U.S. patent application number 13/803392 was filed with the patent office on 2014-06-05 for methods for treating cancer using combination therapies.
The applicant listed for this patent is ENDOCYTE, INC.. Invention is credited to Christopher P. LEAMON, Nikki L. PARKER, Iontcho R. VLAHOV.
Application Number | 20140154702 13/803392 |
Document ID | / |
Family ID | 50825795 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154702 |
Kind Code |
A1 |
PARKER; Nikki L. ; et
al. |
June 5, 2014 |
Methods For Treating Cancer Using Combination Therapies
Abstract
Described are methods, uses, and kits for utilizing a targeted
ligand conjugate in combination with a thiol inhibitor or a system
x.sub.c.sup.- inhibitor for the treatment of cancer or
inflammation. Also described are in vitro assays utilizing a
targeted ligand conjugate in combination with a thiol inhibitor or
a system x.sub.c.sup.- inhibitor.
Inventors: |
PARKER; Nikki L.; (West
Lafayete, IN) ; LEAMON; Christopher P.; (West
Lafayette, IN) ; VLAHOV; Iontcho R.; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENDOCYTE, INC. |
West Lafayette |
IN |
US |
|
|
Family ID: |
50825795 |
Appl. No.: |
13/803392 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61731561 |
Nov 30, 2012 |
|
|
|
Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
A61K 31/454 20130101;
G01N 33/574 20130101; G01N 33/6893 20130101; G01N 2800/7095
20130101; A61K 47/551 20170801 |
Class at
Publication: |
435/7.23 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Claims
1. An in vitro assay for identifying a ligand conjugate suitable
for co-administration to a patient with a thiol inhibitor, the
assay comprising: a) adding the ligand conjugate to the culture
medium of a first sample of cultured cells, wherein the ligand
conjugate comprises a disulfide linkage; b) adding the thiol
inhibitor to the culture medium of the first sample of cultured
cells to provide a test sample; c) adding the ligand conjugate to
the culture medium of a second sample of cultured cells to provide
a control sample; d) measuring the non-ligand-specific activity of
the ligand conjugate or the nonspecific uptake of the drug in the
test sample; e) measuring the non-ligand-specific activity of the
ligand conjugate or the nonspecific uptake of the drug in the
control sample; and f) determining that the ligand conjugate is
suitable for co-administration to the patient with the thiol
inhibitor if the non-ligand-specific activity of the ligand
conjugate and/or the nonspecific uptake of the drug are decreased
in the test sample relative to the control sample, wherein the
ligand conjugate is of the formula BLD.sub.X, wherein B is a cell
surface receptor targeting ligand, D is an independently selected
drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a
releasable polyvalent linker comprising a thiol reactive linkage;
or a pharmaceutically acceptable salt thereof, wherein B is a PSMA
binding ligand, and wherein the PSMA binding ligand is ##STR00211##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C .sctn.119(e)
to U.S. Provisional Application Ser. No. 61/731,561, filed on Nov.
30, 2012, the disclosure of which is herein incorporated by
reference.
TECHNICAL FIELD
[0002] The invention relates to methods, kits, and in vitro assays
utilizing targeted ligand conjugates in combination with a thiol
inhibitor. The invention also relates to methods, kits, and in
vitro assays utilizing targeted ligand conjugates in combination
with a system x.sub.c.sup.- inhibitor.
BACKGROUND AND SUMMARY
[0003] A number of therapeutic agents comprising targeting ligands
linked to a drug contain one or more linkers (e.g., disulfide-based
linkers) placed between the targeting ligand and the
therapeutically active drug. The targeting ligand allows for
specific binding to cells that express a receptor for the ligand
(e.g., cancer cells), resulting in delivery of the therapeutically
active drug to the cells of interest. The disulfide linker allows
for release of the drug inside the cell when the targeting
ligand-drug conjugate is internalized and is exposed to the
reducing environment inside the cells. For example, specific
binding of folate-drug conjugates to the folate receptor (FR) on
cancer cells or inflammatory cells allows for targeted delivery and
specific therapeutic activity directed to the cancer cells or to
sites of inflammation.
[0004] Folate is a member of the B family of vitamins and plays an
essential role in cell survival by participating in the
biosynthesis of nucleic acids and amino acids. This essential
vitamin is also a high affinity ligand that enhances the
specificity of conjugated anti-cancer drugs by targeting, for
example, FR-positive cancer cells or inflammatory cells. The FR, a
glycosylphosphatidyl-inositol anchored protein, can actively
internalize bound folates and folate conjugated compounds via
receptor-mediated endocytosis. It has been found that the FR is
up-regulated in more than 90% of non-mucinous ovarian carcinomas,
and is also found on inflammatory cells. The FR is also found at
high to moderate levels in kidney, brain, lung, and breast
carcinomas while it occurs at low levels in most normal tissues. FR
density appears to increase as the stage of the cancer becomes more
advanced. Folate-targeted drug conjugates have been developed and
are being tested in clinical trials as cancer therapeutics, and in
pre-clinical testing as therapeutics for inflammatory diseases.
[0005] Generally, the disulfide linker of targeted conjugates
remains relatively stable in circulation, but rapidly breaks down
once the conjugate enters the reductive endocytic process within a
targeted cell. However, the possibility exists that a disulfide
linker may be broken down outside cells, resulting in release of
the drug and non-specific toxicity. The current inventors have
hypothesized that instability of the disulfide linker in targeted
conjugates may contribute to non-specific toxicity, and that the
presence of extracellular thiols may play a role in the instability
of the disulfide linker with resulting non-specific toxicity.
[0006] Accordingly, two different approaches were undertaken to
evaluate the impact of extracellular thiols on non-specific
activity of targeted conjugates with disulfide linkers. First,
ligand conjugates and thiol inhibitors were tested together to
evaluate the effects of co-treatment of cells with ligand
conjugates and thiol inhibitors on the nonspecific uptake of the
drug into the target cells and on non-ligand specific activity.
Second, ligand conjugates and system x.sub.c.sup.- inhibitors were
tested together to evaluate the effects of co-treatment of cells
with ligand conjugates and system x.sub.c.sup.- inhibitors on the
nonspecific uptake of the drug into the target cells and on
non-ligand specific activity.
[0007] Surprisingly, the inventors have found that extracellular
thiols (e.g., cysteine) contribute to non-specific toxicity of
targeted conjugates containing one or more disulfide linkers,
especially when the extracellular ligand conjugate concentration is
exceedingly high. Thus, co-administration of thiol inhibitors or
system x.sub.c.sup.- inhibitors to patients along with targeted
conjugates with disulfide linkers may decrease non-specific
toxicity and increase efficacy of targeted therapeutics.
[0008] In one embodiment, a method of treatment of a disease is
provided. The method comprises administering a ligand conjugate to
a patient, wherein the ligand conjugate comprises a disulfide
linkage; and administering a thiol inhibitor to the patient.
[0009] In another embodiment, use of a ligand conjugate in
combination with a thiol inhibitor for the treatment of a disease
is described, wherein the disease is cancer or inflammation, and
wherein the ligand conjugate comprises a disulfide linkage.
[0010] In another embodiment, use of a ligand conjugate for the
manufacture of a medicament for the treatment of a disease is
described, wherein the disease is cancer or inflammation, and
wherein the treatment comprises treating a patient with the ligand
conjugate in combination with a thiol inhibitor, wherein the ligand
conjugate comprises a disulfide linkage.
[0011] In another embodiment, a kit is provided. The kit comprises
a ligand conjugate and one or more thiol inhibitors, wherein the
ligand conjugate comprises a disulfide linkage.
[0012] In another embodiment, an in vitro assay for identifying a
ligand conjugate suitable for co-administration with a thiol
inhibitor to a patient is provided. The assay comprises the steps
of a) adding the ligand conjugate to the culture medium of a first
sample of cultured cells, wherein the ligand conjugate comprises a
disulfide linkage; b) adding the thiol inhibitor to the culture
medium of the first sample of cultured cells to provide a test
sample; c) adding the ligand conjugate to the culture medium of a
second sample of cultured cells to provide a control sample; d)
measuring the non-ligand-specific activity of the ligand conjugate
or the nonspecific uptake of the drug in the test sample; e)
measuring the non-ligand-specific activity of the ligand conjugate
or the nonspecific uptake of the drug in the control sample; and f)
determining that the ligand conjugate is suitable for
co-administration to the patient with the thiol inhibitor if the
non-ligand-specific activity of the ligand conjugate and/or the
nonspecific uptake of the drug are decreased in the test sample
relative to the control sample.
[0013] In yet another embodiment, a method of treatment of a
disease is provided. The method comprises administering a ligand
conjugate to a patient, wherein the ligand conjugate comprises a
disulfide linkage; and administering a system x.sub.c.sup.-
inhibitor to the patient.
[0014] In another embodiment, use of a ligand conjugate in
combination with a system x.sub.c.sup.- inhibitor for the treatment
of a disease is described, wherein the disease is cancer or
inflammation, and wherein the ligand conjugate comprises a
disulfide linkage.
[0015] In another embodiment, use of a ligand conjugate for the
manufacture of a medicament for the treatment of a disease wherein
the disease is cancer or inflammation is described, and wherein the
treatment comprises treating a patient with the ligand conjugate in
combination with a system x.sub.c.sup.- inhibitor, wherein the
ligand conjugate comprises a disulfide linkage.
[0016] In another embodiment, a kit is provided. The kit comprises
a ligand conjugate and one or more system x.sub.c.sup.- inhibitors,
wherein the ligand conjugate comprises a disulfide linkage.
[0017] In another embodiment, an in vitro assay for identifying a
ligand conjugate suitable for co-administration to a patient with a
system x.sub.c.sup.- inhibitor is provided. The assay comprises the
steps of a) adding the ligand conjugate to the culture medium of a
first sample of cultured cells, wherein the ligand conjugate
comprises a disulfide linkage; b) adding the system x.sub.c.sup.-
inhibitor to the culture medium of the first sample of cultured
cells to provide a test sample; c) adding the ligand conjugate to
the culture medium of a second sample of cultured cells to provide
a control sample; d) measuring the non-ligand-specific activity of
the ligand conjugate or the nonspecific uptake of the drug in the
test sample; e) measuring the non-ligand-specific activity of the
ligand conjugate or the nonspecific uptake of the drug in the
control sample; and f) determining that the ligand conjugate is
suitable for co-administration to the patient with the system
x.sub.c.sup.- inhibitor if the non-ligand-specific activity of the
ligand conjugate and/or the nonspecific uptake of the drug are
decreased in the test sample relative to the control sample. Any of
the embodiments described in the following clause list are
considered to be part of the invention.
[0018] 1. A method of treatment of a disease, the method comprising
the steps of: [0019] administering a ligand conjugate to a patient,
wherein the ligand conjugate comprises a disulfide linkage; and
[0020] administering a thiol inhibitor to the patient.
[0021] 2. The method of clause 1, wherein the disease is cancer or
inflammation.
[0022] 3. Use of a ligand conjugate in combination with a thiol
inhibitor for the treatment of a disease wherein the disease is
cancer or inflammation, and wherein the ligand conjugate comprises
a disulfide linkage.
[0023] 4. Use of a ligand conjugate for the manufacture of a
medicament for the treatment of a disease wherein the disease is
cancer or inflammation, and wherein the treatment comprises
treating a patient with the ligand conjugate in combination with a
thiol inhibitor, wherein the ligand conjugate comprises a disulfide
linkage.
[0024] 5. The method or use of any one of clauses 1 to 4, wherein
the disease is inflammation.
[0025] 6. The method or use of any one of clauses 1 to 4, wherein
the disease is cancer.
[0026] 7. The method or use of clause 6, wherein the cancer
comprises a primary tumor.
[0027] 8. The method or use of clause 6, wherein the cancer
comprises metastatic tumor cells.
[0028] 9. The method or use of any one of clauses 1 to 8, wherein
the ligand is folate.
[0029] 10. The method or use of any one of clauses 1 to 8, wherein
the ligand is an antibody.
[0030] 11. The method or use of any one of clauses 1 to 8, wherein
the ligand conjugate is of the formula BLD.sub.X, wherein B is a
cell surface receptor targeting ligand, D is an independently
selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and
L is a releasable polyvalent linker comprising a thiol reactive
linkage; or a pharmaceutically acceptable salt thereof.
[0031] 12. The method or use of clause 11, wherein B is folate.
[0032] 13. The method or use of clause 11, wherein B is
D-folate.
[0033] 14. The method or use of clause 11, wherein B is
L-folate.
[0034] 15. The method or use of clause 11, wherein B is a PSMA
binding ligand.
[0035] 16. The method or use of clause 11, wherein B is a radical
of the formula
##STR00001##
[0036] 17. The method or use of clause 11, wherein B is a radical
of the formula
##STR00002##
[0037] 18. The method or use of clause 11, wherein B is a radical
of the formula
##STR00003##
[0038] 19. The method or use of any one of clauses 11 to 18,
wherein the thiol reactive linkage is a disulfide linkage.
[0039] 20. The method or use of any one of clauses 11 to 19,
wherein L comprises a cysteine disulfide diradical.
[0040] 21. The method or use of any one of clauses 11 to 20,
wherein L further comprises one or more divalent hydrophilic
radicals.
[0041] 22. The method or use of any one of clauses 11 to 21,
wherein D is a cytotoxic agent.
[0042] 23. The method or use of any one of clauses 11 to 22,
wherein D is a cancer treating agent.
[0043] 24. The method or use of any one of clauses 11 to 22,
wherein D is an anti-inflammatory agent.
[0044] 25. The method or use of any one of clauses 11 to 24,
wherein D is a vinca alkaloid.
[0045] 26. The method or use of any one of clauses 11 to 24,
wherein D is desacetylvinblastine monohydrazide.
[0046] 27. The method or use of any one of clauses 11 to 24,
wherein D is a tubulysin.
[0047] 28. The method or use of any one of clauses 11 to 24,
wherein D is tubulysin A.
[0048] 29. The method or use of any one of clauses 11 to 24,
wherein D is tubulysin B.
[0049] 30. The method or use of any one of clauses 11 to 24,
wherein D is tubulysin A hydrazide.
[0050] 31. The method or use of any one of clauses 11 to 24,
wherein D is tubulysin B hydrazide.
[0051] 32. The method or use of any one of clauses 11 to 24,
wherein D is an antifolate.
[0052] 33. The method or use of any one of clauses 11 to 24,
wherein D is an aminopterin.
[0053] 34. The method or use of any one of clauses 11 to 24,
wherein D is a rapamycin.
[0054] 35. The method or use of any one of clauses 11 to 24,
wherein D is a mitomycin.
[0055] 36. The method or use of any one of clauses 11 to 24,
wherein D is a taxane.
[0056] 37. The method or use of any one of clauses 11 to 24,
wherein D is a doxorubicin.
[0057] 38. The method or use of any one of clauses 1 to 9 or 11 to
37, wherein the ligand conjugate is a folate conjugate.
[0058] 39. The method or use of clause 38, wherein the folate
conjugate is
##STR00004##
[0059] 40. The method or use of clause 38, wherein the folate
conjugate is
##STR00005##
[0060] 41. The method or use of any one of clauses 1 to 40, wherein
the thiol inhibitor is selected from the group consisting of
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g.,
N-maleoyl-1'-alanine (N-(2-carboxyethyl) maleimide (NCEM));
p-chloromercuribenzene sulfonate (pCMBS);
4-(N--(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO);
2,2'-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione
(GSSG); vinyl sulfone compounds (e.g.,
methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS).
[0061] 42. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is DTNB.
[0062] 43. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is a maleimide.
[0063] 44. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is NCEM.
[0064] 45. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is pCMBS.
[0065] 46. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is GSAO.
[0066] 47. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is dimesna.
[0067] 48. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is GSSG.
[0068] 49. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is a vinyl sulfone compound.
[0069] 50. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is methoxy-PEG5000-vinylsulfone.
[0070] 51. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is EGCG.
[0071] 52. The method or use of any one of clauses 1 to 41, wherein
the thiol inhibitor is AMS.
[0072] 53. The method or use of any one of clauses 1 to 52, wherein
the ligand conjugate and the thiol inhibitor are in parenteral
dosage forms.
[0073] 54. The method or use of clause 53, wherein the dosage forms
are independently selected from the group consisting of
intradermal, subcutaneous, intramuscular, intraperitoneal,
intravenous, and intrathecal.
[0074] 55. The method or use of any one of clauses 1 to 54, wherein
the ligand conjugate is in a composition and the thiol inhibitor is
in a composition and wherein the compositions further comprise
pharmaceutically acceptable carriers.
[0075] 56. The method or use of clause 55, wherein the
pharmaceutically acceptable carriers are liquid carriers.
[0076] 57. The method or use of clause 56, wherein the liquid
carriers are independently selected from the group consisting of
saline, glucose, alcohols, glycols, esters, amides, and a
combination thereof.
[0077] 58. The method or use of any one of clauses 1 to 57, wherein
the ligand conjugate and the thiol inhibitor are administered in
therapeutically effective amounts.
[0078] 59. The method or use of clause 58, wherein the effective
amounts range from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of
body surface area.
[0079] 60. The method or use of clause 58, wherein the effective
amounts range from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of
body surface area.
[0080] 61. The method or use of clause 58, wherein the effective
amounts range from about 10 .mu.g/kg to about 100 .mu.g/kg of
patient body weight.
[0081] 62. The method or use of any one of clauses 1 to 61, wherein
the ligand conjugate and the thiol inhibitor are in sterile
containers or packages.
[0082] 63. The method or use of any one of clauses 1 to 62, wherein
the ligand conjugate and the thiol inhibitor have a purity of at
least 90% based on weight percentage.
[0083] 64. The method or use of any one of clauses 4 to 63, wherein
the ligand conjugate is in the form of a reconstitutable
lyophilizate.
[0084] 65. The method or use of any one of clauses 1 to 64, wherein
the ligand conjugate and the thiol inhibitor are in sterile,
pyrogen-free aqueous solutions.
[0085] 66. A kit comprising a ligand conjugate and one or more
thiol inhibitors, wherein the ligand conjugate comprises a
disulfide linkage.
[0086] 67. The kit of clause 66, wherein the ligand is folate.
[0087] 68. The kit of clause 66, wherein the ligand is an
antibody.
[0088] 69. The kit of clause 66, wherein the ligand conjugate is of
the formula BLD.sub.X, wherein B is a cell surface receptor
targeting ligand, D is an independently selected drug, x is an
integer selected from 1, 2, 3, 4 and 5; and L is a releasable
polyvalent linker comprising a thiol reactive linkage; or a
pharmaceutically acceptable salt thereof.
[0089] 70. The kit of clause 69, wherein B is folate.
[0090] 71. The kit of clause 69, wherein B is D-folate.
[0091] 72. The kit of clause 69, wherein B is L-folate.
[0092] 73. The kit of clause 69, wherein B is a PSMA binding
ligand.
[0093] 74. The kit of clause 69, wherein B is a radical of the
formula
##STR00006##
[0094] 75. The kit of clause 69, wherein B is a radical of the
formula
##STR00007##
[0095] 76. The kit of clause 69, wherein B is a radical of the
formula
##STR00008##
[0096] 77. The kit of any one of clauses 69 to 76, wherein the
thiol reactive linkage is a disulfide linkage.
[0097] 78. The kit of any one of clauses 69 to 77, wherein L
comprises a cysteine disulfide diradical.
[0098] 79. The kit of any one of clauses 69 to 78, wherein L
further comprises one or more divalent hydrophilic radicals.
[0099] 80. The kit of any one of clauses 69 to 79, wherein D is a
cytotoxic agent.
[0100] 81. The kit of any one of clauses 69 to 80, wherein D is a
cancer treating agent.
[0101] 82. The kit of any one of clauses 69 to 80, wherein D is an
anti-inflammatory agent.
[0102] 83. The kit of any one of clauses 69 to 82, wherein D is a
vinca alkaloid.
[0103] 84. The kit of any one of clauses 69 to 82, wherein D is
desacetylvinblastine monohydrazide.
[0104] 85. The kit of any one of clauses 69 to 82, wherein D is a
tubulysin.
[0105] 86. The kit of any one of clauses 69 to 82, wherein D is
tubulysin A.
[0106] 87. The kit of any one of clauses 69 to 82, wherein D is
tubulysin B.
[0107] 88. The kit of any one of clauses 69 to 82, wherein D is
tubulysin A hydrazide.
[0108] 89. The kit of any one of clauses 69 to 82, wherein D is
tubulysin B hydrazide.
[0109] 90. The kit of any one of clauses 69 to 82, wherein D is an
antifolate.
[0110] 91. The kit of any one of clauses 69 to 82, wherein D is an
aminopterin.
[0111] 92. The kit of any one of clauses 69 to 82, wherein D is a
rapamycin.
[0112] 93. The kit of any one of clauses 69 to 82, wherein D is a
mitomycin.
[0113] 94. The kit of any one of clauses 69 to 82, wherein D is a
taxane.
[0114] 95. The kit of any one of clauses 69 to 82, wherein D is a
doxorubicin.
[0115] 96. The kit of any one of clauses 66 to 67 or 69 to 95,
wherein the ligand conjugate is a folate conjugate.
[0116] 97. The kit of clause 96, wherein the folate conjugate
is
##STR00009##
[0117] 98. The kit of clause 96, wherein the folate conjugate
is
##STR00010##
[0118] 99. The kit of any one of clauses 66 to 98, wherein the
thiol inhibitor is selected from the group consisting of
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g.,
N-maleoyl-1'-alanine (N-(2-carboxyethyl)maleimide (NCEM));
p-chloromercuribenzene sulfonate (pCMBS);
4-(N--(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO);
2,2'-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione
(GSSG); vinyl sulfone compounds (e.g.,
methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS).
[0119] 100. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is DTNB.
[0120] 101. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is a maleimide.
[0121] 102. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is NCEM.
[0122] 103. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is pCMBS.
[0123] 104. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is GSAO.
[0124] 105. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is dimesna.
[0125] 106. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is GSSG.
[0126] 107. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is a vinyl sulfone compound.
[0127] 108. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is methoxy-PEG5000-vinylsulfone.
[0128] 109. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is EGCG.
[0129] 110. The kit of any one of clauses 66 to 99, wherein the
thiol inhibitor is AMS.
[0130] 111. The kit of any one of clauses 66 to 110, wherein the
ligand conjugate and the thiol inhibitor are in parenteral dosage
forms.
[0131] 112. The kit of clause 111, wherein the dosage forms are
independently selected from the group consisting of intradermal,
subcutaneous, intramuscular, intraperitoneal, intravenous, and
intrathecal.
[0132] 113. The kit of any one of clauses 66 to 112, wherein the
ligand conjugate is in a composition and the thiol inhibitor is in
a composition and wherein the compositions further comprise
pharmaceutically acceptable carriers.
[0133] 114. The kit of clause 113, wherein the pharmaceutically
acceptable carriers are liquid carriers.
[0134] 115. The kit of clause 114, wherein the liquid carriers are
independently selected from the group consisting of saline,
glucose, alcohols, glycols, esters, amides, and a combination
thereof.
[0135] 116. The kit of any one of clauses 66 to 115, wherein the
ligand conjugate and the thiol inhibitor are in therapeutically
effective amounts.
[0136] 117. The kit of any one of clauses 66 to 116, wherein the
ligand conjugate and the thiol inhibitor are in sterile containers
or packages.
[0137] 118. The kit of any one of clauses 66 to 117, wherein the
ligand conjugate and the thiol inhibitor have a purity of at least
90% based on weight percentage.
[0138] 119. The kit of any one of clauses 66 to 117, wherein the
ligand conjugate and the thiol inhibitor have a purity of at least
95% based on weight percentage.
[0139] 120. The kit of any one of clauses 66 to 119, wherein the
ligand conjugate is in the form of a lyophilizate.
[0140] 121. The kit of any one of clauses 66 to 120, wherein the
ligand conjugate is in the form of a reconstitutable
lyophilizate.
[0141] 122. The kit of any one of clauses 66 to 121, wherein the
ligand conjugate and the thiol inhibitor are in sterile,
pyrogen-free aqueous solutions.
[0142] 123. An in vitro assay for identifying a ligand conjugate
suitable for co-administration to a patient with a thiol inhibitor,
the assay comprising:
[0143] a) adding the ligand conjugate to the culture medium of a
first sample of cultured cells, wherein the ligand conjugate
comprises a disulfide linkage;
[0144] b) adding the thiol inhibitor to the culture medium of the
first sample of cultured cells to provide a test sample;
[0145] c) adding the ligand conjugate to the culture medium of a
second sample of cultured cells to provide a control sample;
[0146] d) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the test
sample;
[0147] e) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the control
sample; and
[0148] f) determining that the ligand conjugate is suitable for
co-administration to the patient with the thiol inhibitor if the
non-ligand-specific activity of the ligand conjugate and/or the
nonspecific uptake of the drug are decreased in the test sample
relative to the control sample.
[0149] 124. The in vitro assay of clause 123 further comprising
step g) administering the ligand conjugate and the thiol inhibitor
to the patient.
[0150] 125. The in vitro assay of clause 123 or clause 124 wherein
the ligand is folate.
[0151] 126. The in vitro assay of clause 123 or clause 124 wherein
the ligand is an antibody.
[0152] 127. The in vitro assay of clause 123 or clause 124, wherein
the ligand conjugate is of the formula BLD.sub.X, wherein B is a
cell surface receptor targeting ligand, D is an independently
selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and
L is a releasable polyvalent linker comprising a thiol reactive
linkage; or a pharmaceutically acceptable salt thereof.
[0153] 128. The in vitro assay of clause 127, wherein B is
folate.
[0154] 129. The in vitro assay of clause 127, wherein B is
D-folate.
[0155] 130. The in vitro assay of clause 127, wherein B is
L-folate.
[0156] 131. The in vitro assay of clause 127, wherein B is a PSMA
binding ligand.
[0157] 132. The in vitro assay of clause 127, wherein B is a
radical of the formula
##STR00011##
[0158] 133. The in vitro assay of clause 127, wherein B is a
radical of the formula
##STR00012##
[0159] 134. The in vitro assay of clause 127, wherein B is a
radical of the formula
##STR00013##
[0160] 135. The in vitro assay of any one of clauses 127 to 134,
wherein the thiol reactive linkage is a disulfide linkage.
