloxyamino-h- exanoamido]ethyl)amine
(hereinafter "AHAB") to form chemically-defined valency platform
molecules.
[0117] Also provided are chemically-defined, non-polymeric valency
platform molecules of the formulae: 3 G [ 6 ] { O -- C ( -- -- O )
--NR SUB --Q [ 6 ] ( T [ 6 ] ) p [ 6 ] } n6 Formula 6 a G [ 7 ] { O
-- C ( -- -- O ) --N ( Q [ 7 ] ( T [ 7 ] ) p [ 7 ] / 2 ) 2 } n [ 7
] Formula 7 a
[0118] wherein
[0119] each of G.sup.[6] and G.sup.[7], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000, or 10,000 or more, chain atoms selected from the
group C, N, O, Si, P and S; more preferably, each of G.sup.[6] and
G.sup.[7] is a radical derived from a polyalcohol, a polyamine, or
a polyglycol; most preferably, each of G.sup.[6] and G.sup.[7] is
selected from the group --(CH.sub.2).sub.q-- wherein q=0 to 20,
--CH.sub.2(CH.sub.2OCH.sub.2).sub.rCH.sub.2--, wherein r=0 to 300,
and C(CH.sub.2OCH.sub.2CH.sub.2--).sub.s(OH).sub.4-s, wherein s=1
to 4, more preferably s=3 to 4;
[0120] each of the n.sup.[6] x p.sup.[6] moieties shown as
T.sub.[6] and each of the n.sup.[7] x p.sup.[7] moieties shown as
T.sup.[7] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB
(hydrazine), C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER
(activated ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride),
C(.dbd.O)X (acid halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.SUB)NHR.sup.SUB (carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CHCHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsatur- ated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
sulfone);
[0121] more preferably, each of the n.sup.[6], p.sup.[6] moieties
shown as T.sup.[6] and each of the n.sup.[7] x p.sup.[7] moieties
shown as T.sup.[7] is independently chosen from the group
NHR.sup.SUB (amine), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R2 is --C(.dbd.O)CHCHC(.dbd.O)--
(maleimide), C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated carbonyl), R.sup.ALK--Hg--X (alkyl
mercurial), and S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone);
[0122] even more preferably each of the n.sup.[6] x p.sup.[6]
moieties shown as T.sup.[6] and each of the n.sup.[7] x p.sup.[7]
moieties shown as T.sup.[7] is independently chosen from the group
NHR.sup.SUB (amine), C(.dbd.O)CH.sub.2X (haloacetyl),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide), and
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl);
[0123] most preferably, all of the n.sup.[6] x p.sup.[6] moieties
shown as T.sup.[6] and all of the n.sup.[7] x p.sup.[7] moieties
shown as T.sup.[7] are identical;
[0124] wherein
[0125] each X is independently a halogen of atomic number greater
than 16 and less than 54 or other good leaving group;
[0126] each R.sup.ALK is independently a linear, branched, or
cyclic alkyl (1-20C) group;
[0127] each R.sup.SUB is independently H, linear, branched, or
cyclic alkyl (1-20C), aryl (1-20C), or alkaryl (1-30C);
[0128] each R.sup.ESTER is independently N-hydroxysuccinimidyl,
p-nitrophenoxy, pentafluorophenoxy, or other activating group;
[0129] each R.sup.B is independently a radical comprising 1-50
atoms selected from the group C,H,N, O, Si, P and S;
[0130] n.sup.[6]=1 to 32, more preferably n.sup.[6]=1 to 16, even
more preferably n.sup.[6]=1 to 8, yet more preferably n.sup.[6]=1
to 4, most preferably n.sup.[6]=1 to 2;
[0131] p.sup.[6]=1 to 8, more preferably p.sup.[6]=1 to 4, most
preferably p.sup.[6]=1 to 2;
[0132] with the proviso that the product n.sup.[6] x p.sup.[6] be
greater than 1 and less than 33;
[0133] n.sup.[7]=1 to 32, more preferably n.sup.[7]=1 to 16, even
more preferably n.sup.[7]=1 to 8, yet more preferably n.sup.[7]=1
to 4, most preferably n.sup.[7]=1 to 2;
[0134] p.sup.[7]=1 to 8, more preferably p.sup.[7]=1 to 4, most
preferably p.sup.[7]=1 to 2;
[0135] with the proviso that the product n.sup.[7] x p.sup.[7] be
greater than 1 and less than 33;
[0136] each of the n.sup.[6] moieties shown as Q.sup.[6] and each
of the 2x' n.sup.[7] moieties shown as Q.sup.[7] is independently a
radical comprising 1-100 atoms selected from the group C, H, N, O,
Si, P and S, containing attachment sites for at least p.sup.[6])
(for Q.sup.[6]) or p.sup.[7]/2 (for Q.sup.[7], where p.sup.[7]/2 is
an integer) functional groups on alkyl, alkenyl, or aromatic carbon
atoms;
[0137] more preferably, all of the n.sup.6] moieties shown as
Q.sup.[6] are identical;
[0138] more preferably, all of the 2 x n.sup.[7] moieties shown as
Q.sup.[7] are identical;
[0139] more preferably, each of the n.sup.[6] moieties shown as
Q.sup.[6], is independently chosen from the group
CH[(CH.sub.2).sub.r(attachment site)].sub.2 and
CH[(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q(a- ttachment
site)].sub.2, wherein r=1 to 10 and q=1 to 10;
[0140] more preferably, each of the 2 x n.sup.[7] moieties shown as
Q.sup.[7], is independently chosen from the group (CH.sub.2).sub.r,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2).sub.q,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O)(CH-
.sub.2).sub.r,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2CH.sub.2O).sub.-
qCH.sub.2CH.sub.2, and
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2CH.sub.-
2O).sub.qCH.sub.2CH.sub.2, wherein r=1 to 10, more preferably r=2
to 6, and q=1 to 10, more preferably q=1 to 3.
[0141] These chemically defined platform molecules, as described in
U.S. Pat. No. 5,552,391 can be further modified as described herein
to include high molecular weight polyethylene oxide groups.
[0142] Aminooxy Valency Platform Molecules
[0143] In one embodiment, the valency platform molecules comprise
one or more high molecular weight polyethylene oxide groups, as
well as aminooxy groups, for example, 1 to 100, e.g, 1-50, 2-16,
4-16, or e.g., 2, 3, 4, 8, 16, 32 or more aminooxy groups. In one
embodiment, the valency platform molecule has at least 2, 3, 4, 5
or more aminooxy groups. Aminooxy valency platform molecules are
described in U.S. Provisional Patent Application No. 60/138,260,
filed Jun. 8, 1999.
[0144] Also provided are conjugates thereof with other molecules
such as biologically active molecules, and methods for their
synthesis. The aminooxy groups provide attachment sites for the
covalent attachment of other molecules. The molecule may comprise,
e.g., at least 3 aminooxy groups, or 4, 5 or more aminooxy
groups.
[0145] In one embodiment, there is provided a valency platform
molecule having the formula:
R--(ONH.sub.2).sub.m Formula 1b
[0146] wherein in one embodiment:
[0147] m is 1-50 or more, e.g., 3-50; and
[0148] R is an organic moiety comprising 1-10,000 atoms or more
selected from the group consisting of H, C, N, O, P, Si and S
atoms; and
[0149] wherein the valency platform molecule comprises at least one
high molecular weight polyethylene oxide group, for example having
the formula --(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater
than 500, for example 500 to 700, or 600 to 800, or 1000, or
more.
[0150] In another embodiment, there is provided a valency platform
molecule having the formula:
R.sup.c[G.sub.1(ONH.sub.2).sub.n].sub.y; Formula 2b
[0151] wherein in one embodiment:
[0152] y is 1 to 16;
[0153] n is 1 to 32;
[0154] wherein in one embodiment the product of y*n (y multiplied
by n) is at least 3;
[0155] R.sup.c and each G.sub.1 are independently an organic
moiety; and
[0156] wherein the valency platform molecule comprises at least one
high molecular weight polyethylene oxide group, for example having
the formula --(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater
than 500, for example 500 to 700, or 600 to 800, or 1000, or
more.
[0157] In one embodiment, R.sup.c and each G.sub.1 are
independently an organic moiety comprising atoms selected from the
group of H, C, N, O, P, Si and S atoms. The molecules may be
provided for example in a composition having a polydispersity less
than 1.2.
[0158] In another embodiment, a valency platform molecule is
provided having a formula selected from the group consisting
of:
R.sup.c[O--C(.dbd.O)--NR.sup.1-G.sub.2-(ONH.sub.2).sub.n].sub.y
Formula 3b;
R.sup.c[C(.dbd.O)--NR.sup.1-G.sub.2-(ONH.sub.2).sub.n].sub.y
Formula 4b;
R.sup.c[NR.sup.1--C(.dbd.O)-G.sub.2-(ONH.sub.2).sub.n].sub.y
Formula 5b;
R.sup.c[NR.sup.1--C(.dbd.O)--O-G.sub.2-(ONH.sub.2).sub.n].sub.y
Formula 6b;
R.sup.c[R.sup.1C.dbd.N--O-G.sub.2-(ONH.sub.2).sub.n].sub.y Formula
7b; and
R.sup.c[S-G.sub.2(ONH.sub.2).sub.n].sub.y Formula 8b;
[0159] wherein, for example:
[0160] y is 1 to 16;
[0161] n is 1 to 32;
[0162] wherein in one embodiment the product of y*n (y multiplied
by n) is at least 3;
[0163] R.sup.1 is H, alkyl, heteroalkyl, aryl, heteroaryl or
G.sub.2-(ONH.sub.2).sub.n; and
[0164] R.sup.c and each G.sub.2 are independently organic moieties
comprising atoms selected from the group of H, C, N, O, P, Si and S
atoms; and
[0165] wherein the valency platform molecule comprises at least one
high molecular weight polyethylene oxide group, for example having
the formula --(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater
than 500, for example 500 to 700, or 600 to 800, or 1000, or
more.
[0166] In one embodiment, R.sup.C and each G.sub.2 independently
are selected from the group consisting of:
[0167] hydrocarbyl groups consisting only of H and C atoms and
having 1 to 5,000 or 1 to 200 carbon atoms;
[0168] organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having 1-5,000, or 1 to 200 carbon atoms;
[0169] organic groups consisting only of carbon, oxygen, nitrogen,
and hydrogen atoms, and having from 1 to 5,000, or 1 to 200 carbon
atoms;
[0170] organic groups consisting only of carbon, oxygen, sulfur,
and hydrogen atoms, and having from 1 to 5,000, or 1 to 200 carbon
atoms;
[0171] organic groups consisting only of carbon, oxygen, sulfur,
nitrogen and hydrogen atoms and having from 1 to 5,000 or 1 to 200
carbon atoms.
[0172] In one embodiment of the valency platform molecule, R.sup.c
is selected from the group consisting of a C1-200 hydrocarbon
moiety; a C1-200 alkoxy moiety; and a C1-200 hydrocarbon moiety
comprising an aromatic group.
[0173] R.sup.c optionally may comprise an oxyalkylene moiety, such
as an oxyethylene moiety (--CH.sub.2CH.sub.2O--). In one embodiment
R.sup.c comprises oxyethylene units:
--(CH.sub.2CH.sub.2O).sub.n--;
[0174] wherein n is 1-500, e.g., 1-200, 1-100 or 1-20. Optionally n
is greater than 500, for example, 500-700, or 600-800 or 1000 or
more.
