U.S. patent application number 17/492328 was filed with the patent office on 2022-03-17 for patient selection method for inflammation.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Lindsay E. Kelderhouse, Philip S. Low.
Application Number | 20220082553 17/492328 |
Document ID | / |
Family ID | 1000005996768 |
Filed Date | 2022-03-17 |
United States Patent
Application |
20220082553 |
Kind Code |
A1 |
Low; Philip S. ; et
al. |
March 17, 2022 |
PATIENT SELECTION METHOD FOR INFLAMMATION
Abstract
The invention relates to methods and compositions for the
selection of patients for therapy with an anti-inflammatory drug.
More particularly, the invention relates to compositions comprising
folate-imaging agent conjugates for the selection of patients for
therapy with an anti-inflammatory drug, and methods and uses
therefor.
Inventors: |
Low; Philip S.; (West
Lafayette, IN) ; Kelderhouse; Lindsay E.; (Island
Lake, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Family ID: |
1000005996768 |
Appl. No.: |
17/492328 |
Filed: |
October 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15037793 |
May 19, 2016 |
11162937 |
|
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PCT/US2014/066347 |
Nov 19, 2014 |
|
|
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17492328 |
|
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|
61906331 |
Nov 19, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5088 20130101;
A61K 49/0052 20130101; A61K 51/0459 20130101; A61K 49/0032
20130101; G01N 2800/52 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61K 49/00 20060101 A61K049/00; A61K 51/04 20060101
A61K051/04 |
Claims
1. A method for selecting a patient for therapy with an
anti-inflammatory drug, the method comprising the step of
administering to the patient a folate-imaging agent conjugate,
wherein the folate-imaging agent conjugate is used to predict the
response of the patient to the anti-inflammatory drug.
2. The method of claim 1 wherein the folate-imaging agent conjugate
produces a detectable signal in the patient, wherein the signal is
detected, and wherein the detection of the signal is used to
predict the response of the patient to the anti-inflammatory
drug.
3. The method of claim 2 wherein the signal is a radioactive
signal.
4. The method of claim 2 wherein the signal is produced by a
chromophore.
5. The method of claim 4 wherein the chromophore is a
fluorophore.
6. The method of claim 5 wherein the fluorophore is selected from
the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
7. The method of claim 2 wherein the signal is produced as a result
of binding of the folate-imaging agent conjugate to activated
macrophages.
8. The method of claim 1 wherein the administering step comprises a
first administering step and a second administering step.
9. The method of claim 8 wherein the first and second administering
steps produce a first signal and a second signal, respectively.
10. The method of claim 9 wherein the first signal and the second
signal are quantified.
11. The method of claim 9 wherein the first signal is obtained by
administering the folate-imaging agent conjugate prior to
administration of the anti-inflammatory drug.
12. The method of claim 9 wherein the first signal is obtained by
administering the folate-imaging agent conjugate on the same day as
treatment with the anti-inflammatory drug is initiated.
13. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate subsequent to the
administration of the anti-inflammatory drug.
14. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate within about 21
days after administration of the anti-inflammatory drug is
initiated.
15. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate within about 1
week, about 2 weeks, about 3 weeks, or about 4 weeks after the
administration of the anti-inflammatory drug is initiated.
16. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate on any one of the
days within about 21 days after the administration of the
anti-inflammatory drug is initiated.
17. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate on any one of the
days within about 12 weeks after the administration of the
anti-inflammatory drug is initiated.
18. The method of claim 9 wherein the second signal is reduced by
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, or about 100% compared to the
first signal, and wherein the reduction indicates that the patient
should continue to be treated with the anti-inflammatory drug.
19. The method of claim 9 wherein the second signal is obtained by
administering the folate-imaging agent conjugate about 2, about 3,
about 4, about 5, about 6, about 12, about 15, or about 21 days
after the administration of the anti-inflammatory drug is
initiated.
20. The method of claim 1 wherein the inflammatory disease is
selected from the group consisting of arthritis, osteoarthritis,
rheumatoid arthritis, atherosclerosis, psoriasis,
ischemia/reperfusion injury, pulmonary fibrosis, organ transplant
rejection, ulcerative colitis, impact trauma, osteomyelitis,
multiple sclerosis, scleroderma, Crohn's disease, Sjogren's
syndrome, glomerulonephritis, systemic sclerosis, sarcoidosis, an
inflammatory lesion, and chronic inflammation.
21. The method of claim 9 wherein the folate-imaging agent
conjugate has the formula ##STR00065## wherein M is a
radionuclide.
22. The method of claim 21, wherein the radionuclide is selected
from the group consisting of an isotope of gallium, an isotope of
indium, an isotope of copper, an isotope of technetium, and an
isotope of rhenium.
23. The method of claim 22 wherein the radionuclide is an isotope
of technetium.
24. The method of claim 23 wherein the technetium is
.sup.99m-technetium.
25. The method of claim 1 wherein the folate-imaging agent
conjugate is .sup.99mTc-EC20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/037,793, filed May 19, 2016, which is a
U.S. National Stage Entry of PCT/US2014/066347, filed Nov. 19,
2014, which claims, under 35 U.S.C. .sctn. 119(e), the benefit of
and priority to U.S. Provisional Application No. 61/906,331 filed
Nov. 19, 2013, all of which are hereby incorporated by reference
herein.
TECHNICAL FIELD
[0002] The invention relates to methods and compositions for the
selection of patients for therapy with an anti-inflammatory drug.
More particularly, the invention relates to compositions comprising
folate-imaging agent conjugates for the selection of patients for
therapy with an anti-inflammatory drug, and methods and uses
therefor.
BACKGROUND AND SUMMARY
[0003] Biologic therapies (etanercept, infliximab, adalimumab,
rituximab, abatacept, anakinra, efalizumab, etc.) have become the
preferred method for treatment of many autoimmune and inflammatory
diseases, primarily because of their increased efficacy, enhanced
speed of onset, and greater tolerability. However, despite these
obvious benefits, about forty percent of those receiving biologic
therapies still fail to respond. Because no clinical marker
currently exists to accurately predict response to therapy,
physicians have been forced to rely on subjective criteria (i.e.,
assessment of pain, coloration and swelling, radiographic analysis,
etc.) to determine whether to continue with a current treatment or
not. Moreover, since meaningful changes in many subjective
parameters are considered reliable only four to six months after
initiation of therapy, identification of nonresponders is often
possible only after irreversible damage or an ideal "window of
therapeutic opportunity" has transpired. Considering the high costs
of treatment (often upwards of $40,000 per year) and the damage
that can ensue when an initial therapy fails, an ability to quickly
predict a patient's eventual response could reduce both cost and
morbidity associated with a less informed approach to
anti-inflammatory drug selection.
[0004] Activated proinflammatory macrophages have been shown to
constitute key players in the development and progression of a
number of inflammatory and autoimmune diseases, including
rheumatoid arthritis, atherosclerosis, psoriasis,
ischemia/reperfusion injury, pulmonary fibrosis, organ transplant
rejection, ulcerative colitis, impact trauma, multiple sclerosis,
scleroderma, Crohn's disease, Sjogren's syndrome,
glomerulonephritis, and sarcoidosis. Because the folate receptor
f3, a glycosylphosphatidylinositol anchored glycoprotein, is
uniquely over-expressed on the surface of these activated
macrophages, folate receptor-targeted imaging agents have been
recently exploited to visualize sites of activated macrophage
accumulation in both animals and humans. More specifically,
.sup.99mTc-EC20, a folate receptor-targeted radiopharmaceutical,
has proven particularly useful for these imaging applications in
that uptake of .sup.99mTc-EC20 has been observed to correlate
directly with severity of arthritis symptoms.