[0161] 136. The in vitro assay of any one of clauses 127 to 135,
wherein L comprises a cysteine disulfide diradical.
[0162] 137. The in vitro assay of any one of clauses 127 to 136,
wherein L further comprises one or more divalent hydrophilic
radicals.
[0163] 138. The in vitro assay of any one of clauses 127 to 137,
wherein D is a cytotoxic agent.
[0164] 139. The in vitro assay of any one of clauses 127 to 138,
wherein D is a cancer treating agent.
[0165] 140. The in vitro assay of any one of clauses 127 to 138,
wherein D is an anti-inflammatory agent.
[0166] 141. The in vitro assay of any one of clauses 127 to 140,
wherein D is a vinca alkaloid.
[0167] 142. The in vitro assay of any one of clauses 127 to 140,
wherein D is desacetylvinblastine monohydrazide.
[0168] 143. The in vitro assay of any one of clauses 127 to 140,
wherein D is a tubulysin.
[0169] 144. The in vitro assay of any one of clauses 127 to 140,
wherein D is tubulysin A.
[0170] 145. The in vitro assay of any one of clauses 127 to 140,
wherein D is tubulysin B.
[0171] 146. The in vitro assay of any one of clauses 127 to 140,
wherein D is tubulysin A hydrazide.
[0172] 147. The in vitro assay of any one of clauses 127 to 140,
wherein D is tubulysin B hydrazide.
[0173] 148. The in vitro assay of any one of clauses 127 to 140,
wherein D is an antifolate.
[0174] 149. The in vitro assay of any one of clauses 127 to 140,
wherein D is an aminopterin.
[0175] 150. The in vitro assay of any one of clauses 127 to 140,
wherein D is a rapamycin.
[0176] 151. The in vitro assay of any one of clauses 127 to 140,
wherein D is a mitomycin.
[0177] 152. The in vitro assay of any one of clauses 127 to 140,
wherein D is a taxane.
[0178] 153. The in vitro assay of any one of clauses 127 to 140,
wherein D is a doxorubicin.
[0179] 154. The in vitro assay of any one of clauses 123 to 140,
wherein the ligand conjugate is a folate conjugate.
[0180] 155. The in vitro assay of clause 154, wherein the folate
conjugate is
##STR00014##
[0181] 156. The in vitro assay of clause 154, wherein the folate
conjugate is
##STR00015##
[0182] 157. The in vitro assay of any one of clauses 123 to 156,
wherein the thiol inhibitor is selected from the group consisting
of 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g.,
N-maleoyl-1'-alanine (N-(2-carboxyethyl)maleimide (NCEM));
p-chloromercuribenzene sulfonate (pCMBS);
4-(N--(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO);
2,2'-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione
(GSSG); vinyl sulfone compounds (e.g.,
methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS).
[0183] 158. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is DTNB.
[0184] 159. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is a maleimide.
[0185] 160. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is NCEM.
[0186] 161. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is pCMBS.
[0187] 162. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is GSAO.
[0188] 163. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is dimesna.
[0189] 164. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is GSSG.
[0190] 165. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is a vinyl sulfone compound.
[0191] 166. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is methoxy-PEG5000-vinylsulfone.
[0192] 167. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is EGCG.
[0193] 168. The in vitro assay of any one of clauses 123 to 157,
wherein the thiol inhibitor is AMS.
[0194] 169. The in vitro assay of any one of clauses 123 to 168,
wherein the cultured cells are KB cells.
[0195] 170. The in vitro assay of any one of clauses 123 to 168,
wherein the cultured cells are A549 cells.
[0196] 171. The in vitro assay of any one of clauses 123 to 170,
wherein the non-ligand-specific activity of the ligand conjugate is
decreased by the thiol inhibitor.
[0197] 172. The in vitro assay of clause 171, wherein the
non-ligand-specific activity is cytotoxicity.
[0198] 173. The in vitro assay of any one of clauses 123 to 170,
wherein the nonspecific uptake of the drug is decreased by the
thiol inhibitor.
[0199] 174. The in vitro assay of clause 173, wherein the
nonspecific uptake of the drug is measured using competition assays
in the presence and absence of an excess of non-radiolabeled
ligand.
[0200] 175. A method of treatment of a disease, the method
comprising the steps of:
[0201] administering a ligand conjugate to a patient, wherein the
ligand conjugate comprises a disulfide linkage; and
[0202] administering a system x.sub.c.sup.- inhibitor to the
patient.
[0203] 176. The method of clause 175, wherein the disease is cancer
or inflammation.
[0204] 177. Use of a ligand conjugate in combination with a system
x.sub.c.sup.- inhibitor for the treatment of a disease wherein the
disease is cancer or inflammation, and wherein the ligand conjugate
comprises a disulfide linkage.
[0205] 178. Use of a ligand conjugate for the manufacture of a
medicament for the treatment of a disease wherein the disease is
cancer or inflammation, and wherein the treatment comprises
treating a patient with the ligand conjugate in combination with a
system x.sub.c.sup.- inhibitor, wherein the ligand conjugate
comprises a disulfide linkage.
[0206] 179. The method or use of any one of clauses 175 to 178,
wherein the disease is inflammation.
[0207] 180. The method or use of any one of clauses 175 to 178,
wherein the disease is cancer.
[0208] 181. The method or use of clause 180, wherein the cancer
comprises a primary tumor.
[0209] 182. The method or use of clause 180, wherein the cancer
comprises metastatic tumor cells.
[0210] 183. The method or use of any one of clauses 175 to 182,
wherein the ligand is folate.
[0211] 184. The method or use of any one of clauses 175 to 182,
wherein the ligand is an antibody.
[0212] 185. The method or use of any one of clauses 175 to 182,
wherein the ligand conjugate is of the formula BLD.sub.X, wherein B
is a cell surface receptor targeting ligand, D is an independently
selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and
L is a releasable polyvalent linker comprising a thiol reactive
linkage; or a pharmaceutically acceptable salt thereof.
[0213] 186. The method or use of clause 185, wherein B is
folate.
[0214] 187. The method or use of clause 185, wherein B is
D-folate.
[0215] 188. The method or use of clause 185, wherein B is
L-folate.
[0216] 189. The method or use of clause 185, wherein B is a PSMA
binding ligand.
[0217] 190. The method or use of clause 185, wherein B is a radical
of the formula
##STR00016##
[0218] 191. The method or use of clause 185, wherein B is a radical
of the formula
##STR00017##
[0219] 192. The method or use of clause 185, wherein B is a radical
of the formula
##STR00018##
[0220] 193. The method or use of any one of clauses 185 to 192,
wherein the thiol reactive linkage is a disulfide linkage.
[0221] 194. The method or use of any one of clauses 185 to 193,
wherein L comprises a cysteine disulfide diradical.
[0222] 195. The method or use of any one of clauses 185 to 194,
wherein L further comprises one or more divalent hydrophilic
radicals.
[0223] 196. The method or use of any one of clauses 185 to 195,
wherein D is a cytotoxic agent.
[0224] 197. The method or use of any one of clauses 185 to 196,
wherein D is a cancer treating agent.
[0225] 198. The method or use of any one of clauses 185 to 196,
wherein D is an anti-inflammatory agent.
[0226] 199. The method or use of any one of clauses 185 to 198,
wherein D is a vinca alkaloid.
[0227] 200. The method or use of any one of clauses 185 to 198,
wherein D is desacetylvinblastine monohydrazide.
[0228] 201. The method or use of any one of clauses 185 to 198,
wherein D is a tubulysin.
[0229] 202. The method or use of any one of clauses 185 to 198,
wherein D is tubulysin A.
[0230] 203. The method or use of any one of clauses 185 to 198,
wherein D is tubulysin B.
[0231] 204. The method or use of any one of clauses 185 to 198,
wherein D is tubulysin A hydrazide.
[0232] 205. The method or use of any one of clauses 185 to 198,
wherein D is tubulysin B hydrazide.
[0233] 206. The method or use of any one of clauses 185 to 198,
wherein D is an antifolate.
[0234] 207. The method or use of any one of clauses 185 to 198,
wherein D is an aminopterin.
[0235] 208. The method or use of any one of clauses 185 to 198,
wherein D is a rapamycin.
[0236] 209. The method or use of any one of clauses 185 to 198,
wherein D is a mitomycin.
[0237] 210. The method or use of any one of clauses 185 to 198,
wherein D is a taxane.
[0238] 211. The method or use of any one of clauses 185 to 198,
wherein D is a doxorubicin.
[0239] 212. The method or use of any one of clauses 175 to 183 or
185 to 211, wherein the ligand conjugate is a folate conjugate.
[0240] 213. The method or use of clause 212, wherein the folate
conjugate is
##STR00019##
[0241] 214. The method or use of clause 212, wherein the folate
conjugate is
##STR00020##
[0242] 215. The method or use of any one of clauses 175 to 214,
wherein the system x.sub.c.sup.- inhibitor is selected from the
group consisting of sulfasalazine, glutamate; L-quisqualate;
(S)-4-carboxyphenylglycine (4-S-CPG); L-.alpha.-aminoadipic acid;
and L-homocysteic acid.
[0243] 216. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is sulfasalazine.
[0244] 217. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is glutamate.
[0245] 218. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is L-quisqualate.
[0246] 219. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is 4-S-CPG.
[0247] 220. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is L-.alpha.-aminoadipic
acid.
[0248] 221. The method or use of any one of clauses 175 to 215,
wherein the system x.sub.c.sup.- inhibitor is L-homocysteic
acid.
[0249] 222. The method or use of any one of clauses 175 to 221,
wherein the ligand conjugate and the system x.sub.c.sup.- inhibitor
are in parenteral dosage forms.
[0250] 223. The method or use of clause 222, wherein the dosage
forms are independently selected from the group consisting of
intradermal, subcutaneous, intramuscular, intraperitoneal,
intravenous, and intrathecal.
[0251] 224. The method or use of any one of clauses 175 to 223,
wherein the ligand conjugate is in a composition and the system
x.sub.c.sup.- inhibitor is in a composition and wherein the
compositions further comprise pharmaceutically acceptable
carriers.
[0252] 225. The method or use of clause 224, wherein the
pharmaceutically acceptable carriers are liquid carriers.
[0253] 226. The method or use of clause 225, wherein the liquid
carriers are independently selected from the group consisting of
saline, glucose, alcohols, glycols, esters, amides, and a
combination thereof.
[0254] 227. The method or use of any one of clauses 175 to 226,
wherein the ligand conjugate and the system x.sub.c.sup.- inhibitor
are administered in therapeutically effective amounts.
[0255] 228. The method or use of clause 227, wherein the effective
amounts range from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of
body surface area.
[0256] 229. The method or use of clause 227, wherein the effective
amounts range from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of
body surface area.
[0257] 230. The method or use of clause 227, wherein the effective
amounts range from about 10 .mu.g/kg to about 100 .mu.g/kg of
patient body weight.
[0258] 231. The method or use of any one of clauses 175 to 230,
wherein the ligand conjugate and the system x.sub.c.sup.- inhibitor
are in sterile containers or packages.
[0259] 232. The method or use of any one of clauses 175 to 231,
wherein the ligand conjugate and the system x.sub.c.sup.- inhibitor
have a purity of at least 90% based on weight percentage.
[0260] 233. The method or use of any one of clauses 178 to 232,
wherein the ligand conjugate is in the form of a reconstitutable
lyophilizate.
[0261] 234. The method or use of any one of clauses 175 to 233,
wherein the ligand conjugate and the system x.sub.c.sup.- inhibitor
are in sterile, pyrogen-free aqueous solutions.
[0262] 235. A kit comprising a ligand conjugate and one or more
system x.sub.c.sup.- inhibitors, wherein the ligand conjugate
comprises a disulfide linkage.
[0263] 236. The kit of clause 235, wherein the ligand is
folate.
[0264] 237. The kit of clause 235, wherein the ligand is an
antibody.
[0265] 238. The kit of clause 235, wherein the ligand conjugate is
of the formula BLD.sub.X, wherein B is a cell surface receptor
targeting ligand, D is an independently selected drug, x is an
integer selected from 1, 2, 3, 4 and 5; and L is a releasable
polyvalent linker comprising a thiol reactive linkage; or a
pharmaceutically acceptable salt thereof.
[0266] 239. The kit of clause 238, wherein B is folate.
[0267] 240. The kit of clause 238, wherein B is D-folate.
[0268] 241. The kit of clause 238, wherein B is L-folate.
[0269] 242. The kit of clause 238, wherein B is a PSMA binding
ligand.
[0270] 243. The kit of clause 238, wherein B is a radical of the
formula
##STR00021##
[0271] 244. The kit of clause 238, wherein B is a radical of the
formula
##STR00022##
[0272] 245. The kit of clause 238, wherein B is a radical of the
formula
##STR00023##
[0273] 246. The kit of any one of clauses 238 to 245, wherein the
thiol reactive linkage is a disulfide linkage.
[0274] 247. The kit of any one of clauses 238 to 246, wherein L
comprises a cysteine disulfide diradical.
[0275] 248. The kit of any one of clauses 238 to 247, wherein L
further comprises one or more divalent hydrophilic radicals.
[0276] 249. The kit of any one of clauses 238 to 248, wherein D is
a cytotoxic agent.
[0277] 250. The kit of any one of clauses 238 to 249, wherein D is
a cancer treating agent.
[0278] 251. The kit of any one of clauses 238 to 249, wherein D is
an anti-inflammatory agent.
[0279] 252. The kit of any one of clauses 238 to 251, wherein D is
a vinca alkaloid.
[0280] 253. The kit of any one of clauses 238 to 251, wherein D is
desacetylvinblastine monohydrazide.
[0281] 254. The kit of any one of clauses 238 to 251, wherein D is
a tubulysin.
[0282] 255. The kit of any one of clauses 238 to 251, wherein D is
tubulysin A.
[0283] 256. The kit of any one of clauses 238 to 251, wherein D is
tubulysin B.
[0284] 257. The kit of any one of clauses 238 to 251, wherein D is
tubulysin A hydrazide.
[0285] 258. The kit of any one of clauses 238 to 251, wherein D is
tubulysin B hydrazide.
[0286] 259. The kit of any one of clauses 238 to 251, wherein D is
an antifolate.
[0287] 260. The kit of any one of clauses 238 to 251, wherein D is
an aminopterin.
[0288] 261. The kit of any one of clauses 238 to 251, wherein D is
a rapamycin.
[0289] 262. The kit of any one of clauses 238 to 251, wherein D is
a mitomycin.
[0290] 263. The kit of any one of clauses 238 to 251, wherein D is
a taxane.
[0291] 264. The kit of any one of clauses 238 to 251, wherein D is
a doxorubicin.
[0292] 265. The kit of any one of clauses 235 to 236 or 238 to 264,
wherein the ligand conjugate is a folate conjugate.
[0293] 266. The kit of clause 265, wherein the folate conjugate
is
##STR00024##
[0294] 267. The kit of clause 265, wherein the folate conjugate
is
##STR00025##
[0295] 268. The kit of any one of clauses 235 to 267, wherein the
system x.sub.c.sup.- inhibitor is selected from the group
consisting of sulfasalazine, glutamate; L-quisqualate;
(S)-4-carboxyphenylglycine (4-S-CPG); L-.alpha.-aminoadipic acid;
and L-homocysteic acid.
[0296] 269. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is sulfasalazine.
[0297] 270. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is glutamate.
[0298] 271. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is L-quisqualate.
[0299] 272. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is 4-S-CPG.
[0300] 273. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is L-.alpha.-aminoadipic acid.
[0301] 274. The kit of any one of clauses 235 to 268, wherein the
system x.sub.c.sup.- inhibitor is L-homocysteic acid.
[0302] 275. The kit of any one of clauses 235 to 274, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor are in
parenteral dosage forms.
[0303] 276. The kit of clause 275, wherein the dosage forms are
independently selected from the group consisting of intradermal,
subcutaneous, intramuscular, intraperitoneal, intravenous, and
intrathecal.
[0304] 277. The kit of any one of clauses 235 to 276, wherein the
ligand conjugate is in a composition and the system x.sub.c.sup.-
inhibitor is in a composition and wherein the compositions further
comprise pharmaceutically acceptable carriers.
[0305] 278. The kit of clause 277, wherein the pharmaceutically
acceptable carriers are liquid carriers.
[0306] 279. The kit of clause 278, wherein the liquid carriers are
independently selected from the group consisting of saline,
glucose, alcohols, glycols, esters, amides, and a combination
thereof.
[0307] 280. The kit of any one of clauses 235 to 279, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor are in
therapeutically effective amounts.
[0308] 281. The kit of any one of clauses 235 to 280, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor are in
sterile containers or packages.
[0309] 282. The kit of any one of clauses 235 to 281, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor have a
purity of at least 90% based on weight percentage.
[0310] 283. The kit of any one of clauses 235 to 281, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor have a
purity of at least 95% based on weight percentage.
[0311] 284. The kit of any one of clauses 235 to 283, wherein the
ligand conjugate is in the form of a lyophilizate.
[0312] 285. The kit of any one of clauses 235 to 284, wherein the
ligand conjugate is in the form of a reconstitutable
lyophilizate.
[0313] 286. The kit of any one of clauses 235 to 285, wherein the
ligand conjugate and the system x.sub.c.sup.- inhibitor are in
sterile, pyrogen-free aqueous solutions.
[0314] 287. An in vitro assay for identifying a ligand conjugate
suitable for co-administration to a patient with a system
x.sub.c.sup.- inhibitor, the assay comprising:
[0315] a) adding the ligand conjugate to the culture medium of a
first sample of cultured cells, wherein the ligand conjugate
comprises a disulfide linkage;
[0316] b) adding the system x.sub.c.sup.- inhibitor to the culture
medium of the first sample of cultured cells to provide a test
sample;
[0317] c) adding the ligand conjugate to the culture medium of a
second sample of cultured cells to provide a control sample;
[0318] d) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the test
sample;
[0319] e) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the control
sample; and
[0320] f) determining that the ligand conjugate is suitable for
co-administration to the patient with the system x.sub.c.sup.-
inhibitor if the non-ligand-specific activity of the ligand
conjugate and/or the nonspecific uptake of the drug are decreased
in the test sample relative to the control sample.
[0321] 288. The in vitro assay of clause 287 further comprising
step g) administering the ligand conjugate and the system
x.sub.c.sup.- inhibitor to the patient.
[0322] 289. The in vitro assay of clause 287 or clause 288 wherein
the ligand is folate.
[0323] 290. The in vitro assay of clause 287 or clause 288 wherein
the ligand is an antibody.
[0324] 291. The in vitro assay of clause 287 or clause 288, wherein
the ligand conjugate is of the formula BLD.sub.X, wherein B is a
cell surface receptor targeting ligand, D is an independently
selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and
L is a releasable polyvalent linker comprising a thiol reactive
linkage; or a pharmaceutically acceptable salt thereof.
[0325] 292. The in vitro assay of clause 291, wherein B is
folate.
[0326] 293. The in vitro assay of clause 291, wherein B is
D-folate.
[0327] 294. The in vitro assay of clause 291, wherein B is
L-folate.
[0328] 295. The in vitro assay of clause 291, wherein B is a PSMA
binding ligand.
[0329] 296. The in vitro assay of clause 291, wherein B is a
radical of the formula
##STR00026##
[0330] 297. The in vitro assay of clause 291, wherein B is a
radical of the formula
##STR00027##
[0331] 298. The in vitro assay of clause 291, wherein B is a
radical of the formula
##STR00028##
[0332] 299. The in vitro assay of any one of clauses 291 to 298,
wherein the thiol reactive linkage is a disulfide linkage.
[0333] 300. The in vitro assay of any one of clauses 291 to 299,
wherein L comprises a cysteine disulfide diradical.
[0334] 301. The in vitro assay of any one of clauses 291 to 300,
wherein L further comprises one or more divalent hydrophilic
radicals.
[0335] 302. The in vitro assay of any one of clauses 291 to 301,
wherein D is a cytotoxic agent.
[0336] 303. The in vitro assay of any one of clauses 291 to 302,
wherein D is a cancer treating agent.
[0337] 304. The in vitro assay of any one of clauses 291 to 302,
wherein D is an anti-inflammatory agent.
[0338] 305. The in vitro assay of any one of clauses 291 to 304,
wherein D is a vinca alkaloid.
[0339] 306. The in vitro assay of any one of clauses 291 to 304,
wherein D is desacetylvinblastine monohydrazide.
[0340] 307. The in vitro assay of any one of clauses 291 to 304,
wherein D is a tubulysin.
[0341] 308. The in vitro assay of any one of clauses 291 to 304,
wherein D is tubulysin A.
[0342] 309. The in vitro assay of any one of clauses 291 to 304,
wherein D is tubulysin B.
[0343] 310. The in vitro assay of any one of clauses 291 to 304,
wherein D is tubulysin A hydrazide.
[0344] 311. The in vitro assay of any one of clauses 291 to 304,
wherein D is tubulysin B hydrazide.
[0345] 312. The in vitro assay of any one of clauses 291 to 304,
wherein D is an antifolate.
[0346] 313. The in vitro assay of any one of clauses 291 to 304,
wherein D is an aminopterin.
[0347] 314. The in vitro assay of any one of clauses 291 to 304,
wherein D is a rapamycin.
[0348] 315. The in vitro assay of any one of clauses 291 to 304,
wherein D is a mitomycin.
[0349] 316. The in vitro assay of any one of clauses 291 to 304,
wherein D is a taxane.
[0350] 317. The in vitro assay of any one of clauses 291 to 304,
wherein D is a doxorubicin.
[0351] 318. The in vitro assay of any one of clauses 287 to 290,
wherein the ligand conjugate is a folate conjugate.
[0352] 319. The in vitro assay of clause 318, wherein the folate
conjugate is
##STR00029##
[0353] 320. The in vitro assay of clause 318, wherein the folate
conjugate is
##STR00030##
[0354] 321. The in vitro assay of any one of clauses 287 to 320,
wherein the system x.sub.c.sup.- inhibitor is selected from the
group consisting of sulfasalazine, glutamate; L-quisqualate;
(S)-4-carboxyphenylglycine (4-S-CPG); L-.alpha.-aminoadipic acid;
and L-homocysteic acid.
[0355] 322. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is sulfasalazine.
[0356] 323. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is glutamate.
[0357] 324. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is L-quisqualate.
[0358] 325. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is 4-S-CPG.
[0359] 326. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is L-.alpha.-aminoadipic
acid.
[0360] 327. The in vitro assay of any one of clauses 287 to 321,
wherein the system x.sub.c.sup.- inhibitor is L-homocysteic
acid.
[0361] 328. The in vitro assay of any one of clauses 287 to 327,
wherein the cultured cells are KB cells.
[0362] 329. The in vitro assay of any one of clauses 287 to 327,
wherein the cultured cells are A549 cells.
[0363] 330. The in vitro assay of any one of clauses 287 to 329,
wherein the non-ligand-specific activity of the ligand conjugate is
decreased by the system x.sub.c.sup.- inhibitor.
[0364] 331. The in vitro assay of clause 330, wherein the
non-ligand-specific activity is cytotoxicity.
[0365] 332. The in vitro assay of any one of clauses 287 to 330,
wherein the nonspecific uptake of the drug is decreased by the
system x.sub.c.sup.- inhibitor.
[0366] 333. The in vitro assay of clause 332, wherein the
nonspecific uptake of the drug is measured using competition assays
in the presence and absence of an excess of non-radiolabeled
ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0367] FIG. 1 shows the amount of extracellular thiol activity
(nmol of thiol/mg of protein) per conditioning time in conditioned
culture media over an 8 hour period. Data obtained for KB cells
(closed circles) and A549 cells (closed squares) are shown.
[0368] FIG. 2 shows the amount of extracellular thiol activity
(nmol of thiol/mg of protein) for various cell lines, as evaluated
using RPMI cell culture media and thiol-free (SH-free) RPMI cell
culture media. Data obtained for each cell line shown on the X-axis
incubated in RPMI medium are shown (left-hand bar in each set of
two bars), and data obtained for each cell line using thiol-free
RPMI cell culture media are also shown (right-hand bar in each set
of two bars).
[0369] FIG. 3 shows the correlation between the amount of
extracellular thiol activity (nmol of thiol/mg of protein) and the
non-specific activity of the folate conjugate EC0531 (IC.sub.50
(nM), adjusted for tubulysin B hydrazide sensitivity) in various
cell lines.
[0370] FIG. 4 shows the specific and non-specific activity of
EC0531 (% .sup.3H-Thymidine incorporation measured in counts per
minute (CPM)) at varying concentrations of EC0531 (Log M) applied
to KB cells or A549 cells. The specific activity of EC0531
administered to KB cells is shown (closed circles). The
non-specific activity of EC0531 in the presence of excess folic
acid administered to KB cells is also shown (open circles), along
with the specific activity of EC0531 administered to A549 cells
(closed squares).
[0371] FIG. 5 shows the percent inhibition of non-FR-specific
activity of EC0531 (% .sup.3H-Thymidine incorporation measured in
counts per minute (CPM)) following co-administration of thiol
inhibitors and the folate conjugate EC0531 to the culture medium of
KB cells. Data obtained using the thiol inhibitors DTNB (closed
circles), NCEM (closed squares), and folate-maleimide (closed
triangles) are shown.
[0372] FIG. 6 shows the lack of inhibition of non-FR-specific
activity of EC0531 (% .sup.3H-Thymidine incorporation measured in
counts per minute (CPM)) by pre-treating KB cells with thiol
inhibitors prior to administration of both EC0531 and excess folic
acid. The first bar shows data obtained with no pretreatment of KB
cells with thiol inhibitors prior to administration of EC0531 and
folic acid. The second bar shows data obtained with pretreatment of
KB cells with DTNB prior to administration of EC0531 and excess
folic acid. The third bar shows data obtained with pretreatment of
KB cells with NCEM prior to administration of EC0531 and excess
folic acid.