[0175] As used herein "oxyethylene, oxypropylene and oxyalkylene"
are used interchangably with "ethylene oxide, propylene oxide and
alkylene oxide".
[0176] In one embodiment, each G.sub.2 independently comprises a
functional group selected from the group consisting of alkyl,
heteroalkyl, aryl, and heteroaryl.
[0177] In another embodiment, each G.sub.2 independently comprises
a functional group selected from the group consisting of a C1-200
hydrocarbon moiety; a C1-200 alkoxy moiety; and a C1-200
hydrocarbon moiety comprising an aromatic group.
[0178] Each G.sub.2 independently can comprise an oxyalkylene
moiety, such as an oxyethylene moiety (--CH.sub.2CH.sub.2O--). In
one embodiment, each G.sub.2 independently comprises oxyethylene
units:
--(CH.sub.2CH.sub.2O).sub.n--;
[0179] wherein n is 1-500, e.g., 1-200, 1-100 or 1-20. Optionally n
is greater than 500, for example, 500-700, or 600-800 or 1000 or
more.
[0180] In one embodiment of the valency platform molecule each
G.sub.2 independently comprises a functional group selected from
the group consisting of amine; amide; ester; ether; ketone;
aldehyde; carbamate; thioether; piperazinyl; piperidinyl; alcohol;
polyamine; polyether; hydrazide; hydrazine; carboxylic acid;
anhydride; halo; sulfonyl; sulfonate; sulfone; cyanate; isocyanate;
isothiocyanate; formate; carbodiimide; thiol; oxime; imine;
aminooxy; and maleimide.
[0181] In another embodiment, compounds of Formulas 9-13 shown in
FIG. 8 are provided. In Formulas 9-13, in one embodiment, R.sub.c
and G.sub.2 are as defined above, and n is about 1-500, e.g.,
1-200, 1-100 or 1-50. Optionally n is greater than 500, for
example, 500-700, or 600-800 or 1000 or more.
[0182] In one embodiment, in Formulas 3b-8b and 9-13, at least one
of R.sub.c and G.sub.2 comprises at least one high molecular weight
polyethylene oxide group, for example having the formula
--(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater than 500, for
example 500 to 700, or 600 to 800, or 1000, or more.
[0183] Valency Platform Molecules Comprising Carbamate Linkages
[0184] In another embodiment, valency platform molecules comprising
carbamate linkages, as described in PCT US99/29338 and U.S. Ser.
No. 09/47,607, filed Dec. 8, 1999 can be modified to include high
molecular weight polyethylene oxide.
[0185] In one embodiment, the valency platform compounds comprise a
carbamate linkage, for example having the structure shown in
Formulae Ic, IIc, and IIIc, IVc, Vc, and VIc, wherein the valency
platform compound further comprises at least one high molecular
weight polyethylene oxide group: 1
[0186] wherein
[0187] n is a positive integer from 1 to 10;
[0188] y.sup.1, Y.sup.2, and y.sup.3 are independently 1 or 2;
[0189] J independently denotes either an oxygen atom or a covalent
bond;
[0190] R.sup.C is selected from the group consisting of:
[0191] hydrocarbyl groups having from 1 to 20 carbon atoms;
[0192] organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms;
[0193] organic groups consisting only of carbon, oxygen, nitrogen,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0194] organic groups consisting only of carbon, oxygen, sulfur,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0195] each G.sup.1, G.sup.2, and G.sup.3 is independently selected
from the group consisting of:
[0196] hydrocarbyl groups having from 1 to 20 carbon atoms;
[0197] organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms;
[0198] organic groups consisting only of carbon, oxygen, nitrogen,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0199] each R.sup.N is independently selected from the group
consisting of:
[0200] hydrogen;
[0201] linear or branched alkyl groups having from 1 to 15 carbon
atoms;
[0202] alkyl groups comprising an alicyclic structure and having
from 1 to 15 carbon atoms;
[0203] aromatic groups having from 6 to 20 carbon atoms;
[0204] heteroaromatic groups having from 3 to 20 carbon atoms;
[0205] each Z is independently selected from the group consisting
of:
[0206] --H
[0207] --C(.dbd.O)OR.sup.CARB
[0208] --C(.dbd.O)R.sup.ESTER
[0209] --C(.dbd.O)NR.sup.AR.sup.B
[0210] wherein:
[0211] each R.sup.CARB is organic groups comprising from 1 to about
20 carbon atoms;
[0212] each R.sup.ESTER is organic groups comprising from 1 to
about 20 carbon atoms;
[0213] each group --NR.sup.AR.sup.B is independently selected from
the group consisting of:
[0214] --NH.sub.2
[0215] --NHR.sup.A
[0216] --NR.sup.AR.sup.B
[0217] --NR.sup.AB
[0218] wherein each monovalent R.sup.A and R.sup.B and each
divalent R.sup.AB is independently an organic group comprising from
1 to 20 carbon atoms, and further comprising a reactive conjugating
functional group; and
[0219] wherein the valency platform molecule comprises at least one
high molecular weight polyethylene oxide group, for example having
the formula --(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater
than 500, for example 500 to 700, or 600 to 800, or 1000, or
more.
[0220] In one embodiment, said compound has the structure of
Formula Ic. In one embodiment, said compound has the structure of
Formula IIc. In one embodiment, said compound has the structure of
Formula IIIc. In one embodiment, said compound has the structure of
Formula IVc. In one embodiment, n is a positive integer from 2 to
4. In one embodiment, y.sup.1, y.sup.2, and y.sup.3 are each 2. In
one embodiment, J is an oxygen atom. In one embodiment, J is a
covalent bond. In one embodiment, R.sup.C is selected from the
group consisting of hydrocarbyl groups having from 1 to 20 carbon
atoms. In one embodiment, R.sup.C is selected from the group
consisting of: 2
[0221] In one embodiment, R.sup.C is selected from the group
consisting of organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms. In one
embodiment, R.sup.C is: 3
[0222] wherein p is a positive integer from 2 to 20. In one
embodiment, each G.sup.1, G.sup.2, and G.sup.3 is independently
selected from the group consisting of hydrocarbyl groups having
from 1 to 20 carbon atoms. In one embodiment, each G.sup.1,
G.sup.2, and G.sup.3is --(CH.sub.2).sub.q-- wherein q is a positive
integer from 1 to 20. In one embodiment, each G.sup.1, G.sup.2, and
G.sup.3 is independently selected from the group consisting of
organic groups consisting only of carbon, oxygen, and hydrogen
atoms, and having from 1 to 20 carbon atoms. In one embodiment,
each G.sup.1, G.sup.2, and G.sup.3 is: 4
[0223] wherein p is a positive integer from 2 to 20. In one
embodiment, R.sup.N is independently selected from the group
consisting of --H, --CH.sub.3, and --CH.sub.2CH.sub.3. In one
embodiment, each group --NR.sup.AR.sup.B is independently selected
from the group consisting of: 5
[0224] Another aspect of the present invention pertains to a
valency platform compound having the structure of one of the
following formulae: 6
[0225] wherein:
[0226] n is a positive integer from 1 to 10;
[0227] y.sup.1, y.sup.2, and y.sup.3 are independently a positive
integer from 1 to 10;
[0228] J independently denotes either an oxygen atom or a covalent
bond;
[0229] R.sup.C is selected from the group consisting of:
[0230] hydrocarbyl groups having from 1 to 20 carbon atoms;
[0231] organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms;
[0232] organic groups consisting only of carbon, oxygen, nitrogen,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0233] organic groups consisting only of carbon, oxygen, sulfur,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0234] each G.sup.1, G.sup.2, and G.sup.3 is independently selected
from the group consisting of:
[0235] hydrocarbyl groups having from 1 to 20 carbon atoms;
[0236] organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms;
[0237] organic groups consisting only of carbon, oxygen, nitrogen,
and hydrogen atoms, and having from 1 to 20 carbon atoms;
[0238] each R.sup.N is independently selected from the group
consisting of:
[0239] hydrogen;
[0240] linear or branched alkyl groups having from 1 to 15 carbon
atoms;
[0241] alkyl groups comprising an alicyclic structure and having
from 1 to 15 carbon atoms;
[0242] aromatic groups having from 6 to 20 carbon atoms;
[0243] heteroaromatic groups having from 3 to 20 carbon atoms;
[0244] each Z is independently selected from the group consisting
of:
[0245] --H
[0246] --C(.dbd.O)OR.sup.CARB
[0247] --C(.dbd.O)R.sup.ESTER
[0248] --C(.dbd.O)NR.sup.AR.sup.B
[0249] wherein:
[0250] each R.sup.CARB is organic groups comprising from 1 to about
20 carbon atoms;
[0251] each R.sup.ESTER is organic groups comprising from 1 to
about 20 carbon atoms;
[0252] each group --NR.sup.AR.sup.B is independently selected from
the group consisting of:
[0253] --NH.sub.2
[0254] --NHR.sup.A
[0255] --NR.sup.AR.sup.B
[0256] --NR.sup.AB
[0257] wherein each monovalent R.sup.A and R.sup.B and each
divalent R.sup.AB is independently an organic group comprising from
1 to 20 carbon atoms, and further comprising a reactive conjugating
functional group; and
[0258] wherein the valency platform molecule comprises at least one
high molecular weight polyethylene oxide group, for example having
the formula --(CH.sub.2CH.sub.2)).sub.n--, wherein n is greater
than 500, for example 500 to 700, or 600 to 800, or 1000, or
more.
[0259] In one embodiment, said compound has the structure of
Formula IVc. In one embodiment, said compound has the structure of
Formula Vc. In one embodiment, said compound has the structure of
Formula VIc. In one embodiment, n is a positive integer from 2 to
4. In one embodiment, y.sup.1, y.sup.2, and y.sup.3 are each 2. In
one embodiment, J is an oxygen atom. In one embodiment, J is a
covalent bond. In one embodiment, R.sup.C is selected from the
group consisting of hydrocarbyl groups having from 1 to 20 carbon
atoms. In one embodiment, R.sup.C is selected from the group
consisting of:
[0260] --CH.sub.2--;
[0261] --CH.sub.2CH.sub.2--;
[0262] --CH.sub.2CH.sub.2CH.sub.2--; 7
[0263] In one embodiment, R.sup.C is selected from the group
consisting of organic groups consisting only of carbon, oxygen, and
hydrogen atoms, and having from 1 to 20 carbon atoms. In one
embodiment, R.sup.C is: 8
[0264] wherein p is a positive integer from 2 to 20. In one
embodiment, each G.sup.1, G.sup.2, and G.sup.3 is independently
selected from the group consisting of hydrocarbyl groups having
from 1 to 20 carbon atoms. In one embodiment, each G.sup.1,
G.sup.2, and G.sup.3 is selected from the group consisting of:
9
[0265] In one embodiment, each G.sup.1, G.sup.2, and G.sup.3 is
independently selected from the group consisting of organic groups
consisting only of carbon, oxygen, and hydrogen atoms, and having
from 1 to 20 carbon atoms. In one embodiment, each R.sup.N is
independently selected from the group consisting of --H,
--CH.sub.3, and --CH.sub.2CH.sub.3. In one embodiment, each group
--NR.sup.AR.sup.B is independently selected from the group
consisting of: 10
[0266] In Formulas Ic, IIc, IIIc, IVc, Vc, and VIc, in one
embodiment at least one of R.sup.c, G.sup.1, G.sup.2 and G.sup.3
comprise at least one high molecular weight polyethylene oxide
group, for example having the formula
--(CH.sub.2CH.sub.2O).sub.n--, wherein n is greater than 500, for
example 500 to 700, or 600 to 800, or 1000, or more.