[0005] Applicants have surprisingly found that reduction in uptake
of folate-imaging agent conjugates, such as .sup.99mTc-EC20,
dramatically precedes observable changes in symptomology for
patients with inflammatory disease being treated with
anti-inflammatory drugs. This reduction in uptake occurs regardless
which specific anti-inflammatory drug is being employed. This
surprising observation resulted in Applicants' invention which is a
method that utilizes a folate-targeted imaging agent, such as
.sup.99mTc-EC20, to predict an eventual response to therapy for
inflammatory diseases. Applicants have demonstrated that uptake of
folate receptor-targeted imaging agents at sites of inflammatory
disease shortly after initiation of therapy can accurately forecast
a subsequent response to an anti-inflammatory drug long before
clinical changes can be detected. Accordingly, Applicants have
found that folate-imaging agent conjugates are useful as clinical
tools for the selection of patients who will eventually benefit
from treatment with a particular anti-inflammatory drug.
[0006] Several embodiments of the invention are described by the
following enumerated clauses:
[0007] 1. A method for selecting a patient for therapy with an
anti-inflammatory drug, the method comprising the steps of
administering to the patient a folate-imaging agent conjugate, and
using the folate-imaging agent conjugate to predict the response of
the patient to the anti-inflammatory drug.
[0008] 2. Use of a folate-imaging agent conjugate for selecting a
patient for therapy with an anti-inflammatory drug wherein the
folate-imaging agent conjugate is administered to the patient and
is used to predict the response of the patient to the
anti-inflammatory drug.
[0009] 3. Use of a folate-imaging agent conjugate in the
manufacture of a medicament for selecting a patient for therapy
with an anti-inflammatory drug wherein the folate-imaging agent
conjugate is administered to the patient and is used to predict the
response of the patient to the anti-inflammatory drug.
[0010] 4. The method or use of any one of clauses 1 to 3 wherein
the folate-imaging agent conjugate produces a detectable signal in
the patient, wherein the signal is detected, and wherein the
detection of the signal is used to predict the response of the
patient to the anti-inflammatory drug.
[0011] 5. The method or use of clause 4 wherein the signal is a
radioactive signal.
[0012] 6. The method or use of clause 4 wherein the signal is
produced by a chromophore.
[0013] 7. The method or use of clause 6 wherein the chromophore is
a fluorophore.
[0014] 8. The method or use of clause 7 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0015] 9. The method or use of any one of clauses 4 to 8 wherein
the signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0016] 10. The method or use of any one of clauses 1 to 9 wherein
the administering step comprises a first administering step and a
second administering step.
[0017] 11. The method or use of clause 10 wherein the first and
second administering steps produce a first signal and a second
signal, respectively.
[0018] 12. The method or use of clause 11 wherein the first signal
and the second signal are quantified.
[0019] 13. The method or use of clause 11 wherein the first signal
is obtained by administering the folate-imaging agent conjugate
prior to administration of the anti-inflammatory drug.
[0020] 14. The method or use of clause 11 wherein the first signal
is obtained by administering the folate-imaging agent conjugate on
the same day as treatment with the anti-inflammatory drug is
initiated.
[0021] 15. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0022] 16. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
within about 21 days after administration of the anti-inflammatory
drug is initiated.
[0023] 17. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
within about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks
after the administration of the anti-inflammatory drug is
initiated.
[0024] 18. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate on
any one of the days within about 21 days after the administration
of the anti-inflammatory drug is initiated.
[0025] 19. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate on
any one of the days within about 12 weeks after the administration
of the anti-inflammatory drug is initiated.
[0026] 20. The method or use of clause 11 wherein the second signal
is reduced by about 10%, about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, or about 100%
compared to the first signal, and wherein the reduction indicates
that the patient should continue to be treated with the
anti-inflammatory drug.
[0027] 21. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
about 2, about 3, about 4, about 5, about 6, about 12, about 15, or
about 21 days after the administration of the anti-inflammatory
drug is initiated.
[0028] 22. The method or use of any one of clauses 1 to 21 wherein
the inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0029] 23. The method or use of any one of clauses 1 to 22 wherein
the folate-imaging agent conjugate is in a parenteral dosage
form.
[0030] 24. The method or use of clause 23 wherein the dosage form
is selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0031] 25. The method or use of any one of clauses 1 to 24 wherein
the folate-imaging agent conjugate is in a composition and wherein
the composition further comprises a pharmaceutically acceptable
carrier.
[0032] 26. The method or use of clause 25 wherein the
pharmaceutically acceptable carrier is a liquid carrier.
[0033] 27. The method or use of clause 26 wherein the liquid
carrier is selected from the group consisting of saline, glucose,
alcohols, glycols, esters, amides, and a combination thereof.
[0034] 28. The method or use of any one of clauses 1 to 27 wherein
the folate-imaging agent conjugate is administered in an effective
amount.
[0035] 29. The method or use of clause 28 wherein the effective
amount ranges from about 1 ng to about 1 mg per kilogram of body
weight of the patient.
[0036] 30. The method or use of clause 28 wherein the effective
amount ranges from about 100 ng to about 500 .mu.g per kilogram of
body weight of the patient.
[0037] 31. The method or use of clause 28 wherein the effective
amount ranges from about 100 ng to about 25 .mu.g per kilogram of
body weight of the patient.
[0038] 32. The method or use of clause 28 wherein the effective
amount ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of
body surface area of the patient.
[0039] 33. The method or use of clause 28 wherein the effective
amount ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of
body surface area of the patient.
[0040] 34. The method or use of clause 28 wherein the effective
amount ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of
patient body weight.
[0041] 35. The method or use of any one of clauses 1 to 34 further
comprising the step of administering unlabeled folic acid to the
patient.
[0042] 36. The method or use of clause 35 wherein the unlabeled
folic acid is administered before administration of the
folate-imaging agent conjugate.
[0043] 37. The method or use of any one of clauses 1 to 5 or 9 to
36 wherein the folate-imaging agent conjugate has the formula
##STR00001##
wherein M is a radionuclide.
[0044] 38. The method or use of any one of clauses 1 to 5 or 9 to
37 wherein the folate-imaging agent conjugate has the formula
##STR00002##
[0045] wherein M is a radionuclide.
[0046] 39. The method or use of clause 37 or 38 wherein the
radionuclide is selected from the group consisting of an isotope of
gallium, an isotope of indium, an isotope of copper, an isotope of
technetium, and an isotope of rhenium.
[0047] 40. The method or use of clause 39 wherein the radionuclide
is an isotope of technetium.
[0048] 41. The method or use of clause 40 wherein the technecium is
.sup.99m-technetium.
[0049] 42. The method or use of any one of clauses 1 to 5 or 9 to
41 wherein the folate-imaging agent conjugate is
.sup.99mTc-EC20.
[0050] 43. The method or use of any one of clauses 1 to 5 or 9 to
42 wherein the signal is detected using scintigraphic imaging.
[0051] 44. The method or use of any one of clauses 1 to 43 wherein
the patient is a human patient.
[0052] 45. The method or use of any one of clauses 1 to 43 wherein
the patient is a veterinary patient.
[0053] 46. The method or use of any one of clauses 1 to 5 or 9 to
45 wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0054] 47. The method or use of any one of clauses 1 to 46 wherein
the folate-imaging agent conjugate is in the form of a
reconstituted lyophilizate.
[0055] 48. The method or use of any one of clauses 1 to 47 wherein
the folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0056] 49. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00003##
[0057] or a pharmaceutically acceptable salt of any of these
compounds.
[0058] 50. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00004##
[0059] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.f comprising a radiophore or a precursor to a radiophore.
[0060] 51. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00005##
[0061] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0062] 52. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00006##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.f is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0063] 53. A method for selecting a patient for therapy with an
anti-inflammatory drug, the method comprising the steps of
assessing whether the patient is in need of therapy with the
anti-inflammatory drug by relying on the results obtained by means
for detecting a signal produced in the patient by a folate-imaging
agent conjugate administered to the patient; and prescribing or
continuing to prescribe the anti-inflammatory drug to treat the
patient assessed to be in need of the anti-inflammatory drug.
[0064] 54. The method of clause 54 wherein the folate-imaging agent
conjugate produces a detectable signal in the patient, wherein the
signal is detected, and wherein the detection of the signal is used
to assess whether the patient is in need of therapy with the
anti-inflammatory drug.
[0065] 55. The method of clause 54 wherein the signal is a
radioactive signal.