[0373] FIG. 7 shows the amount of uptake of .sup.3H-EC0531
(molecules per cell.times.10.sup.6) at various concentrations of
administered conjugates in both KB cells and A549 cells. FIG. 7(a)
shows the uptake of .sup.3H-EC0531 compared to the uptake of
.sup.3H-folic acid in KB cells. FIG. 7(b) shows the uptake of
.sup.3H-EC0531 compared to the uptake of .sup.3H-folic acid in A549
cells. Data obtained with .sup.3H-EC0531 (closed squares),
.sup.3H-EC0531 plus excess folic acid (open squares), .sup.3H-folic
acid (closed triangles) and .sup.3H-folic acid plus excess folic
acid (open triangles) are shown.
[0374] FIG. 8 shows the percent uptake of .sup.3H-EC0531 following
co-administration of thiol inhibitors and the folate conjugate
EC0531 in both KB cells and A549 cells. Left-hand set of bars in
each graph--The open bars show data obtained with no thiol
inhibitor administered. The middle bar in each set of three data
points shows data obtained with co-administraton of 10 .mu.M DTNB
and .sup.3H-EC0531. The last bar in each set of three data points
shows data obtained with co-administraton of 100 .mu.M DTNB and
.sup.3H-EC0531. Right-hand set of bars in each graph--The open bars
show data obtained with no thiol inhibitor administered. The middle
bar in each set of three data points shows data obtained with
co-administraton of 10 .mu.M DTNB and .sup.3H-folate and excess
folic acid. The last bar in each set of three data points shows
data obtained with co-administraton of 100 .mu.M DTNB and
.sup.3H-folate and excess folic acid.
[0375] FIG. 9 shows the percent uptake of .sup.3H-EC0531 following
co-administration of thiol inhibitors and the folate conjugate
EC0531 in KB cells (with excess folic acid for the left-hand graph)
and A549 cells. In addition, the graphs show the percent uptake of
.sup.3H-EC0531 following co-administration of methotrexate and
EC0531 in both KB cells and A549 cells under the above-described
conditions.
[0376] FIG. 10 shows the non-FR-specific activity of EC0531 (%
.sup.3H-Thymidine incorporation measured in counts per minute
(CPM)) in KB cells following replacement of cell culture medium in
the cell culture under the above-described conditions. The
non-specific activity of EC0531 administered to KB cells is shown
in the left pair of bars. The specific activity of EC0531
administered to KB cells in the presence of excess folic acid is
shown in the right pair of bars. Data obtained using a two hour
pulse (i.e., no replacement of cell culture medium) are shown in
the left-most bars in each pair. Data obtained using four separate
30 minute pulses (i.e., four replacements of cell culture medium)
are shown in the right-most bars in each pair.
[0377] FIG. 11 shows the amount of extracellular thiol (i.e., SH
concentration (.mu.M)) for A549 cells following addition of GSSG
and/or cysteine. The first bar shows data obtained with thiol-free
RPMI cell culture media (SH-free RPMI) with no addition of thiol.
The second bar shows data obtained with SH-free RPMI cell culture
media with addition of GSSG (1.6 .mu.M). The third bar shows data
obtained with SH-free RPMI cell culture media with addition of
cysteine (189 .mu.M). The fourth bar shows data obtained with
SH-free RPMI cell culture media with addition of GSSG (1.6 .mu.M)
and cysteine (189 .mu.M). The fifth bar shows data obtained with
RPMI cell culture media containing thiols (SH-containing RPMI).
[0378] FIG. 12 shows the amount of extracellular thiol (i.e., SH
concentration (.mu.M)) in A549 cells following addition of system
x.sub.c.sup.- inhibitors. The first bar shows data obtained with no
addition of system x.sub.c.sup.- inhibitor. The second bar shows
data obtained with addition of glutamate (5 mM). The third bar
shows data obtained with addition of quisqualic acid (0.3 mM). The
fourth bar shows data obtained with addition of quisqualic acid (1
mM).
[0379] FIG. 13 shows the percent inhibition of non-specific
activity of EC0531 (% .sup.3H-Thymidine incorporation measured in
counts per minute (CPM)) following co-administration of varying
concentrations of the system x.sub.c.sup.- inhibitor sulfasalazine
and one concentration of the folate conjugate EC0531 along with
excess folic acid to the culture medium of KB cells.
[0380] FIG. 14 shows the percent inhibition of non-specific
activity of EC0531 (% .sup.3H-Thymidine incorporation measured in
counts per minute (CPM)) following co-administration of system
x.sub.c.sup.- inhibitors and varying concentrations of the folate
conjugate EC0531 to the culture medium of A549 cells. The specific
activity of EC0531 is shown (closed squares). Data obtained using
the system x.sub.c.sup.- inhibitors glutamate (closed triangles,
uppermost line with an inhibitor) and sulfasalazine (closed
triangles, lowermost line with an inhibitor) are also shown.
[0381] FIG. 15 shows the percent uptake of .sup.3H-EC0531 following
co-administration of the system x.sub.c.sup.- inhibitor
sulfasalazine and the folate conjugate EC0531 in the presence of
excess folic acid for KB cells and without excess folic acid for
A549 cells. In addition, the figure shows the percent uptake of
.sup.3H-EC0531 following co-administration of methotrexate (MTX)
and EC0531 for both types of cells, as well as the percent uptake
of .sup.3H-EC0531 following co-administration of probenecid and
EC0531 for both types of cells.
[0382] FIG. 16 shows the amount of extracellular thiol activity
(percentage of UTC (nmol of thiol/mg protein)) in KB cells and in
A549 cells following addition of xCT siRNA to the cell culture
medium. The extracellular thiol activity in A549 cells is shown in
the left pair of bars. The extracellular thiol activity for KB
cells is shown in the right pair of bars. The first bar in each
pair shows data for cells administered xCT siRNA. The second bar in
each pair shows data for cells administered a non-specific (NS)
siRNA.
DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
[0383] As used herein, the term "thiol inhibitor" means an agent
that inhibits disulfide reduction, for example by blocking free
thiols.
[0384] As used herein, the term "system x.sub.c.sup.- inhibitor"
means an agent that inhibits the cystine/glutamate antiporter of
cells.
[0385] For all of the embodiments described herein, any applicable
combination of embodiments is contemplated. Any applicable
combination of the embodiments described below is considered to be
in accordance with the invention. Any combination of the
embodiments described below with the embodiments described in the
Summary of Invention section, including the clause list, is
considered to be part of the invention.
[0386] In one embodiment described herein, a method of treatment of
a disease is provided. The method comprises administering a ligand
conjugate to a patient, wherein the ligand conjugate comprises a
disulfide linkage; and administering a thiol inhibitor to the
patient.
[0387] In illustrative embodiments, the disease can be cancer or
inflammation.
[0388] In another embodiment, use of a ligand conjugate in
combination with a thiol inhibitor for the treatment of a disease
is described, wherein the disease is cancer or inflammation, and
wherein the ligand conjugate comprises a disulfide linkage.
[0389] In yet another embodiment, use of a ligand conjugate for the
manufacture of a medicament for the treatment of a disease is
described, wherein the disease is cancer or inflammation, and
wherein the treatment comprises treating a patient with the ligand
conjugate in combination with a thiol inhibitor, wherein the ligand
conjugate comprises a disulfide linkage.
[0390] In another embodiment, a kit is provided. The kit comprises
a ligand conjugate and one or more thiol inhibitors, wherein the
ligand conjugate comprises a disulfide linkage.
[0391] In various embodiments of the methods and uses described
herein, the disease is inflammation. In other embodiments, the
disease is cancer. In some embodiments, the cancer comprises a
primary tumor. In yet other embodiments, the cancer comprises
metastatic tumor cells. The methods and uses described herein can
be utilized to treat such cancers as carcinomas, sarcomas,
lymphomas, Hodgekin's disease, melanomas, mesotheliomas, Burkitt's
lymphoma, nasopharyngeal carcinomas, leukemias, and myelomas. The
cancers can also be oral, thyroid, endocrine, skin, gastric,
esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian,
cervical, uterine, breast, testicular, prostate, rectal, kidney,
liver, or lung cancers.
[0392] Illustratively, the conjugates described herein can be
prepared using synthetic procedures described in WO2007/022493,
WO2007/022494, WO2008/101231, WO2008/112873, WO2011/069116,
WO2010/033733, WO2012/047525, WO2003/097647, WO2009/002993,
WO2004/069159, WO2009/026177, and WO2011/106639, the contents of
each which are incorporated by reference herein in its
entirety.
[0393] Acceptable ligands include folate, folic acid, analogs of
folic acid and other folate receptor-binding molecules, other
vitamins, peptide ligands identified from library screens,
tumor-specific peptides, tumor-specific aptamers, tumor-specific
carbohydrates, antibodies, tumor-specific monoclonal or polyclonal
antibodies, Fab or scFv (i.e., a single chain variable region)
fragments of antibodies such as, for example, an Fab fragment of an
antibody, small organic molecules derived from combinatorial
libraries, growth factors, such as EGF, FGF, insulin, and
insulin-like growth factors, and homologous polypeptides,
somatostatin and its analogs, transferrin, lipoprotein complexes,
bile salts, selectins, steroid hormones, Arg-Gly-Asp containing
peptides, retinoids, various Galectins, .delta.-opioid receptor
ligands, cholecystokinin A receptor ligands, ligands specific for
angiotensin ATI or AT2 receptors, peroxisome proliferator-activated
receptor .gamma. ligands, .beta.-lactam antibiotics, small organic
molecules including antimicrobial drugs, and other molecules that
bind specifically to a receptor preferentially expressed on the
surface of tumor cells or inflammatory cells, or fragments of any
of these molecules.
[0394] Another acceptable ligand is a prostate-specific membrane
antigen (PSMA). PMSA is a biomarker that is overexpressed on
prostate cancer. PSMA is also expressed on the neovasculature
within many non-prostate solid tumors. PSMA is over-expressed in
malignant prostate tissues when compared to other organs in the
human body such as kidney, proximal small intestine, and salivary
glands.
[0395] In any embodiment described herein, a ligand conjugate of
the formula BLD.sub.X can be used, wherein B is a cell surface
receptor targeting ligand, D is an independently selected drug, x
is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable
polyvalent linker comprising a thiol reactive linkage; or a
pharmaceutically acceptable salt thereof.
[0396] Also described is an embodiment where in the preceding
embodiment, the thiol reactive linkage is a disulfide linkage.
[0397] Also described is an embodiment where in any of the
preceding embodiments, B is a folate receptor binding moiety.
[0398] Also described is an embodiment where in any of the
preceding embodiments, B is a folate.
[0399] Also described is an embodiment where in any of the
preceding embodiments, B is a folate comprising D-glutamyl.
[0400] Also described is an embodiment where in any of the
preceding embodiments, B is folate.
[0401] Also described is an embodiment where in any of the
preceding embodiments, B is an unnatural folate radical of the
formula
##STR00031##
[0402] Also described is an embodiment where in any of the
preceding embodiments, B is a folate radical of the formula
##STR00032##
[0403] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand.
[0404] Also described is an embodiment where in any of the
preceding embodiments, the PSMA ligand is a thiourea or a urea of
two amino acids.
[0405] Also described is an embodiment where in any of the
preceding embodiments, the PSMA ligand is a urea of two amino
acids.
[0406] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a thiourea or
urea of lysine and glutamate.
[0407] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a urea of lysine
and glutamate.
[0408] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a thiourea or
urea of glutamate and glutamate.
[0409] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a urea of
glutamate and glutamate.
[0410] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a thiourea or
urea of cysteine and glutamate.
[0411] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA ligand that is a urea of
cysteine and glutamate.
[0412] Also described is an embodiment where in any of the
preceding embodiments, B is a PSMA binding moiety.
[0413] Also described is an embodiment where in any of the
preceding embodiments, B is a radical of the formula
##STR00033##
[0414] Also described is an embodiment where in any of the
preceding embodiments, B is a radical of the formula
##STR00034##
[0415] Also described is an embodiment where in any of the
preceding embodiments, B is a radical of the formula
##STR00035##
[0416] Also described is an embodiment where in any of the
preceding embodiments, L comprises one or more aspartic acid
diradicals.
[0417] Also described is an embodiment where in any of the
preceding embodiments, L comprises one or more aspartic acid
diradicals.
[0418] Also described is an embodiment where in any of the
preceding embodiments, the aspartic acid diradicals are L-aspartic
acid diradicals.
[0419] Also described is an embodiment where in any of the
preceding embodiments, L comprises a cysteine diradical.
[0420] Also described is an embodiment where in any of the
preceding embodiments, L comprises a L-cysteine diradical.
[0421] Also described is an embodiment where in any of the
preceding embodiments, L comprises L-Asp-L-Asp-L-Cys.
[0422] Also described is an embodiment where in any of the
preceding embodiments, L is a releasable linker.
[0423] Also described is an embodiment where in any of the
preceding embodiments, L comprises a disulfide.
[0424] Also described is an embodiment where in any of the
preceding embodiments, L comprises a cysteine disulfide
diradical.
[0425] Also described is an embodiment where in any of the
preceding embodiments, L comprises a L-cysteine disulfide
diradical.
[0426] Also described is an embodiment where in any of the
preceding embodiments, L comprises L-Asp-L-Asp-L-Cys(S--S).
[0427] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
O--C(O)--N.
[0428] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
O--C(O)--NH.
[0429] Also described is an embodiment where in any of the
preceding embodiments, L and at least one D taken together comprise
a diradical of the formula O--C(O)--N.
[0430] Also described is an embodiment where in any of the
preceding embodiments, L and at least one D taken together comprise
a diradical of the formula O--C(O)--NH.
[0431] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
S--(CH.sub.2).sub.m--O, where m is 2, 3, or 4.
[0432] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
S--(CH.sub.2).sub.m--O--C(O)--N, where m is 2, 3, or 4.
[0433] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
S--(CH.sub.2).sub.m--O--C(O)--NH, where m is 2, 3, or 4.
[0434] Also described is an embodiment where in any of the
preceding embodiments, L and at least one D taken together comprise
a diradical of the formula S--(CH.sub.2).sub.m--O--C(O)--N, where m
is 2, 3, or 4.
[0435] Also described is an embodiment where in any of the
preceding embodiments, L and at least one D taken together comprise
a diradical of the formula S--(CH.sub.2).sub.m--O--C(O)--NH, where
m is 2, 3, or 4.
[0436] Also described is an embodiment where in any of the
preceding embodiments, m is 2.
[0437] Also described is an embodiment where in any of the
preceding embodiments, L comprises a chain of at least about 7
atoms, at least about 8 atoms, at least about 9 atoms, at least
about 10, atoms, at least about 11, atoms, at least about 12 atoms,
at least about 13 atoms, at least about 14 atoms, or at least about
15 atoms.
[0438] Also described is an embodiment where in any of the
preceding embodiments, L comprises a chain of at least about 16
atoms, at least about 17 atoms, at least about 18 atoms, at least
about 19, atoms, at least about 20, atoms, at least about 21 atoms,
at least about 22 atoms, at least about 23 atoms, at least about 24
atoms, at least about 25 atoms, or at least about 26 atoms.
[0439] Also described is an embodiment where in any of the
preceding embodiments, L comprises a chain of between about 7 and
about 35 atoms, between about 7 and about 30 atoms, or between
about 7 and about 26 atoms.
[0440] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
##STR00036##
[0441] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
##STR00037##
[0442] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
##STR00038##
[0443] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
##STR00039##
[0444] Also described is an embodiment where in any of the
preceding embodiments, L comprises at least one unnatural amino
acid which is selected from D-alanine, D-aspartic acid,
D-asparagine, D-cysteine, D-glutamic acid, D-phenylalanine,
D-histidine, D-isoleucine, D-lysine, D-leucine, D-methionine,
D-proline, D-glutamine, D-arginine, D-serine, D-threonine,
D-valine, D-tryptophan, D-tyrosine, and D-ornithine, or a
derivative thereof.
[0445] Also described is an embodiment where in any of the
preceding embodiments, L comprises two or more unnatural amino
acids.
[0446] Also described is an embodiment where in any of the
preceding embodiments, L comprises three or more unnatural amino
acids.
[0447] Also described is an embodiment where in any of the
preceding embodiments, L comprises four or more unnatural amino
acids.
[0448] Also described is an embodiment where in any of the
preceding embodiments, L further comprises one or more
disulfides.
[0449] Also described is an embodiment where in any of the
preceding embodiments, at least one disulfide comprises
D-cysteinyl.
[0450] Also described is an embodiment where in any of the
preceding embodiments, L further comprises one or more divalent
hydrophilic radicals.
[0451] Also described is an embodiment where in any of the
preceding embodiments, L further comprises two or more divalent
hydrophilic radicals.
[0452] Also described is an embodiment where in any of the
preceding embodiments, L further comprises three or more divalent
hydrophilic radicals.
[0453] Also described is an embodiment where in any of the
preceding embodiments, L further comprises four or more divalent
hydrophilic radicals.
[0454] Also described is an embodiment where in any of the
preceding embodiments, L further comprises one or more divalent
polyoxy radicals.
[0455] Also described is an embodiment where in any of the
preceding embodiments, L further comprises two or more divalent
polyoxy radicals.
[0456] Also described is an embodiment where in any of the
preceding embodiments, L further comprises three or more divalent
polyoxy radicals.
[0457] Also described is an embodiment where in any of the
preceding embodiments, L further comprises four or more divalent
polyoxy radicals.
[0458] Also described is an embodiment where in any of the
preceding embodiments, L further comprises one or more divalent
polyhydroxy radicals.
[0459] Also described is an embodiment where in any of the
preceding embodiments, L further comprises two or more divalent
polyhydroxy radicals.
[0460] Also described is an embodiment where in any of the
preceding embodiments, L further comprises three or more divalent
polyhydroxy radicals.
[0461] Also described is an embodiment where in any of the
preceding embodiments, L further comprises four or more divalent
polyhydroxy radicals.
[0462] Also described is an embodiment where in any of the
preceding embodiments, at least one unnatural amino acid comprises
a polyhydroxy radical.
[0463] Also described is an embodiment where in any of the
preceding embodiments, at least two unnatural amino acids comprise
a polyhydroxy radical.
[0464] Also described is an embodiment where in any of the
preceding embodiments, at least three unnatural amino acids
comprise a polyhydroxy radical.
[0465] Also described is an embodiment where in any of the
preceding embodiments, at least four unnatural amino acids comprise
a polyhydroxy radical.
[0466] Also described is an embodiment where in any of the
preceding embodiments, at least one of the polyhydroxy radicals is
of the formula
CH.sub.2--(CH(OH)).sub.n--CH.sub.2--OH
where n is selected from 1, 2, 3, 4, 5, and 6.
[0467] Also described is an embodiment where in any of the
preceding embodiments, n is selected from 1, 2, 3, and 4.
[0468] Also described is an embodiment where in any of the
preceding embodiments, n is selected from 3 and 4.
[0469] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent polyglutamic acid
radical, where at least one glutamic acid forms an amide with an
aminopolyhydroxy radical.
[0470] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent polyglutamic acid
radical, where at least two glutamic acids form an amide with an
aminopolyhydroxy radical.
[0471] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent polyglutamic acid
radical, where at least three glutamic acids form an amide with an
aminopolyhydroxy radical.
[0472] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent polyglutamic acid
radical, where at least four glutamic acids form an amide with an
aminopolyhydroxy radical.
[0473] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent poly(D-glutamic acid)
radical, where at least one glutamic acid forms an amide with an
aminopolyhydroxy radical.
[0474] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent poly(D-glutamic acid)
radical, where at least two glutamic acids form an amide with an
aminopolyhydroxy radical.
[0475] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent poly(D-glutamic acid)
radical, where at least three glutamic acids form an amide with an
aminopolyhydroxy radical.
[0476] Also described is an embodiment where in any of the
preceding embodiments, L comprises a divalent poly(D-glutamic acid)
radical, where at least four glutamic acids form an amide with an
aminopolyhydroxy radical.
[0477] Also described is an embodiment where in any of the
preceding embodiments, at least one of the aminopolyhydroxy
radicals is of the formula
NH--CH.sub.2--(CH(OH)).sub.m--CH.sub.2--OH
where m is selected from 1, 2, 3, 4, 5, and 6.
[0478] Also described is an embodiment where in any of the
preceding embodiments, L comprises a diradical of the formula
##STR00040##
[0479] Also described is an embodiment where in any of the
preceding embodiments, L comprises at least one unnatural amino
acid having the D-configuration.
[0480] Also described is an embodiment where in any of the
preceding embodiments, x is 3.
[0481] Also described is an embodiment where in any of the
preceding embodiments, x is 2.
[0482] Also described is an embodiment where in any of the
preceding embodiments, x is 1.
[0483] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a cytotoxic agent.
[0484] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a cancer treating
agent.
[0485] Also described is an embodiment where in any of the
preceding embodiments, at least one D is an anti-inflammatory
agent.
[0486] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a vinca alkaloid.
[0487] Also described is an embodiment where in any of the
preceding embodiments, at least one D is desacetylvinblastine
monohydrazide.
[0488] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a tubulysin.
[0489] Also described is an embodiment where in any of the
preceding embodiments, at least one D is tubulysin A.
[0490] Also described is an embodiment where in any of the
preceding embodiments, at least one D is tubulysin B.
[0491] Also described is an embodiment where in any of the
preceding embodiments, at least one D is tubulysin A hydrazide.
[0492] Also described is an embodiment where in any of the
preceding embodiments, at least one D is tubulysin B hydrazide.
[0493] Also described is an embodiment where in any of the
preceding embodiments, at least one D is an antifolate.
[0494] Also described is an embodiment where in any of the
preceding embodiments, at least one D is an aminopterin.
[0495] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a rapamycin.
[0496] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a mitomycin.
[0497] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a taxane.
[0498] Also described is an embodiment where in any of the
preceding embodiments, at least one D is a doxorubicin.
[0499] It is to be understood that every combination of the various
embodiments of each of B, L, D, and x described herein form
illustrative embodiments of the conjugates of the invention,
whether those various embodiments of each of B, L, D are species,
subgenera, or genera. It is to be further understood that each of
those additional illustrative embodiments of compounds may be used
in any of the kits, methods and/or uses described herein.
[0500] In some embodiments, the ligand is a vitamin analog or a
vitamin derivative. Illustrative embodiments of vitamin analogs
and/or derivatives include folate and analogs and derivatives of
folate such as folinic acid, pteropolyglutamic acid, and folate
receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, tetrahydrofolates, and their deaza and dideaza
analogs. The terms "deaza" and "dideaza" analogs refer to the
art-recognized analogs having a carbon atom substituted for one or
two nitrogen atoms in the naturally occurring folic acid structure,
or analog or derivative thereof. For example, the deaza analogs
include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza
analogs of folate, folinic acid, pteropolyglutamic acid, and folate
receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, and tetrahydrofolates. The dideaza analogs include,
for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and
5,8-dideaza analogs of folate, folinic acid, pteropolyglutamic
acid, and folate receptor-binding pteridines such as
tetrahydropterins, dihydrofolates, and tetrahydrofolates. Other
folates useful as complex forming ligands for this invention are
the folate receptor-binding analogs aminopterin, amethopterin (also
known as methotrexate), N.sup.10-methylfolate,
2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin
or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed "folates," reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein.
[0501] In another embodiment, the ligand is capable of binding or
targeting PSMA. In another embodiment, the ligand capable of
binding or targeting PSMA is a phosphoric, phosphonic, or
phosphinic acid or derivative thereof. In one aspect, the
phosphoric, phosphonic, or phosphinic acid or derivative thereof
includes one or more carboxylic acid groups. In another aspect, the
phosphoric, phosphonic, or phosphinic acid or derivative thereof
includes one or more thiol groups or derivatives thereof. In
another aspect, the phosphoric, phosphonic, or phosphinic acid or
derivative thereof includes one or more carboxylic acid
bioisosteres, such as an optionally substituted tetrazole, and the
like.
[0502] In another embodiment, the PSMA ligand is a derivative of
pentanedioic acid. Illustratively, the pentanedioic acid derivative
is a compound of the formula:
##STR00041##
wherein X is RP(O)(OH)CH.sub.2-- (see, e.g., U.S. Pat. No.
5,968,915 incorporated herein by reference);
RP(O)(OH)N(R.sup.1)--(see, e.g., U.S. Pat. No. 5,863,536
incorporated herein by reference); RP(O)(OH)O-- (see, e.g., U.S.
Pat. No. 5,795,877 incorporated herein by reference); RN(OH)C(O)Y--
or RC(O)NH(OH)Y, wherein Y is --CR.sub.1R.sub.2--, --NR.sub.3-- or
--O-- (see, e.g., U.S. Pat. No. 5,962,521 incorporated herein by
reference); RS(O)Y, RSO.sub.2Y, or RS(O)(NH)Y, wherein Y is
--CR.sub.1R.sub.2--, --NR.sub.3-- or --O--(see, e.g., U.S. Pat. No.
5,902,817 incorporated herein by reference); and RS-alkyl, wherein
R is for example hydrogen, alkyl, aryl, or arylalkyl, each of which
may be optionally substituted (see, e.g., J. Med. Chem.
46:1989-1996 (2003) incorporated herein by reference).
[0503] In each of the foregoing formulae, R, R.sub.1, R.sub.2, and
R.sub.3 are each independently selected from hydrogen,
C.sub.1-C.sub.9 straight or branched chain alkyl, C.sub.2-C.sub.9
straight or branched chain alkenyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.5-C.sub.7 cycloalkenyl, and aryl. In addition, in each case,
each of R, R.sub.1, R.sub.2, and R.sub.3 may be optionally
substituted, such as with one or more groups selected from
C.sub.3-C.sub.8 cycloalkyl, C.sub.5-C.sub.7 cycloalkenyl, halo,
hydroxy, nitro, trifluoromethyl, C.sub.1-C.sub.6 straight or
branched chain alkyl, C.sub.2-C.sub.6 straight or branched chain
alkenyl, C.sub.1-C.sub.4 alkoxy, C.sub.2-C.sub.4 alkenyloxy,
phenoxy, benzyloxy, amino, aryl. In one aspect, aryl is selected
from 1-naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
benzyl, and phenyl, and in each case aryl may be optionally
substituted with one or more, illustratively with one to three,
groups selected from halo, hydroxy, nitro, trifluoromethyl,
C.sub.1-C.sub.6 straight or branched chain alkyl, C.sub.2-C.sub.6
straight or branched chain alkenyl, C.sub.1-C.sub.4 alkoxy,
C.sub.2-C.sub.4 alkenyloxy, phenoxy, benzyloxy, and amino. In one
variation of each of the above formulae, R is not hydrogen.