[0267] Preparation of Valency Platform Molecules
[0268] Methods known in the art to make valency platform molecules
may be modified to permit the incorporation of high molecular
weight polyethylene oxides in the molecule. Methods for making
valency platform molecules are described, for example, in U.S. Pat.
Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512; 5,726,329;
5,268,454; 5,552,391; 5,606,047; 5,663,395; and 5,874,409; as well
as U.S. Ser. No. 60/111,641; U.S. Ser. No. 09/457,607; PCT WO
00/34231; PCT US97/10075; U.S. Ser. No. 09/590,592; and
PCT/US/00/15968.
[0269] Methods known the art for making valency platform molecules,
include, for example, a propagation method, or segmental approach.
Such methods can be modified, using the appropriate reagents, to
provide polyethylene oxide groups on the resulting molecule.
Exemplary methods are demonstrated in the Examples herein.
[0270] The valency platforms can be prepared from a segmental
approach in which segments are independently synthesized and
subsequently attached to a core group. An alternative to the core
propagation process is an iterative process that may be used to
generate a dendritic structure.
[0271] Examples of core compounds include alcohol containing core
compounds methanol, ethanol, propanol, isopropanol, and
methoxypolyethylene glycol, monohydroxylamines, ethylene glycol,
diethylene glycol, triethylene glycol, tetraethylene glycol,
pentaethylene glycol, hexaethylene glycol,
1,4-bis-(hydroxymethyl)benzene and polyethylene glycol
HO(CH.sub.2CH.sub.2O).sub.nH, wherein, for example, n is about
1-200, e.g., 1-10, or 1 to 5, or primary or secondary amines having
two hydroxyl groups. Core compounds also may be high molecular
weight polyethylene oxide molecules as disclosed herein.
[0272] The following are exemplary syntheses of valency platform
molecules which include high molecular weight polyethylene oxide
groups, and conjugates thereof. These examples involve the
preparation of a polyethylene oxide-containing platform
intermediate with amino groups at the temini. These amino groups
are derivatized with appropriate reactive groups for conjugation
with biologically active molecules.
[0273] FIG. 1 shows how a tetrameric platform is synthesized with
the terminal amino groups protected with Cbz and an Fmoc-protected
amino group in the core. Compound 1 is obtained as described in
U.S. Pat. No. 5,552,391. The Fmoc group is removed and replaced
with a methoxy-PEG chain. Removal of the Cbz groups yields the free
tetra-amine, compound 6, which is converted to the
tetra-chloroacetyl derivative, 7. Compound 7 can be reacted with
any thiol containing molecule to form a tetavalent conjugate
compound 8. In FIG. 1, n is, for example, greater than 500, e.g.,
500-800 or 500-1000.
[0274] FIG. 2 shows how the tetra-amine, 6, can be modified with
serine to give compound 9. The serines of compound 9 undergo
oxidative cleavage with periodate to provide terminal aldehyde
groups which can react with biologically active molecules
containing aminooxy groups, or other groups which form stable
imines (hydrazides, semicarbazides, carbazides, etc.), to form
oxime conjugates represented by 11. In FIG. 2, n is, for example,
greater than 500, e.g., 500-800 or 500-1000.
[0275] FIG. 3 demonstrates how a high molecular weight PEG platform
can be prepared with PEG placed internally, as part of the core of
the platform. In this embodiment, compound 14 is prepared by
reacting two equivalents of compound 13 (prepared as described in
U.S. Pat. No. 5,552,391) with one equivalent of compound 12
(PEG.sub.20K-bis-BTC, Shearwater Polymers). The Cbz-protecting
groups are removed by hydrogenation or acidolysis to provide the
tetra-amine, compound 15. Chloroacetylation with chloroacetic
anhydride yields the chloroacetylated platform, compound 16.
Compound 16 is treated with a thiol containing biologically active
molecule to provide 17, a tetravalent platform conjugate of the
biologically active molecule. In FIG. 3, n is, for example, greater
than 500, e.g., 500-800 or 500-1000.
[0276] FIG. 4 demonstrates how a high molecular weight PEG platform
can be prepared with PEG placed internally in the arms of the
platform. "PEG" or "polyethylene glycol" or "polyethylene oxide"
are used interchangably herein to refer to polymers of ethylene
oxide. The Boc-protecting groups are removed from compound 20 with
TFA, and the resulting tetra-amine, compound 21, is
chloroacetylated with chloroacetic anhydride to yield the
chloroacetylated platform, compound 22. Compound 22 is treated with
a thiol containing biologically active molecule to provide 23 a
tetravalent platform conjugate of the biologically active molecule.
In FIG. 4, n is, for example, greater than 500, e.g., 500-800 or
500-1000.
[0277] As shown in FIG. 14, a bPEG 8-mer platform, M is synthesized
by a process wherein a octameric PNP carbonate ester (compound 50a)
is reacted with compound 133 resulting in the formation of compound
K. The Boc-protecting groups are removed from compound K, and the
resulting octa-amine is treated with compound 106 resulting in the
formation of compound L. Removal of the Boc-protecting groups from
compound M results in the formation of compound M.
[0278] A tetravalent aminooxy platform with two PEG chains attached
is synthesized as shown in FIG. 15 from intermediate 122 which has
two PEG chains attached. Thus compound 122 is reacted with NHS
ester O (Shearwater Polymers) to form platform P.
[0279] Conjugates, Methods of Preparation, and Uses Thereof
[0280] The term "biologically active molecule" is used herein to
refer to molecules which have biological activity, preferably in
vivo. For example, a biological activity includes binding to a
target. In one embodiment, the biologically active molecule is one
which interacts specifically with receptor proteins. In another
embodiment, the biologically active molecule binds to an antibody
which, if used in vivo, may be circulating or on a cell surface,
such as a B cell surface. Biologically active molecules include one
or more nucleic acids of any length (polynucleotides) including
oligonucleotides; peptides; polypeptides; proteins; antibodies of
any type (such as monoclonal, polyclonal, and anti-idiotype)
including fragments thereof; saccharides; polysaccharides;
epitopes; mimotopes; enzymes (including domains thereof); hormones;
drugs; lipids; fatty acids; and mixtures thereof.
[0281] Depending on the valency of the platform, the platform
molecule conjugate may include, for example, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12 or more biologically active molecules, or e.g.,
16, 18, 32, 36 or more.
[0282] The conjugates of biologically active molecules and valency
platform molecules comprising high molecular weight polyethylene
oxide may be used as toleragens (which cause reduction and/or
stabilization of the extent of an immune response to an immunogen)
and may be used in a variety of applications for lowering levels of
an antibody associated with disease and thus the treatment of
antibody mediated diseases. Examples of conjugates for treating
lupus, which are useful as B cell toleragens, are shown in FIG. 5.
In these embodiments, the biologically active molecule generally
comprises a nucleic acid which specifically binds to an anti-double
stranded DNA antibody, as described, for example, in U.S. Pat. No.
5,552,391. Examples of conjugates for use in xenotransplantation,
which are useful as B cell toleragens, are shown in FIG. 6 and in
PCT WO 00/34296. Alternatively the conjugates disclosed herein can
include other biologically active molecules such as active domain 1
.beta..sub.2 GPI polypeptide sequences.
[0283] As used herein "an effective amount for treatment" refers to
an amount sufficient to palliate, ameliorate, stabilize, reverse,
slow or delay progression of a condition such as a disease
state.
[0284] In one embodiment, the biologically active molecule
comprises a domain 1 polypeptide of .beta..sub.2GPI or analog
thereof, as described, e.g., in U.S. Ser. No. 60/103,088, U.S. Ser.
No. 09/328,199, filed Jun. 8, 1999 and PCT WO 99/64595. Exemplary
structures of conjugates are shown in FIG. 7. In one embodiment,
the polypeptide does not include domain 4 of .beta..sub.2GPI.
[0285] The domain 1 .beta..sub.2GPI polypeptide contains at least
five (or more) contiguous amino acids of FIG. 19 (SEQ ID NO:2),
which depicts domain 1. In one embodiment, the domain 1
.beta..sub.2GPI polypeptide consists of (or, in some embodiments,
consisting essentially of) the amino acid sequence shown in FIG. 19
(SEQ ID NO:2), which represents domain 1.
[0286] The domain 1 .beta..sub.2GPI polypeptide preferably
specifically binds to a .beta..sub.2GPI-dependent antiphospholipid
antibody. In some embodiments, the polypeptide comprises fragments
of domain 1. In other embodiments, the polypeptide comprises a
conformational epitope. In yet other embodiments, the polypeptide
consists of domain 1. There further is provided a polypeptide
comprising a domain 1 .beta..sub.2GPI polypeptide, wherein the
polypeptide lacks a (detectable) T cell epitope capable of
activating T cells in an individual having .beta..sub.2GPI
dependent antiphospholipid antibodies.
[0287] The domain 1 .beta..sub.2GPI polypeptide may, for example,
range from (a) about the first cysteine to about the fourth
cysteine (when determined from the N-terminus); (b) about the N
terminus to about the fifth cysteine (more precisely, the last
amino acid before the fifth cysteine); (c) about the first cysteine
to about the fifth cysteine. In some embodiments, an additional
cysteine may be added in any suitable position, to serve as a
reactive group for conjugation. Accordingly, an additional cysteine
(which in some embodiments is the fifth cysteine of
.beta..sub.2GPI) may be included in any position, particularly near
or at the C terminus or N terminus. A domain 1 .beta..sub.2GPI
polypeptide may also comprise (or consist of, or consist
essentially of) any of the following: (a) amino acid 1 to amino
acid 59 of SEQ ID NO:2; (b) amino acid 2 to amino acid 60 of SEQ ID
NO:2; (c) amino acid 2 to amino acid 63 of SEQ ID NO:2; (d) about
amino acid 1 to about amino acid 60 of SEQ ID NO:2; (e) amino acid
1 to amino acid 61 of SEQ ID NO:2; and (f) amino acid 1 to amino
acid 62 of SEQ ID NO:2. Domain 2 .beta..sub.2GPI polypeptides which
contain the fifth cysteine are particularly convenient for
conjugation.
[0288] The domain 1 .beta..sub.2GPIpolypeptide specifically binds
to a .beta..sub.2GPI-dependent antiphospholipid antibody. The
domain 1 .beta..sub.2GPI polypeptide need only bind to one
.beta..sub.2GPI-depende- nt antiphospholipid antibody, although it
may be desirable (for example, in the detection context), for the
domain 1 .beta..sub.2GPI polypeptide to bind to more than one
.beta..sub.2GPI-dependent antiphospholipid antibody.
[0289] The size of a domain 1 .beta..sub.2GPI polypeptide may vary
widely, as long as the requisite functionality (based on specific
binding to a .beta..sub.2GPI-dependent antiphospholipid antibody is
met. For example, the length sufficient to effect specific binding
to a .beta..sub.2GPI-dependent antiphospholipid antibody could be
as small as, for example, a 5-mer amino acid sequence. In some
embodiments, the domain 1 .beta..sub.2GPI polypeptide is less than
about 350 amino acids in length; less than about 250 amino acids in
length; less than about 150 amino acids in length, less than about
100 amino acids in length; less than about 50 amino acids in
length; less than about 25 amino acids in length; less than about
15 amino acids in length; or less than about 10 amino acids in
length.