[0066] 56. The method of clause 54 wherein the signal is produced
by a chromophore.
[0067] 57. The method of clause 56 wherein the chromophore is a
fluorophore.
[0068] 58. The method of clause 57 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0069] 59. The method of any one of clauses 53 to 58 wherein the
signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0070] 60. The method of any one of clauses 53 to 59 wherein the
administration comprises a first administering step and a second
administering step.
[0071] 61. The method of clause 60 wherein the first and second
administering steps produce a first signal and a second signal,
respectively.
[0072] 62. The method of clause 61 wherein the first signal and the
second signal are quantified.
[0073] 63. The method of clause 61 wherein the first signal is
obtained by administering the folate-imaging agent conjugate prior
to administration of the anti-inflammatory drug.
[0074] 64. The method of clause 61 wherein the first signal is
obtained by administering the folate-imaging agent conjugate on the
same day as treatment with the anti-inflammatory drug is
initiated.
[0075] 65. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0076] 66. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 21 days after administration of the anti-inflammatory drug is
initiated.
[0077] 67. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after
the administration of the anti-inflammatory drug is initiated.
[0078] 68. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 21 days after the administration of
the anti-inflammatory drug is initiated.
[0079] 69. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 12 weeks after the administration of
the anti-inflammatory drug is initiated.
[0080] 70. The method of clause 61 wherein the second signal is
reduced by about 10%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, or about 100% compared
to the first signal, and wherein the reduction indicates that the
patient should continue to be treated with the anti-inflammatory
drug.
[0081] 71. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate about
2, about 3, about 4, about 5, about 6, about 12, about 15, or about
21 days after the administration of the anti-inflammatory drug is
initiated.
[0082] 72. The method of any one of clauses 53 to 71 wherein the
inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0083] 73. The method of any one of clauses 53 to 72 wherein the
folate-imaging agent conjugate is in a parenteral dosage form.
[0084] 74. The method of clause 73 wherein the dosage form is
selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0085] 75. The method of any one of clauses 53 to 74 wherein the
folate-imaging agent conjugate is in a composition and wherein the
composition further comprises a pharmaceutically acceptable
carrier.
[0086] 76. The method of clause 75 wherein the pharmaceutically
acceptable carrier is a liquid carrier.
[0087] 77. The method of clause 76 wherein the liquid carrier is
selected from the group consisting of saline, glucose, alcohols,
glycols, esters, amides, and a combination thereof.
[0088] 78. The method of any one of clauses 53 to 77 wherein the
folate-imaging agent conjugate is administered in an effective
amount.
[0089] 79. The method of clause 78 wherein the effective amount
ranges from about 1 ng to about 1 mg per kilogram of body weight of
the patient.
[0090] 80. The method of clause 78 wherein the effective amount
ranges from about 100 ng to about 500 .mu.g per kilogram of body
weight of the patient.
[0091] 81. The method of clause 78 wherein the effective amount
ranges from about 100 ng to about 25 .mu.g per kilogram of body
weight of the patient.
[0092] 82. The method of clause 78 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of body
surface area of the patient.
[0093] 83. The method of clause 78 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of body
surface area of the patient.
[0094] 84. The method of clause 78 wherein the effective amount
ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of patient body
weight.
[0095] 85. The method of any one of clauses 53 to 84 further
comprising the step of administering unlabeled folic acid to the
patient.
[0096] 86. The method of clause 85 wherein the unlabeled folic acid
is administered before administration of the folate-imaging agent
conjugate.
[0097] 87. The method of any one of clauses 53 to 55 or 59 to 86
wherein the folate-imaging agent conjugate has the formula
##STR00007##
wherein M is a radionuclide.
[0098] 88. The method of any one of clauses 53 to 55 or 59 to 87
wherein the folate-imaging agent conjugate has the formula
##STR00008##
[0099] wherein M is a radionuclide.
[0100] 89. The method of clause 87 or 88 wherein the radionuclide
is selected from the group consisting of an isotope of gallium, an
isotope of indium, an isotope of copper, an isotope of technetium,
and an isotope of rhenium.
[0101] 90. The method of clause 89 wherein the radionuclide is an
isotope of technetium.
[0102] 91. The method of clause 90 wherein the technecium is
.sup.99m-technetium.
[0103] 92. The method of any one of clauses 53 to 55 or 59 to 91
wherein the folate-imaging agent conjugate is .sup.99mTc-EC20.
[0104] 93. The method of any one of clauses 53 to 55 or 59 to 92
wherein the signal is detected using scintigraphic imaging.
[0105] 94. The method of any one of clauses 53 to 93 wherein the
patient is a human patient.
[0106] 95. The method of any one of clauses 53 to 93 wherein the
patient is a veterinary patient.
[0107] 96. The method of any one of clauses 53 to 55 or 59 to 95
wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0108] 97. The method of any one of clauses 53 to 96 wherein the
folate-imaging agent conjugate is in the form of a reconstituted
lyophilizate.
[0109] 98. The method of any one of clauses 53 to 97 wherein the
folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0110] 99. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00009##
[0111] or a pharmaceutically acceptable salt of any of these
compounds.
[0112] 100. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00010##
[0113] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.f comprising a radiophore or a precursor to a radiophore.
[0114] 101. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00011##
[0115] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0116] 102. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00012##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.f is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0117] 103. A method for predicting the efficacy of an
anti-inflammatory drug in a patient, the method comprising the
steps of administering to the patient a folate-imaging agent
conjugate, and using the folate-imaging agent conjugate to predict
efficacy of the anti-inflammatory drug in the patient.
[0118] 104. The method of clause 103 wherein the folate-imaging
agent conjugate produces a detectable signal in the patient,
wherein the signal is detected, and wherein the detection of the
signal is used to assess whether the patient is in need of therapy
with the anti-inflammatory drug.
[0119] 105. The method of clause 104 wherein the signal is a
radioactive signal.
[0120] 106. The method of clause 104 wherein the signal is produced
by a chromophore.
[0121] 107. The method of clause 106 wherein the chromophore is a
fluorophore.
[0122] 108. The method of clause 107 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0123] 109. The method of any one of clauses 104 to 108 wherein the
signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0124] 110. The method of any one of clauses 103 to 109 wherein the
administering step comprises a first administering step and a
second administering step.
[0125] 111. The method of clause 110 wherein the first and second
administering steps produce a first signal and a second signal,
respectively.
[0126] 112. The method of clause 111 wherein the first signal and
the second signal are quantified.
[0127] 113. The method of clause 111 wherein the first signal is
obtained by administering the folate-imaging agent conjugate prior
to administration of the anti-inflammatory drug.
[0128] 114. The method of clause 111 wherein the first signal is
obtained by administering the folate-imaging agent conjugate on the
same day as treatment with the anti-inflammatory drug is
initiated.
[0129] 115. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0130] 116. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 21 days after administration of the anti-inflammatory drug is
initiated.
[0131] 117. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after
the administration of the anti-inflammatory drug is initiated.
[0132] 118. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 21 days after the administration of
the anti-inflammatory drug is initiated.
[0133] 119. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 12 weeks after the administration of
the anti-inflammatory drug is initiated.
[0134] 120. The method of clause 111 wherein the second signal is
reduced by about 10%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, or about 100% compared
to the first signal, and wherein the reduction indicates that the
patient should continue to be treated with the anti-inflammatory
drug.
[0135] 121. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate about
2, about 3, about 4, about 5, about 6, about 12, about 15, or about
21 days after the administration of the anti-inflammatory drug is
initiated.
[0136] 122. The method of any one of clauses 103 to 121 wherein the
inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0137] 123. The method of any one of clauses 103 to 122 wherein the
folate-imaging agent conjugate is in a parenteral dosage form.
[0138] 124. The method of clause 123 wherein the dosage form is
selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0139] 125. The method of any one of clauses 103 to 124 wherein the
folate-imaging agent conjugate is in a composition and wherein the
composition further comprises a pharmaceutically acceptable
carrier.
[0140] 126. The method of clause 125 wherein the pharmaceutically
acceptable carrier is a liquid carrier.