[0504] Illustrative PSMA ligands described in U.S. Pat. No.
5,968,915 include 2-[[methylhydroxyphosphinyl]methyl]pentanedioic
acid; 2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;
2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;
2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;
2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;
2-[[phenylhydroxyphosphinyl]methyl]pentanedioic acid;
2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]pentanedioic
acid;
2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]pentanedioic
acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid; 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid; 2-[[phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid;
2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid;
2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid;
2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid;
2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic
acid; 2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]pentanedioic
acid; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic
acid.
[0505] Illustrative PSMA ligands described in U.S. Pat. No.
5,863,536 include N-[methylhydroxyphosphinyl]glutamic acid;
N-[ethylhydroxyphosphinyl]glutamic acid;
N-[propylhydroxyphosphinyl]glutamic acid;
N-[butylhydroxyphosphinyl]glutamic acid;
N-[phenylhydroxyphosphinyl]glutamic acid;
N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid;
N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid; and
N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid.
[0506] Illustrative PSMA ligands described in U.S. Pat. No.
5,795,877 include 2-[[methylhydroxyphosphinyl]oxy]pentanedioic
acid; 2-[[ethylhydroxyphosphinyl]oxy]pentanedioic acid;
2-[[propylhydroxyphosphinyl]oxy]pentanedioic acid;
2-[[butylhydroxyphosphinyl]oxy]pentanedioic acid;
2-[[phenylhydroxyphosphinyl]oxy]pentanedioic acid;
2-[[((4-pyridyl)methyphydroxyphosphinyl]oxy]pentanedioic acid;
2-[[((2-pyridyl)methyphydroxyphosphinyl]oxy]pentanedioic acid;
2-[[(phenylmethyl)hydroxyphosphinyl]oxy]pentanedioic acid; and
2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]pentanedioic
acid.
[0507] Illustrative PSMA ligands described in U.S. Pat. No.
5,962,521 include 2-[[(N-hydroxy)carbamoyl]methyl]pentanedioic
acid; 2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-butyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid;
2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-ethyl-N-hydroxy)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy-N-4-pyridyl)carbamoyl]methyl]pentanedioic acid;
2-[[(N-hydroxy)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(methyl)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(benzyl)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(phenyl)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(2-phenylethyl)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(ethyl)carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy(propyl) carboxamido]methyl]pentanedioic acid;
2-[[N-hydroxy (3-phenylpropyl)carboxamido]methyl]pentanedioic acid;
and 2-[[N-hydroxy(4-pyridyl)carboxamido]methyl]pentanedioic
acid.
[0508] Illustrative PSMA ligands described in U.S. Pat. No.
5,902,817 include 2-[(sulfinyl)methyl]pentanedioic acid;
2-[(methylsulfinyemethyl]pentanedioic acid;
2-[(ethylsulfinyl)methyl]pentanedioic acid;
2-[(propylsulfinyl)methyl]pentanedioic acid;
2-[(butylsulfinyl)methyl]pentanedioic acid;
2-[(phenylsulfinyl)methyl]pentanedioic acid;
2-[[(2-phenylethyl)sulfinyl]methyl]pentanedioic acid;
2-[[(3-phenylpropyl)sulfinyl]methyl]pentanedioic acid;
2-[[(4-pyridyl)sulfinyl]methyl]pentanedioic acid;
2-[(benzylsulfinyl)methyl]pentanedioic acid;
2-[(sulfonyl)methyl]pentanedioic acid;
2-[(methylsulfonyl)methyl)]pentanedioic acid;
2-[(ethylsulfonyl)methyl]pentanedioic acid;
2-[(propylsulfonyl)methyl]pentanedioic acid;
2-[(butylsulfonyl)methyl]pentanedioic acid;
2-[(phenylsulfonyl)methyl]pentanedioic acid;
2-[[(2-phenylethyl)sulfonyl]methyl]pentanedioic acid;
2-[[(3-phenylpropyl)sulfonyl]methyl]pentanedioic acid;
2-[[(4-pyridyl)sulfonyl]methyl]pentanedioic acid;
2-[(benzylsulfonyl)methyl]pentanedioic acid;
2-[(sulfoximinyl)methyl]pentanedioic acid;
2-[(methylsulfoximinyl)methyl]pentanedioic acid;
2-[(ethylsulfoximinyl)methyl]pentanedioic acid;
2-[(propylsulfoximinyl)methyl]pentanedioic acid;
2-[(butylsulfoximinyemethyl)pentanedioic acid;
2-[(phenylsulfoximinyl]methyl]pentanedioic acid;
2-[[(2-phenylethyl)sulfoximinyl]methyl]pentanedioic acid;
2-[[(3-phenylpropyl)sulfoximinyl]methyl]pentanedioic acid;
2-[[(4-pyridyesulfoximinyl]methyl]pentanedioic acid; and
2-[(benzylsulfoximinyl)methyl]pentanedioic acid.
[0509] Pentanedioic acid derivatives described herein have been
reported to have high binding affinity at PSMA, including but not
limited to the following phosphonic and phosphinic acid
derivatives
##STR00042##
[0510] In another illustrative embodiment, the pentanedioic acid
derivative includes a thiol group, such as compounds of the
following formulae:
##STR00043##
with configuration (R,S), (R), and (S).
[0511] In another embodiment, the PSMA ligand is a urea of two
amino acids. In one aspect, the amino acids include one or more
additional carboxylic acids. In another aspect, the amino acids
include one or more additional phosphoric, phosphonic, phosphinic,
sulfinic, sulfonic, or boronic acids. In another aspect, the amino
acids include one or more thiol groups or derivatives thereof. In
another aspect, the amino acids include one or more amino groups or
derivatives thereof. In another aspect, the amino acids includes
one or more carboxylic acid bioisosteres, such as tetrazoles and
the like.
[0512] In another embodiment, the PSMA ligand is a aminocarbonyl
derivative of pentanedioic acid. Illustratively, the
aminocarbonylpentanedioic acid derivative is a compound of the
formula:
##STR00044##
wherein R.sup.1 and R.sup.2 are each selected from hydrogen,
optionally substituted carboxylic acids, such as thiolacetic acids,
thiolpropionic acids, and the like; malonic acids, succinic acids,
glutamic acids, adipic acids, and the like; and others.
Illustrative aminocarbonylpentanedioic acid derivatives are
described in J. Med. Chem. 44:298-301 (2001) and J. Med. Chem.
47:1729-38 (2004), the disclosures of which are incorporated herein
by reference.
[0513] In another embodiment, the PSMA ligand is a compound of the
formula:
TABLE-US-00001 ##STR00045## R.sup.1 ##STR00046## (R = H) (R =
tert-Bu) ##STR00047## ##STR00048## (R = H) (R = OH) ##STR00049## (R
= H) (R = OH) ##STR00050## (R = H) (R = CH.sub.2CH.sub.2CN)
##STR00051##
[0514] It is appreciated that the urea compounds described herein
may also be advantageous in the preparation of the ligands also
described herein due to the sub-nanomolar potency, water
solubility, and/or long term stability of these compounds. The urea
compounds described herein may generally be prepared from
commercially available starting materials as described herein.
[0515] It is appreciated that in each of the above illustrative
pentanedioic acid compounds and urea compounds, there is at least
one asymmetric carbon atom. Accordingly, the above illustrative
formulae are intended to refer both individually and collectively
to all stereoisomers as pure enantiomers, or mixtures of
enantiomers and/or diastereomers, including but not limited to
racemic mixtures, mixtures that include one epimer at a first
asymmetric carbon but allow mixtures at other asymmetric carbons,
including racemic mixtures, and the like.
[0516] In another illustrative embodiment, the ligand is a urea of
an amino dicarboxylic acid, such as aspartic acid, glutamic acid,
and the like, and another amino dicarboxylic acid, or an analog
thereof, such as a ligand of the formulae
##STR00052##
wherein Q is a an amino dicarboxylic acid, such as aspartic acid,
glutamic acid, or an analog thereof, n and m are each selected from
an integer between 1 and about 6, and (*) represents the point of
attachment for the linker L.
[0517] In another illustrative embodiment, the ligand is a thiourea
of an amino dicarboxylic acid, such as aspartic acid, glutamic
acid, and the like, and another amino dicarboxylic acid, or an
analog thereof, such as a ligand of the formulae
##STR00053##
wherein Q is a an amino dicarboxylic acid, such as aspartic acid,
glutamic acid, or an analog thereof, n and m are each selected from
an integer between 1 and about 6, and (*) represents the point of
attachment for the linker L.
[0518] In another embodiment, the PSMA ligand includes at least
four carboxylic acid groups, or at least three free carboxylic acid
groups after the PSMA ligand is conjugated to the ligand or linker.
It is understood that as described herein, carboxylic acid groups
on the PSMA ligand include bioisosteres of carboxylic acids.
[0519] Illustratively, the PSMA ligand is a compound of the
formulae:
##STR00054## ##STR00055## ##STR00056## ##STR00057##
[0520] In another embodiment, the PSMA ligand is
2-[3-(1-Carboxy-2-mercapto-ethyl)-ureido]-pentanedioic acid (MUPA)
or 2-[3-(1,3-Dicarboxy-propyl)-ureido]-pentanedioic acid
(DUPA).
[0521] In another embodiment, the PSMA ligand is a urea or thiourea
of lysine and glutamate, or one or more carboxylic acid derivatives
thereof. In another embodiment, the PSMA ligand is a urea of lysine
and glutamate. In another embodiment, the PSMA ligand is a urea or
thiourea of L-lysine and L-glutamate, or one or more carboxylic
acid derivatives thereof. In another embodiment, the PSMA ligand is
a urea of L-lysine and L-glutamate.
[0522] Other illustrative examples of PSMA ligands include peptide
analogs such as quisqualic acid, aspartate glutamate (Asp-Glu),
Glu-Glu, Gly-Glu, .gamma.-Glu-Glu,
beta-N-acetyl-L-aspartate-L-glutamate (.beta.-NAAG), and the
like.
[0523] As used herein, the term "linker" includes is a chain of
atoms that connects two or more functional parts of a molecule to
form a conjugate. As used herein, the terms "linker," "linkers,"
and "L" are used interchangeably. Illustratively, the chain of
atoms is selected from C, N, O, S, Si, and P, or C, N, O, S, and P,
C, N, O, and S. The chain of atoms covalently connects different
functional capabilities of the conjugate, such as ligands and
drugs. The linker may have a wide variety of lengths, such as in
the range from about 2 to about 100 atoms in the contiguous
backbone. The atoms used in forming the linker may be combined in
all chemically relevant ways, such as chains of carbon atoms
forming alkylene, alkenylene, and alkynylene groups, and the like;
chains of carbon and oxygen atoms forming ethers, polyoxyalkylene
groups, or when combined with carbonyl groups forming esters and
carbonates, and the like; chains of carbon and nitrogen atoms
forming amines, imines, polyamines, hydrazines, hydrazones, or when
combined with carbonyl groups forming amides, ureas,
semicarbazides, carbazides, and the like; chains of carbon,
nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines, or
when combined with carbonyl groups forming urethanes, amino acids,
acyloxylamines, hydroxamic acids, and the like; and many others. In
addition, it is to be understood that the atoms forming the chain
in each of the foregoing illustrative embodiments may be either
saturated or unsaturated, thus forming single, double, or triple
bonds, such that for example, alkanes, alkenes, alkynes, imines,
and the like may be radicals that are included in the linker. In
addition, it is to be understood that the atoms forming the linker
may also be cyclized upon each other or be part of cyclic structure
to form divalent cyclic structures that form the linker, including
cyclo alkanes, cyclic ethers, cyclic amines, and other
heterocycles, arylenes, heteroarylenes, and the like in the linker.
In this latter arrangement, it is to be understood that the linker
length may be defined by any pathway through the one or more cyclic
structures. Illustratively, the linker length is defined by the
shortest pathway through the each one of the cyclic structures. It
is to be understood that the linkers may be optionally substituted
at any one or more of the open valences along the chain of atoms,
such as optional substituents on any of the carbon, nitrogen,
silicon, or phosphorus atoms. It is also to be understood that the
linker may connect the two or more functional parts of a molecule
to form a conjugate at any open valence, and it is not necessary
that any of the two or more functional parts of a molecule forming
the conjugate are attached at any apparent end of the linker.
[0524] In any of the embodiments described herein heteroatom
linkers can be --NR.sup.1R.sup.2--, oxygen, sulfur, and the
formulae --(NHR.sup.1NHR.sup.2)--, --SO--, --(SO.sub.2)--, and
--N(R.sup.3)O--, wherein R.sup.1, R.sup.2, and R.sup.3 are each
independently selected from hydrogen, alkyl, aryl, arylalkyl,
substituted aryl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, and alkoxyalkyl.
[0525] The releasable linkers can be methylene, 1-alkoxyalkylene,
1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl,
oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl,
iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
below.
[0526] In any of the embodiments described herein, the heteroatom
linker can be oxygen, and the releasable linkers can be methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the oxygen to
form an acetal or ketal. Alternatively, the heteroatom linker can
be oxygen, and the releasable linker can be methylene, wherein the
methylene is substituted with an optionally-substituted aryl, and
the releasable linker is bonded to the oxygen to form an acetal or
ketal. Further, the heteroatom linker can be oxygen, and the
releasable linker can be sulfonylalkyl, and the releasable linker
is bonded to the oxygen to form an alkylsulfonate.
[0527] In another embodiment of the above releasable linker
embodiment, the heteroatom linker can be nitrogen, and the
releasable linkers can be iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the nitrogen
to form an hydrazone. In an alternate configuration, the hydrazone
may be acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form various acylhydrazone
releasable linkers.
[0528] Alternatively, the heteroatom linker can be oxygen, and the
releasable linkers can be alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below, and the releasable
linker is bonded to the oxygen to form a silanol.
[0529] In the above releasable linker embodiment, the drug can
include a nitrogen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom nitrogen
to form an amide, and also bonded to the drug nitrogen to form an
amide
[0530] In the above releasable linker embodiment, the drug can
include an oxygen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom linker
nitrogen to form an amide, and also bonded to the drug oxygen to
form an ester.
[0531] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the heteroatom linker can be
nitrogen, and the substituent X.sup.2 and the heteroatom linker can
be taken together with the releasable linker to which they are
bound to form an heterocycle.
[0532] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0533] In one aspect of the various conjugates described herein,
the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkyloxymethyloxy moiety, where the methyl is
optionally substituted with alkyl or substituted aryl.
[0534] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms an
acylaziridine with the drug, or analog or derivative thereof.
[0535] In another aspect, the polyvalent linker comprises a
1-alkoxycycloalkylenoxy moiety.
[0536] In another aspect, the polyvalent linker comprises an
alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
[0537] In another aspect, the polyvalent linker comprises a
dithioalkylcarbonylhydrazide, where the hydrazide forms an
hydrazone with the drug, or analog or derivative thereof.
[0538] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide
forms a hydrazone with the drug, or analog or derivative
thereof.
[0539] In another aspect, the polyvalent linker comprises a
3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the
disubstituted silyl is substituted with alkyl or optionally
substituted aryl.
[0540] In another aspect, the polyvalent linker comprises a
plurality of spacer linkers selected from the group consisting of
the naturally occurring amino acids and stereoisomers thereof.
[0541] In another aspect, the polyvalent linker comprises a
2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with
the drug, or analog or derivative thereof.
[0542] In another aspect, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug, or analog or derivative thereof, and the aryl is
optionally substituted.
[0543] In another aspect, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate
with the drug, or analog or derivative thereof, and the aryl is
optionally substituted.
[0544] In another aspect, the polyvalent linker comprises a
3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the
alkylidene forms an hydrazone with the drug, or analog or
derivative thereof, each alkyl is independently selected, and the
oxyalkyloxy is optionally substituted with alkyl or optionally
substituted aryl.
[0545] In another aspect, the polyvalent linker comprises a
2-dithioalkyloxycarbonylhydrazide.
[0546] In another aspect, the polyvalent linker comprises a 2- or
3-dithioalkylamino, where the amino forms a vinylogous amide with
the drug, or analog or derivative thereof.
[0547] In another aspect, the polyvalent linker comprises a
2-dithioalkylamino, where the amino forms a vinylogous amide with
the drug, or analog or derivative thereof, and the alkyl is
ethyl.
[0548] In another aspect, the polyvalent linker comprises a 2- or
3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate
with the drug, or analog or derivative thereof.
[0549] In another aspect, the polyvalent linker comprises a
releasable linker, a spacer linker, and a releasable linker taken
together to form 2-dithioalkylaminocarbonyl, where the carbonyl
forms a carbamate with the drug, or analog or derivative thereof,
and the alkyl is ethyl.
[0550] In another aspect, the polyvalent linker comprises a
2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug, or analog or derivative
thereof.
[0551] In another aspect, the polyvalent linker comprises a
4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate
or a carbamoylaziridine with the drug, or analog or derivative
thereof.
[0552] In one embodiment, the polyvalent linkers described herein
comprise divalent linkers of formulae (II)
##STR00058##
where n is an integer selected from 1 to about 4; R.sup.a and
R.sup.b are each independently selected from the group consisting
of hydrogen and alkyl, including lower alkyl such as
C.sub.1-C.sub.4 alkyl that are optionally branched; or R.sup.a and
R.sup.b are taken together with the attached carbon atom to form a
carbocyclic ring; R is an optionally substituted alkyl group, an
optionally substituted acyl group, or a suitably selected nitrogen
protecting group; and (*) indicates points of attachment for the
drug, vitamin, imaging agent, diagnostic agent, other bivalent
linkers, or other parts of the conjugate.
[0553] In another embodiment, the polyvalent linkers described
herein comprise divalent linkers of formulae (III)
##STR00059##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, other bivalent
linkers, or other parts of the conjugate.
[0554] In another embodiment, the polyvalent linkers described
herein comprise divalent linkers of formulae (IV)
##STR00060##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, ligand, other divalent
linkers, or other parts of the conjugate.
[0555] In another embodiment, the polyvalent linkers described
herein comprise one or more hydrophilic linkers. It is appreciated
that the arrangement and/or orientation of the various hydrophilic
linkers may be in a linear or branched fashion, or both. For
example, the hydrophilic linkers may form the backbone of the
linker forming the conjugate between the binding ligand and the
drug. Alternatively, the hydrophilic portion of the linker may be
pendant to or attached to the backbone of the chain of atoms
connecting the binding ligand B to the agent A. In this latter
arrangement, the hydrophilic portion may be proximal or distal to
the backbone chain of atoms.
[0556] In another embodiment, the linker is more or less linear,
and the hydrophilic groups are arranged largely in a series to form
a chain-like linker in the conjugate. Said another way, the
hydrophilic groups form some or all of the backbone of the linker
in this linear embodiment.
[0557] In another embodiment, the linker is branched with
hydrophilic groups. In this branched embodiment, the hydrophilic
groups may be proximal to the backbone or distal to the backbone.
In each of these arrangements, the linker is more spherical or
cylindrical in shape. In one variation, the linker is shaped like a
bottle-brush. In one aspect, the backbone of the linker is formed
by a linear series of amides, and the hydrophilic portion of the
linker is formed by a parallel arrangement of branching side
chains, such as by connecting monosaccharides, sulfonates, and the
like, and derivatives and analogs thereof.
[0558] It is understood that the linker may be neutral or ionizable
under certain conditions, such as physiological conditions
encountered in vivo. For ionizable linkers, under the selected
conditions, the linker may deprotonate to form a negative ion, or
alternatively become protonated to form a positive ion. It is
appreciated that more than one deprotonation or protonation event
may occur. In addition, it is understood that the same linker may
deprotonate and protonate to form inner salts or zwitterionic
compounds.
[0559] In another embodiment, the hydrophilic spacer linkers are
neutral, i.e. under physiological conditions, the linkers do not
significantly protonate nor deprotonate. In another embodiment, the
hydrophilic spacer linkers may be protonated to carry one or more
positive charges. It is understood that the protonation capability
is condition dependent. In one aspect, the conditions are
physiological conditions, and the linker is protonated in vivo. In
another embodiment, the spacers include both regions that are
neutral and regions that may be protonated to carry one or more
positive charges. In another embodiment, the spacers include both
regions that may be deprotonated to carry one or more negative
charges and regions that may be protonated to carry one or more
positive charges. It is understood that in this latter embodiment
that zwitterions or inner salts may be formed.
[0560] In one aspect, the regions of the linkers that may be
deprotonated to carry a negative charge include carboxylic acids,
such as aspartic acid, glutamic acid, and longer chain carboxylic
acid groups, and sulfuric acid esters, such as alkyl esters of
sulfuric acid. In another aspect, the regions of the linkers that
may be protonated to carry a positive charge include amino groups,
such as polyaminoalkylenes including ethylene diamines, propylene
diamines, butylene diamines and the like, and/or heterocycles
including pyrollidines, piperidines, piperazines, and other amino
groups, each of which is optionally substituted. In another
embodiment, the regions of the linkers that are neutral include
poly hydroxyl groups, such as sugars, carbohydrates, saccharides,
inositols, and the like, and/or polyether groups, such as
polyoxyalkylene groups including polyoxyethylene, polyoxypropylene,
and the like.
[0561] In one embodiment, the hydrophilic spacer linkers described
herein include are formed primarily from carbon, hydrogen, and
oxygen, and have a carbon/oxygen ratio of about 3:1 or less, or of
about 2:1 or less. In one aspect, the hydrophilic linkers described
herein include a plurality of ether functional groups. In another
aspect, the hydrophilic linkers described herein include a
plurality of hydroxyl functional groups. Illustrative fragments
that may be used to form such linkers include polyhydroxyl
compounds such as carbohydrates, polyether compounds such as
polyethylene glycol units, and acid groups such as carboxyl and
alkyl sulfuric acids. In one variation, oligoamide spacers, and the
like may also be included in the linker.
[0562] Illustrative carbohydrate spacers include saccharopeptides
as described herein that include both a peptide feature and sugar
feature; glucuronides, which may be incorporated via [2+3] Huisgen
cyclization, also known as click chemistry; .beta.-alkyl
glycosides, such as of 2-deoxyhexapyranoses (2-deoxyglucose,
2-deoxyglucuronide, and the like), and .beta.-alkyl
mannopyranosides. Illustrative PEG groups include those of a
specific length range from about 4 to about 20 PEG groups.
Illustrative alkyl sulfuric acid esters may also be introduced with
click chemistry directly into the backbone. Illustrative oligoamide
spacers include EDTA and DTPA spacers, .beta.-amino acids, and the
like.
[0563] In another embodiment, the hydrophilic spacer linkers
described herein include a polyether, such as the linkers of the
following formulae:
##STR00061##
where m is an integer independently selected in each instance from
1 to about 8; p is an integer selected 1 to about 10; and n is an
integer independently selected in each instance from 1 to about 3.
In one aspect, m is independently in each instance 1 to about 3. In
another aspect, n is 1 in each instance. In another aspect, p is
independently in each instance about 4 to about 6. Illustratively,
the corresponding polypropylene polyethers corresponding to the
foregoing are contemplated herein and may be included in the
conjugates as hydrophilic spacer linkers. In addition, it is
appreciated that mixed polyethylene and polypropylene polyethers
may be included in the conjugates as hydrophilic spacer linkers.
Further, cyclic variations of the foregoing polyether compounds,
such as those that include tetrahydrofuranyl, 1,3-dioxanes,
1,4-dioxanes, and the like are contemplated herein.
[0564] In another illustrative embodiment, the hydrophilic spacer
linkers described herein include a plurality of hydroxyl functional
groups, such as linkers that incorporate monosaccharides,
oligosaccharides, polysaccharides, and the like. It is to be
understood that the polyhydroxyl containing spacer linkers
comprises a plurality of --(CROH)-- groups, where R is hydrogen or
alkyl.
[0565] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00062## ##STR00063##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, p is an
integer from 1 to about 5, and r is an integer selected from 1 to
about 3. In one aspect, the integer n is 3 or 4. In another aspect,
the integer p is 3 or 4. In another aspect, the integer r is 1.
[0566] In another embodiment, the spacer linker includes one or
more of the following cyclic polyhydroxyl groups:
##STR00064## ##STR00065##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4. In one aspect,
the integer n is 3 or 4. In another aspect, the integer p is 3 or
4. In another aspect, the integer r is 2 or 3. It is understood
that all stereochemical forms of such sections of the linkers are
contemplated herein. For example, in the above formula, the section
may be derived from ribose, xylose, glucose, mannose, galactose, or
other sugar and retain the stereochemical arrangements of pendant
hydroxyl and alkyl groups present on those molecules. In addition,
it is to be understood that in the foregoing formulae, various
deoxy compounds are also contemplated. Illustratively, compounds of
the following formulae are contemplated:
##STR00066##
wherein n is equal to or less than r, such as when r is 2 or 3, n
is 1 or 2, or 1, 2, or 3, respectively.
[0567] In another embodiment, the spacer linker includes a
polyhydroxyl compound of the following formula:
##STR00067##
wherein n and r are each an integer selected from 1 to about 3. In
one aspect, the spacer linker includes one or more polyhydroxyl
compounds of the following formulae:
##STR00068##
It is understood that all stereochemical forms of such sections of
the linkers are contemplated herein. For example, in the above
formula, the section may be derived from ribose, xylose, glucose,
mannose, galactose, or other sugar and retain the stereochemical
arrangements of pendant hydroxyl and alkyl groups present on those
molecules.