[0290] It is also understood that certain sequence variations may
be introduced into a domain 1 .beta..sub.2GPI polypeptide which may
preserve or enhance its reactivity. These variant and modified
sequences are collectively denoted as "functionally equivalent
variants", which may have the same, enhanced, or diminished binding
when compared to another domain 1 .beta..sub.2GPI polypeptide, and
are denoted "equivalent" because they maintain the ability to
specifically bind to a .beta..sub.2GPI-dependent antiphospholipid
antibody.
[0291] The domain 1 conjugates can be used in methods for detection
of a .beta..sub.2GPI-dependent antiphospholipid antibody (or an
antibody that specifically binds to a domain 1 .beta..sub.2GPI
polypeptide) in a sample by contacting antibody in the sample with
the conjugate under conditions that permit the formation of a
stable antigen-antibody complex; and detecting stable complex
formed if any. The conjugates also can be used in methods of
inducing tolerance in an individual which comprise administering an
effective amount of a conjugate to an individual, particularly a
conjugate comprising a domain 1 .beta..sub.2GPI polypeptide(s) that
lacks a T cell epitope, wherein an effective amount is an amount
sufficient to induce tolerance. The compositions may be used, for
example, in the treatment of antibody mediated thrombosis.
[0292] In another embodiment, there is provided a conjugate of a
valency platform molecule and at least one .alpha.Gal epitope or
analog thereof that specifically binds to an anti-.alpha.Gal
antibody. In another aspect, a method of reducing circulating
levels of anti-.alpha.Gal antibodies in an individual is provided
comprising administering an effective amount of the conjugate to
the individual, wherein an effective amount is an amount sufficient
to reduce the circulating levels of anti-.alpha.Gal antibodies, or
to neutralize circulating levels of anti-.alpha.Gal antibodies. In
another aspect, a method of inducing immunological tolerance
(generally to a xenotransplantation antigen, more specifically to
.alpha.Gal), is provided, the method comprising administering an
effective amount of the conjugate comprising the .alpha.Gal epitope
or analog thereof. The conjugates also can be used to detect the
presence and/or amount of anti-.alpha.Gal antibody in a biological
sample. Methods of performing a xenotransplantation in an
individual also are provided, comprising administering a conjugate
to the individual; and introducing xenotissue to the individual. In
another aspect, methods of suppressing rejection of a transplanted
tissue are provided comprising administering the conjugate to the
individual in an amount sufficient to suppress rejection. These
methods are described generally in PCT US99/29338.
[0293] The conjugates also may be used for immunotolerance
treatment of lupus, optionally based on assessment of initial
affinity of antibody from the individual (i.e., antibody associated
with lupus, namely, anti double stranded DNA antibodies) and used
as a basis for selecting the individual for treatment, or in
methods of identifying individuals suitable (or unsuitable) for
treatment based on assessing antibody affinity. Methods of treating
systemic lupus erythematosus (SLE) in an individual comprise
administering to the individual a conjugate comprising (a) a
non-immunogenic valency platform molecule and (b) two or more
polynucleotides or analogs thereof which specifically bind to an
antibody from the individual which specifically binds to double
stranded DNA. These methods are described generally in PCT
US99/29336.
[0294] Thus, the valency platform may be covalently linked with one
or more biologically active molecules to form a conjugate.
Biologically active molecules include one or more nucleic acids of
any length (polynucleotides) including oligonucleotides; peptides;
polypeptides; proteins; antibodies of any type (such as monoclonal,
polyclonal, and anti-idiotype) including fragments thereof;
saccharides; polysaccharides; epitopes; mimotopes; enzymes
(including domains thereof); hormones; drugs; lipids; fatty acids;
and mixtures thereof.
[0295] The terms "protein", "polypeptide", and "peptide" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
It also may be modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, myristylation,
acetylation, alkylation, phosphorylation or dephosphorylation. Also
included within the definition are polypeptides containing one or
more analogs of an amino acid (including, for example, unnatural
amino acids) as well as other modifications known in the art.
[0296] One advantage of the conjugates of valency platforms is the
ability to introduce enhanced affinity of the tethered biologically
active molecules for their binding partners, for example when the
binding partners are associated in a cluster. The covalent
attachment of plural biological molecules to the valency platform
molecule provides an enhanced local concentration of the
biomolecules as they are associated together for example on the
platform molecule. Another advantage of the valency platforms is
the ability to facilitate binding of multiple ligands, as is useful
in B cell tolerance. For example, the conjugates can be used as
toleragens to present multivalent epitopes to induce clustering on
the surface of a B cell. Thus, for use as a toleragen, it is
preferred that the conjugate includes two or more biologically
active molecules, or epitopes. Another advantage of the valency
platforms is the ability to include functionality on the "core"
that can be independently modified to enable the preparation of
conjugates which can be tailored for specific purposes.
[0297] To form a conjugate, in general a molecule comprising a
reactive group such as an aminooxy group is reacted with a second
molecule comprising a reactive group such as a carbonyl group,
e.g., an aldehyde or ketone, to form, for example, an oxime
conjugate.
[0298] In one embodiment, a method of preparing chemically defined
multivalent conjugates of native polypeptides or proteins with
multivalent preferably non-immunogenic valency platform molecules
comprising aminooxy groups is provided, wherein, if needed, the
polypeptide is selectively modified to generate an aldehyde or
ketone moiety at a specific position on the polypeptide. The
polypeptide then is reacted with the multivalent valency platform
molecule which contains aminooxy groups to form one or more oxime
linkages between the platform and the polypeptide. Amines, for
example at the N-terminus, of virtually any polypeptide or other
molecule can be converted to an aldehyde or a ketone by a reaction
which is known in the art as a transamination reaction.
[0299] Another way to generate an glyoxyl group at the N-terminus
is to oxidize an N-terminal serine or threonine with sodium
periodate. This oxidation cleaves the carbon-carbon bond between
the hydroxyl and amino groups of the N-terminal serine or threonine
providing a glyoxyl group. Thus in one embodiment, polypeptides can
be site specifically modified by forming a ketone or aldehyde at
the N-terminus. Synthetic polypeptides and other drugs or
biologically active molecules can be modified similarly to include
aldehydes or ketones which can be used to form oxime linkages.
[0300] Multivalent platforms containing aminooxy reactive groups
permit covalent attachment of the selectively modified polypeptides
to the platforms. The aminooxy groups may be, e.g., aminooxyacetyl
groups or aminooxyalkyl groups. In one preferred embodiment, the
aminooxy groups in the platform molecule are aminooxyalkyl groups,
such as --CH.sub.2CH.sub.2ONH.sub.2.
[0301] Attachment of biomolecules with aldehyde or ketone
functionality to aminooxy platforms via oxime bond formation can be
implemented. Transaminated polypeptides, or polypeptides otherwise
modified with aldehyde or ketone groups can be reacted with
aminooxy platforms. In one embodiment, transaminated Domain 1 is
attached to tetravalent platforms by treating the platforms with
the glyoxyl-polypeptide in acidic aqueous solution. A preferred
acidic condition is 100 mM pH 4.6 sodium acetate. In the case of
making a tetravalent Domain 1 conjugate, an excess of four
equivalents, for example six equivalents, of transaminated Domain 1
is used.
[0302] The conjugates of FIG. 5 are prepared as described for
compound 20-II in FIG. 6B of U.S. Pat. No. 5,552,391. The
appropriate haloacetylated platform 7, 17, or 23 is treated with
oligonucleotide with thiol linker at the 5'-end
(HS(CH.sub.2).sub.6OPO.sub.3.sup.-(CA).sub.10)- , prepared as
described in Example 5 of U.S. Pat. No. 5,552,391. The resulting
oligonucleotide conjugate is annealed with a complimentary strand
((TG).sub.10) to provide the corresponding tetrameric
double-stranded oligonucleotide conjugate. In FIG. 5, n is, for
example, greater than 500, e.g., 500-800 or 500-1000.
[0303] The conjugates of FIG. 6 are prepared as described for the
conjugates of FIG. 5 using 2-[2-(2-thioethoxy)ethoxy]ethyl
3-O-(.alpha.-D-galactopyranosyl)-.beta.-D-galactopyranoside
(described in PCT US99/29338) instead of an oligonucleotide with
thiol linker at the 5'-end. Thus the appropriate haloacetylated
platform 7, 17, or 23 is treated with
2-[2-(2-thioethoxy)ethoxy]ethyl 3-O-(.alpha.-D-galactopyrano-
syl)-.beta.-D-galactopyranoside to provide the corresponding
.alpha.-1,3-digal conjugate. In FIG. 6, n is, for example, greater
than 500, e.g., 500-800 or 500-1000.
[0304] The disclosures of all publications, patents, patent
applications and published patent applications referred to herein
by an identifying citation are hereby incorporated herein by
reference in their entirety.
[0305] The invention will be further understood by the following
nonlimiting examples.
EXAMPLES
[0306] In the following examples, the following abbreviations are
used: DCC, 1,3-dicyclohexylcarbodiimide; DIC,
1,3-diisopropylcarbodiimide; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; NHS, N-hydroxysuccinimide;
HOBt, 1-hydroxybenzotriazole; DMF, dimethylformamide.
Example 1
Transamination of a Domain 1 Polypeptide
[0307] Synthesis of Transaminated Domain 1 (TA/D1): The synthesis
is shown in FIG. 20. Water and sodium acetate buffer is sparged
with helium before use. A Domain 1 .beta.2GPI polypeptide having
the sequence of amino acids 1 to 63 of SEQ ID NO.:2 shown in FIG.
19 is used, which is described in U.S. Provisional Appl. Ser. No.
60/103,088, filed Jun. 9, 1998; U.S. Ser. No. 09/328,199, filed
Jun. 8, 1999; and PCT WO 99/64595. Domain 1 has an N-terminal
glycine. The Domain 1 polypeptide (10.55 mg, 1.49 .mu.mol) is
dissolved in 0.5 mL of H.sub.2O in a polypropylene tube, and 4.0 mL
of 2 M pH 5.5 NaOAc buffer is added. A solution of 3.73 mg (14.9
.mu.mol) of CuSO.sub.4 in 0.5 mL of H.sub.2O is added to the
mixture, followed by a solution of 2.75 mg (29.9 .mu.mol) of
glyoxylic acid in 0.5 mL of 2 M pH 5.5 NaOAc buffer. The mixture is
kept under nitrogen atmosphere and agitated gently for 18 h at
which time the reaction appears complete by analytical HPLC using a
4.6 mm.times.250 mm, 300 .ANG., 5 .mu.m, diphenyl column (Vydac,
Hesperia, Calif.) with detection at 280 nM (1 mL/min; gradient
25%-45% B, 0-20 min, A=0.1% TFA/H.sub.2O, B=0.1% TFA/CH.sub.3CN).
Approximate retention times are as follows: D1, 13.2 min; TA/D1,
13.7 min; oxidized TA/D1, 13.4 min). The mixture is diluted to a
volume of 20 mL with H.sub.2O, filtered, and purified by HPLC (22.4
mm.times.250 mm, 300 .ANG., 10 .mu.m, diphenyl column (Vydac) (12
mumin; gradient 25%-40% B, 0-40 min, A=0.1% TFA/H.sub.2O, B=0.1%
TFA/CH.sub.3CN). Fractions containing pure TA/D1, as evidenced by
analytical HPLC, are pooled and lyophilized to provide 5.0 mg (48%)
of TA/D1. The reaction converts the N-terminal glycine to an
N-terminal glyoxyl in TA D1, thus the D1 portion has the sequence
of amino acids 2 to 63 of SEQ ID NO.:2 (see FIG. 20).