[0141] 127 The method of clause 126 wherein the liquid carrier is
selected from the group consisting of saline, glucose, alcohols,
glycols, esters, amides, and a combination thereof.
[0142] 128. The method of any one of clauses 103 to 127 wherein the
folate-imaging agent conjugate is administered in an effective
amount.
[0143] 129. The method of clause 128 wherein the effective amount
ranges from about 1 ng to about 1 mg per kilogram of body weight of
the patient.
[0144] 130. The method of clause 128 wherein the effective amount
ranges from about 100 ng to about 500 .mu.g per kilogram of body
weight of the patient.
[0145] 131. The method of clause 128 wherein the effective amount
ranges from about 100 ng to about 25 .mu.g per kilogram of body
weight of the patient.
[0146] 132. The method of clause 128 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of body
surface area of the patient.
[0147] 133. The method of clause 128 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of body
surface area of the patient.
[0148] 134. The method of clause 128 wherein the effective amount
ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of patient body
weight.
[0149] 135. The method of any one of clauses 103 to 134 further
comprising the step of administering unlabeled folic acid to the
patient.
[0150] 136. The method of clause 135 wherein the unlabeled folic
acid is administered before administration of the folate-imaging
agent conjugate.
[0151] 137. The method of any one of clauses 103 to 105 or 109 to
136 wherein the folate-imaging agent conjugate has the formula
##STR00013##
wherein M is a radionuclide.
[0152] 138. The method of any one of clauses 103 to 105 or 109 to
137 wherein the folate-imaging agent conjugate has the formula
##STR00014##
wherein M is a radionuclide.
[0153] 139. The method of clause 137 or 138 wherein the
radionuclide is selected from the group consisting of an isotope of
gallium, an isotope of indium, an isotope of copper, an isotope of
technetium, and an isotope of rhenium.
[0154] 140. The method of clause 139 wherein the radionuclide is an
isotope of technetium.
[0155] 141. The method of clause 140 wherein the technecium is
.sup.99m-technetium.
[0156] 142. The method of any one of clauses 103 to 105 or 109 to
141 wherein the folate-imaging agent conjugate is
.sup.99mTc-EC20.
[0157] 143. The method of any one of clauses 103 to 105 or 109 to
142 wherein the signal is detected using scintigraphic imaging.
[0158] 144. The method of any one of clauses 103 to 143 wherein the
patient is a human patient.
[0159] 145. The method of any one of clauses 103 to 143 wherein the
patient is a veterinary patient.
[0160] 146. The method of any one of clauses 103 to 105 or 109 to
145 wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0161] 147. The method of any one of clauses 103 to 146 wherein the
folate-imaging agent conjugate is in the form of a reconstituted
lyophilizate.
[0162] 148. The method of any one of clauses 103 to 147 wherein the
folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0163] 149. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00015##
[0164] or a pharmaceutically acceptable salt of any of these
compounds.
[0165] 150. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00016##
[0166] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.t comprising a radiophore or a precursor to a radiophore.
[0167] 151. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00017##
[0168] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0169] 152. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00018##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.t is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0170] 153. The method of use of any one of clauses 1 to 152
wherein the folate portion of the folate-imaging agent conjugate
comprises a compound of the formula:
##STR00019##
[0171] 154. The method or use of any one of clauses 1-4, 9-36,
44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136, or
144-148 wherein the folate-imaging agent conjugate has the
formula:
##STR00020##
[0172] wherein:
[0173] X is an amino acid or a derivative thereof, and
[0174] Y is a dye that has a fluorescence excitation and emission
spectra in the near infrared range, and said compound maintains or
enhances the fluorescence of Y.
[0175] 155. The method or use of any one of clauses 1-4, 9-36,
44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136, or
144-148 wherein the imaging agent Y has the formula:
##STR00021##
[0176] wherein:
[0177] X' is independently selected from the group consisting of O,
S, N and C, and
[0178] R' is independently selected from the group consisting of
CH2 and CH2CH2. In some embodiments, the dye Y is selected from the
group consisting of LS288, IR800, SP054, S0121, KODAK IRD28, S2076,
S0456 and derivatives thereof.
[0179] 156. The method or use of any one of clauses of clauses 1-4,
9-36, 44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136,
or 144-148 wherein the folate-imaging agent conjugate has the
formula:
##STR00022##
wherein W, X, Y, Z each are H, Na.sup.+, K.sup.+ or
NH.sub.4.sup.+.
BRIEF DESCRIPTION OF THE DRAWINGS
[0180] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0181] FIG. 1. Analysis of .sup.99mTc-EC20 accumulation in CIA mice
treated with methotrexate or dexamethasone. A: Radioimage of mice
injected with .sup.99mTc-EC20 three days after initiation of
treatment with methotrexate (left), saline (middle) or
dexamethasone (right). B and C: Arthritis scores and paw thickness
measurements of mice in each of the following treatment groups:
disease control (up triangle), methotrexate (diamond),
dexamethasone (down triangle), and healthy control (square).
[0182] FIG. 2. Analysis of .sup.99mTc-EC20 uptake in CIA mice
treated with etanercept or abatacept. A: Radioimages of mice
injected with .sup.99mTc-EC20 three days after initiation of
treatment with etanercept (left), saline (middle) or abatacept
(right). B and C: Arthritis scores and paw thickness measurements
of mice in each of the following treatment groups: disease control
(up triangle), etanercept (down triangle), abatacept (diamond), and
healthy control (square).
[0183] FIG. 3. Analysis of .sup.99mTc-EC20 accumulation in CIA mice
treated with naproxen. A: Radioimage of mice injected with
.sup.99mTc-EC20 three days after initiation of treatment with
naproxen (left), or saline (right). B and C: Arthritis scores and
paw thickness measurements of mice for the following treatment
groups: disease control (up triangle), naproxen (down triangle),
and healthy control (square).
[0184] FIG. 4. Analysis of .sup.99mTc-EC20 accumulation in mice
with ulcerative colitis treated with cimetidine or sulphasalazine.
A: Radioimages of mice injected with .sup.99mTc-EC20 four days
after initiation of treatment. From left to right, healthy control,
treatment with saline, treatment with cimetidine and treatment with
sulphasalazine. B: Clinical colitis scores of mice were recorded
daily for each treatment group: disease control (up triangle),
cimetidine (diamond), sulphasalazine (down triangle), and healthy
control (square). C. Colons were removed after 4 days of treatment
(left columns) and 8 days of treatment (right columns), and the
lengths were measured using calipers.
[0185] FIG. 5. Analysis of .sup.99mTc-EC20 accumulation in the
aortic sinus of ApoE-/- mice treated with valsartan or fluvastatin.
A: Radioimages of mice injected with .sup.99mTc-EC20 3 weeks after
initiation of treatment. From left to right: healthy (C57BL/6)
control, ApoE-/- mouse treated with saline, ApoE-/- mouse treated
with valsartan, ApoE-/- mouse treated with fluvastatin. B. H&E
staining of arterial walls from the aortic sinus of each of the
mice imaged directly overhead. RBCs=red blood cells.
[0186] FIG. 6. Analysis of .sup.99mTc-EC20 accumulation in mice
with pulmonary fibrosis treated with etanercept or dexamethasone.
A: Radioimages of mice injected with .sup.99m Tc-EC20 after six
days of treatment. From left to right, healthy control, treatment
with saline, treatment with dexamethasone and treatment with
etanercept. B: Lung hydroxyproline content of lungs analyzed after
15 days of treatment. C: Total bronchoalveolar lavage fluid cell
counts after 15 days of treatment. D. H&E staining of lung
tissue, from left to right: healthy control, treatment with saline,
treatment with dexamethasone and treatment with etanercept.
[0187] FIG. 7. Analysis of .sup.99mTc-EC20 accumulation in CIA mice
treated with methotrexate or dexamethasone. A: Radioimage of mice
injected with .sup.99mTc-EC20 11 days after initiation of treatment
with methotrexate (left), saline (middle) or dexamethasone
(right).