[0568] In another configuration, the hydrophilic linkers L
described herein include polyhydroxyl groups that are spaced away
from the backbone of the linker. In one embodiment, such
carbohydrate groups or polyhydroxyl groups are connected to the
back bone by a triazole group, forming triazole-linked hydrophilic
spacer linkers. Illustratively, such linkers include fragments of
the following formulae:
##STR00069##
wherein n, m, and r are integers and are each independently
selected in each instance from 1 to about 5. In one illustrative
aspect, m is independently 2 or 3 in each instance. In another
aspect, r is 1 in each instance. In another aspect, n is 1 in each
instance. In one variation, the group connecting the polyhydroxyl
group to the backbone of the linker is a different heteroaryl
group, including but not limited to, pyrrole, pyrazole,
1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole,
isothiazole, oxadiazole, and the like. Similarly, divalent
6-membered ring heteroaryl groups are contemplated. Other
variations of the foregoing illustrative hydrophilic spacer linkers
include oxyalkylene groups, such as the following formulae:
##STR00070##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0569] In another embodiment, such carbohydrate groups or
polyhydroxyl groups are connected to the back bone by an amide
group, forming amide-linked hydrophilic spacer linkers.
Illustratively, such linkers include fragments of the following
formulae:
##STR00071##
wherein n is an integer selected from 1 to about 3, and m is an
integer selected from 1 to about 22. In one illustrative aspect, n
is 1 or 2. In another illustrative aspect, m is selected from about
6 to about 10, illustratively 8. In one variation, the group
connecting the polyhydroxyl group to the backbone of the linker is
a different functional group, including but not limited to, esters,
ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic
variations are contemplated. Other variations of the foregoing
illustrative hydrophilic spacer linkers include oxyalkylene groups,
such as the following formulae:
##STR00072##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0570] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00073## ##STR00074##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0571] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00075## ##STR00076## ##STR00077##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0572] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00078## ##STR00079## ##STR00080##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0573] In another embodiment, the hydrophilic spacer linker is a
combination of backbone and branching side motifs such as is
illustrated by the following formulae
##STR00081##
wherein n is an integer independently selected in each instance
from 0 to about 3. The above formula are intended to represent 4,
5, 6, and even larger membered cyclic sugars. In addition, it is to
be understood that the above formula may be modified to represent
deoxy sugars, where one or more of the hydroxy groups present on
the formulae are replaced by hydrogen, alkyl, or amino. In
addition, it is to be understood that the corresponding carbonyl
compounds are contemplated by the above formulae, where one or more
of the hydroxyl groups is oxidized to the corresponding carbonyl.
In addition, in this illustrative embodiment, the pyranose includes
both carboxyl and amino functional groups and (a) can be inserted
into the backbone and (b) can provide synthetic handles for
branching side chains in variations of this embodiment. Any of the
pendant hydroxyl groups may be used to attach other chemical
fragments, including additional sugars to prepare the corresponding
oligosaccharides. Other variations of this embodiment are also
contemplated, including inserting the pyranose or other sugar into
the backbone at a single carbon, i.e. a spiro arrangement, at a
geminal pair of carbons, and like arrangements. For example, one or
two ends of the linker, or the ligand may be connected to the sugar
to be inserted into the backbone in a 1,1; 1,2; 1,3; 1,4; 2,3, or
other arrangement.
[0574] In another embodiment, the hydrophilic spacer linkers
described herein include are formed primarily from carbon,
hydrogen, and nitrogen, and have a carbon/nitrogen ratio of about
3:1 or less, or of about 2:1 or less. In one aspect, the
hydrophilic linkers described herein include a plurality of amino
functional groups.
[0575] In another embodiment, the spacer linkers include one or
more amino groups of the following formulae:
##STR00082##
where n is an integer independently selected in each instance from
1 to about 3. In one aspect, the integer n is independently 1 or 2
in each instance. In another aspect, the integer n is 1 in each
instance.
[0576] In another embodiment, the hydrophilic spacer linker is a
sulfuric acid ester, such as an alkyl ester of sulfuric acid.
Illustratively, the spacer linker is of the following formula:
##STR00083##
where n is an integer independently selected in each instance from
1 to about 3. Illustratively, n is independently 1 or 2 in each
instance.
[0577] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0578] In each of the foregoing illustrative examples of linkers L,
there are also included in some cases additional spacer linkers,
and/or additional releasable linkers. Those spacer linker and
releasable linkers also may include asymmetric carbon atoms. It is
to be further understood that the stereochemical configurations
shown herein are merely illustrative, and other stereochemical
configurations are contemplated. For example in one variation, the
corresponding unnatural amino acid configurations may be included
in the conjugated described herein as follows:
##STR00084##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4, as described
above.
[0579] It is to be further understood that in the foregoing
embodiments, open positions, such as (*) atoms are locations for
attachment of the ligand or the drug. In addition, it is to be
understood that such attachment of either or both of B and A may be
direct or through an intervening linker. Intervening linkers
include other spacer linkers and/or releasable linkers.
Illustrative additional spacer linkers and releasable linkers that
are included in the conjugated described herein are described in
U.S. patent application Ser. No. 10/765,335, the disclosure of
which is incorporated herein by reference.
[0580] In one embodiment, the hydrophilic spacer linker comprises
one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises at least three carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more carbohydrate
containing or polyhydroxyl group containing linkers, and one or
more aspartic acids. In another embodiment, the hydrophilic spacer
linker comprises one or more carbohydrate containing or
polyhydroxyl group containing linkers, and one or more glutamic
acids. In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers, one or more glutamic acids, one or more
aspartic acids, and one or more beta amino alanines. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0581] In another embodiment, the hydrophilic spacer linker
comprises one or more divalent 1,4-piperazines that are included in
the chain of atoms connecting at least one of the ligands with at
least one of the drugs. In one variation, the hydrophilic spacer
linker includes one or more carbohydrate containing or polyhydroxyl
group containing linkers. In another variation, the hydrophilic
spacer linker includes one or more carbohydrate containing or
polyhydroxyl group containing linkers and one or more aspartic
acids. In another variation, the hydrophilic spacer linker includes
one or more carbohydrate containing or polyhydroxyl group
containing linkers and one or more glutamic acids. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0582] In another embodiment, the hydrophilic spacer linker
comprises one or more oligoamide hydrophilic spacers, such as but
not limited to aminoethylpiperazinylacetamide.
[0583] In another embodiment, the hydrophilic spacer linker
comprises one or more triazole linked carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more amide linked
carbohydrate containing or polyhydroxyl group containing linkers.
In another embodiment, the hydrophilic spacer linker comprises one
or more PEG groups and one or more cysteines. In another
embodiment, the hydrophilic spacer linker comprises one or more
EDTE derivatives.
[0584] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00085##
wherein m, n, and q are integers that are independently selected
from the range of 0 to about 8; AA is an amino acid, R.sup.1 is
hydrogen, alkyl, or a nitrogen protecting group, and drugs are
optionally attached at the (*) atoms. In one aspect, AA is a
naturally occurring amino acid of either the natural or unnatural
configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integer o is 1 or greater. In
another aspect, the integer m is 2 or greater. The drugs, or
analogs or derivatives thereof, and optionally additional linkers
and additional ligands may be connected to the above formula at the
free NH side chains of the 2,.omega.-diaminoalkanoic acid
fragments, or at the terminal carboxylate as indicated by the free
valences therein.
[0585] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00086##
wherein m, n, q, and p are integers that are independently selected
from the range of 0 to about 8; AA is an amino acid, R.sup.1 is
hydrogen, alkyl, or a nitrogen protecting group, and drugs are
optionally attached at the (*) atoms. In one aspect, AA is as a
naturally occurring amino acid of either the natural or unnatural
configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integers o and p are 1 or
greater. In another aspect, the integer m is 2 or greater. The
drugs, or analogs or derivatives thereof, and optionally additional
linkers and additional ligands may be connected to the above
formula at the free NH side chains of the 2,.omega.-diaminoalkanoic
acid fragments, at the cysteinyl thiol groups, or at the terminal
carboxylate, as indicated by the free valences therein.
[0586] In another embodiment, a folate ligand intermediate is
described having the following formula
##STR00087##
wherein m, n, q, p, and r are integers that are independently
selected from the range of 0 to about 8; AA is an amino acid,
R.sup.1 is hydrogen, alkyl, or a nitrogen protecting group, and
drugs are optionally attached at the (*) atoms. In one aspect, AA
is as a naturally occurring amino acid of either the natural or
unnatural configuration. In another aspect, one or more of AA is a
hydrophilic amino acid. In another aspect, one or more of AA is Asp
and/or Arg. In another aspect, the integers o, p, and r are 1 or
greater. In another aspect, the integer m is 2 or greater. The
drugs, or analogs or derivatives thereof, and optionally additional
linkers and additional ligands may be connected to the above
formula at the free NH side chains of the 2,.omega.-diaminoalkanoic
acid fragments, at the cyteinyl thiol groups, at the serinyl
hydroxy groups, or at the terminal carboxylate, as indicated by the
free valences therein.
[0587] In another embodiment, the compound of any of the embodiment
described herein wherein L comprises a divalent linker of the
formula
##STR00088##
[0588] wherein * indicates the point of attachment to a folate and
** indicates the point of attachment to a drug; and F and G are
each independently 1, 2, 3 or 4 are described.
[0589] In another embodiment, the of any one of the embodiments
described herein wherein L is a linker comprises a divalent linker
of the formula
##STR00089##
[0590] wherein *, **, *** each indicate points of attachment to the
ligand, and the one or more drugs D. It is to be understood that
when there are fewer drugs, *, **, *** are substituted with
hydrogen or a heteroatom. F and G are each independently 1, 2, 3 or
4; and W.sup.1 is NH or O is described. In another aspect, m.sup.1
is 0 or 1.
[0591] In another embodiment, amino acid refers to beta, gamma, and
longer amino acids, such as amino acids of the formula:
--N(R)--(CR'R'').sub.q--C(O)--
[0592] where R is hydrogen, alkyl, acyl, or a suitable nitrogen
protecting group, R' and R'' are hydrogen or a substituent, each of
which is independently selected in each occurrence, and q is an
integer such as 1, 2, 3, 4, or 5. Illustratively, R' and/or R''
independently correspond to, but are not limited to, hydrogen or
the side chains present on naturally occurring amino acids, such as
methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl,
carboxylmethyl, guanidinopropyl, and the like, and derivatives and
protected derivatives thereof. The above described formula includes
all stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like.
[0593] As used herein, the term "alkyl" includes a chain of carbon
atoms, which is optionally branched. As used herein, the term
"alkenyl" and "alkynyl" includes a chain of carbon atoms, which is
optionally branched, and includes at least one double bond or
triple bond, respectively. It is to be understood that alkynyl may
also include one or more double bonds. It is to be further
understood that in certain embodiments, alkyl is advantageously of
limited length, including C.sub.1-C.sub.24, C.sub.1-C.sub.12,
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4.
Illustratively, such particularly limited length alkyl groups,
including C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may
be referred to as lower alkyl. It is to be further understood that
in certain embodiments alkenyl and/or alkynyl may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenyl and/or alkynyl groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenyl and/or alkynyl. It is appreciated herein that shorter
alkyl, alkenyl, and/or alkynyl groups may add less lipophilicity to
the compound and accordingly will have different pharmacokinetic
behavior. In embodiments of the invention described herein, it is
to be understood, in each case, that the recitation of alkyl refers
to alkyl as defined herein, and optionally lower alkyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkenyl refers to
alkenyl as defined herein, and optionally lower alkenyl. In
embodiments of the invention described herein, it is to be
understood, in each case, that the recitation of alkynyl refers to
alkynyl as defined herein, and optionally lower alkynyl.
Illustrative alkyl, alkenyl, and alkynyl groups are, but not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl,
hexyl, heptyl, octyl, and the like, and the corresponding groups
containing one or more double and/or triple bonds, or a combination
thereof.
[0594] As used herein, the term "alkylene" includes a divalent
chain of carbon atoms, which is optionally branched. As used
herein, the term "alkenylene" and "alkynylene" includes a divalent
chain of carbon atoms, which is optionally branched, and includes
at least one double bond or triple bond, respectively. It is to be
understood that alkynylene may also include one or more double
bonds. It is to be further understood that in certain embodiments,
alkylene is advantageously of limited length, including
C.sub.1-C.sub.24, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, and C.sub.1-C.sub.4. Illustratively, such
particularly limited length alkylene groups, including
C.sub.1-C.sub.8, C.sub.1-C.sub.6, and C.sub.1-C.sub.4 may be
referred to as lower alkylene. It is to be further understood that
in certain embodiments alkenylene and/or alkynylene may each be
advantageously of limited length, including C.sub.2-C.sub.24,
C.sub.2-C.sub.12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, and
C.sub.2-C.sub.4. Illustratively, such particularly limited length
alkenylene and/or alkynylene groups, including C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 may be referred to as lower
alkenylene and/or alkynylene. It is appreciated herein that shorter
alkylene, alkenylene, and/or alkynylene groups may add less
lipophilicity to the compound and accordingly will have different
pharmacokinetic behavior. In embodiments of the invention described
herein, it is to be understood, in each case, that the recitation
of alkylene, alkenylene, and alkynylene refers to alkylene,
alkenylene, and alkynylene as defined herein, and optionally lower
alkylene, alkenylene, and alkynylene. Illustrative alkyl groups
are, but not limited to, methylene, ethylene, n-propylene,
isopropylene, n-butylene, isobutylene, sec-butylene, pentylene,
1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, and
the like.
[0595] As used herein, the term "cycloalkyl" includes a chain of
carbon atoms, which is optionally branched, where at least a
portion of the chain in cyclic. It is to be understood that
cycloalkylalkyl is a subset of cycloalkyl. It is to be understood
that cycloalkyl may be polycyclic. Illustrative cycloalkyl include,
but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl,
2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like.
As used herein, the term "cycloalkenyl" includes a chain of carbon
atoms, which is optionally branched, and includes at least one
double bond, where at least a portion of the chain in cyclic. It is
to be understood that the one or more double bonds may be in the
cyclic portion of cycloalkenyl and/or the non-cyclic portion of
cycloalkenyl. It is to be understood that cycloalkenylalkyl and
cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be
understood that cycloalkyl may be polycyclic. Illustrative
cycloalkenyl include, but are not limited to, cyclopentenyl,
cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to
be further understood that chain forming cycloalkyl and/or
cycloalkenyl is advantageously of limited length, including
C.sub.3-C.sub.24, C.sub.3-C.sub.12, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, and C.sub.5-C.sub.6. It is appreciated herein that
shorter alkyl and/or alkenyl chains forming cycloalkyl and/or
cycloalkenyl, respectively, may add less lipophilicity to the
compound and accordingly will have different pharmacokinetic
behavior.
[0596] As used herein, the term "heteroalkyl" includes a chain of
atoms that includes both carbon and at least one heteroatom, and is
optionally branched. Illustrative heteroatoms include nitrogen,
oxygen, and sulfur. In certain variations, illustrative heteroatoms
also include phosphorus, and selenium. As used herein, the term
"cycloheteroalkyl" including heterocyclyl and heterocycle, includes
a chain of atoms that includes both carbon and at least one
heteroatom, such as heteroalkyl, and is optionally branched, where
at least a portion of the chain is cyclic. Illustrative heteroatoms
include nitrogen, oxygen, and sulfur. In certain variations,
illustrative heteroatoms also include phosphorus, and selenium.
Illustrative cycloheteroalkyl include, but are not limited to,
tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,
morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the
like.
[0597] As used herein, the term "aryl" includes monocyclic and
polycyclic aromatic carbocyclic groups, each of which may be
optionally substituted. Illustrative aromatic carbocyclic groups
described herein include, but are not limited to, phenyl, naphthyl,
and the like. As used herein, the term "heteroaryl" includes
aromatic heterocyclic groups, each of which may be optionally
substituted. Illustrative aromatic heterocyclic groups include, but
are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,
tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl,
pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl,
isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,
benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and
the like.
[0598] As used herein, the term "amino" includes the group
NH.sub.2, alkylamino, and dialkylamino, where the two alkyl groups
in dialkylamino may be the same or different, i.e. alkylalkylamino.
Illustratively, amino includes methylamino, ethylamino,
dimethylamino, methylethylamino, and the like. In addition, it is
to be understood that when amino modifies or is modified by another
term, such as aminoalkyl, or acylamino, the above variations of the
term amino are included therein. Illustratively, aminoalkyl
includes H.sub.2N-alkyl, methylaminoalkyl, ethylaminoalkyl,
dimethylaminoalkyl, methylethylaminoalkyl, and the like.
Illustratively, acylamino includes acylmethylamino, acylethylamino,
and the like.
[0599] As used herein, the term "amino and derivatives thereof"
includes amino as described herein, and alkylamino, alkenylamino,
alkynylamino, heteroalkylamino, heteroalkenylamino,
heteroalkynylamino, cycloalkylamino, cycloalkenylamino,
cycloheteroalkylamino, cycloheteroalkenylamino, arylamino,
arylalkylamino, arylalkenylamino, arylalkynylamino,
heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino,
heteroarylalkynylamino, acylamino, and the like, each of which is
optionally substituted. The term "amino derivative" also includes
urea, carbamate, and the like.
[0600] As used herein, the term "hydroxy and derivatives thereof"
includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy,
heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy,
cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy,
arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy,
heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like,
each of which is optionally substituted. The term "hydroxy
derivative" also includes carbamate, and the like.
[0601] As used herein, the term "thio and derivatives thereof"
includes SH, and alkylthio, alkenylthio, alkynylthio,
heteroalkylthio, heteroalkenylthio, heteroalkynylthio,
cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio,
cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio,
arylalkynylthio, heteroarylthio, heteroarylalkylthio,
heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the
like, each of which is optionally substituted. The term "thio
derivative" also includes thiocarbamate, and the like.
[0602] As used herein, the term "acyl" includes formyl, and
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,
cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,
arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl,
heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl,
heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of
which is optionally substituted.
[0603] As used herein, the term "carbonyl and derivatives thereof"
includes the group C(O), C(S), C(NH) and substituted amino
derivatives thereof. As used herein, the term "carboxylic acid and
derivatives thereof" includes the group CO.sub.2H and salts
thereof, and esters and amides thereof, and CN.
[0604] As used herein, the term "sulfinic acid or a derivative
thereof" includes SO.sub.2H and salts thereof, and esters and
amides thereof.
[0605] As used herein, the term "sulfonic acid or a derivative
thereof" includes SO.sub.3H and salts thereof, and esters and
amides thereof.
[0606] As used herein, the term "sulfonyl" includes alkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, heteroalkylsulfonyl,
heteroalkenylsulfonyl, heteroalkynylsulfonyl, cycloalkylsulfonyl,
cycloalkenylsulfonyl, cycloheteroalkylsulfonyl,
cycloheteroalkenylsulfonyl, arylsulfonyl, arylalkylsulfonyl,
arylalkenylsulfonyl, arylalkynylsulfonyl, heteroarylsulfonyl,
heteroarylalkylsulfonyl, heteroarylalkenylsulfonyl,
heteroarylalkynylsulfonyl, acylsulfonyl, and the like, each of
which is optionally substituted.
[0607] As used herein, the term "phosphinic acid or a derivative
thereof" includes P(R)O.sub.2H and salts thereof, and esters and
amides thereof, where R is alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteroalkyl,
cycloheteroalkenyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, each of which is optionally substituted.
[0608] As used herein, the term "phosphonic acid or a derivative
thereof" includes PO.sub.3H.sub.2 and salts thereof, and esters and
amides thereof.
[0609] As used herein, the term "hydroxylamino and derivatives
thereof" includes NHOH, and alkyloxylNH alkenyloxylNH alkynyloxylNH
heteroalkyloxylNH heteroalkenyloxylNH heteroalkynyloxylNH
cycloalkyloxylNH cycloalkenyloxylNH cycloheteroalkyloxylNH
cycloheteroalkenyloxylNH aryloxylNH arylalkyloxylNH
arylalkenyloxylNH arylalkynyloxylNH heteroaryloxylNH
heteroarylalkyloxylNH heteroarylalkenyloxylNH
heteroarylalkynyloxylNH acyloxy, and the like, each of which is
optionally substituted.
[0610] As used herein, the term "hydrazino and derivatives thereof"
includes alkylNHNH, alkenylNHNH, alkynylNHNH, heteroalkylNHNH,
heteroalkenylNHNH, heteroalkynylNHNH, cycloalkylNHNH,
cycloalkenylNHNH, cycloheteroalkylNHNH, cycloheteroalkenylNHNH,
arylNHNH, arylalkylNHNH, arylalkenylNHNH, arylalkynylNHNH,
heteroarylNHNH, heteroarylalkylNHNH, heteroarylalkenylNHNH,
heteroarylalkynylNHNH, acylNHNH, and the like, each of which is
optionally substituted.
[0611] The term "optionally substituted" as used herein includes
the replacement of hydrogen atoms with other functional groups on
the radical that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0612] As used herein, the terms "optionally substituted aryl" and
"optionally substituted heteroaryl" include the replacement of
hydrogen atoms with other functional groups on the aryl or
heteroaryl that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
heteroarylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the like.
Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl,
heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is
optionally substituted.
[0613] Illustrative substituents include, but are not limited to, a
radical --(CH.sub.2).sub.xZ.sup.X, where x is an integer from 0-6
and Z.sup.X is selected from halogen, hydroxy, alkanoyloxy,
including C.sub.1-C.sub.6 alkanoyloxy, optionally substituted
aroyloxy, alkyl, including C.sub.1-C.sub.6 alkyl, alkoxy, including
C.sub.1-C.sub.6 alkoxy, cycloalkyl, including C.sub.3-C.sub.8
cycloalkyl, cycloalkoxy, including C.sub.3-C.sub.8 cycloalkoxy,
alkenyl, including C.sub.2-C.sub.6 alkenyl, alkynyl, including
C.sub.2-C.sub.6 alkynyl, haloalkyl, including C.sub.1-C.sub.6
haloalkyl, haloalkoxy, including C.sub.1-C.sub.6 haloalkoxy,
halocycloalkyl, including C.sub.3-C.sub.8 halocycloalkyl,
halocycloalkoxy, including C.sub.3-C.sub.8 halocycloalkoxy, amino,
C.sub.1-C.sub.6 alkylamino, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)amino, alkylcarbonylamino, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylamino, aminoalkyl, C.sub.1-C.sub.6
alkylaminoalkyl, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z.sup.X is
selected from --CO.sub.2R.sup.4 and --CONR.sup.5R.sup.6, where
R.sup.4, R.sup.5, and R.sup.6 are each independently selected in
each occurrence from hydrogen, C.sub.1-C.sub.6 alkyl,
aryl-C.sub.1-C.sub.6 alkyl, and heteroaryl-C.sub.1-C.sub.6
alkyl.
[0614] The term "prodrug" as used herein generally refers to any
compound that when administered to a biological system generates a
biologically active compound as a result of one or more spontaneous
chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or
metabolic chemical reaction(s), or a combination thereof. In vivo,
the prodrug is typically acted upon by an enzyme (such as
esterases, amidases, phosphatases, and the like), simple biological
chemistry, or other process in vivo to liberate or regenerate the
more pharmacologically active drug. This activation may occur
through the action of an endogenous host enzyme or a non-endogenous
enzyme that is administered to the host preceding, following, or
during administration of the prodrug. Additional details of prodrug
use are described in U.S. Pat. No. 5,627,165; and Pathalk et al.,
Enzymic protecting group techniques in organic synthesis,
Stereosel. Biocatal. 775-797 (2000). It is appreciated that the
prodrug is advantageously converted to the original drug as soon as
the goal, such as targeted delivery, safety, stability, and the
like is achieved, followed by the subsequent rapid elimination of
the released remains of the group forming the prodrug.
[0615] Prodrugs may be prepared from the compounds described herein
by attaching groups that ultimately cleave in vivo to one or more
functional groups present on the compound, such as --OH--, --SH,
--CI.sub.2H, --NR.sub.2. Illustrative prodrugs include but are not
limited to carboxylate esters where the group is alkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, acyloxyalkyl,
alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and
amines where the group attached is an acyl group, an
alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrative
esters, also referred to as active esters, include but are not
limited to 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such as
acetoxymethyl, pivaloyloxymethyl, .beta.-acetoxyethyl,
.beta.-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl,
(1-aminoethyl)carbonyloxymethyl, and the like;
alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl,
.alpha.-ethoxycarbonyloxyethyl, .beta.-ethoxycarbonyloxyethyl, and
the like; dialkylaminoalkyl groups, including di-lower alkylamino
alkyl groups, such as dimethylaminomethyl, dimethylaminoethyl,
diethylaminomethyl, diethylaminoethyl, and the like;
2-(alkoxycarbonyl)-2-alkenyl groups such as
2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and
the like; and lactone groups such as phthalidyl,
dimethoxyphthalidyl, and the like. Further illustrative prodrugs
contain a chemical moiety, such as an amide or phosphorus group
functioning to increase solubility and/or stability of the
compounds described herein. Further illustrative prodrugs for amino
groups include, but are not limited to, (C.sub.3-C.sub.20)alkanoyl;
halo-(C.sub.3-C.sub.20)alkanoyl; (C.sub.3-C.sub.20)alkenoyl;
(C.sub.4-C.sub.7)cycloalkanoyl;
(C.sub.3-C.sub.6)-cycloalkyl(C.sub.2-C.sub.16)alkanoyl; optionally
substituted aroyl, such as unsubstituted aroyl or aroyl substituted
by 1 to 3 substituents selected from the group consisting of
halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with one or more of 1 to 3
halogen atoms; optionally substituted
aryl(C.sub.2-C.sub.26)alkanoyl and optionally substituted
heteroaryl(C.sub.2-C.sub.16)alkanoyl, such as the aryl or
heteroaryl radical being unsubstituted or substituted by 1 to 3
substituents selected from the group consisting of halogen,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with 1 to 3 halogen atoms; and
optionally substituted heteroarylalkanoyl having one to three
heteroatoms selected from O, S and N in the heteroaryl moiety and 2
to 10 carbon atoms in the alkanoyl moiety, such as the heteroaryl
radical being unsubstituted or substituted by 1 to 3 substituents
selected from the group consisting of halogen, cyano,
trifluoromethanesulphonyloxy, (C.sub.1-C.sub.3)alkyl, and
(C.sub.1-C.sub.3)alkoxy, each of which is optionally further
substituted with 1 to 3 halogen atoms. The groups illustrated are
exemplary, not exhaustive, and may be prepared by conventional
processes.