Example 2
Synthesis of Compound 125
[0308] Compound 115: To a solution of 8.00 g (13.4 mmol) of
compound 13 (prepared as described in U.S. Pat. No. 5,552,391) in
80 mL of anhydrous DMF was added 4.00 g (1 6.1 mmol) of
N-(benzyloxycarbonyloxy)succinimide (Aldrich Chemical Co.). The
mixture was stirred for 2 hours under nitrogen at room temperature,
at which time it was poured into 600 mL of ice water and extracted
with four 100 mL portions of CH.sub.2Cl.sub.2. The combined
CH.sub.2Cl.sub.2 layers were washed with 100 mL of H.sub.2O, dried
(Na.sub.2SO.sub.4), filtered, and concentrated. Concentration from
heptane helped to solidify the crude product. Recrystallization
from EtOAc gave compound 115 as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 1.26 (m, 4H), 1.43-1.62 (m, 8H), 2.05 (m, 4H),
3.16 (q, 4H), 3.40 (brd s, 8H), 4.98 (s, 2H) overlapped with 5.08
(s, 4H) and 5.11 (s, 2H), 6.31 (s, 1H), 6.44 (s, 1H), 7.26-7.38 (m,
15H).
[0309] Synthesis of triamine, compound 116: A solution of 9.0 g
(12.3 mmol) of compound 115 in 18 mL of cyclohexane and 36 mL of
anhydrous ethanol was deoxygenated by bubbling N.sub.2 gas through
it. To the solution was added 1.80 g of 10% Pd/C, and the mixture
was heated at reflux for 3 hours. When cool, the mixture was
filtered through Celite.RTM. using MeOH to rinse. The filtrate was
concentrated, and the concentrate was concentrated from
CH.sub.2Cl.sub.2 to provide 4.20 g (87%) of compound 1 6 as an off
white solid.
[0310] Synthesis of compound 117: To a solution of 5.39 g (21.8
mmol) of compound 105 in 10 mL of anhydrous acetonitrile was added
3.02 g (23.9 mmol) of CDI (carbonyldiimidazole), and the mixture
was stirred for 1.5 hours under nitrogen atmosphere. The resulting
solution was added to a solution of 4.20 g (10.7 mmol) of compound
116 in 15 mL of anhydrous DMF, and the mixture was stirred for 2
hours and poured into 500 mL of ice water. The resulting mixture
was extracted with four 100 mL portions of CH.sub.2Cl.sub.2. The
combined CH.sub.2Cl.sub.2 layers were washed with 100 mL of
H.sub.2O, dried (Na.sub.2SO.sub.4), filtered, and concentrated. The
resulting semisolid residue was crystallized from 10% isopropyl
alcohol/EtOAc to provide 4.0 g (44%) of 117 as a white solid:
.sup.1H NMR CDCl.sub.3 (.delta.) 1.35 (m, 4H), 1.42 (m, 4H), 1.49
(s, 18H), 1.63 (m, 16H), 2.01 (brd s, 1H), 2.20 (t, 4H), 3.23 (m,
4H), 3.34 (m, 4H), 3.85 (t, 4H), 6.34 (t, 2H), 6.70 (t, 2H), 7.98
(brd s, 1H).
[0311] Compound 119: To a solution of 3.65 g (14.11 mmol) of
9-fluorenylmethylchloroformate (Fmoc-Cl) in 15 mL of dioxane was
added a solution of 3.00 g (15.5 mmol) of compound 118 (Bondunov et
al., J. Org. Chem. 1995, Vol. 60, pp. 6097-6102) in 15 mL of
dioxane followed by a solution of 1.95 g (14.11 mmol) of potassium
carbonate in 30 mL of H.sub.2O. The mixture was stirred for 18
hours at room temperature and concentrated. The resulting oil was
partitioned between 50 mL of 1 N NaOH solution and three 150 mL
portions of CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers
were dried (MgSO4), filtered, and concentrated to a yellow oil.
Purification by silica gel chromatography (step gradient; 90/10
EtOAc/AcOH to 90/10/1 EtOAc/AcOH/MeOH) to give 3.85 g (66%) of 119
as a viscous oil: .sup.1H NMR CDCl.sub.3 (.delta.) 3.26 (m, 4H),
3.39 (m, 2H), 3.49 (m, 2H), 3.59 (m, 2H), 3.65 (m, 4H), 3.69 (m,
2H), 4.25 (t, 1H), 4.60 (d, 2H), 7.35 (t, 2H), 7.41 (t, 2H), 7.59
(d, 2H), 7.78 9d, 2H).
[0312] Compound 120: To a solution of 3.77 g (9.08 mmol) of
compound 119 and 7.32 g (36.3 mmol) of 4-nitrophenylchloroformate
in 50 mL of CH.sub.2Cl.sub.2 at 0.degree. C. was added 5.88 mL
(5.75 g, 72.6 mmol) of pyridine. The mixture was stirred at room
temperature under nitrogen atmosphere for 72 hours, and the mixture
was partitioned between 200 mL of CH.sub.2Cl.sub.2 and four 100 ml
portions of 10% aqueous sodium bicarbonate solution. The
CH.sub.2Cl.sub.2 layer was washed successively with 100 mL of
H.sub.2O, 100 mL of 1 N HCl, then 100 mL of brine. The solution was
dried (MgSO.sub.4), filtered, and concentrated to yield an orange
oil. Purification by silica gel chromatography (15/50/35/1
EtOAc/CH.sub.2Cl.sub.2/ hexane/AcOH) to provide 2.67 g (39%) of
compound 120 as a yellow gum: .sup.1H NMR (CDCl.sub.3) .delta. 3.32
(m, 4H), 3.52 (m, 2H). 3.60 (m, 4H), 3.74 (m, 2H), 4.23 (t, 1H),
4.38 (m, 2H), 4.41 (m, 2H), 4.57 (d, 2H), 7.37 (m, 8H), 7.59 (d,
2H), 7.78 (d, 2H), 8.26 (overlapping d, 4H); mass spectrum (ESI)
(M+H) calculated for C.sub.37H.sub.36N.sub.3O.sub.14: 746. Found
746.
[0313] Compound 121: To a solution of 482 mg (0.612 mmol) of
compound 117 in 5 mL of CH.sub.2Cl.sub.2 was added 182 mg (0.245
mmol) of compound 120 followed by 171 .mu.L (124 mg, 1.22 mmol) of
Et.sub.3N and 26 mg (0.490 mmol) of HOBt. The mixture was stirred
at room temperature until the reaction was complete as judged by
TLC (1/9 MeOH/CH.sub.2Cl.sub.2). The mixture was partitioned
between 300 mL of CH.sub.2Cl.sub.2 and three 50 mL portions of 1 N
HCl. The CH.sub.2Cl.sub.2 layer was washed with brine, dried
(MgSO.sub.4), filtered, and concentrated to a yellow oil.
Purification by silica gel chromatography (multiple step gradient;
5/1/94 to 10/1/89 to 15/1/84 to 20/1/79 MeOH/HOAc/CH.sub.2Cl.sub.2)
to provide 317 mg (63%) of compound 121 as a sticky white solid:
.sup.1H NMR (CD.sub.3OD) .delta. 1.34 (m, 16H), 1.43 (m, 8H), 1.48
(s, 36H), 1.64 (m, 24H), 2.20 (m, 16H), 3.19 (m, 12H), 3.25-3.52
(m, 18H), 3.55 (m, 2H), 3.79 (t, 8H), 4.16 (m, 4H), 4.28 (t, 1H),
4.59 (d, 2H), 7.33 (t, 2H), 7.41 (t, 2H), 7.60 (d, 2H), 7.84 (d,
2H); .sup.13C NMR (CD.sub.3OD) .delta. 14.6, 23.8, 26.7, 26.7,
26.9, 27.7, 28.8, 28.9, 30.3, 37.1, 38.8, 39.1, 40.3, 65.8, 66.0,
68.1, 70.2, 70.3, 77.3, 82.0, 121.2, 126.0, 128.4, 129.0, 142.9,
145.6, 157.9, 158.2, 159.2, 176.1, 176.3; mass spectrum (ESI)
(M+2Na)/2 calculated for C.sub.101H.sub.171Na.sub.2N.sub.1-
5O.sub.28: 1044. Found 1044.
[0314] Compound 122: To a solution of 163 mg (79.8 mmol) of
compound 121 in 2.4 mL of DMF was added 600 .mu.L of diethylamine.
The mixture was stirred for 3 hours and concentrated. Purification
by silica gel chromatography (multi-step gradient; 10/1/89 to
12.5/6/86.5/ to 15/1/84 MeOH/con NH4OH/CH.sub.2Cl.sub.2) gave 127
mg (81%) of compound 122 as a glassy gum: .sup.1H NMR (CD.sub.3OD)
.delta. 1.38 (m, 16H), 1.48 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H),
2.83 (t, 4H), 3.17 (t, 8H), 3.38 (m, 16H), 3.63 (t, 4H), 3.69 (t,
4H), 3.78 (t, 4H), 4.21 (m, 4H); .sup.13C NMR (CD.sub.3OD) .delta.
26.7, 27.9, 27.8, 28.8, 28.9, 30.3, 37.1, 38.8, 39.1, 40.3, 49.9,
66.0, 70.4, 70.9, 77.3, 82.0, 158.2, 159.2, 176.1, 176.3; mass
spectrum (ESI) (M+H) calculated for C.sub.86H.sub.162N.sub.15-
O.sub.26: 1821. Found 1821.
[0315] Compound 124b: To a solution of 20 mg (11.0 .mu.mol) of
compound 122 in 5 mL of DMF was added 103 mg (8.8 .mu.mol) of
methoxypolyethyleneglycol benzotriazolylcarbonate of molecular
weight 11,690 g/mol (mPEG.sub.12K-BTC, compound 123b, Shearwater
Polymers) followed by 5 .mu.L (3.6 mg, 35.9 .mu.mol) of Et.sub.3N.
The mixture was stirred at room temperature for 18 hours and
concentrated. The residue was purified by silica gel chromatography
(multi-step gradient; 5/95 to 15/85 to 20/80 MeOH/CH.sub.2Cl.sub.2)
to provide 109 mg of compound 124b as a waxy off white solid:
.sup.1H NMR (CDCl.sub.3) .delta. 1.37 (m, 16H), 1.49 (m, 44H), 1.65
(m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H),
3.68 (m, approximately 1056H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m,
4H).
[0316] Compound 124a: This compound was prepared using essentially
the same procedure used for the preparation of compound 124b;
however, methoxypolyethyleneglycol benzotriazolylcarbonate of
molecular weight 5,215 g/mol (mPEG.sub.5K-BTC, compound 123a,
Shearwater Polymers) was used: .sup.1H NMR (4:1
CDCL.sub.3/CD.sub.3OD) .delta. 1.37 (m, 16H), 1.49 (m, 44H), 1.65
(m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H),
3.68 (m, approximately 468H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m,
4H).