[0188] FIG. 8. Analysis of .sup.99mTc-EC20 uptake in CIA mice
treated with etanercept or abatacept. A: Radioimages of mice
injected with .sup.99mTc-EC20 11 days after initiation of treatment
with etanercept (left), saline (middle) or abatacept (right).
[0189] FIG. 9. Analysis of .sup.99mTc-EC20 accumulation in mice
with ulcerative colitis treated with cimetidine or sulphasalazine.
A: Radioimages of mice injected with .sup.99mTc-EC20 8 days after
initiation of treatment. From left to right, healthy control,
treatment with saline, treatment with cimetidine and treatment with
sulphasalazine.
[0190] FIG. 10. Analysis of .sup.99mTc-EC20 accumulation in the
aortic sinus of ApoE-/- mice treated with valsartan or fluvastatin.
A: Radioimages of mice injected with .sup.99mTc-EC20 3 weeks after
initiation of treatment. From left to right: healthy (C57BL/6)
mouse, ApoE-/- mouse treated with saline, ApoE-/- mouse treated
with valsartan, ApoE-/- mouse treated with fluvastatin.
[0191] FIG. 11. Analysis of .sup.99mTc-EC20 accumulation in mice
with pulmonary fibrosis treated with etanercept or dexamethasone.
A: Radioimages of mice injected with .sup.99mTc-EC20 after 15 days
of treatment. From left to right, healthy control mouse, mouse
treated with saline, mouse treated with dexamethasone, and mouse
treated with etanercept.
[0192] FIG. 12. Flow cytometric analysis of macrophage accumulation
in mice with ulcerative colitis treated with cimetidine or
sulphasalazine.
[0193] FIG. 13. Analysis of OTL-38 accumulation in CIA mice treated
with orencia or dexamethasone. Clinical scores are shown for mice
in each of the following treatment groups: healthy control, disease
control, orencia, and dexamethasone.
[0194] FIG. 14. Analysis of OTL-38 accumulation (Day 3) in CIA mice
treated with orencia or dexamethasone. Clinical scores are shown
for mice in each of the following treatment groups: healthy
control, disease control, orencia, and dexamethasone.
[0195] FIG. 15. Analysis of OTL-38 accumulation (Day 11) in CIA
mice treated with orencia or dexamethasone. Clinical scores are
shown for mice in each of the following treatment groups: healthy
control, disease control, orencia, and dexamethasone.
[0196] FIG. 16. Analysis of OTL-38 accumulation in CIA mice treated
with orencia or dexamethasone. Paw thickness measurements of mice
in each of the following treatment groups are shown: healthy
control, disease control, orencia, and dexamethasone.
[0197] FIG. 17. Analysis of OTL-38 accumulation in CIA mice treated
with orencia or dexamethasone. Imaging of mice injected with OTL-38
three days after initiation of treatment is shown: dexamethasone
(left), saline (middle) or orencia (right).
[0198] FIG. 18. Analysis of OTL-38 accumulation in CIA mice treated
with orencia or dexamethasone. Imaging of mice injected with OTL-38
eleven days after initiation of treatment is shown: dexamethasone
(left), saline (middle) or orencia (right).
[0199] FIG. 19. Analysis of OTL-38 accumulation in mice with
ulcerative colitis treated with sulphasalazine or cimetidine.
Clinical scores are shown for mice in each of the following
treatment groups: healthy control, disease control, sulphasalazine,
and cimetidine.
[0200] FIG. 20. Analysis of OTL-38 accumulation (Day 4) in mice
with ulcerative colitis treated with sulphasalazine or cimetidine.
Imaging of mice injected with OTL-38 four days after initiation of
treatment is shown: healthy control (far left), disease control
(middle left), cimetidine (middle right), and sulphasalazine (far
right).
[0201] FIG. 21. Analysis of OTL-38 accumulation (Day 4) in mice
with ulcerative colitis treated with sulphasalazine or cimetidine.
Imaging of colons of mice injected with OTL-38 four days after
initiation of treatment is shown: healthy control (far left),
disease control (middle left), cimetidine (middle right), and
sulphasalazine (far right).
[0202] FIG. 22. Analysis of OTL-38 accumulation (Day 4) in mice
with ulcerative colitis treated with s sulphasalazine or
cimetidine. Imaging of colons of mice injected with OTL-38 four
days after initiation of treatment is shown: healthy control (far
left), disease control (middle left), cimetidine (middle right),
and sulphasalazine (far right).
[0203] FIG. 23. Analysis of OTL-38 accumulation in mice with
ulcerative colitis treated with sulphasazine or cimetidine. Colon
length is shown for mice in each of the following treatment groups
on day 4 of treatment: healthy control, disease control,
sulphasalazine, and cimetidine.
[0204] FIG. 24. Analysis of OTL-38 accumulation (Day 10) in mice
with ulcerative colitis treated with sulphasalazine or cimetidine.
Imaging of mice injected with OTL-38 ten days after initiation of
treatment is shown: healthy control (far left), disease control
(middle left), cimetidine (middle right), and sulphasalazine (far
right).
[0205] FIG. 25. Analysis of OTL-38 accumulation (Day 10) in mice
with ulcerative colitis treated with sulphasalazine or cimetidine.
Imaging of colons of mice injected with OTL-38 ten days after
initiation of treatment is shown: healthy control (far left),
disease control (middle left), cimetidine (middle right), and
sulphasalazine (far right).
[0206] FIG. 26. Analysis of OTL-38 accumulation (Day 10) in mice
with ulcerative colitis treated with sulphasalazine or cimetidine.
Imaging of colons of mice injected with OTL-38 ten days after
initiation of treatment is shown: healthy control (far left),
disease control (middle left), cimetidine (middle right), and
sulphasalazine (far right).
[0207] FIG. 27. Analysis of OTL-38 accumulation in mice with
ulcerative colitis treated with sulphasalazine or cimetidine. Colon
length is shown for mice in each of the following treatment groups
on day 10 of treatment: healthy control, disease control,
sulphasalazine, and cimetidine.
[0208] FIG. 28. Analysis of OTL-38 accumulation (Day 21) in mice
with atherosclerosis treated with valsartan or fluvastatin. Imaging
of mice injected with OTL-38 at 21 days after initiation of
treatment is shown: healthy control (far left), disease control
(middle left), valsartan (middle right), and fluvastatin (far
right).
[0209] FIG. 29. Analysis of OTL-38 accumulation (Day 6) in mice
with pulmonary fibrosis treated with etanercept or dexamethasone.
Imaging of mice injected with OTL-38 at 6 days after initiation of
treatment is shown: healthy control (far left), disease control
(middle left), etanercept (middle right), and dexamethasone (far
right).
DEFINITIONS
[0210] As used herein, the term "signal" means any detectable
signal produced by a medical imaging agent that is capable of being
detected using a medical imaging procedure. Examples of medical
imaging procedures include, but are not limited to, the use of
radioactive imaging agents in imaging procedures, the use of
fluorescent imaging agents or other types of dyes in medical
imaging, positron emission tomography, magnetic resonance imaging,
computed tomography, scintigraphic imaging, optical imaging,
ultrasound, and the like.
[0211] As used herein, the phrases "selecting a patient for therapy
with an anti-inflammatory drug," "selection of a patient for
therapy with an anti-inflammatory drug," "selection of patients for
therapy with an anti-inflammatory drug," and similar phrases mean
that the determination is made that a patient should be treated
with or should continue to be treated with a particular
anti-inflammatory drug.
[0212] As used herein the phrase "predict the response of the
patient to the anti-inflammatory drug," means that a determination
is made whether the patient is expected to benefit from initiation
of treatment or continuation of treatment with an anti-inflammatory
drug or is not expected to benefit from initiation of treatment or
continuation of treatment with an anti-inflammatory drug.
[0213] As used herein, the term "folate-imaging agent conjugate"
means a folate ligand linked to an imaging agent.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0214] Several embodiments of the invention are described by the
following enumerated clauses and each of the embodiments described
in this Detailed Description section of this application apply to
each of the following embodiments:
[0215] 1. A method for selecting a patient for therapy with an
anti-inflammatory drug, the method comprising the steps of
administering to the patient a folate-imaging agent conjugate, and
using the folate-imaging agent conjugate to predict the response of
the patient to the anti-inflammatory drug.