[0616] It is understood that the prodrugs themselves may not
possess significant biological activity, but instead undergo one or
more spontaneous chemical reaction(s), enzyme-catalyzed chemical
reaction(s), and/or metabolic chemical reaction(s), or a
combination thereof after administration in vivo to produce the
compound described herein that is biologically active or is a
precursor of the biologically active compound. However, it is
appreciated that in some cases, the prodrug is biologically active.
It is also appreciated that prodrugs may often serves to improve
drug efficacy or safety through improved oral bioavailability,
pharmacodynamic half-life, and the like. Prodrugs also refer to
derivatives of the compounds described herein that include groups
that simply mask undesirable drug properties or improve drug
delivery. For example, one or more compounds described herein may
exhibit an undesirable property that is advantageously blocked or
minimized may become pharmacological, pharmaceutical, or
pharmacokinetic barriers in clinical drug application, such as low
oral drug absorption, lack of site specificity, chemical
instability, toxicity, and poor patient acceptance (bad taste,
odor, pain at injection site, and the like), and others. It is
appreciated herein that a prodrug, or other strategy using
reversible derivatives, can be useful in the optimization of the
clinical application of a drug.
[0617] The drug can be any molecule capable of modulating or
otherwise modifying cell function, including pharmaceutically
active compounds. Suitable molecules can include, but are not
limited to: peptides, oligopeptides, retro-inverso oligopeptides,
proteins, protein analogs in which at least one non-peptide linkage
replaces a peptide linkage, apoproteins, glycoproteins, enzymes,
coenzymes, enzyme inhibitors, amino acids and their derivatives,
receptors and other membrane proteins; antigens and antibodies
thereto; haptens and antibodies thereto; hormones, lipids,
phospholipids, liposomes; toxins; antibiotics; analgesics;
bronchodilators; beta-blockers; antimicrobial agents;
antihypertensive agents; cardiovascular agents including
antiarrhythmics, cardiac glycosides, antianginals and vasodilators;
central nervous system agents including stimulants, psychotropics,
antimanics, and depressants; antiviral agents; antihistamines;
cancer drugs including chemotherapeutic agents; tranquilizers;
anti-depressants; H-2 antagonists; anticonvulsants; antinauseants;
prostaglandins and prostaglandin analogs; muscle relaxants;
anti-inflammatory substances; immunosuppressants, stimulants;
decongestants; antiemetics; diuretics; antispasmodics;
antiasthmatics; anti-Parkinson agents; expectorants; cough
suppressants; mucolytics; and mineral and nutritional
additives.
[0618] Further, the drug can be any drug known in the art which is
cytotoxic, enhances tumor permeability, inhibits tumor cell
proliferation, promotes apoptosis, decreases anti-apoptotic
activity in target cells, enhances an endogenous immune response
directed to the pathogenic cells, or is useful for treating a
disease state caused by any type of pathogenic cell. Drugs suitable
for use in accordance with this invention include adrenocorticoids
and corticosteroids, alkylating agents, antiandrogens,
antiestrogens, androgens, aclamycin and aclamycin derivatives,
estrogens, antimetabolites such as cytosine arabinoside, purine
analogs, pyrimidine analogs, and methotrexate, busulfan,
carboplatin, chlorambucil, cisplatin and other platinum compounds,
tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,
Taxotere.RTM., cyclophosphamide, daunomycin, rhizoxin, T2 toxin,
plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins,
discodermolides, microtubule inhibitors, epothilones, tubulysins,
cyclopropyl benz[e]indolone, seco-cyclopropyl benz[e]indolone,
O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vinca alkaloids, such
as vincristine, vinblastine, vindesine, vinorelbine and analogs and
derivative thereof such as deacetylvinblastine monohydrazide
(DAVLBH), colchicine, colchicine derivatives, allocolchicine,
thiocolchicine, trityl cysteine, halicondrin B, dolastatins such as
dolastatin 10, amanitins such as .alpha.-amanitin, camptothecin,
irinotecan, and other camptothecin derivatives thereof,
geldanamycin and geldanamycin derivatives, estramustine,
nocodazole, MAP4, colcemid, inflammatory and proinflammatory
agents, peptide and peptidomimetic signal transduction inhibitors,
and any other art-recognized drug or toxin. Other drugs that can be
used in accordance with the invention include rapamycins, such as
sirolimus or everolimus, penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and
any other art-recognized antimicrobial compound.
[0619] In another embodiment, the drug is selected from a
cryptophycin, bortezomib, thiobortezomib, a tubulysin, aminopterin,
rapamycin, paclitaxel, docetaxel, doxorubicin, daunorubicin,
everolimus, .alpha.-amanatin, verucarin, didemnin B, geldanomycin,
purvalanol A, everolimus, ispinesib, budesonide, dasatinib, an
epothilone, a maytansine, and a tyrosine kinase inhibitor,
including analogs and derivatives of the foregoing. In another
embodiment, the ligand conjugate includes at least two drugs (D)
selected illustratively from a vinca alkaloid, a cryptophycin,
bortezomib, thiobortezomib, a tubulysin, aminopterin, a rapamycin,
such as everolimus or sirolimus, paclitaxel, docetaxel,
doxorubicin, daunorubicin, everolimus, .alpha.-amanatin, verucarin,
didemnin B, geldanomycin, purvalanol A, ispinesib, budesonide,
dasatinib, an epothilone, a maytansine, and a tyrosine kinase
inhibitor, including analogs and derivatives of the foregoing. In
one variation, the drugs (D) are the same. In another variation,
the drugs (D) are different.
[0620] As used herein, tubulysins refer generally to tetrapeptide
compounds of the formula
##STR00090##
and pharmaceutical salts thereof, where
[0621] n is 1-3;
[0622] V is H, OR.sup.2, or halo, and W is H, OR.sup.2, or alkyl,
where R.sup.2 is independently selected in each instance from H,
alkyl, and C(O)R.sup.3, where R.sup.3 is alkyl, cycloalkyl,
alkenyl, aryl, or arylalkyl, each of which is optionally
substituted; providing that R.sup.2 is not H when both V and
[0623] W are OR.sup.2; or V and W are taken together with the
attached carbon to form a carbonyl; X.dbd.H, C.sub.1-4 alkyl,
alkenyl, each of which is optionally substituted, or
CH.sub.2QR.sup.9; where Q is --N--, --O--, or --S--; R.sup.9=H,
C.sub.1-4 alkyl, alkenyl, aryl, or C(O)R.sup.10; and
R.sup.10=C.sub.1-6 alkyl, alkenyl, aryl, or heteroaryl, each of
which is optionally substituted;
[0624] Z is alkyl and Y is O; or Z is alkyl or C(O)R.sup.4, and Y
is absent, where R.sup.4 is alkyl, CF.sub.3, or aryl;
[0625] R.sup.1 is H, or R.sup.1 represents 1 to 3 substituents
selected from halo, nitro, carboxylate or a derivative thereof,
cyano, hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, and
OR.sup.6, where R.sup.6 is hydrogen or optionally substituted aryl,
a phenol protecting group, a prodrug moiety, alkyl, arylalkyl,
C(O)R.sup.7, P(O)(OR.sup.8).sub.2, or SO.sub.3R.sup.8, where
R.sup.7 and R.sup.8 are independently selected in each instance
from H, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and
arylalkyl, each of which is optionally substituted, or R.sup.8 is a
metal cation; and
[0626] R is OH or a leaving group, or R forms a carboxylic acid
derivative.
[0627] Conjugates of each of the foregoing tubulysins are described
herein. In one variation, Z is methyl. In another variation,
R.sup.1 is H. In another variation, R.sup.1 is OR.sup.6 at C(4),
where R.sup.6 is H, alkyl, or COR.sup.7. In another variation, V is
H, and W is OC(O)R.sup.3.
[0628] Natural tubulysins are generally linear tetrapeptides
consisting of N-methyl pipecolic acid (Mep), isoleucine (Ile), an
unnatural aminoacid called tubuvalin (Tuv), and either an unnatural
aminoacid called tubutyrosine (Tut, an analog of tyrosine) or an
unnatural aminoacid called tubuphenylalanine (Tup, an analog of
phenylalanine). In another embodiment, naturally occurring
tubulysins, and analogs and derivatives thereof, of the following
general formula are described
##STR00091##
and pharmaceutical salts thereof, where R, R.sup.1, and R.sup.10
are as described in the various embodiments herein. Conjugates of
each of the foregoing tubulysins are described herein.
[0629] In another embodiment, conjugates of naturally occurring
tubulysins of the following general formula are described
TABLE-US-00002 ##STR00092## Factor R.sup.10 R.sup.1 A
(CH.sub.3).sub.2CHCH.sub.2 OH B CH.sub.3(CH.sub.2).sub.2 OH C
CH.sub.3CH.sub.2 OH D (CH.sub.3).sub.2CHCH.sub.2 H E
CH.sub.3(CH.sub.2).sub.2 H F CH.sub.2CH.sub.3 H G
(CH.sub.3).sub.2C.dbd.CH OH H CH.sub.3 H I CH.sub.3 OH
[0630] and pharmaceutical salts thereof.
[0631] In another embodiment, the drug has the formula
##STR00093##
wherein
[0632] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0633] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0634] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug foming group,
and C(O)R.sup.D, where R.sup.D is in each instance independently
selected from the group consisting of hydrogen, and alkyl, alkenyl,
heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,
heteroaryl, and heteroarylalkyl, each of which is optionally
substituted is described.
[0635] The ligand conjugates described herein can be administered
in a combination therapy with any other known drug whether or not
the additional drug is targeted. Illustrative additional drugs
include, but are not limited to, peptides, oligopeptides,
retro-inverso oligopeptides, proteins, protein analogs in which at
least one non-peptide linkage replaces a peptide linkage,
apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors,
amino acids and their derivatives, receptors and other membrane
proteins, antigens and antibodies thereto, haptens and antibodies
thereto, hormones, lipids, phospholipids, liposomes, toxins,
antibiotics, analgesics, bronchodilators, beta-blockers,
antimicrobial agents, antihypertensive agents, cardiovascular
agents including antiarrhythmics, cardiac glycosides, antianginals,
vasodilators, central nervous system agents including stimulants,
psychotropics, antimanics, and depressants, antiviral agents,
antihistamines, cancer drugs including chemotherapeutic agents,
tranquilizers, anti-depressants, H-2 antagonists, anticonvulsants,
antinauseants, prostaglandins and prostaglandin analogs, muscle
relaxants, anti-inflammatory substances, stimulants, decongestants,
antiemetics, diuretics, antispasmodics, antiasthmatics,
anti-Parkinson agents, expectorants, cough suppressants,
mucolytics, and mineral and nutritional additives.
[0636] Illustrative examples of ligand conjugates are described
below:
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108##
##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113##
##STR00114## ##STR00115##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00116##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00117##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00118##
herein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00119##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00120##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00121##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00122##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00123##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00124##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00125##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00126##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00127##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00128##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00129##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00130##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00131##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00132##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00133##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00134##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00135##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00136##
wherein A is N.sup.9--CH.sub.3-5-d(i)PteGlu, 5-d(i)PteGlu, IAHQ,
BW1843U89, 2-NH.sub.2-ZD1694, ZD1694, CB3717, 5-dH.sub.4PteAPBA,
5-dPteHCysA, or DDATHF,
##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141##
##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146##
##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151##
##STR00152## ##STR00153## ##STR00154## ##STR00155## ##STR00156##
##STR00157## ##STR00158## ##STR00159## ##STR00160## ##STR00161##
##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166##
##STR00167## ##STR00168## ##STR00169## ##STR00170## ##STR00171##
##STR00172## ##STR00173## ##STR00174## ##STR00175## ##STR00176##
##STR00177## ##STR00178## ##STR00179## ##STR00180## ##STR00181##
##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186##
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## ##STR00193## ##STR00194## ##STR00195## ##STR00196##
##STR00197## ##STR00198## ##STR00199## ##STR00200## ##STR00201##
##STR00202## ##STR00203## ##STR00204## ##STR00205## ##STR00206##
##STR00207## ##STR00208## ##STR00209## ##STR00210##
[0637] In various embodiments of the methods, uses, and kits
described herein, the thiol inhibitor is selected from the group
consisting of 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB);
maleimides (e.g., N-maleoyl-1'-alanine (N-(2-carboxyethyl)maleimide
(NCEM)); p-chloromercuribenzene sulfonate (pCMBS);
4-(N--(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO);
2,2'-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione
(GSSG); vinyl sulfone compounds (e.g.,
methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). In
one embodiment, the thiol inhibitor is DTNB. In another embodiment,
the thiol inhibitor is a maleimide. In yet another embodiment, the
thiol inhibitor is NCEM. In one embodiment, the thiol inhibitor is
pCMBS. In another embodiment, the thiol inhibitor is GSAO. In yet
another embodiment, the thiol inhibitor is dimesna. In one
embodiment, the thiol inhibitor is GSSG. In another embodiment, the
thiol inhibitor is a vinyl sulfone compound. In yet another
embodiment, the thiol inhibitor is methoxy-PEG5000-vinylsulfone. In
still another embodiment, the thiol inhibitor is EGCG. In another
embodiment, the thiol inhibitor is AMS. Any combinations of these
thiol inhibitors are contemplated in accordance with the
invention.
[0638] In one aspect, the ligand conjugate and the thiol inhibitor
are in parenteral dosage forms and may be administered directly
into the blood stream, into muscle, or into an internal organ.
Suitable routes for such parenteral administration include
intravenous, intraarterial, intraperitoneal, intrathecal,
intradermal, epidural, intracerebroventricular, intraurethral,
intrasternal, intracranial, intratumoral, intramuscular and
subcutaneous delivery. Suitable means for parenteral administration
include needle (including microneedle) injectors, needle-free
injectors and infusion techniques.
[0639] In one illustrative aspect, parenteral formulations are
typically aqueous solutions which may contain carriers or
excipients such as salts, carbohydrates and buffering agents
(preferably at a pH of from 3 to 9), but, for some applications,
they may be more suitably formulated as a sterile non-aqueous
solution or as a dried form to be used in conjunction with a
suitable vehicle such as sterile, pyrogen-free water. In other
embodiments, any of the liquid formulations described herein may be
adapted for parenteral administration of the conjugates or
additional chemotherapeutic agents described herein. The
preparation of parenteral formulations under sterile conditions,
for example, by lyophilization under sterile conditions, may
readily be accomplished using standard pharmaceutical techniques
well-known to those skilled in the art.
[0640] In various embodiments of the methods, uses, and kits
described herein, the ligand conjugate is in a composition and the
thiol inhibitor is in a composition and the compositions may
further comprise pharmaceutically acceptable carriers. The carriers
can be excipients. In some embodiments, the pharmaceutically
acceptable carriers are liquid carriers. In various embodiments,
the liquid carriers are independently selected from the group
consisting of saline, glucose, alcohols, glycols, esters, amides,
and a combination thereof.
[0641] The choice of carrier will to a large extent depend on
factors such as the particular mode of administration, the effect
of the carrier on solubility and stability, and the nature of the
dosage form. Pharmaceutical compositions suitable for the delivery
of ligand conjugates or thiol inhibitors described herein and
methods for their preparation will be readily apparent to those
skilled in the art. Such compositions and methods for their
preparation may be found, for example, in Remington: The Science
& Practice of Pharmacy, 21st Edition (Lippincott Williams &
Wilkins, 2005), incorporated herein by reference.
[0642] In one illustrative aspect, the pharmaceutically acceptable
carrier may be any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, and combinations thereof, that are
physiologically compatible. In some embodiments, the carrier is
suitable for parenteral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. Supplementary active compounds
can also be incorporated into compositions of the invention.
[0643] In various embodiments of the methods, uses, and kits
described herein, the ligand conjugate and the thiol inhibitor are
administered in therapeutically effective amounts. The unitary
daily dosage of the ligand conjugate and the thiol inhibitor can
vary significantly depending on the patient condition, the disease
state being treated, the purity of the compounds and their route of
administration and tissue distribution, and the possibility of
co-usage of other therapeutic treatments, such as radiation
therapy. The effective amount to be administered to a patient is
based on body surface area, mass, and physician assessment of
patient condition. Effective doses can range, for example, from
about 1 ng/kg to about 1 mg/kg, from about 1 .mu.g/kg to about 500
.mu.g/kg, and from about 1 .mu.g/kg to about 100 .mu.g/kg. These
doses are based on an average patient weight of about 70 kg, and
the kg are kg of patient body weight (mass).
[0644] The ligand conjugate and the thiol inhibitor can each be
administered in a dose of from about 1.0 ng/kg to about 1000
.mu.g/kg, from about 10 ng/kg to about 1000 .mu.g/kg, from about 50
ng/kg to about 1000 .mu.g/kg, from about 100 ng/kg to about 1000
.mu.g/kg, from about 500 ng/kg to about 1000 .mu.g/kg, from about 1
ng/kg to about 500 .mu.g/kg, from about 1 ng/kg to about 100
.mu.g/kg, from about 1 .mu.g/kg to about 50 .mu.g/kg, from about 1
.mu.g/kg to about 10 .mu.g/kg, from about 5 .mu.g/kg to about 500
.mu.g/kg, from about 10 .mu.g/kg to about 100 .mu.g/kg, from about
20 .mu.g/kg to about 200 .mu.g/kg, from about 10 .mu.g/kg to about
500 .mu.g/kg, or from about 50 .mu.g/kg to about 500 .mu.g/kg. The
total dose may be administered in single or divided doses and may,
at the physician's discretion, fall outside of the typical range
given herein. These dosages are based on an average patient weight
of about 70 kg and the "kg" are kilograms of patient body weight.
The physician will readily be able to determine doses for subjects
whose weight falls outside this range, such as infants and the
elderly.
[0645] In another embodiment, the ligand conjugate and the thiol
inhibitor can each be administered in a dose of from about 1
.mu.g/m.sup.2 to about 500 mg/m.sup.2, from about 1 .mu.g/m.sup.2
to about 300 mg/m.sup.2, or from about 100 .mu.g/m.sup.2 to about
200 mg/m.sup.2. In other embodiments, the ligand conjugate and the
thiol inhibitor can each be administered in a dose of from about 1
mg/m.sup.2 to about 500 mg/m.sup.2, from about 1 mg/m.sup.2 to
about 300 mg/m.sup.2, from about 1 mg/m.sup.2 to about 200
mg/m.sup.2, from about 1 mg/m.sup.2 to about 100 mg/m.sup.2, from
about 1 mg/m.sup.2 to about 50 mg/m.sup.2, or from about 1
mg/m.sup.2 to about 600 mg/m.sup.2. The total dose may be
administered in single or divided doses and may, at the physician's
discretion, fall outside of the typical range given herein. These
dosages are based on m.sup.2 of body surface area.
[0646] The ligand conjugate and the thiol inhibitor described
herein may contain one or more chiral centers, or may otherwise be
capable of existing as multiple stereoisomers. Accordingly, it is
to be understood that the present invention includes pure
stereoisomers as well as mixtures of stereoisomers, such as
enantiomers, diastereomers, and enantiomerically or
diastereomerically enriched mixtures. The ligand conjugate and the
thiol inhibitor described herein may be capable of existing as
geometric isomers. Accordingly, it is to be understood that the
present invention includes pure geometric isomers or mixtures of
geometric isomers.
[0647] It is appreciated that the ligand conjugate and the thiol
inhibitor described herein may exist in unsolvated forms as well as
solvated forms, including hydrated forms. In general, the solvated
forms are equivalent to unsolvated forms and are encompassed within
the scope of the present invention. The ligand conjugate and the
thiol inhibitor described herein may exist in multiple crystalline
or amorphous forms. In general, all physical forms are equivalent
for the uses contemplated by the present invention and are intended
to be within the scope of the present invention.
[0648] In another embodiment, compositions and/or dosage forms for
administration of ligand conjugate and the thiol inhibitor are
prepared from compounds with a purity of at least about 90%, or
about 95%, or about 96%, or about 97%, or about 98%, or about 99%,
or about 99.5%. In another embodiment, compositions and or dosage
forms for administration of ligand conjugate and the thiol
inhibitor are prepared from compounds with a purity of at least
90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 99.5%.
[0649] As used herein, purity determinations may be based on weight
percentage, mole percentage, and the like. In addition, purity
determinations may be based on the absence or substantial absence
of certain predetermined components, such as, but not limited to,
folic acid, disulfide containing components not containing a drug,
oxidation products, disulfide components not containing a folate,
and the like. It is also to be understood that purity
determinations are applicable to solutions of the compounds and
compositions purified by the methods described herein. In those
instances, purity measurements, including weight percentage and
mole percentage measurements, are related to the components of the
solution exclusive of the solvent.
[0650] The purity of the ligand conjugate and the thiol inhibitor
may be measured using any conventional technique, including various
chromatography or spectroscopic techniques, such as high pressure
or high performance liquid chromatography (HPLC), nuclear magnetic
resonance spectroscopy, TLC, UV absorbance spectroscopy,
fluorescence spectroscopy, and the like.
[0651] In some embodiments of the methods, uses, and kits described
herein, the ligand conjugate and the thiol inhibitor are in sterile
containers or packages. In other embodiments of the methods, uses,
and kits described herein, the ligand conjugate and the thiol
inhibitor are in sterile, pyrogen-free aqueous solutions. In some
embodiments of the uses described herein, the ligand conjugate is
in the form of a reconstitutable lyophilizate. The methods, uses,
and kits described herein can be for both human clinical medicine
and veterinary applications. Thus, the patient can be human or, in
the case of veterinary applications, can be a laboratory,
agricultural, domestic, or wild animal. The methods, uses, and kits
described herein can be applied to humans (i.e., a human patient),
laboratory animals such rodents (e.g., mice, rats, hamsters, etc.),
rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats,
and rabbits, agricultural animals such as cows, horses, pigs,
sheep, goats, and wild animals in captivity such as bears, pandas,
lions, tigers, leopards, elephants, zebras, giraffes, gorillas,
dolphins, and whales.
[0652] In another embodiment described herein, an in vitro assay
for identifying a ligand conjugate suitable for co-administration
to a patient with a thiol inhibitor is provided. The assay
comprises the steps of:
[0653] a) adding the ligand conjugate to the culture medium of a
first sample of cultured cells, wherein the ligand conjugate
comprises a disulfide linkage;
[0654] b) adding the thiol inhibitor to the culture medium of the
first sample of cultured cells to provide a test sample;
[0655] c) adding the ligand conjugate to the culture medium of a
second sample of cultured cells to provide a control sample;
[0656] d) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the test
sample;
[0657] e) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the control
sample; and
[0658] f) determining that the ligand conjugate is suitable for
co-administration to the patient with the thiol inhibitor if the
non-ligand-specific activity of the ligand conjugate and/or the
nonspecific uptake of the drug are decreased in the test sample
relative to the control sample.
[0659] As used herein, the term "in vitro assay" refers to any
assay that may be performed using cultured cells. The term
"suitable for co-administration" refers to a ligand conjugate that
would advantageously benefit from co-administration with a thiol
inhibitor. Such benefit can be observed via a reduction in
non-ligand-specific activity of the ligand conjugate, a reduction
in nonspecific uptake of the drug portion of the ligand conjugate
into the cells, or any other suitable assay.
[0660] As used herein, the term "adding" refers to placing a
solution comprising the ligand conjugate or the thiol inhibitor, or
both, into the culture medium in which the cells are incubated.
Methods of "adding," such as pipeting, are known to a practitioner
skilled in the art.
[0661] As used herein, the term "culture medium" means the
extracellular medium containing the nutrients and other
constituents supporting the growth of cells. Such media can be
prepared in the laboratory by methods known to a skilled artisan
and are also commercially available. In some embodiments, the
culture medium contains thiols. In other embodiments, the culture
medium does not contain thiols (i.e., is thiol-free). In some
embodiments, the culture medium is Roswell Park Memorial Institute
(RPMI) medium (Cellgro, Manassas, Va.).
[0662] Cultured cells according to the present invention may
include any cell type known in the art that can be cultured in
vitro. In some embodiments, the cultured cells express folate
receptors on the cell surface. In other embodiments, the cultured
cells do not express folate receptors on the cell surface. In some
embodiments, the cultured cells are KB cells. In other embodiments,
the cultured cells are A549 cells. In yet other embodiments, the
cultured cells can be, for example, H23 cells, AN3CA cells, HepG2
cells, RAW264.7 cells, MDA-MB-468 cells, or MDA-MB-231 cells.
[0663] As used herein, the term "measuring" refers to determining
the amount quantitatively, of the non-ligand-specific activity,
non-specific uptake, or other activity of interest. Measuring can
be done directly or indirectly. Indirect methods of measuring
include measuring of cellular responses such as .sup.3H-thymidine
incorporation as a measure of cytotoxicity, or, for example,
enzymatic reaction products. Direct measuring includes such assays
as measuring uptake of radiolabeled molecules into cells.
[0664] As used herein, the term "non-ligand-specific activity"
refers to activity that is not the result of ligand binding to the
cells of interest. When the ligand is folate and the ligand
receptor is a folate receptor (FR), the "non-FR-specific activity"
refers to activity that is not the result of binding to folate
receptors on the cell surface. In some embodiments,
non-ligand-specific activity is determinied by measuring
.sup.3H-thymidine incorporation as a measure of cytotoxicity. As
used herein, the term "cytotoxicity" refers to cell killing or
inhibition of cell growth or division as a result of toxicity.
[0665] As used herein, the term "nonspecific uptake" refers to
uptake of the drug moiety of the ligand conjugate into a cell where
the uptake is not the result of ligand binding to the cells of
interest. For example, when the ligand is folate and the ligand
receptor is a folate receptor (FR), the "nonspecific uptake" of the
drug refers to uptake that is not the result of binding of folate
to the folate receptors on the cells. In some embodiments,
nonspecific uptake is measured using competition assays.
Competition assays are known to a skilled artisan. In some
embodiments, the competition assay may include use of a
radiolabeled ligand conjugate, and in some embodiments, the
radiolabel is .sup.3H-thymidine. In some embodiments, a
non-radiolabeled ligand is also added to the assay in excess and
the uptake of the radiolabeled conjugate (i.e., the drug portion of
the conjugate) that is not competed by the excess non-radiolabeled
ligand is non-specific uptake.