[0317] Compound 124c: This compound was prepared using essentially
the same procedure used for the preparation of compound 124b;
however, methoxypolyethyleneglycol benzotriazolylcarbonate of
molecular weight 22,334 g/mol (mPEG.sub.20K-BTC, compound 123c,
Shearwater Polymers) was used: .sup.1H NMR (5:1
CDC.sub.3/CD.sub.3OD) .delta. 1.37 (m, 16H), 1.49 (m, 44H), 1.65
(m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H),
3.68 (m, approximately 2024H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m,
4H).
[0318] Analogously, the synthesis can be conducted with mPEG-BTC of
the desired molecular weight, for example, 23,000, 25,000, 40,000
or more. Thus, in FIG. 9, n is optionally greater than 500, for
example, 550, 600, 800 or more.
[0319] Compound 125b: The Boc-protecting groups are removed from
compounds 124a-c in a manner essentially similar to that described
for the preparation of compound 16 to provide compounds 125a-c.
[0320] The reaction scheme is shown in FIG. 9.
Example 3
Synthesis of Compound 129
[0321] Compound 126: To a solution of 14 mg (18.6 .mu.mol) of
compound 120 and 29 mg (186.3 .mu.mol) of HOBT in 5 mL of anhydrous
DMF was added 56 .mu.L (38 mg, 373 .mu.mol) of Et.sub.3N. The
mixture was stirred for 1 hour and a solution of 85 mg (46.6
.mu.mol) of compound 122 in 1 mL of DMF was added. The mixture was
stirred at room temperature for 5 hours and partitioned between 150
mL of CH.sub.2Cl.sub.2 and 50 mL of 1 N HCl. The CH.sub.2Cl.sub.2
layer was washed with brine, dried (MgSO.sub.4), filtered, and
concentrated. Purification by silica gel chromatography provided 34
mg (44%) of compound 126 as a waxy white solid: .sup.1H NMR
(CD.sub.3O(D) .delta. 1.37 (m, 32H), 1.49 (m overlapping s at 1.48,
88H) 1.62 (m, 48H), 2.20 (t, 32H), 3.18 (t, 16H), 3.36 (m, 32H),
3.50 (m, 12H), 3.64 (m, 24H), 3.79 (t, 16H) 4.17 (m, 12H), 4.29 (t,
1H), 4.60 (d, 2H), 7.37 (t, 2H), 7.43 (t, 2H), 7.65 (d, 2H), 7.84
(d, 2H); mass spectrum (ESI) (M+3Na)/3 calculated for
C.sub.197H.sub.347Na.sub.3N.sub.3- 1O.sub.60: 1393. Found 1393.
[0322] Alternative synthesis of compound 126: Pyridine (5 mL) was
added to a flask charged with 136 mg (74.7 umol) of compound 122,
22.3 mg (29.9 umol) of compound 120, and 18.3 mg (119.5 umol) of
hydroxybenzotriazole monohydrate (HOBt). The resulting solution was
stirred for 18 hours, and the pyridine was removed by rotory
evaporation under vacuum. Purification by silica gel chromatography
(gradient 1/13/86 to 1/20/79 AcOH/MeOH/CH.sub.2Cl.sub.2) provided
104 mg (85%) of compound 126 as a white solid.
[0323] Compound 127: To a solution of 34 mg (8.27 .mu.mol) of
compound 126 in 1.6 mL of DMF was added 400 .mu.L of diethylamine.
The mixture was stirred at room temperature for 4 hours and
concentrated. The concentrate was purified by silica gel
chromatography (1/10/89 con NH.sub.4OH/MeOH/CH.sub.2Cl.sub.2) to
provide 13 mg (40%) of compound 127: .sup.1H NMR (CD.sub.3OD)
.delta. 1.35 (m, 32H), 1.49 (m overlapping s at 1.48, 88H), 1.63
(m, 48H), 2.19 (t, 32H), 3.08 (brd t, 4H) 3.17 (t, 16H), 3.38 (m,
36H), 3.52 (m, 8H), 3.63 (t, 8H), 3.70 (m, 12H), 3.78 (t, 16H),
4.21 (m, 12H); mass spectrum (ESI) (M+3Na)/3 calculated for
C.sub.182H.sub.337Na.sub.3N.sub.31O.sub.58: 1319. Found 1319.
[0324] Compound 128: To a solution of 13 mg (3.34 .mu.mol) of
compound 127 in 5 mL of pyridine was added 60 mg (2.68 .mu.mol) of
methoxypolyethyleneglycol benzotriazolylcarbonate of molecular
weight 22,334 g/mol (mPEG.sub.20K-BTC, Shearwater Polymers)
followed by 5 .mu.L (3.6 mg, 35.9 .mu.mol) of Et.sub.3N. The
mixture was stirred at room temperature for 18 hours and
concentrated. The residue was purified by silica gel chromatography
(multi-step gradient; 10/90 to 15/85 to 20/80
MeOH/CH.sub.2Cl.sub.2) to provide 45 mg of compound 128 as a waxy
solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.30 (m, 32H), 1.50 (m
overlapping s at 1.48, 88H), 1.67 (m, 48H), 2.24 (t, 32H), 3.23 (m,
16H), 3.41 (m, 32H), 3.65 (m, approximately 2024H), 3.70 (t, 24H),
3.89 (m, 16H), 4.21 (m, 12H).
[0325] Analogously, the synthesis can be conducted with mPEG-BTC of
the desired molecular weight, for example, 23,000, 25,000, 40,000
or more. Thus, in FIG. 10, n is optionally greater than 500, for
example, 550, 600, 800 or more.
[0326] Compound 129: The Boc-protecting groups are removed from
compound 128 in a manner essentially similar to that described for
the preparation of compound 16 to provide compounds 129, as shown
in FIG. 10.
Example 4
Synthesis of Compound 132
[0327] Compound 131: To a solution of 22 mg (27.3 .mu.mol) of
compound 117 in 5 mL of pyridine was added 236 mg (10.9 .mu.mol) of
polyethyleneglycol bis-benzotriazolylcarbonate of molecular weight
21,529 g/mol (PEG.sub.20K-bis-BTC, compound 130, Shearwater
Polymers) followed by 8 .mu.L (5.8 mg, 57.4 .mu.mol) of Et.sub.3N.
The mixture was stirred at room temperature for 18 hours and
concentrated. The residue was purified by silica gel chromatography
(multi-step gradient; 5/95 to 10/90 to 15/85 to 20/80
MeOH/CH.sub.2Cl.sub.2) to provide 242 mg (96%) of compound 131 as a
white solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.35 (m, 16H), 1.48
(m, 44H), 1.61 (m, 24H), 2.20 (m, 16H), 3.22 (m, 8H), 3.52-3.96 (m,
approximately 2000H), 4.23 (m, 4H).
[0328] Analogously, the synthesis can be conducted with PEG-bis-BTC
of the desired molecular weight, for example, 23,000, 25,000,
40,000 or more. Thus, in FIG. 11, n is optionally greater than 500,
for example, 550, 600, 800 or more.
[0329] Compound 132: The Boc-protecting groups are removed from
compound 131 in a manner essentially similar to that described for
the preparation of compound 16 to provide compound 132.
[0330] The reaction scheme is shown in FIG. 11.
Example 5
Synthesis of Compound 136
[0331] Compound 134: To a solution of 3.87 mg (4.85 .mu.mol) of
pentaerythritol tetrakis-(4-nitrophenylcarbonate ester),(prepared
by reaction of pentaerythritol with para-nitrophenylchloroformate
to yield the tetra para-nitrophenylcarbonate compound), in 5 mL of
pyridine was added 124 mg (24.2 .mu.mol) of mono-Boc-protected
diaminopolyethylene glycol of molecular weight 5094 g/mol (compound
133, BocNH-PEG.sub.(5K)-NH.sub.2, Shearwater), and 5 .mu.L (3.63
mg, 35.9 .mu.mol) of Et.sub.3N. The mixture was stirred for 18
hours and concentrated. The residue was purified by silica gel
chromatography (step gradient; 5/95 to 15/85 MeOH/CH.sub.2Cl.sub.2)
to provide 77 mg (77%) of compound 134 as a white solid: .sup.1H
NMR (CDCl.sub.3) .delta. 1.48 (s, 36H), 3.32 (m, 16H), 3.52-3.96
(m, approximately 1818H), 4.10 (m, 8H).
[0332] Analogously, the synthesis can be conducted with
BocNH-PEG-NH.sub.2 of the desired molecular weight, for example,
23,000, 25,000, 40,000 or more. Thus, in FIG. 12, n is optionally
greater than 500, for example, 550, 600, 800 or more.
[0333] Synthesis of compound 106: To a magnetically stirred
solution of 5.2g (20.2 mmol, 1.1 eq.) of di-succinimidyl carbonate
in 200 mL of acetonitrile was added 4.54g (18.3 mmol, 1.0 eq.) of a
solution of compound 105 in 100 mL of acetonitrile. Pyridine (2.67
mL, 2.61 g, 33.03 mmol, 1.8 eq.) was added, and the mixture was
stirred overnight. The mixture was concentrated, and the residue
was dissolved in 20 mL of dichloromethane. The organic layer was
washed with two 20 mL portions of 1 N HCl and 20 mL sat. aq. NaCl.
The organic layer was dried (MgSO.sub.4), filtered, and
concentrated to provide 5.7g of yellow oil. A portion of this
material (2.732 g) was further purified by silica gel
chromatography (15/85 acetone/toluene) to give 4.38 g (68%) of
compound 106 as a colorless oil. .sup.1H NMR CDCl.sub.3 (.delta.)
1.48 (s, 9H), 1.51 (m, 2H), 1.68 (p, 2H), 1.79 (p, 2H), 2.63 (t,
2H), 2.84 (br. s, 4H), 3.87 (t, 2H), 7.18 (br. s, 1H); .sup.13C NMR
CDCl.sub.3 (.delta.) 24.32, 25.07, 25.53, 27.42, 28.16, 30.77,
76.13, 81.51, 156.89, 168.45, 169.15.
[0334] Compound 135: Compound 134 (77 mg, 3.73 .mu.mol) was
dissolved in 5 mL of trifluoroacetic acid, and the mixture was
allowed to stand for three hours. The TFA was removed under a
stream of N.sub.2 and the residue was dissolved in 5 mL of
CH.sub.2Cl.sub.2. To the resulting solution was added a solution of
7.72 mg (22.4 .mu.mol) of compound 106 in 5 mL of CH.sub.2Cl.sub.2
followed by 35 .mu.L (25.4 mg, 251 .mu.mol) of Et.sub.3N. (Note:
The pH of the mixture should be checked and adjusted accordingly
with Et.sub.3N to make sure it is basic.) The mixture was stirred
under nitrogen for 18 hours. The mixture was partitioned between 50
mL of CH.sub.2Cl.sub.2 and three 25 mL portions of 1 N HCl. The
CH.sub.2Cl.sub.2 layer was washed with brine, dried (MgSO.sub.4),
filtered and concentrated. Purification by silica gel
chromatography (step gradient; 5/95 to 10/90 MeOH/CH.sub.2Cl.sub.2)
provided 42 mg (53%) of compound 135 as waxy solid: .sup.1H NMR
(CDCl.sub.3) .delta. 1.40 (m, 8H), 1.48 (s, 36H), 1.66 (m, 16H),
2.18 (t, 8H), 3.32 (m, 16H), 3.38-3.89 (m, approximately 1818H),
4.10 (m, 8H), 4.97 (t, 4H), 6.43 (t, 4H), 7.47 (s, 4H).