[0216] 2. Use of a folate-imaging agent conjugate for selecting a
patient for therapy with an anti-inflammatory drug wherein the
folate-imaging agent conjugate is administered to the patient and
is used to predict the response of the patient to the
anti-inflammatory drug.
[0217] 3. Use of a folate-imaging agent conjugate in the
manufacture of a medicament for selecting a patient for therapy
with an anti-inflammatory drug wherein the folate-imaging agent
conjugate is administered to the patient and is used to predict the
response of the patient to the anti-inflammatory drug.
[0218] 4. The method or use of any one of clauses 1 to 3 wherein
the folate-imaging agent conjugate produces a detectable signal in
the patient, wherein the signal is detected, and wherein the
detection of the signal is used to predict the response of the
patient to the anti-inflammatory drug.
[0219] 5. The method or use of clause 4 wherein the signal is a
radioactive signal.
[0220] 6. The method or use of clause 4 wherein the signal is
produced by a chromophore.
[0221] 7. The method or use of clause 6 wherein the chromophore is
a fluorophore.
[0222] 8. The method or use of clause 7 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0223] 9. The method or use of any one of clauses 4 to 8 wherein
the signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0224] 10. The method or use of any one of clauses 1 to 9 wherein
the administering step comprises a first administering step and a
second administering step.
[0225] 11. The method or use of clause 10 wherein the first and
second administering steps produce a first signal and a second
signal, respectively.
[0226] 12. The method or use of clause 11 wherein the first signal
and the second signal are quantified.
[0227] 13. The method or use of clause 11 wherein the first signal
is obtained by administering the folate-imaging agent conjugate
prior to administration of the anti-inflammatory drug.
[0228] 14. The method or use of clause 11 wherein the first signal
is obtained by administering the folate-imaging agent conjugate on
the same day as treatment with the anti-inflammatory drug is
initiated.
[0229] 15. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0230] 16. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
within about 21 days after administration of the anti-inflammatory
drug is initiated.
[0231] 17. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
within about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks
after the administration of the anti-inflammatory drug is
initiated.
[0232] 18. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate on
any one of the days within about 21 days after the administration
of the anti-inflammatory drug is initiated.
[0233] 19. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate on
any one of the days within about 12 weeks after the administration
of the anti-inflammatory drug is initiated.
[0234] 20. The method or use of clause 11 wherein the second signal
is reduced by about 10%, about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, or about 100%
compared to the first signal, and wherein the reduction indicates
that the patient should continue to be treated with the
anti-inflammatory drug.
[0235] 21. The method or use of clause 11 wherein the second signal
is obtained by administering the folate-imaging agent conjugate
about 2, about 3, about 4, about 5, about 6, about 12, about 15, or
about 21 days after the administration of the anti-inflammatory
drug is initiated.
[0236] 22. The method or use of any one of clauses 1 to 21 wherein
the inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0237] 23. The method or use of any one of clauses 1 to 22 wherein
the folate-imaging agent conjugate is in a parenteral dosage
form.
[0238] 24. The method or use of clause 23 wherein the dosage form
is selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0239] 25. The method or use of any one of clauses 1 to 24 wherein
the folate-imaging agent conjugate is in a composition and wherein
the composition further comprises a pharmaceutically acceptable
carrier.
[0240] 26. The method or use of clause 25 wherein the
pharmaceutically acceptable carrier is a liquid carrier.
[0241] 27. The method or use of clause 26 wherein the liquid
carrier is selected from the group consisting of saline, glucose,
alcohols, glycols, esters, amides, and a combination thereof.
[0242] 28. The method or use of any one of clauses 1 to 27 wherein
the folate-imaging agent conjugate is administered in an effective
amount.
[0243] 29. The method or use of clause 28 wherein the effective
amount ranges from about 1 ng to about 1 mg per kilogram of body
weight of the patient.
[0244] 30. The method or use of clause 28 wherein the effective
amount ranges from about 100 ng to about 500 .mu.g per kilogram of
body weight of the patient.
[0245] 31. The method or use of clause 28 wherein the effective
amount ranges from about 100 ng to about 25 .mu.g per kilogram of
body weight of the patient.
[0246] 32. The method or use of clause 28 wherein the effective
amount ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of
body surface area of the patient.
[0247] 33. The method or use of clause 28 wherein the effective
amount ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of
body surface area of the patient.
[0248] 34. The method or use of clause 28 wherein the effective
amount ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of
patient body weight.
[0249] 35. The method or use of any one of clauses 1 to 34 further
comprising the step of administering unlabeled folic acid to the
patient.
[0250] 36. The method or use of clause 35 wherein the unlabeled
folic acid is administered before administration of the
folate-imaging agent conjugate.
[0251] 37. The method or use of any one of clauses 1 to 5 or 9 to
36 wherein the folate-imaging agent conjugate has the formula
##STR00023##
wherein M is a radionuclide.
[0252] 38. The method or use of any one of clauses 1 to 5 or 9 to
37 wherein the folate-imaging agent conjugate has the formula
##STR00024##
wherein M is a radionuclide.
[0253] 39. The method or use of clause 37 or 38 wherein the
radionuclide is selected from the group consisting of an isotope of
gallium, an isotope of indium, an isotope of copper, an isotope of
technetium, and an isotope of rhenium.
[0254] 40. The method or use of clause 39 wherein the radionuclide
is an isotope of technetium.
[0255] 41. The method or use of clause 40 wherein the technecium is
.sup.99m-technetium.
[0256] 42. The method or use of any one of clauses 1 to 5 or 9 to
41 wherein the folate-imaging agent conjugate is
.sup.99mTc-EC20.
[0257] 43. The method or use of any one of clauses 1 to 5 or 9 to
42 wherein the signal is detected using scintigraphic imaging.
[0258] 44. The method or use of any one of clauses 1 to 43 wherein
the patient is a human patient.
[0259] 45. The method or use of any one of clauses 1 to 43 wherein
the patient is a veterinary patient.
[0260] 46. The method or use of any one of clauses 1 to 5 or 9 to
45 wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0261] 47. The method or use of any one of clauses 1 to 46 wherein
the folate-imaging agent conjugate is in the form of a
reconstituted lyophilizate.
[0262] 48. The method or use of any one of clauses 1 to 47 wherein
the folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0263] 49. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00025##
[0264] or a pharmaceutically acceptable salt of any of these
compounds.
[0265] 50. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00026##
[0266] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.f comprising a radiophore or a precursor to a radiophore.
[0267] 51. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00027##
[0268] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0269] 52. The method or use of any one of clauses 1-5, 9 to 36, or
44 to 48 wherein the folate-imaging agent conjugate has the
formula:
##STR00028##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.f is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0270] 53. A method for selecting a patient for therapy with an
anti-inflammatory drug, the method comprising the steps of
assessing whether the patient is in need of therapy with the
anti-inflammatory drug by relying on the results obtained by means
for detecting a signal produced in the patient by a folate-imaging
agent conjugate administered to the patient; and prescribing or
continuing to prescribe the anti-inflammatory drug to treat the
patient assessed to be in need of the anti-inflammatory drug.
[0271] 54. The method of clause 54 wherein the folate-imaging agent
conjugate produces a detectable signal in the patient, wherein the
signal is detected, and wherein the detection of the signal is used
to assess whether the patient is in need of therapy with the
anti-inflammatory drug.
[0272] 55. The method of clause 54 wherein the signal is a
radioactive signal.
[0273] 56. The method of clause 54 wherein the signal is produced
by a chromophore.
[0274] 57. The method of clause 56 wherein the chromophore is a
fluorophore.
[0275] 58. The method of clause 57 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0276] 59. The method of any one of clauses 53 to 58 wherein the
signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0277] 60. The method of any one of clauses 53 to 59 wherein the
administration comprises a first administering step and a second
administering step.
[0278] 61. The method of clause 60 wherein the first and second
administering steps produce a first signal and a second signal,
respectively.
[0279] 62. The method of clause 61 wherein the first signal and the
second signal are quantified.