[0666] The previously described embodiments of the ligand conjugate
and the thiol inhibitor are applicable to the in vitro assay
described herein. In various embodiments of the in vitro assay
described herein, the assay further comprises step g) administering
the ligand conjugate and the thiol inhibitor to the patient. The
term "administering" refers to any suitable means of delivering the
ligand conjugate, the thiol inhibitor, or both, to the patient. In
some embodiments, the administration is a parenteral
administration. Suitable routes for such parenteral administration
include intravenous, intraarterial, intraperitoneal, intrathecal,
intradermal, epidural, intracerebroventricular, intraurethral,
intrasternal, intracranial, intratumoral, intramuscular and
subcutaneous delivery. Suitable means for parenteral administration
include needle (including microneedle) injectors, needle-free
injectors and infusion techniques.
[0667] In various embodiments of the in vitro assay described
herein, the non-ligand-specific activity of the ligand conjugate is
decreased by the thiol inhibitor. In other embodiments of the in
vitro assay described herein, the non-ligand-specific activity is
cytotoxicity. In various embodiments of the in vitro assay
described herein, the nonspecific uptake of the drug is decreased
by the thiol inhibitor. In yet other embodiments of the in vitro
assay described herein, the nonspecific uptake of the ligand
conjugate is measured using competition assays in the presence and
absence of an excess of non-radiolabeled ligand.
[0668] In one embodiment described herein, a method of treatment of
a disease is provided. The method comprises administering a ligand
conjugate to a patient, wherein the ligand conjugate comprises a
disulfide linkage; and administering a system x.sub.c.sup.-
inhibitor to the patient. In some embodiments, the disease is
cancer or inflammation.
[0669] In another embodiment, use of a ligand conjugate in
combination with a system x.sub.c.sup.- inhibitor for the treatment
of a disease wherein the disease is cancer or inflammation, and
wherein the ligand conjugate comprises a disulfide linkage is
described.
[0670] In yet another embodiment, use of a ligand conjugate for the
manufacture of a medicament for the treatment of a disease wherein
the disease is cancer or inflammation, and wherein the treatment
comprises treating a patient with the ligand conjugate in
combination with a system x.sub.c.sup.- inhibitor, wherein the
ligand conjugate comprises a disulfide linkage is described.
[0671] In another embodiment, a kit is provided. The kit comprises
a ligand conjugate and one or more system x.sub.c.sup.- inhibitor,
wherein the ligand conjugate comprises a disulfide linkage.
[0672] In various embodiments of the methods and uses described
herein, the disease is inflammation. In other embodiments, the
disease is cancer. In some embodiments, the cancer comprises a
primary tumor. In yet other embodiments, the cancer comprises
metastatic tumor cells. The methods and uses described herein can
be utilized to treat such cancers as carcinomas, sarcomas,
lymphomas, Hodgekin's disease, melanomas, mesotheliomas, Burkitt's
lymphoma, nasopharyngeal carcinomas, leukemias, and myelomas. The
cancers can also be oral, thyroid, endocrine, skin, gastric,
esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian,
cervical, uterine, breast, testicular, prostate, rectal, kidney,
liver, or lung cancers.
[0673] The previously described embodiments of the ligand conjugate
are applicable to the methods, uses, and kits utilizing the system
x.sub.c.sup.- inhibitor described herein.
[0674] In various embodiments of the methods, uses, and kits
described herein, the system x.sub.c.sup.- inhibitor is selected
from the group consisting of sulfasalazine, glutamate;
L-quisqualate; (S)-4-carboxyphenylglycine (4-S-CPG);
L-.alpha.-aminoadipic acid; and L-homocysteic acid. In one
embodiment, the system x.sub.c.sup.- inhibitor is sulfasalazine. In
another embodiment, the system x.sub.c.sup.- inhibitor is
glutamate. In yet another embodiment, the system x.sub.c.sup.-
inhibitor is L-quisqualate. In one embodiment, the system
x.sub.c.sup.- inhibitor is 4-S-CPG. In another embodiment, the
system x.sub.c.sup.- inhibitor is L-.alpha.-aminoadipic acid. In
yet another embodiment, the system x.sub.c.sup.- inhibitor is
L-homocysteic acid. Any combinations of these system x.sub.c.sup.-
inhibitors are contemplated in accordance with the invention.
[0675] In one aspect, the ligand conjugate and the system
x.sub.c.sup.- inhibitor are in parenteral dosage forms and may be
administered directly into the blood stream, into muscle, or into
an internal organ. Suitable routes for such parenteral
administration include intravenous, intraarterial, intraperitoneal,
intrathecal, intradermal, epidural, intracerebroventricular,
intraurethral, intrasternal, intracranial, intratumoral,
intramuscular and subcutaneous delivery. Suitable means for
parenteral administration include needle (including microneedle)
injectors, needle-free injectors and infusion techniques.
[0676] In one illustrative aspect, parenteral formulations are
typically aqueous solutions which may contain carriers or
excipients such as salts, carbohydrates and buffering agents
(preferably at a pH of from 3 to 9), but, for some applications,
they may be more suitably formulated as a sterile non-aqueous
solution or as a dried form to be used in conjunction with a
suitable vehicle such as sterile, pyrogen-free water. In other
embodiments, any of the liquid formulations described herein may be
adapted for parenteral administration of the conjugates or
additional chemotherapeutic agents described herein. The
preparation of parenteral formulations under sterile conditions,
for example, by lyophilization under sterile conditions, may
readily be accomplished using standard pharmaceutical techniques
well-known to those skilled in the art.
[0677] In various embodiments of the methods, uses, and kits
described herein, the ligand conjugate is in a composition and the
system x.sub.c.sup.- inhibitor is in a composition and the
compositions may further comprise pharmaceutically acceptable
carriers. The carriers can be excipients. In some embodiments, the
pharmaceutically acceptable carriers are liquid carriers. In
various embodiments, the liquid carriers are independently selected
from the group consisting of saline, glucose, alcohols, glycols,
esters, amides, and a combination thereof.
[0678] The choice of carrier will to a large extent depend on
factors such as the particular mode of administration, the effect
of the carrier on solubility and stability, and the nature of the
dosage form. Pharmaceutical compositions suitable for the delivery
of ligand conjugates or system x.sub.c.sup.- inhibitors described
herein and methods for their preparation will be readily apparent
to those skilled in the art. Such compositions and methods for
their preparation may be found, for example, in Remington: The
Science & Practice of Pharmacy, 21st Edition (Lippincott
Williams & Wilkins, 2005), incorporated herein by
reference.
[0679] In one illustrative aspect, the pharmaceutically acceptable
carrier may be any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, and combinations thereof, that are
physiologically compatible. In some embodiments, the carrier is
suitable for parenteral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. Supplementary active compounds
can also be incorporated into compositions of the invention.
[0680] In various embodiments of the methods, uses, and kits
described herein, the ligand conjugate and the system x.sub.c.sup.-
inhibitor are administered in therapeutically effective amounts.
The unitary daily dosage of the ligand conjugate and the system
x.sub.c.sup.- inhibitor can vary significantly depending on the
patient condition, the disease state being treated, the purity of
the compounds and their route of administration and tissue
distribution, and the possibility of co-usage of other therapeutic
treatments, such as radiation therapy. The effective amount to be
administered to a patient is based on body surface area, mass, and
physician assessment of patient condition. Effective doses can
range, for example, from about 1 ng/kg to about 1 mg/kg, from about
1 .mu.g/kg to about 500 .mu.g/kg, and from about 1 .mu.g/kg to
about 100 .mu.g/kg. These doses are based on an average patient
weight of about 70 kg, and the kg are kg of patient body weight
(mass).
[0681] The ligand conjugate and the system x.sub.c.sup.- inhibitor
can each be administered in a dose of from about 1.0 ng/kg to about
1000 .mu.g/kg, from about 10 ng/kg to about 1000 .mu.g/kg, from
about 50 ng/kg to about 1000 .mu.g/kg, from about 100 ng/kg to
about 1000 .mu.g/kg, from about 500 ng/kg to about 1000 .mu.g/kg,
from about 1 ng/kg to about 500 .mu.g/kg, from about 1 ng/kg to
about 100 .mu.g/kg, from about 1 .mu.g/kg to about 50 .mu.g/kg,
from about 1 .mu.g/kg to about 10 .mu.g/kg, from about 5 .mu.g/kg
to about 500 .mu.g/kg, from about 10 .mu.g/kg to about 100
.mu.g/kg, from about 20 .mu.g/kg to about 200 .mu.g/kg, from about
10 .mu.g/kg to about 500 .mu.g/kg, or from about 50 .mu.g/kg to
about 500 .mu.g/kg. The total dose may be administered in single or
divided doses and may, at the physician's discretion, fall outside
of the typical range given herein. These dosages are based on an
average patient weight of about 70 kg and the "kg" are kilograms of
patient body weight. The physician will readily be able to
determine doses for subjects whose weight falls outside this range,
such as infants and the elderly.
[0682] In another embodiment, the ligand conjugate and the system
x.sub.c.sup.- inhibitor can each be administered in a dose of from
about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2, from about 1
.mu.g/m.sup.2 to about 300 mg/m.sup.2, or from about 100
.mu.g/m.sup.2 to about 200 mg/m.sup.2. In other embodiments, the
ligand conjugate and the system x.sub.c.sup.- inhibitor can each be
administered in a dose of from about 1 mg/m.sup.2 to about 500 m
g/m.sup.2, from about 1 mg/m.sup.2 to about 300 mg/m.sup.2, from
about 1 mg/m.sup.2 to about 200 mg/m.sup.2, from about 1 mg/m.sup.2
to about 100 mg/m.sup.2, from about 1 mg/m.sup.2 to about 50
mg/m.sup.2, or from about 1 mg/m.sup.2 to about 600 mg/m.sup.2. The
total dose may be administered in single or divided doses and may,
at the physician's discretion, fall outside of the typical range
given herein. These dosages are based on m.sup.2 of body surface
area.
[0683] The ligand conjugate and the system x.sub.c.sup.- inhibitor
described herein may contain one or more chiral centers, or may
otherwise be capable of existing as multiple stereoisomers.
Accordingly, it is to be understood that the present invention
includes pure stereoisomers as well as mixtures of stereoisomers,
such as enantiomers, diastereomers, and enantiomerically or
diastereomerically enriched mixtures. The ligand conjugate and the
system x.sub.c.sup.- inhibitor described herein may be capable of
existing as geometric isomers. Accordingly, it is to be understood
that the present invention includes pure geometric isomers or
mixtures of geometric isomers.
[0684] It is appreciated that the ligand conjugate and the system
x.sub.c.sup.- inhibitor described herein may exist in unsolvated
forms as well as solvated forms, including hydrated forms. In
general, the solvated forms are equivalent to unsolvated forms and
are encompassed within the scope of the present invention. The
ligand conjugate and the system x.sub.c.sup.- inhibitor described
herein may exist in multiple crystalline or amorphous forms. In
general, all physical forms are equivalent for the uses
contemplated by the present invention and are intended to be within
the scope of the present invention.
[0685] In another embodiment, compositions and/or dosage forms for
administration of ligand conjugate and the system x.sub.c.sup.-
inhibitor are prepared from compounds with a purity of at least
about 90%, or about 95%, or about 96%, or about 97%, or about 98%,
or about 99%, or about 99.5%. In another embodiment, compositions
and or dosage forms for administration of ligand conjugate and the
system x.sub.c.sup.- inhibitor are prepared from compounds with a
purity of at least 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or
99.5%.
[0686] As used herein, purity determinations may be based on weight
percentage, mole percentage, and the like. In addition, purity
determinations may be based on the absence or substantial absence
of certain predetermined components, such as, but not limited to,
folic acid, disulfide containing components not containing a drug,
oxidation products, disulfide components not containing a folate,
and the like. It is also to be understood that purity
determinations are applicable to solutions of the compounds and
compositions purified by the methods described herein. In those
instances, purity measurements, including weight percentage and
mole percentage measurements, are related to the components of the
solution exclusive of the solvent.
[0687] The purity of the ligand conjugate and the system
x.sub.c.sup.- inhibitor may be measured using any conventional
technique, including various chromatography or spectroscopic
techniques, such as high pressure or high performance liquid
chromatography (HPLC), nuclear magnetic resonance spectroscopy,
TLC, UV absorbance spectroscopy, fluorescence spectroscopy, and the
like.
[0688] In some embodiments of the methods, uses, and kits described
herein, the ligand conjugate and the system x.sub.c.sup.- inhibitor
are in sterile containers or packages. In other embodiments of the
methods, uses, and kits described herein, the ligand conjugate and
the system x.sub.c.sup.- inhibitor are in sterile, pyrogen-free
aqueous solutions. In some embodiments of the uses described
herein, the ligand conjugate is in the form of a reconstitutable
lyophilizate. The methods, uses, and kits described herein can be
for both human clinical medicine and veterinary applications. Thus,
the patient can be human or, in the case of veterinary
applications, can be a laboratory, agricultural, domestic, or wild
animal. The methods, uses, and kits described herein can be applied
to humans (i.e., a human patient), laboratory animals such rodents
(e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees,
domestic animals such as dogs, cats, and rabbits, agricultural
animals such as cows, horses, pigs, sheep, goats, and wild animals
in captivity such as bears, pandas, lions, tigers, leopards,
elephants, zebras, giraffes, gorillas, dolphins, and whales.
[0689] In another embodiment described herein, an in vitro assay
for identifying a ligand conjugate suitable for co-administration
to a patient with a system x.sub.c.sup.- inhibitor is provided. The
assay comprises the steps of:
[0690] a) adding the ligand conjugate to the culture medium of a
first sample of cultured cells, wherein the ligand conjugate
comprises a disulfide linkage;
[0691] b) adding the system x.sub.c.sup.- inhibitor to the culture
medium of the first sample of cultured cells to provide a test
sample;
[0692] c) adding the ligand conjugate to the culture medium of a
second sample of cultured cells to provide a control sample;
[0693] d) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the test
sample;
[0694] e) measuring the non-ligand-specific activity of the ligand
conjugate or the nonspecific uptake of the drug in the control
sample; and
[0695] f) determining that the ligand conjugate is suitable for
co-administration to the patient with the system x.sub.c.sup.-
inhibitor if the non-ligand-specific activity of the ligand
conjugate and/or the nonspecific uptake of the drug are decreased
in the test sample relative to the control sample.
[0696] As used herein, the term "in vitro assay" refers to any
assay that may be performed using cultured cells. The term
"suitable for co-administration" refers to a ligand conjugate that
would advantageously benefit from co-administration with a system
x.sub.c.sup.- inhibitor. Such benefit can be observed via a
reduction in non-ligand-specific activity of the ligand conjugate,
a reduction in nonspecific uptake of the drug portion of the ligand
conjugate into the cells, or any other suitable assay.
[0697] As used herein, the term "adding" refers to placing a
solution comprising the ligand conjugate or the system
x.sub.c.sup.- inhibitor, or both, into the culture medium in which
the cells are incubated. Methods of "adding," such as pipeting, are
known to a practitioner skilled in the art.
[0698] As used herein, the term "culture medium" means the
extracellular medium containing the nutrients and other
constituents supporting the growth of cells. Such media can be
prepared in the laboratory by methods known to a skilled artisan
and are also commercially available. In some embodiments, the
culture medium contains thiols. In other embodiments, the culture
medium does not contain thiols (i.e., is thiol-free). In some
embodiments, the culture medium is Roswell Park Memorial Institute
(RPMI) medium (Cellgro, Manassas, Va.).
[0699] Cultured cells according to the present invention may
include any cell type known in the art that can be cultured in
vitro. In some embodiments, the cultured cells express folate
receptors on the cell surface. In other embodiments, the cultured
cells do not express folate receptors on the cell surface. In some
embodiments, the cultured cells are KB cells. In other embodiments,
the cultured cells are A549 cells. In yet other embodiments, the
cultured cells can be, for example, H23 cells, AN3CA cells, HepG2
cells, RAW264.7 cells, MDA-MB-468 cells, or MDA-MB-231 cells.
[0700] As used herein, the term "measuring" refers to determining
the amount quantitatively, of the non-ligand-specific activity,
non-specific uptake, or other activity of interest. Measuring can
be done directly or indirectly. Indirect methods of measuring
include measuring of cellular responses such as .sup.3H-thymidine
incorporation as a measure of cytotoxicity, or, for example,
enzymatic reaction products. Direct measuring includes such assays
as measuring uptake of radiolabeled molecules into cells.
[0701] As used herein, the term "non-ligand-specific activity"
refers to activity that is not the result of ligand binding to the
cells of interest. When the ligand is folate and the ligand
receptor is a folate receptor (FR), the "non-FR-specific activity"
refers to activity that is not the result of binding to folate
receptors on the cell surface. In some embodiments,
non-ligand-specific activity is determinied by measuring
.sup.3H-thymidine incorporation as a measure of cytotoxicity. As
used herein, the term "cytotoxicity" refers to cell killing or
inhibition of cell growth or division as a result of toxicity.
[0702] As used herein, the term "nonspecific uptake" refers to
uptake of the drug moiety of the ligand conjugate into a cell where
the uptake is not the result of ligand binding to the cells of
interest. For example, when the ligand is folate and the ligand
receptor is a folate receptor (FR), the "nonspecific uptake" of the
drug refers to uptake that is not the result of binding of folate
to the folate receptors on the cells. In some embodiments,
nonspecific uptake is measured using competition assays.
Competition assays are known to a skilled artisan. In some
embodiments, the competition assay may include use of a
radiolabeled ligand conjugate, and in some embodiments, the
radiolabel is .sup.3H-thymidine. In some embodiments, a
non-radiolabeled ligand is also added to the assay in excess and
the uptake of the radiolabeled conjugate (i.e., the drug portion of
the conjugate) that is not competed by the excess non-radiolabeled
ligand is non-specific uptake.
[0703] The previously described embodiments of the ligand conjugate
and the system x.sub.c.sup.- inhibitor are applicable to the in
vitro assay described herein. In various embodiments of the in
vitro assay described herein, the assay further comprises step g)
administering the ligand conjugate and the system x.sub.c.sup.-
inhibitor to the patient. The term "administering" refers to any
suitable means of delivering the ligand conjugate, the system
x.sub.c.sup.- inhibitor, or both, to the patient. In some
embodiments, the administration is a parenteral administration.
Suitable routes for such parenteral administration include
intravenous, intraarterial, intraperitoneal, intrathecal,
intradermal, epidural, intracerebroventricular, intraurethral,
intrasternal, intracranial, intratumoral, intramuscular and
subcutaneous delivery. Suitable means for parenteral administration
include needle (including microneedle) injectors, needle-free
injectors and infusion techniques.
[0704] The previously described embodiments of the in vitro assay
utilizing the system x.sub.c.sup.- inhibitor are applicable to the
in vitro assay utilizing the system x.sub.c.sup.- inhibitor
described herein. In various embodiments of the in vitro assay
described herein, the assay further comprises step g) administering
the ligand conjugate and the system x.sub.c.sup.- inhibitor to the
patient.
[0705] In some embodiments of the in vitro assay described herein,
the cultured cells are KB cells. In other embodiments of the in
vitro assay described herein, the cultured cells arA549 cells.
[0706] In various embodiments of the in vitro assay described
herein, the non-ligand-specific activity of the ligand conjugate is
decreased by the system x.sub.c.sup.- inhibitor. In other
embodiments of the in vitro assay described herein, the
non-ligand-specific activity is cytotoxicity. In various
embodiments of the in vitro assay described herein, the nonspecific
uptake of the drug is decreased by the system x.sub.c.sup.-
inhibitor. In yet other embodiments of the in vitro assay described
herein, the nonspecific uptake of the ligand conjugate is measured
using competition assays in the presence and absence of an excess
of non-radiolabeled ligand.
[0707] In another embodiment, the methods, uses, compositions,
pharmaceutical compositions, combinations, or kits described herein
include the following examples. The examples further illustrate
additional features of the various embodiments of the invention
described herein. However, it is to be understood that the examples
are illustrative and are not to be construed as limiting other
embodiments of the invention described herein. In addition, it is
appreciated that other variations of the examples are included in
the various embodiments of the invention described herein.
Example 1
[0708] The presence of extracellular thiols can undesirably affect
the activity and uptake of ligand conjugates. For example, an
increase in extracellular thiols in cell culture media can
demonstrate such undesired effects in vitro, and an increase in
extracellular thiols in interstitial fluids can have undesirable
effects in vivo.
[0709] The in vitro extracellular thiol activity can be evaluated
in conditioned culture media of the KB cell line and the A549 cell
line. Each type of cells was plated in 24-well tissue
cultured-treated plates at 1.times.10.sup.5 cells per well in
folate-deficient RPMI/10% heat-inactivated fetal bovine serum
(FDRPMI/HaBS; KB cells) or in RPMI medium with folate/10% HIFBS
(RPMI+FA/HIFBS; A549 cells) and the cells were allowed to attach to
the plates overnight. The cells were rinsed one time with PBS, pH
7.4, and the time course for thiol activity was initiated by adding
500 .mu.L of RPMI medium without phenol red (PR) or serum to each
well. The cells were then incubated at 37.degree. C. in a 5%
CO.sub.2/95% humidified air incubator for increasing periods of
time. At each time point, 55 .mu.L of a 4 mM solution of DTNB
(Sigma) was added to the appropriate wells (n=3) to achieve a final
DTNB concentration of 400 .mu.M. Plates were rocked gently to mix
and incubated for 5 minutes at room temperature to allow the DTNB
to react with extracellular thiols. The solutions were then removed
from the cells, and absorbance was determined at a wavelength of
412 nm Background absorbance (determined from an aliquot of DTNB
solution incubated in an empty well of the tissue culture plate)
was subtracted from each value. Thiol concentrations were
calculated based on an extinction coefficient of 14,150
M.sup.-1cm.sup.-1. Protein concentrations of cell lysates were
determined by the BCA Assay method (Pierce, Rockford, Ill.).
[0710] As shown in FIG. 1, a linear increase in in vitro
extracellular thiol activity (nmol of thiol/mg of protein) is
observed in conditioned culture media for the KB cell line and the
A549 cell line over an 8 hour period.
Example 2
[0711] Furthermore, the degree of extracellular thiol activity
varies among different cell lines. Cells from various cell lines
(i.e., H23, AN3CA, HepG2, RAW264.7, MDA-MB-468, MDA-MB-231, KB, and
A549 cell lines) were each plated in 24-well tissue
cultured-treated plates at 2.times.10.sup.5 cells per well in
FDRPMI/HIFBS (FR+cells) or in RPMI+FA/HIFBS (FR-cells) and were
allowed to attach to the plates overnight. The cells were rinsed
one time with PBS, pH 7.4, and then a 200 .mu.M solution of DTNB in
PR-free RPMI or in PR-free RPMI without cystine and glutathione
(SH-free) was added to each well (n=3). The cells were incubated
for 2 h at 37.degree. C., the solutions were removed from the
cells, and absorbance was determined at a wavelength of 412 nm
Background absorbance determined from an aliquot of DTNB solution
incubated in an empty well of the tissue culture plate was
subtracted from each value. Thiol concentrations were calculated
based on an extinction coefficient of 14,150 M.sup.-1 cm.sup.-1.
Protein concentrations of cell lysates were determined by the BCA
Assay method (Pierce, Rockford, Ill.).
[0712] As shown in FIG. 2, degree of extracellular thiol activity
(nmol of thiol/mg of protein) varies among different cell lines, as
evaluated using RPMI cell culture media and thiol-free RPMI cell
culture media. A549 cells exhibit the highest amount of
extracellular thiol activity. In contrast, RAQ264.7 cells exhibit
the lowest amount of extracellular thiol activity. Furthermore, as
shown in FIG. 2, removal of thiols from the culture medium
virtually eliminates the extracellular release of thiols.
Example 3
[0713] In addition, the amount of thiol activity correlates with
the non-ligand-specific activity of ligand conjugates. In this
example, the non-FR-specific activity of an exemplary folate
conjugate (EC0531) was investigated in MDA-MD-468, H23, AN3CA,
MDA-MB-231, KB, and A549 cells.
[0714] Extracellular thiol activity was determined using the DTNB
method described above. Non-FR-specific activity of EC0531 was
determined using a .sup.3H-thymidine incorporation assay. Cells
were seeded in 24-well tissue culture-treated plates at
1.times.10.sup.5 cells per well and allowed to attach overnight at
37.degree. C. Serial dilutions of EC0531 were prepared in
FDRPMI/HIFBS, and each well received 0.5 mL of EC0531 solution. To
assess non-FR-targeted activity in FR+cells, 100 .mu.M FA was
included as a competitor along with the drug in the treatment
solutions. Cells were incubated for 2 hours in the presence of
drug, washed 3 times with media, and then chased in 0.5 mL of
FDRPMI/HIFBS (FR+cells) or RPMI+FA/HIFBS (FR-cells) to 72 h at
37.degree. C. Spent medium was then aspirated from the wells, and
cells were incubated with 1 .mu.Ci/mL .sup.3H-thymidine for 4 hours
at 37.degree. C., washed two times with PBS, pH 7.4, then treated
with 0.4 mL 5% trichloroacetic acid per well. After 15 minutes, the
trichloroacetic acid was aspirated from the wells, and cells were
solubilized in 0.5 mL 0.25 N sodium hydroxide. Each sample (450
.mu.L) was transferred to a scintillation vial containing 3 mL of
Ecolite+scintillation cocktail and then counted in a liquid
scintillation counter (LSC). Final results were expressed as
percentage of .sup.3H-thymidine incorporation relative to an
untreated control (non-competed groups) or FA control (competed
groups). Sensitivity to the base drug, tubulysin B hydrazide, was
determined using the .sup.3H-thymidine incorporation assay and the
same incubation conditions as described for EC0531.
[0715] As shown in FIG. 3, the extracellular thiol activity
(IC.sub.50 (nM), adjusted for tubulysin B hydrazide sensitivity) of
various cell lines correlates with the non-FR-specific activity of
the folate conjugate EC0531 FIG. 3 demonstrates the correlation
between extracellular thiol activity and non-FR-specific activity
of EC0531 (data adjusted for tubulysin B hydrazide
sensitivity).