[0335] Compound 136: The Boc-protecting groups are removed from
compound 135 in a manner essentially similar to that described for
the preparation of compound 16 to provide compound 136, as shown in
FIG. 12.
Example 6
Synthesis of Compound 143
[0336] Compound 137: To a 0.degree. C. solution of 200 mg (1.11
mmol) of ethyl 3,5-diaminobenzoate in 5 mL of CH.sub.2Cl.sub.2
under nitrogen atmosphere was added 928 .mu.L (674 mg, 6.66 mmol)
of Et.sub.3N. To the mixture was added dropwise a solution of 510
.mu.L (710 mg, 3.33 mmol) of 6-bromohexanoyl chloride in 5 mL of
CH.sub.2Cl.sub.2. The mixture was stirred at room temperature for
1.5 hours and partitioned between 50 mL of 1 N HCl and two 50 mL
portions of CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layers were
washed with saturated sodium bicarbonate solution, dried
(MgSO.sub.4), filtered and concentrated. The product was purified
by silica gel chromatography (6/4 hexane/EtOAc) to give 554 mg
(93%) of compound 137 as an oil: .sup.1H NMR (CDCl.sub.3):
.delta.1.39 (t, 3H), 1.52 (m, 4H), 1.75 (m, 4H), 1.90 (m, 4H), 2.40
(t, 4H), 3.42 (t, 4H), 4.36 (q, 2H), 7.60 (s, 2H), 7.88 (s, 2H),
8.17 (s, 1H).
[0337] Compound 138: DBU (612 .mu.L, 623 mg, 4.01 mmol) was added
to a solution of 547 mg (1.02 mmol) of compound 137 and 272 mg
(2.05 mmol) of N-(tert-butyloxycarbonyl)hydroxylamine (Aldrich
Chemical Co.). The mixture was stirred for 18 hours at room
temperature and partitioned between 50 mL of 1 N HCl and three 50
mL portions of CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2
layers were dried (MgSO.sub.4), filtered, and concentrated. The
product was purified by silica gel chromatography (1/1
hexane/EtOAc) to give 216 mg (33%) of compound 138 as awhite solid:
mp 55-60.degree. C.; .sup.1H NMR (CDCl.sub.3): .delta. 1.38 (t,
3H), 1.48 (s, 18H; burried m, 4H), 1.60 (m, 4H), 1.73 (m, 4H), 2.40
(m, 4H), 3.86 (t, 4H), 4.36 (q, 2H), 7.41 (s, 2H), 7.90 (s, 2H),
8.06 (s, 2H), 8.11 (s, 1H); mass spectrum (ESI) (M+Na) calculated
for C.sub.31H.sub.50NaN.sub.4O- .sub.10: 661. Found 661.
[0338] Compound 139: To a solution of 205 mg (0.32 mmol) of
compound 138 in 1/1 acetone/EtOH was added 256 .mu.L (2.56 mmol) of
10 N NaOH, and the mixture was heated to 60.degree. C. for 4 hours.
When cool, the mixture was partitioned between 50 mL of 1 N HCl and
four 50 mL portions of 4/1 CH.sub.2Cl.sub.2/MeOH. The combined
organic layers were dried (MgSO.sub.4), filtered, and concentrated.
The product was purified by silica gel chromatography (3/97/1
MeOH/CH.sub.2Cl.sub.2/HOAc) to give 184 mg (94%) of compound 139 as
a viscous oil: .sup.1H NMR (CDCl.sub.3): .delta. 1.38 (m, 4H), 1.42
(s, 18H), 1.60 (m, 4H), 1.70 (m, 4H), 2.38 (m, 4H), 3.80 (t, 4H),
7.77 (s, 2H), 8.00 (s, 2H), 8.11 (s, 1H), 8.91 (s, 2H); mass
spectrum (ESI) (M+Na) calculated for C.sub.29H.sub.46NaN.sub.4O-
.sub.10: 633. Found 633.
[0339] Compound 140: To a 0.degree. C. solution of 164 mg (0.268
mmol) of compound 139 in 2.0 mL of dry THF was added 31 mg (0.268
mmol) of N-hydroxysuccinimide, followed by 83 mg (0.403 mmol) of
DCC. The mixture was allowed to come to room temperature and
stirred for 18 hours under nitrogen atmosphere, and 200 .mu.L of
HOAc was added. The mixture was stirred for another hour, diluted
with approximately 5 mL of EtOAc, and allowed to stand for an hour.
The resulting precipitate was removed by filtration, and the
filtrate was concentrated. Purification by silica gel
chromatography (3/97/MeOH/CH.sub.2Cl.sub.2) provided 129 mg (68%)
of compound 140 as a white solid: .sup.1H NMR (CDCl.sub.3): .delta.
1.40 (m, 4H), 1.43 (s, 18H), 1.65 (m, 4H), 1.80 (m, 4H), 2.34 (m,
4H), 2.93 (s, 4H), 3.85 (t, 4H), 7.68 (s, 2H), 7.87 (s, 2H), 8.36
(s, 1H), 8.61 (s, 2H).
[0340] Compound 142: To a solution of 60 mg (0.85 mmol) of compound
140 in 0.5 mL of CH.sub.2Cl.sub.2 was added 14 .mu.L (13.3 mg,
0.168 mmol) of pyridine. The mixture was cooled to 0.degree. C. and
a solution of 71 mg (0.021 mmol) of diamino-PEG, compound 141, in
0.5 mL of CH.sub.2Cl.sub.2 was added. The mixture was stirred under
nitrogen atmosphere at room temperature for 18 hours, and
partitioned between 10 mL of 1 N HCl and three 10 mL portions of
CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers were dried
(MgSO.sub.4), filtered, and concentrated. Purification by silica
gel chromatography (step gradient 5/95 MeOH/CH.sub.2Cl.sub.2 to
10/90 MeOH/CH.sub.2Cl.sub.2) provided 66 mg (69%) of compound 142
as a viscous oil: .sup.1H NMR (CDCl.sub.3): .delta. 1.45 (s, 36H),
1.60-1.80 (m, 24H), 2.39 (t, 8H), 3.39 (m, 8H), 3.50-3.80 (brd s,
approx. 318H), 3.87 (t, 8H), 4.22 (t, 4H), 7.50 (brd s, 2H), 7.63
(s, 4H), 7.77 (s, 2H), 8.08 (s, 2H), 8.60 (s, 2H); mass spectrum
(MALDI) (M+H) calculated for C.sub.207H.sub.389N.sub.12O.sub.93:
4535. Found distribution centered at approximately 4324.
[0341] Analogously, the synthesis can be conducted with compound
141 of the desired molecular weight, for example, where n is
optionally greater than 500, for example, 550, 600, 800 or more, as
shown in FIG. 13.
[0342] Compound 143: The Boc-protecting groups are removed from
compound 142 in a manner essentially similar to that described for
the preparation of compound 16 to provide 143, as shown in FIG.
13.
[0343] Synthesis of compound 302: To a solution of 209 mg (9.36
.mu.mol) of compound 123c and 8.84 mg (11.2 .mu.mol) of compound
117 in 5 mL of anhydrous pyridine was added 3.26 uL (2.36 mg, 23.4
.mu.mol) of triethylamine. The mixture was stirred for 18 hours
under a N.sub.2 atmosphere, the pyridine was removed by rotory
evaporation under vacuum, and the residue was purified by
preparative HPLC on a 22 mm.times.250 mm aminopropyl silica column
(gradient, 10% A to 30% B over 60 minutes; A=H.sub.2O,
B=CH.sub.3CN) to provide 200 mg (93%) of compound 302.
[0344] Synthesis of compound 303: The Boc-protecting groups are
removed from compound 302 in a manner essentially similar to that
described for the preparation of compound 16 to provide compound
303.
[0345] Synthesis of compound 304: To a solution of 4.43 mL (4.48 g,
30.2 mmol) of 2,2'-(ethylenedioxy)bis(ethylamine) (Aldrich Chemical
Co.) in 50 mL of EtOAc was added a solution of 1.04 g (3.0 mmol) of
compound 106. A precipitate formed which was removed by filtration.
The filtrate was concentrated and the residue was dissolved in 100
mL of CH.sub.2Cl.sub.2 and shaken with 50 mL of 1 N NaOH. The
aqueous layer was extracted with four 50 mL portions of
CH.sub.2Cl.sub.2, and all the CH.sub.2Cl.sub.2 extracts were
combined and concentrated to a yellow oil. Purification by silica
gel chromatography (1.25/13.75/85 con NH.sub.4OH/MeOH/CH.sub.2Cl.s-
ub.2) provided 520 mg (46%) of compound 304 as a yellowish oil.
[0346] Synthesis of compound 305: To a solution of 500 mg (23.2
umol) of compound 130 in 9 mL of anhydrous pyridine was added a
solution of 21.9 mg (58.1 umol) of compound 304 in 1 mL of pyridine
followed by 26 uL (18.8 mg, 186 umol) of triethylamine, and the
mixture was stirred under nitrogen atmosphere for 18 hours. Tthe
pyridine was removed by rotory evaporation under vacuum, and the
residue was purified by preparative HPLC on a 22 mm.times.250 mm
aminopropyl silica column (gradient, 13% A to 33% B over 60
minutes; A=H.sub.2O, B=CH.sub.3CN) to provide 428 mg (84%) of
compound 305.
[0347] Synthesis of compound 306: The Boc-protecting groups are
removed from compound 305 in a manner essentially similar to that
described for the preparation of compound 16 to provide compound
306 (FIG. 17).
Example 7
Method of Preparation of Conjugates
[0348] Conjugates 200, 201, 202, 203, 204, and 205 (FIG. 7) were
prepared as follows.
[0349] Compound 200: To a solution of 68.8 mg (9.74 .mu.mol 6
equivalents) of TA/D1 in 10 mL of helium sparged 0.1 M, pH 4.6
sodium acetate buffer was added a solution of 36.8 mg (1.62
.mu.mol) of compound 125c in 6.15 mL of 1/1 acetonitrile/0.1 M, pH
8.0 tris acetate buffer. Care was taken to keep the mixture under
nitrogen atmosphere while stirring at room temperature for 18
hours. When the reaction was complete, it was directly purified by
cation exchange chromatography using a PolyCat A WCX column
manufactured by PolyLC Inc. (gradient 10% B to 25% B, A 10 mM
sodium phosphate pH 7 in 1/9 acetonitrile/H.sub.2O) to provide 57
mg (40%) of compound 200.
[0350] Compound 201: Compound 201 was prepared in a manner
essentially similar to compound 200. Thus, to an approximately 1 mM
solution of 6 equivalents of TA/D1 in helium sparged 0.1 M, pH 4.6
sodium acetate buffer was added 1 equivalent of compound 125a as a
0.25 to 10 mM solution in 1/1 acetonitrile/0.1 M, pH 8.0 tris
acetate buffer. Care was taken to keep the mixture under nitrogen
atmosphere while stirring at room temperature for 18 hours. When
the reaction was complete, it was directly purified by cation
exchange chromatography to provide compound 201.
[0351] Compound 202: Compound 202 was prepared in a manner
essentially similar to 200. Thus, to an approximately 1 mM solution
of 6 equivalents of TA/D1 in helium sparged 0.1 M, pH 4.6 sodium
acetate buffer was added 1 equivalent of compound 132 as a 0.25 to
10 mM solution in 1/1 acetonitrile/0.1 M, pH 8.0 tris acetate
buffer. Care was taken to keep the mixture under nitrogen
atmosphere while stirring at room temperature for 18 hours. When
the reaction was complete, it was directly purified by cation
exchange chromatography to provide compound 202.