[0280] 63. The method of clause 61 wherein the first signal is
obtained by administering the folate-imaging agent conjugate prior
to administration of the anti-inflammatory drug.
[0281] 64. The method of clause 61 wherein the first signal is
obtained by administering the folate-imaging agent conjugate on the
same day as treatment with the anti-inflammatory drug is
initiated.
[0282] 65. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0283] 66. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 21 days after administration of the anti-inflammatory drug is
initiated.
[0284] 67. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after
the administration of the anti-inflammatory drug is initiated.
[0285] 68. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 21 days after the administration of
the anti-inflammatory drug is initiated.
[0286] 69. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 12 weeks after the administration of
the anti-inflammatory drug is initiated.
[0287] 70. The method of clause 61 wherein the second signal is
reduced by about 10%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, or about 100% compared
to the first signal, and wherein the reduction indicates that the
patient should continue to be treated with the anti-inflammatory
drug.
[0288] 71. The method of clause 61 wherein the second signal is
obtained by administering the folate-imaging agent conjugate about
2, about 3, about 4, about 5, about 6, about 12, about 15, or about
21 days after the administration of the anti-inflammatory drug is
initiated.
[0289] 72. The method of any one of clauses 53 to 71 wherein the
inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0290] 73. The method of any one of clauses 53 to 72 wherein the
folate-imaging agent conjugate is in a parenteral dosage form.
[0291] 74. The method of clause 73 wherein the dosage form is
selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0292] 75. The method of any one of clauses 53 to 74 wherein the
folate-imaging agent conjugate is in a composition and wherein the
composition further comprises a pharmaceutically acceptable
carrier.
[0293] 76. The method of clause 75 wherein the pharmaceutically
acceptable carrier is a liquid carrier.
[0294] 77. The method of clause 76 wherein the liquid carrier is
selected from the group consisting of saline, glucose, alcohols,
glycols, esters, amides, and a combination thereof.
[0295] 78. The method of any one of clauses 53 to 77 wherein the
folate-imaging agent conjugate is administered in an effective
amount.
[0296] 79. The method of clause 78 wherein the effective amount
ranges from about 1 ng to about 1 mg per kilogram of body weight of
the patient.
[0297] 80. The method of clause 78 wherein the effective amount
ranges from about 100 ng to about 500 .mu.g per kilogram of body
weight of the patient.
[0298] 81. The method of clause 78 wherein the effective amount
ranges from about 100 ng to about 25 .mu.g per kilogram of body
weight of the patient.
[0299] 82. The method of clause 78 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of body
surface area of the patient.
[0300] 83. The method of clause 78 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of body
surface area of the patient.
[0301] 84. The method of clause 78 wherein the effective amount
ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of patient body
weight.
[0302] 85. The method of any one of clauses 53 to 84 further
comprising the step of administering unlabeled folic acid to the
patient.
[0303] 86. The method of clause 85 wherein the unlabeled folic acid
is administered before administration of the folate-imaging agent
conjugate.
[0304] 87. The method of any one of clauses 53 to 55 or 59 to 86
wherein the folate-imaging agent conjugate has the formula
##STR00029##
wherein M is a radionuclide.
[0305] 88. The method of any one of clauses 53 to 55 or 59 to 87
wherein the folate-imaging agent conjugate has the formula
##STR00030##
wherein M is a radionuclide.
[0306] 89. The method of clause 87 or 88 wherein the radionuclide
is selected from the group consisting of an isotope of gallium, an
isotope of indium, an isotope of copper, an isotope of technetium,
and an isotope of rhenium.
[0307] 90. The method of clause 89 wherein the radionuclide is an
isotope of technetium.
[0308] 91. The method of clause 90 wherein the technecium is
.sup.99m-technetium.
[0309] 92. The method of any one of clauses 53 to 55 or 59 to 91
wherein the folate-imaging agent conjugate is .sup.99mTc-EC20.
[0310] 93. The method of any one of clauses 53 to 55 or 59 to 92
wherein the signal is detected using scintigraphic imaging.
[0311] 94. The method of any one of clauses 53 to 93 wherein the
patient is a human patient.
[0312] 95. The method of any one of clauses 53 to 93 wherein the
patient is a veterinary patient.
[0313] 96. The method of any one of clauses 53 to 55 or 59 to 95
wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0314] 97. The method of any one of clauses 53 to 96 wherein the
folate-imaging agent conjugate is in the form of a reconstituted
lyophilizate.
[0315] 98. The method of any one of clauses 53 to 97 wherein the
folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0316] 99. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00031##
[0317] or a pharmaceutically acceptable salt of any of these
compounds.
[0318] 100. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00032##
[0319] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.f comprising a radiophore or a precursor to a radiophore.
[0320] 101. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00033##
[0321] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0322] 102. The method of any one of clauses 53-55, 59 to 86, or 94
to 98 wherein the folate-imaging agent conjugate has the
formula:
##STR00034##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.f is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0323] 103. A method for predicting the efficacy of an
anti-inflammatory drug in a patient, the method comprising the
steps of administering to the patient a folate-imaging agent
conjugate, and using the folate-imaging agent conjugate to predict
efficacy of the anti-inflammatory drug in the patient.
[0324] 104. The method of clause 103 wherein the folate-imaging
agent conjugate produces a detectable signal in the patient,
wherein the signal is detected, and wherein the detection of the
signal is used to assess whether the patient is in need of therapy
with the anti-inflammatory drug.
[0325] 105. The method of clause 104 wherein the signal is a
radioactive signal.
[0326] 106. The method of clause 104 wherein the signal is produced
by a chromophore.
[0327] 107. The method of clause 106 wherein the chromophore is a
fluorophore.
[0328] 108. The method of clause 107 wherein the fluorophore is
selected from the group consisting of a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a
cyanine.
[0329] 109. The method of any one of clauses 104 to 108 wherein the
signal is produced as a result of binding of the folate-imaging
agent conjugate to activated macrophages.
[0330] 110. The method of any one of clauses 103 to 109 wherein the
administering step comprises a first administering step and a
second administering step.
[0331] 111. The method of clause 110 wherein the first and second
administering steps produce a first signal and a second signal,
respectively.
[0332] 112. The method of clause 111 wherein the first signal and
the second signal are quantified.
[0333] 113. The method of clause 111 wherein the first signal is
obtained by administering the folate-imaging agent conjugate prior
to administration of the anti-inflammatory drug.
[0334] 114. The method of clause 111 wherein the first signal is
obtained by administering the folate-imaging agent conjugate on the
same day as treatment with the anti-inflammatory drug is
initiated.
[0335] 115. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug.
[0336] 116. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 21 days after administration of the anti-inflammatory drug is
initiated.
[0337] 117. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate within
about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after
the administration of the anti-inflammatory drug is initiated.
[0338] 118. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 21 days after the administration of
the anti-inflammatory drug is initiated.
[0339] 119. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 12 weeks after the administration of
the anti-inflammatory drug is initiated.
[0340] 120. The method of clause 111 wherein the second signal is
reduced by about 10%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, or about 100% compared
to the first signal, and wherein the reduction indicates that the
patient should continue to be treated with the anti-inflammatory
drug.
[0341] 121. The method of clause 111 wherein the second signal is
obtained by administering the folate-imaging agent conjugate about
2, about 3, about 4, about 5, about 6, about 12, about 15, or about
21 days after the administration of the anti-inflammatory drug is
initiated.
[0342] 122. The method of any one of clauses 103 to 121 wherein the
inflammatory disease is selected from the group consisting of
arthritis, osteoarthritis, rheumatoid arthritis, atherosclerosis,
psoriasis, ischemia/reperfusion injury, pulmonary fibrosis, organ
transplant rejection, ulcerative colitis, impact trauma,
osteomyelitis, multiple sclerosis, scleroderma, Crohn's disease,
Sjogren's syndrome, glomerulonephritis, systemic sclerosis,
sarcoidosis, an inflammatory lesion, and chronic inflammation.
[0343] 123. The method of any one of clauses 103 to 122 wherein the
folate-imaging agent conjugate is in a parenteral dosage form.