Example 4
[0716] The presence of extracellular thiols can affect the
non-ligand-specific activity of ligand conjugates. In this example,
the non-FR-specific activity of an exemplary folate conjugate
(EC0531) was investigated. The non-FR-specific activity of EC0531
was evaluated in both KB cells (cells known to be FR positive) and
in A549 cells (cells known to be FR negative). FR-specific and
non-FR-specific activity of EC0531 was determined by the
.sup.3H-thymidine incorporation assay using the methods described
in Example 3 above. As shown in FIG. 4, the non-FR-specific
activity of EC0531 is observed at concentrations of about 0.3-1
.mu.M in both KB cells and A549 cells.
[0717] The effects of three different cell-impermeable thiol
inhibitors on the non-FR-specific activity of EC0531 were evaluated
in this example: 1) 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB); 2)
N-(2-carboxyethyl)maleimide (NCEM), and 3) folate-maleimide. Each
thiol inhibitor was separately co-administered with EC0531 (1
.mu.M) and folic acid (100 .mu.M), and the agents were incubated
for 2 hours, followed by a 72 hour chase. The inhibition of
non-FR-specific activity (i.e., cytotoxicity) was evaluated for
each thiol inhibitor.
[0718] As shown in FIG. 5, DTNB, NCEM, and folate-maleimide all
exhibit a dose-responsive inhibition of non-FR-specific activity in
KB cells. DTNB exhibited an IC.sub.50 of 4.6 .mu.M, NCEM exhibited
an IC.sub.50 of 17 .mu.M, and folate-maleimide exhibited an
IC.sub.50 of 3.5 .mu.M.
[0719] However, pre-treatment of cells with thiol inhibitors prior
to the administration of EC0531 did not inhibit the non-FR-specific
activity. Cells were seeded in 24-well tissue culture-treated
plates at 1.times.10.sup.5 cells per well and allowed to attach
overnight at 37.degree. C. To assess the effect of various
inhibitors on non-targeted activity of EC0531 in the FR+KB cell
line, cells were treated concurrently with DTNB (100 .mu.M) or NCEM
(10 mM) and 1 .mu.M EC0531 plus 100 .mu.M FA to block all
FR-specific drug uptake. Cells were incubated for 2 hours in the
presence of drug, competitor, and inhibitor, washed 3 times with
media, and then chased in 0.5 mL of FDRPMI/HIFBS to 72 hours at
37.degree. C. Cells were then treated as described above to
determine .sup.3H-thymidine incorporation. Final results were
expressed as percentage of .sup.3H-thymidine incorporation relative
to an untreated control (non-competed groups) or FA control
(competed groups). As shown in FIG. 6, pre-treatment of KB cells
with DTNB or NCEM prior to administration of EC0531 and folic acid
is not effective to inhibit the non-FR-specific activity in the
cells.
[0720] Various thiol inhibitors were tested to evaluate their
effectiveness in inhibiting the non-FR-specific activity following
co-administration with EC0531. The results are shown in Table
1.
TABLE-US-00003 TABLE 1 Inhibition Concentration of EC0531 Action
Inhibitor (.mu.M) Nonspecific Activity Membrane- DTNB 10 .mu.M Full
inhibition impermeable NCEM 100 .mu.M Full inhibition sulfhydryl
Folate-maleimide 100 .mu.M Full inhibition blockers pCMBS 100 .mu.M
>50% inhibition EC1277 (GSAO) 3 mM Full inhibition SH-reactive
GSSG 10 mM Full inhibition agents Dimesna 1 mM Full inhibition
Methoxy-PEG5000- 3 mM >50% inhibition vinylsulfone EGCg 100
.mu.M >50% inhibition Nonspecific DIDS 1 mM Full inhibition
anion BSP 1 mM <50% inhibition transport inhibitors
Example 5
[0721] The presence of extracellular thiols can also affect the
nonspecific uptake of ligand conjugates. In this example, the
nonspecific uptake of an exemplary folate conjugate (EC0531;
specifically .sup.3H-EC0531) was investigated. The nonspecific
uptake of EC0531 was evaluated in both KB cells and in A549
cells.
[0722] For .sup.3H-EC0531 uptake studies, cells were seeded at
2.times.10.sup.5 cells per well of a 24-well tissue culture plate
and allowed to attach to plates overnight. Increasing
concentrations of 3H-EC0531 and .sup.3H-FA were prepared in
FFRPMI/HIFBS. To determine non-FR-targeted uptake, 100 .mu.M FA was
included in the uptake solutions. Cells were incubated with 0.5 mL
of uptake solution for 2 h at 37.degree. C., washed 3 times with
ice-cold PBS, and were solubilized in 0.5 mL 1% SDS in PBS, pH 7.4.
Cell lysates (450 .mu.L) were added to 3 mL Ecolite+scintillation
cocktail and counted in a liquid scintillation counter. Protein
concentrations of the remaining lysates were determined using the
BCA Protein Assay (Pierce, Rockford, Ill.). Molecules per cell were
calculated based on DPM values and previously determined cellular
protein conversion factors (2.82.times.10.sup.-7 and
2.23.times.10.sup.-7 mg protein per cell for KB and A549,
respectively).
[0723] As shown in FIG. 7, the nonspecific uptake of .sup.3H-EC0531
(molecules per cell.times.10.sup.6) is observed at concentrations
of about 1 .mu.M in both KB cells and A549 cells.
[0724] The effects of two different concentrations of DTNB (10
.mu.M and 100 .mu.M) on the reduction of nonspecific uptake of
.sup.3H-EC0531 into cells were also evaluated in this example
Inhibition of non-FR-targeted .sup.3H-EC0531 uptake was assessed by
incubating .sup.3H-EC0531 in the presence of inhibitors. Cells were
seeded at 4.times.10.sup.5 cells per well of a 12-well tissue
culture plate, and were treated the next day with 1 .mu.M EC0531
plus the indicated concentrations of inhibitors in 1 mL
FFRPMI/HIFBS for 2 hours at 37.degree. C. FA at a concentration of
100 .mu.M was used as a competitor to determine non-FR-specific
uptake in KB cells. Wells were then washed 3 times with 1 mL of
ice-cold PBS, pH 7.4 and were processed for LSC analysis as
described above.
[0725] As shown in FIG. 8, DTNB exhibited the ability to reduce the
nonspecific uptake of .sup.3H-EC0531 in KB cells and in A549 cells.
In comparison, the nonspecific uptake of .sup.3H-folic acid was
mostly unaffected.
[0726] The effects of three thiol inhibitors (DTNB, NCEM, and DIDS)
on the reduction of nonspecific uptake of .sup.3H-EC0531 into cells
were also evaluated in this example. In addition to the thiol
inhibitors, the effects of a reduced folate carrier inhibitor
(e.g., methotrexate) on the reduction of nonspecific uptake of
.sup.3H-EC0531 were evaluated for comparison. Methods were as
described above in this example.
[0727] As shown in FIG. 9, DTNB, NCEM, and DIDS reduced the
nonspecific uptake of .sup.3H-EC0531 in KB cells and in A549 cells.
In comparison, reduced folate carrier inhibitors (e.g.,
methotrexate) were ineffective to reduce the nonspecific uptake of
.sup.3H-EC0531 in KB cells and in A549 cells.
Example 6
[0728] The presence of thiols in cell culture media can affect the
non-ligand-specific activity of ligand conjugates. In this example,
the exemplary folate conjugate investigated was EC0531. The
non-FR-specific activity of EC0531 due to the presence of thiols in
cell culture media was evaluated in both KB cells and in A549
cells.
KB Cells
[0729] KB cells were treated in four different types of culture
media: 1) FDRPMI with no serum added; 2) FDRPMI with 10% FBS added;
3) thiol-free FDRPMI with no serum added; and 4) thiol-free FDRPMI
with 10% FBS added. The KB cells in each type of culture media were
administered two different treatments. First, EC0531 was
administered to KB cells in the various culture media, and the
IC.sub.50 values were evaluated. Second, EC0531 plus folic acid was
administered to KB cells in the various culture media, and the
IC.sub.50 values were evaluated.
[0730] A modified RPMI medium (SH-free RPMI), in which glutathione
(GSH) and cystine were omitted, was prepared to evaluate the
activity of EC0531 in the absence of low molecular weight thiols.
Additionally, the effect of high molecular weight thiols (from
serum proteins) on EC0531 activity was assessed in medium
containing no HIFBS. A dilution series of EC0531 was prepared in
RPMI medium with and without 10% HIFBS or SH-free RPMI medium with
and without 10% HIFBS. To assess non-FR targeted activity in the
FR+KB cells, 100 .mu.M FA was included in the solutions with
EC0531. Cells (1.times.10.sup.5/well) were incubated for 2 hours in
the presence of drug, washed 3 times with media, and then chased in
0.5 mL of FDRPMI/HIFBS to 72 hours at 37.degree. C.
.sup.3H-thymidine incorporation was determined as described. The
non-FR-specific activity of EC0531 was subsequently evaluated in
each type of culture media.
A549 Cells
[0731] A549 cells were treated in four different types of culture
media: 1) FDRPMI with no serum added; 2) FDRPMI with 10% FBS added;
3) thiol-free FDRPMI with no serum added; and 4) thiol-free FDRPMI
with 10% FBS added. The non-FR-specific activity of EC0531 was
subsequently evaluated in each type of culture media. The A549
cells in each type of culture media were administered one
treatment. EC0531 was administered to A549 cells in the various
culture media, and the IC.sub.50 values were evaluated. The
administration of EC0531 plus folic acid was not evaluated, as A549
cells do not have folate receptors.
[0732] A modified RPMI medium (SH-free RPMI), in which glutathione
(GSH) and cystine were omitted, was prepared to evaluate the
activity of EC0531 in the absence of low molecular weight thiols.
Additionally, the effect of high molecular weight thiols (from
serum proteins) on EC0531 activity was assessed in medium
containing no HIFBS. A dilution series of EC0531 was prepared in
RPMI medium with and without 10% HIFBS or SH-free RPMI medium with
and without 10% HIFBS. To assess non-FR targeted activity in the
FR+KB cells, 100 .mu.M FA was included in the solutions with
EC0531. Cells (1.times.10.sup.5/well) were incubated for 2 hours in
the presence of drug, washed 3 times with media, and then chased in
0.5 mL of FDRPMI/HIFBS RPMI+FA/HIFBS to 72 hours at 37.degree. C.
.sup.3H-thymidine incorporation was determined as described. The
non-FR-specific activity of EC0531 was subsequently evaluated in
each type of culture media.
[0733] As shown in Table 2, the presence of thiols in culture media
increases the non-FR-specific activity of folate conjugates in KB
cells and in A549 cells. Elimination of cystine and glutathione
from the incubation medium significantly attenuated the nonspecific
activity of EC0531.
TABLE-US-00004 TABLE 2 EC0531 IC.sub.50 EC0531 + Folic Acid Cell
Line Treatment (nM) IC.sub.50 (nM) KB FDRPMI/no serum 7.0 540
FDRPMI/10% FBS 6.7 948 SH-free FDRPMI/no 7.8 830 serum SH-free
FDRPMI/ 9.2 2323 10% FBS Conditioned 8.3 57 FDRPMI/10% FBS A549
FDRPMI/no serum 164 FDRPMI/10% FBS 295 SH-free FDRPMI/no 575 serum
SH-free FDRPMI/ 1133 10% FBS
Example 7
[0734] Because the presence of thiols in cell culture media can
affect the non-ligand-specific activity of ligand conjugates, this
example evaluated if the replacement of cell culture media would
affect the nonspecific activity. In this example, the exemplary
folate conjugate EC0531 was investigated in KB cells.
[0735] KB cells were suspended in FDRPMI/HIFBS, seeded at
1.times.10.sup.5 cells/well in 24-well tissue culture plates, and
were allowed to attach overnight. The following day, cells were
incubated in 500 .mu.L of a solution containing 1 .mu.M EC0531 plus
100 .mu.M FA in FDRPMI/HIFBS to assess non-FR-targeted activity.
The treatment solution was removed every 30 minutes and replaced
with fresh medium containing 1 .mu.M EC0531 plus 100 .mu.M FA for a
cumulative 2 hour incubation. Alternatively, one group of cells was
treated with the same concentrations of EC0531 and FA continuously
without media changes. Total activity of EC0531 (i.e. in the
absence of excess FA) was also assessed using both pulse
conditions. After the drug incubations, cells were washed,
replenished with fresh media, and processed for .sup.3H-thymidine
incorporation as described above. Final results were expressed as
percentage of .sup.3H-thymidine incorporation relative to an
untreated control (non-competed groups) or FA control (competed
groups). The total treatment time for each group was 2 hours,
followed by a 72 hour chase.
[0736] Four groups of cells were evaluated in the example and are
shown in Table 3.
TABLE-US-00005 TABLE 3 Amount of Amount of Folic EC0531 Acid
Administered Administered Number of Time of Each Group (.mu.M)
(.mu.M) Pulses Pulse 1 1 0 1 2 hours 2 1 0 4 30 minutes 3 1 100 1 2
hours 4 1 100 4 30 minutes
[0737] As shown in FIG. 10, replacing the cell culture media in KB
cell culture every 30 minutes eliminates the non-FR-specific
activity of EC0531 following co-administration of EC0531 and folic
acid. In particular, replacing the cell culture media in KB cell
culture every 30 minutes eliminates the non-FR-specific activity of
EC0531. In other words, when the "reducing" medium is replaced with
a fresh oxidized medium every 30 minutes, the non-FR-specific
activity of EC0531 is diminished.
Example 8
[0738] Various thiols can be tested in vitro to determine their
effects on extracellular thiol activity in cells. In this example,
the extracellular thiol activity of A549 cells was evaluated.
[0739] A549 cells were incubated in five different groups: 1)
SH-free RPMI medium (negative control); 2) SH-free RPMI medium plus
GSSG (1.6 .mu.M); 3) SH-free RPMI medium plus cysteine (189 .mu.M);
4) SH-free RPMI medium plus GSSG (1.6 .mu.M) plus cysteine (189
.mu.M); and normal RPMI medium including SH (positive control). The
cells underwent 2 hours of continuous incubation.
[0740] A549 cells were plated in 24-well tissue cultured-treated
plates at 2.times.10.sup.5 cells per well in RPMI+FA/HIFBS and were
allowed to attach to the plates overnight. A 200 .mu.M solution of
DTNB in the indicated PR-free media was prepared. The cells were
rinsed one time with PBS, pH 7.4, and then 500 .mu.L of the
appropriate DTNB solution were added to each well (n=3). The cells
were incubated for 2 hours at 37.degree. C. The solutions were then
removed from the cells, and absorbance was determined at a
wavelength of 412 nm Background absorbance determined from an
aliquot of DTNB solution incubated in an empty well of the tissue
culture plate was subtracted from each value. Thiol concentrations
were calculated based on an extinction coefficient of 14,150
M.sup.-1 cm.sup.-1. Protein concentrations of cell lysates were
determined by the BCA Assay method (Pierce, Rockford, Ill.).
[0741] FIG. 11 shows the individual effect of GSSG (1.6 .mu.M) and
cysteine (189 .mu.M) on the extracellular thiol activity in A549
cells, as measured by thiol (i.e., SH) concentration (.mu.M).
Thiol-free RPMI cell culture media was used as a negative control,
and RPMI cell culture media containing thiols was used as a
positive control. Cysteine appears to be responsible for
stimulation of the extracellular thiol activity in A549 cells, but
GSSG appears to have no effect.
[0742] In addition, cysteine is the predominant thiol released by
cells, at levels reaching 11 to 42 .mu.M (for KB and A549 cells,
respectively) following only a 2 hour conditioning period at
37.degree. C. Cells were plated in 24-well tissue cultured-treated
plates at 2.times.10.sup.5 cells per well in FDRPMI/HIFBS (KB
cells) or RPMI+FA/HIFBS (A549 cells) and were allowed to attach to
the plates overnight. The conditioning time course was initiated by
adding 500 .mu.L of fresh FDRPMI/HIFBS to each well. Plates were
incubated at 37.degree. C. for the indicated time periods. At each
time point, 450 .mu.L medium was removed (n=3) from the appropriate
wells and added to a tube containing 50 .mu.L of 100 mM NCEM to
quench all thiol reactions. Quantitation of cysteine, glutathione,
homocysteine, and cysteinylglycine was done by LC/MS/MS analysis.
Analytes were extracted from the FDRPMI/HIFBS using a 96-well
protein precipitation plate and acidified acetonitrile.
L-cysteine-.sup.13C.sub.3, .sup.15N was added as an internal
standard during the extraction. Following centrifugation, the
supernatants were collected, evaporated to dryness, and
reconstituted in 0.2% formic acid. The extracted samples were
injected into a UPLC/MS/MS system using an HSS T3 C18 reverse phase
column implementing a mobile phase gradient. Detection was
conducted by monitoring the NCEM conjugates of the thiol compounds,
and quantitation was achieved using standard calibrators of the
NCEM conjugates. The effects were observed independently of cell
type or FR expression status.
Example 9
[0743] System x.sub.c.sup.- is a cystine/glutamate antiporter, and
has been shown to be involved in regulating the extracellular redox
state of the cysteine/cystine couple in vitro. In this example, the
effect of system x.sub.c.sup.- inhibitors on extracellular thiol
activity in A549 cells was evaluated.
[0744] A549 cells were administered system x.sub.c.sup.- inhibitors
in four different groups: 1) no inhibitor (negative control); 2)
glutamate (5 mM); 3) quisqualic acid (0.3 mM); and 4) quisqualic
acid (1 mM). A549 cells were plated in 24-well tissue
cultured-treated plates at 2.times.10.sup.5 cells per well in
RPMI+FA/HIFBS and were allowed to attach to the plates overnight.
Solutions of DTNB with and without the indicated concentrations of
system x.sub.c.sup.- inhibitors in PR-free RPMI were prepared. The
cells were rinsed one time with PBS, pH 7.4, followed by the
addition of 500 .mu.L of DTNB solution+/-system x.sub.c.sup.-
inhibitor to each appropriate well (n=3). The cells were then
incubated for 2 hours at 37.degree. C., the solutions were removed
from the cells, and absorbances were determined at a wavelength of
412 nm Background absorbance determined from an aliquot of DTNB
solution incubated in an empty well of the tissue culture plate was
subtracted from each value. Thiol concentrations were calculated
based on an extinction coefficient of 14,150 M.sup.-1cm.sup.-1.
Protein concentrations of cell lysates were determined by the BCA
Assay method (Pierce, Rockford, Ill.).
[0745] As shown in FIG. 12, system x.sub.c.sup.- inhibitors
glutamate (5 mM) and quisqualic acid (0.3 mM and 1 mM)
significantly reduce the release of extracellular thiols in A549
cells as measured by thiol (i.e., SH) concentration (.mu.M).
Furthermore, quisqualic acid demonstrated a dose-dependent
reduction of the release of extracellular thiols.
Example 10
[0746] The presence of extracellular thiols can affect the
non-ligand-specific activity of ligand conjugates. In this example,
the non-FR-specific activity of an exemplary folate conjugate
(EC0531) was investigated. The non-FR-specific activity of EC0531
was evaluated in KB cells.
[0747] The effects of the system x.sub.c.sup.- inhibitor
sulfasalazine on the non-FR-specific activity of EC0531 were
evaluated in this example. Sulfasalazine was co-administered with
EC0531 (1 .mu.M) and folic acid (100 .mu.M). The inhibition of
non-FR-specific activity (i.e., cytotoxicity) was evaluated. KB
cells were seeded in 24-well tissue culture-treated plates at
1.times.10.sup.5 cells per well in FDRPMI/HIFBS and allowed to
attach to the plates overnight at 37.degree. C. A dilution series
of sulfasalazine was prepared in FDRPMI/HIFBS medium containing a
final concentration of 1 .mu.M EC0531 and 100 .mu.M FA. Cells were
incubated for 2 hours in the presence of drug, FA competitor, and
inhibitor, washed 3 times with media, then chased in 0.5 mL of
FDRPMI/HIFBS to 72 hours at 37.degree. C. Cells were then treated
as described above to determine .sup.3H-thymidine incorporation.
Final results were expressed as percentage of .sup.3H-thymidine
incorporation relative to an untreated control (non-competed
groups) or FA control (competed groups).
[0748] As shown in FIG. 13, the system x.sub.c.sup.- inhibitor
sulfasalazine exhibits a dose-responsive inhibition of
non-FR-specific activity in KB cells. Sulfasalazine exhibited an
IC.sub.50 of 170 .mu.M.
Example 11
[0749] Furthermore, among various system x.sub.c.sup.- inhibitors,
sulfasalazine appears to be a more potent inhibitor than glutamate.
In this example, the non-FR-specific activity of an exemplary
folate conjugate (EC0531) was investigated. The non-FR-specific
activity of EC0531 was evaluated in A549 cells.
[0750] The effects of the system x.sub.c.sup.- inhibitors
sulfasalazine and glutamate on the non-FR-specific activity of
EC0531 were evaluated in this example.
[0751] A549 cells were seeded in 24-well tissue culture-treated
plates at 1.times.10.sup.5 cells per well in RPMI+FA/HIFBS and
allowed to attach overnight at 37.degree. C. The next day, cells
were treated concurrently with increasing concentrations of EC0531
and a constant concentration of inhibitor (1 mM sulfasalazine or 5
mM glutamate in FDRPMI/HIFBS). Another group received no inhibitor
(i.e. EC0531 only) and was also included. Cells were incubated for
2 hours in the presence of drug and inhibitor, washed 3 times with
media, and then chased in 0.5 mL of FDRPMI/HIFBS to 72 hours at
37.degree. C. Cells were then treated as described above to
determine .sup.3H-thymidine incorporation. The inhibition of
non-FR-specific activity (i.e., cytotoxicity) was evaluated for
each system x.sub.c.sup.- inhibitor. Final results were expressed
as percentage of .sup.3H-thymidine incorporation relative to an
untreated control.
[0752] As shown in FIG. 14, both sulfasalazine (1 mM) and glutamate
(5 mM) inhibit the non-PR-specific activity of EC0531 in A549
cells, as measured by percentage of .sup.3H-Thymidine incorporated
in cells (counts per minute). Sulfasalazine exhibited a greater
inhibition of non-FR-specific activity of EC0531 in A549 cells
compared to glutamate.
Example 12
[0753] The presence of extracellular thiols can affect the
nonspecific uptake of ligand conjugates. In this example, the
nonspecific uptake of an exemplary folate conjugate (EC0531;
specifically 3H-EC0531) was investigated. The nonspecific uptake of
EC0531 was evaluated in both KB cells and in A549 cells.
[0754] The effects of the system x.sub.c.sup.- inhibitor
sulfasalazine on the reduction of nonspecific uptake of
.sup.3H-EC0531 into cells were evaluated in this example. In
addition to sulfasalazine, the effects of a reduced folate carrier
inhibitor (e.g., methotrexate) on the reduction of nonspecific
uptake of .sup.3H-EC0531 were evaluated for comparison.
[0755] Inhibition of non-FR-targeted .sup.3H-EC0531 uptake was
assessed by incubating .sup.3H-EC0531 in the presence of
inhibitors. Cells were seeded at 4.times.10.sup.5 cells per well of
a 12-well tissue culture plate, and were treated the next day with
1 .mu.M EC0531 plus the indicated concentrations of inhibitors in 1
mL FFRPMI/HIFBS for 2 h at 37.degree. C. Wells were then washed 3
times with 1 mL of ice-cold PBS, pH 7.4 and were processed for LSC
analysis as described above.
[0756] As shown in FIG. 15, sulfasalazine reduced the nonspecific
uptake of .sup.3H-EC0531 in KB cells and in A549 cells. In
comparison, reduced folate carrier inhibitors (e.g., methotrexate)
were ineffective to reduce the nonspecific uptake of .sup.3H-EC0531
in KB cells and in A549 cells.
Example 13
[0757] In the system x.sub.c.sup.- pathway, xCT is known to be the
functional subunit of system x.sub.c.sup.-. siRNA-mediated
knockdown of xCT shows effects on extracellular thiol activity. In
this example, the non-FR-specific activity of an exemplary folate
conjugate (EC0531) was investigated. The non-FR-specific activity
of EC0531 was evaluated in KB cells and in A549 cells.
[0758] Knockdown of xCT by siRNA transfection of A549 and KB cells
was achieved by incubating cells for 48 hours in the presence of
xCT siRNA (Ambion). Transfection complexes were formed by preparing
a solution of 25 nM xCT siRNA and 10 .mu.L RNAiMAX reagent
(Invitrogen) per mL Opti-MEM medium (Gibco). Additionally, a
nonspecific (NS) siRNA (Ambion) was also prepared accordingly and
used as a control for all assays. Transfection complexes were
allowed to form for 15 min at room temperature, and then 100 .mu.L
of transfection complex (2.5 pmol siRNA and 1 .mu.L RNAiMAX) were
added per well to 24-well tissue culture-treated plates. Controls
that received Opti-MEM medium only (i.e. no siRNA) were also
prepared. Next, A549 cells and KB cells were suspended in
RPMI+FA/HIFBS (A549) or FDRPMI/HIFBS (KB) and were added to the
wells (5.times.10.sup.4 cells/well). Plates were placed in an
incubator at 37.degree. C., and transfection was allowed to proceed
for 48 hours. After the transfection period, wells were washed two
times with FDRPMI/HIFBS, and EC0531 activity (i.e. cytotoxicity)
and extracellular thiol activity (DTNB assay) were assessed as
described above.
[0759] As shown in FIG. 16, siRNA-mediated knockdown of xCT causes
a reduction of extracellular thiol activity in both KB cells and in
A549 cells, as measured by percentage of UTC (nmol of thiol/mg of
protein).
[0760] Furthermore, as shown in Table 4, the siRNA-mediated
knockdown of xCT reduced non-FR-specific activity of EC0531 in both
KB cells and in A549 cells.
TABLE-US-00006 TABLE 4 EC0531 + Folic Acid Cell Line Treatment
EC0531 IC.sub.50 (nM) IC.sub.50 (nM) KB No siRNA 54.8 258 NS siRNA
49.6 434 xCT siRNA 252 1775 A549 No siRNA 121 NS siRNA 128 xCT
siRNA 3797
* * * * *