[0352] Also isolated were two lower valent species as minor
impurities. A compound with three domain 1 polypeptides attached,
as evidenced by polyacrylamide gel electrophoresis and mass
spectroscopy, was isolated and is referred to as 202 trimer. A
compound with two domain 1 poly peptides attached, as evidenced by
polyacrylamide gel electrophoresis and mass spectroscopy, was
isolated and is referred to as 202 dimer.
[0353] Compound 203: Compound 203 was prepared in a manner
essentially similar to 200. Thus, to an approximately 1 mM solution
of 6 equivalents of TA/D1 in helium sparged 0.1 M, pH 4.6 sodium
acetate buffer was added 1 equivalent of compound 136 as a 0.25 to
10 mM solution in 1/1 acetonitrile/0.1 M, pH 8.0 tris acetate
buffer. Care was taken to keep the mixture under nitrogen
atmosphere while stirring at room temperature for 18 hours. When
the reaction was complete, it was directly purified by cation
exchange chromatography to provide compound 203.
[0354] Compound 204: Compound 204 was prepared in a manner
essentially similar to 200. Thus, to an approximately 1 mM solution
of 6 equivalents of TA/D1 in helium sparged 0.1 M, pH 4.6 sodium
acetate buffer was added 1 equivalent of compound 143 as a 0.25 to
10 mM solution in 1/1 acetonitrile/0.1 M, pH 8.0 tris acetate
buffer. Care was taken to keep the mixture under nitrogen
atmosphere while stirring at room temperature for 18 hours. When
the reaction was complete, it was directly purified by cation
exchange chromatography to provide compound 204.
[0355] Compound 205: Compound 205 was prepared in a manner
essentially similar to 200. Thus, to an approximately 1 mM solution
of 6 equivalents of TA/D1 in helium sparged 0.1 M, pH 4.6 sodium
acetate buffer was added 1 equivalent of compound 125b as a 0.25 to
10 mM solution in 1/1 acetonitrile/0.1 M, pH 8.0 tris acetate
buffer. Care was taken to keep the mixture under nitrogen
atmosphere while stirring at room temperature for 18 hours. When
the reaction was complete, it was directly purified by cation
exchange chromatography to provide compound 205.
[0356] Synthesis of compound 300: Compound 300 was prepared in a
manner essentially similar to compound 200. Thus, to an
approximately 1 mM solution of 12 equivalents of TA/D1 in helium
sparged 0.1 M, pH 4.6 sodium acetate buffer was added 1 equivalent
of compound 129 as a 0.25 to 10 mM solution in 1/1 acetonitrile/0.1
M pH 8.0 tris acetate buffer. Care was taken to keep the mixture
under nitrogen atmosphere while stirring at room temperature for 18
hours. When the reaction was complete, it was directly purified by
cation exchange chromatography to provide compound 300 (FIG.
16).
[0357] Synthesis of compound 309: Compound 309 was prepared in a
manner essentially similar to compound 200. Thus, to an
approximately 1 mM solution of 3 equivalents of TA/D1 in helium
sparged 0.1 M, pH 4.6 sodium acetate buffer was added 1 equivalent
of compound 303 as a 0.25 to 10 mM solution in 1/1 acetonitrile/0.1
M pH 8.0 tris acetate buffer. Care was taken to keep the mixture
under nitrogen atmosphere while stirring at room temperature for 18
hours. When the reaction was complete, it was directly purified by
cation exchange chromatography to provide compound 303.
[0358] Synthesis of compound 310: Compound 310 was prepared in a
manner essentially similar to compound 200. Thus, to an
approximately 1 MM solution of 3 equivalents of TA/D1 in helium
sparged 0.1 M, pH 4.6 sodium acetate buffer was added 1 equivalent
of compound 306 as a 0.25 to 10 mM solution in 1/1 acetonitrile/0.1
M pH 8.0 tris acetate buffer. Care was taken to keep the mixture
under nitrogen atmosphere while stirring at room temperature for 18
hours. When the reaction was complete, it was directly purified by
cation exchange chromatography to provide compound 310 (FIG.
18).
Example 8
Evaluation of Toleragen Efficiency and Serum Half-Life
[0359] Domain 1--keyhole limpet hemocyanin conjugate (D1 CYS--KLH)
was prepared for use in animal immunization. Recombinant Domain 1
with a fifth cysteine was expressed as a glutathione mixed
disulfide in insect cells using the baculovirus expression vector
system. The structure consists of the first 66 amino-terminal amino
acids present in native human .beta..sub.2-glycoprotein I followed
by a C-terminal leu-(his).sub.5 expression tag. The polyhistidine
expression tag at the C-terminus was the basis for a purification
procedure by nickel affinity chromatography. Iverson et al. (1998)
Proc. Nat'l. Acad. Sci. 95: 15542-15546.
[0360] The resulting Domain 1 with a free sulfhydryl (D1 CYS--SH)
was alkylated by maleimidyl-KLH. Maleimidyl-activated KLH (Pierce
Chemical Co.; Rockford, Ill.) was dissolved at 10 mg/mL in water as
per the manufacturer's instructions. Immediately, the KLH was added
to D1 CYS--SH at a ratio of 1.27 mg per mg D1-SH. The tube
containing the KLH and D1 CYS was mixed by rotation at 2
h.times.RT. At the end of the incubation the contents were dialyzed
against cold PBS at 4.degree. C. using a >25,000 MW cut-off
membrane for the removal of unconjugated D1 CYS. An aliquot of the
dialyzed sample was removed and tested for the presence of
immunoreactive D1 by an ELISA with patient-derived affinity
purified antiphospholipid antibodies (aPL).
[0361] An immunized rat model was used for measuring toleragen
efficacy. Lewis rats (Harlan Sprague Dawley, Indianapolis, Ind.)
were immunized i.p. with 10 .mu.g of D1 CYS--KLH in alum with
pertussis adjuvant. Three weeks after priming, groups of four
animals were treated i.v. with toleragen or PBS control. Five days
after treatment animals were boosted i.p. with 10 .mu.g D1
CYS--KLH, and sera samples were collected seven days after
boost.
[0362] An ELISA was used for detection of anti-domain 1 antibody in
rat sera. Nunc Maxisorp Immunoplates (Nalge Nunc International,
Rochester, N.Y.) were coated overnight with 50 .mu.l of 5 82 g/ml
recombinant human .beta..sub.2-GPI in carbonate buffer (Sigma, St.
Louis, Mo.) pH 9.6 at 4.degree. C. Subsequent steps were carried
out at room temperature. Plates were washed 3.times. with phosphate
buffered saline (PBS), then blocked 1 h with 250 .mu.l 2% nonfat
dry milk (Carnation, Solon, Ohio) in PBS. After washing, wells were
incubated 1 h with 50 .mu.l serial dilutions in PBS of each sera
sample in triplicate. Non-immunized serum was used as control, and
a pool of sera from immunized animals was used to generate a
standard curve. After washing, the wells were incubated 1 h with 50
.mu.l alkaline phosphatase-conjugated goat anti-rat IgG (Jackson
ImmunoResearch, West Grove, Pa.) diluted 1:2000 in PBS/0.1% BSA.
Wells were washed 3.times. with dIH.sub.2O and were developed 20
minutes with PPMP solution ((10 gm phenolphthalein mono-phosphate
(Sigma, St. Louis Mo.), 97.4 ml 2-amino-2-methyl-1-propanol
(Sigma), 9.62 ml dIH.sub.2O, 21 ml HCl)). Color development was
stopped with 50 .mu.l 0.2 M Na.sub.2HPO.sub.4 and the OD.sub.550
was read on a Bio-Tek Instruments PowerWave 340 Microplate
Spectrophotometer (Winooski, Vt.). Nominal antibody units were
assigned to the standard pool and the concentration of anti-domain
1 antibody (units/ml) in test sera were derived from the standard
curve. Percent suppression of anti-domain 1 antibody by multivalent
platform conjugate, using conjugates 200, 201, 202 and 203
treatment was calculated by comparison to PBS-treated controls. The
results are shown in Table 1, below.
1TABLE 1 Percent Suppression of Anti-Domain 1 Antibody in Immunized
Rats nanomoles drug/rat Compound 0.17 1.7 17 2.4 3.5 200 61 82 89
201 34 73 86 202 72 89 96 203 73 93 94 202 trimer 83 202 dimer 70
By definition, PBS control = 0% suppression
[0363] The plasma pharmacokinetics of the compounds were measured
in mice, rats and macaques. The compounds were radiolabeled with
1251 by the iodine monochloride method. Contreras et al., 1983,
Methods in Enzymology 92: 277-292. The labeled compound was
injected i.v. into female Balb/c mice, male Sprague-Dawley rats and
female Cynomologous macaques. Plasma samples were collected over
one hour in mice and over twenty-four hours in rats and macaques.
The amount of radiolabeled drug was detected using a Packard
Instruments Cobra Gamma counter (Dowers Grove, Ill.)
Pharmacokinetic parameters were calculated using Pharsight
WinNonLin software (Mountain View, Calif.) The overall plasma half
life was calculated using the formula t.sub.1/2=In2(AUC/0.5*Cmax).
The Clearance rate (CL, ml/hour and half-life (t.sub.1/2, hours)
for all three species are shown below in Table 2.
2TABLE 2 Pharmacokinetic Parameters in mice, rats and macaques Mice
Rats Macaques # Cl t.sub.1/2 Cl t.sub.1/2 Cl t.sub.1/2 204 62 0.05
1.70 8.0 64 3.3 200 1.2 0.61 0.67 20.2 34 7.5 201 9.0 0.28 1.48 9.8
89 2.2 205 6.7 0.30 0.78 14.0 48 4.7 202 2.3 0.73 0.69 18.4 28 5.7
203 1.5 1.84 0.65 20.0 12 13.2 300 4.2 0.27 0.98 14.7 ND ND 301 1.1
2.07 0.52 37.7 ND ND
[0364]
Sequence CWU 1
1
2 1 192 DNA Homo Sapien 1 ggacggacct gtcccaagcc agatgattta
ccattttcca cagtggtccc gttaaaaaca 60 ttctatgagc caggagaaga
gattacgtat tcctgcaagc cgggctatgt gtcccgagga 120 gggatgagaa
agtttatctg ccctctcaca ggactgtggc ccatcaacac tctgaaatgt 180
acacccagag ta 192 2 64 PRT Homo Sapien 2 gga cgg acc tgt ccc aag
cca gat gat tta cca ttt tcc aca gtg gtc 48 Gly Arg Thr Cys Pro Lys
Pro Asp Asp Leu Pro Phe Ser Thr Val Val 1 5 10 15 ccg tta aaa aca
ttc tat gag cca gga gaa gag att acg tat tcc tgc 96 Pro Leu Lys Thr
Phe Tyr Glu Pro Gly Glu Glu Ile Thr Tyr Ser Cys 20 25 30 aag ccg
ggc tat gtg tcc cga gga ggg atg aga aag ttt atc tgc cct 144 Lys Pro
Gly Tyr Val Ser Arg Gly Gly Met Arg Lys Phe Ile Cys Pro 35 40 45
ctc aca gga ctg tgg ccc atc aac act ctg aaa tgt aca ccc aga gta 192
Leu Thr Gly Leu Trp Pro Ile Asn Thr Leu Lys Cys Thr Pro Arg Val 50
55 60
* * * * *