[0344] 124. The method of clause 123 wherein the dosage form is
selected from the group consisting of an intradermal, a
subcutaneous, an intramuscular, an intraperitoneal, an intravenous,
and an intrathecal dosage form.
[0345] 125. The method of any one of clauses 103 to 124 wherein the
folate-imaging agent conjugate is in a composition and wherein the
composition further comprises a pharmaceutically acceptable
carrier.
[0346] 126. The method of clause 125 wherein the pharmaceutically
acceptable carrier is a liquid carrier.
[0347] 127. The method of clause 126 wherein the liquid carrier is
selected from the group consisting of saline, glucose, alcohols,
glycols, esters, amides, and a combination thereof.
[0348] 128. The method of any one of clauses 103 to 127 wherein the
folate-imaging agent conjugate is administered in an effective
amount.
[0349] 129. The method of clause 128 wherein the effective amount
ranges from about 1 ng to about 1 mg per kilogram of body weight of
the patient.
[0350] 130. The method of clause 128 wherein the effective amount
ranges from about 100 ng to about 500 .mu.g per kilogram of body
weight of the patient.
[0351] 131. The method of clause 128 wherein the effective amount
ranges from about 100 ng to about 25 .mu.g per kilogram of body
weight of the patient.
[0352] 132. The method of clause 128 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 500 mg/m.sup.2 of body
surface area of the patient.
[0353] 133. The method of clause 128 wherein the effective amount
ranges from about 1 .mu.g/m.sup.2 to about 300 mg/m.sup.2 of body
surface area of the patient.
[0354] 134. The method of clause 128 wherein the effective amount
ranges from about 10 .mu.g/kg to about 100 .mu.g/kg of patient body
weight.
[0355] 135. The method of any one of clauses 103 to 134 further
comprising the step of administering unlabeled folic acid to the
patient.
[0356] 136. The method of clause 135 wherein the unlabeled folic
acid is administered before administration of the folate-imaging
agent conjugate.
[0357] 137. The method of any one of clauses 103 to 105 or 109 to
136 wherein the folate-imaging agent conjugate has the formula
##STR00035##
wherein M is a radionuclide.
[0358] 138. The method of any one of clauses 103 to 105 or 109 to
137 wherein the folate-imaging agent conjugate has the formula
##STR00036##
wherein M is a radionuclide.
[0359] 139. The method of clause 137 or 138 wherein the
radionuclide is selected from the group consisting of an isotope of
gallium, an isotope of indium, an isotope of copper, an isotope of
technetium, and an isotope of rhenium.
[0360] 140. The method of clause 139 wherein the radionuclide is an
isotope of technetium.
[0361] 141. The method of clause 140 wherein the technecium is
.sup.99m-technetium.
[0362] 142. The method of any one of clauses 103 to 105 or 109 to
141 wherein the folate-imaging agent conjugate is
.sup.99mTc-EC20.
[0363] 143. The method of any one of clauses 103 to 105 or 109 to
142 wherein the signal is detected using scintigraphic imaging.
[0364] 144. The method of any one of clauses 103 to 143 wherein the
patient is a human patient.
[0365] 145. The method of any one of clauses 103 to 143 wherein the
patient is a veterinary patient.
[0366] 146. The method of any one of clauses 103 to 105 or 109 to
145 wherein the folate-imaging agent conjugate has a radiochemical
purity of at least 90% based on weight percentage.
[0367] 147. The method of any one of clauses 103 to 146 wherein the
folate-imaging agent conjugate is in the form of a reconstituted
lyophilizate.
[0368] 148. The method of any one of clauses 103 to 147 wherein the
folate-imaging agent conjugate is in a sterile, pyrogen-free
aqueous solution.
[0369] 149. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00037##
[0370] or a pharmaceutically acceptable salt of any of these
compounds.
[0371] 150. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00038##
[0372] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.t comprising a radiophore or a precursor to a radiophore.
[0373] 151. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00039##
[0374] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0375] 152. The method of any one of clauses 103-105, 109 to 136,
or 144 to 148 wherein the folate-imaging agent conjugate has the
formula:
##STR00040##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.t is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0376] 153. The method of use of any one of clauses 1 to 152
wherein the folate portion of the folate-imaging agent conjugate
comprises a compound of the formula:
##STR00041##
[0377] 154. The method or use of any one of clauses 1-4, 9-36,
44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136, or
144-148 wherein the folate-imaging agent conjugate has the
formula:
##STR00042##
[0378] wherein:
[0379] X is an amino acid or a derivative thereof, and
[0380] Y is a dye that has a fluorescence excitation and emission
spectra in the near infrared range, and said compound maintains or
enhances the fluorescence of Y.
[0381] 155. The method or use of any one of clauses 1-4, 9-36,
44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136, or
144-148 wherein the imaging agent Y has the formula:
##STR00043##
[0382] wherein:
[0383] X' is independently selected from the group consisting of O,
S, N and C, and
[0384] R' is independently selected from the group consisting of
CH2 and CH2CH2. In some embodiments, the dye Y is selected from the
group consisting of LS288, IR800, SP054, S0121, KODAK IRD28, S2076,
S0456 and derivatives thereof.
[0385] 156. The method or use of any one of clauses of clauses 1-4,
9-36, 44-45, 47-48, 53-54, 59-86, 94-95, 97-98, 103-104, 109-136,
or 144-148 wherein the folate-imaging agent conjugate has the
formula:
##STR00044##
wherein W, X, Y, Z each are H, Na.sup.+, K.sup.+ or NH.sub.4.sup.+.
This compound is referred to herein as OTL-38 or OTL-0038.
[0386] In any of the various embodiments described herein, the
following features may be present where applicable, providing
additional embodiments of the invention. For all of the embodiments
described herein, any applicable combination of embodiments is also
contemplated. Any applicable combination of the above-described
embodiments is also considered to be in accordance with the
invention.
[0387] The invention relates to methods and compositions for the
selection of patients for therapy with an anti-inflammatory drug.
More particularly, the invention relates to compositions comprising
folate-imaging agent conjugates for the selection of patients for
therapy with an anti-inflammatory drug, and methods and uses
therefor.
[0388] In one embodiment, a method is provided for selecting a
patient for therapy with an anti-inflammatory drug. The method
comprises the steps of administering to the patient a
folate-imaging agent conjugate, and using the folate-imaging agent
conjugate to predict the response of the patient to the
anti-inflammatory drug.
[0389] In another embodiment, a use is provided of a folate-imaging
agent conjugate for selecting a patient for therapy with an
anti-inflammatory drug. The folate-imaging agent conjugate is
administered to the patient and is used to predict the response of
the patient to the anti-inflammatory drug.
[0390] In yet another embodiment, a use is provided of a
folate-imaging agent conjugate in the manufacture of a medicament
for selecting a patient for therapy with an anti-inflammatory drug.
The folate-imaging agent conjugate is administered to the patient
and is used to predict the response of the patient to the
anti-inflammatory drug.
[0391] In another illustrative embodiment, a method for selecting a
patient for therapy with an anti-inflammatory drug is provided. The
method comprises the steps of assessing whether the patient is in
need of therapy with the anti-inflammatory drug by relying on the
results obtained by means for detecting a signal produced in the
patient by a folate-imaging agent conjugate administered to the
patient; and prescribing or continuing to prescribe the
anti-inflammatory drug to treat the patient assessed to be in need
of the anti-inflammatory drug.
[0392] The folate-imaging agent conjugate produces a detectable
signal in the patient, the signal is detected, and detection of the
signal is used to predict the response of the patient to the
anti-inflammatory drug. Without being bound by theory, the signal
may be produced as a result of the binding of folate-imaging agent
conjugates to activated macrophages at a site of inflammation,
and/or may result from accumulation of folate-imaging agent
conjugates in activated macrophages at a site of inflammation.
[0393] In all of the above-described embodiments, the
folate-imaging agent conjugate is administered to the patient and
is used to predict the response of the patient to the
anti-inflammatory drug. The administering step can comprise a first
administering step and a second administering step. The first and
second administering steps can produce a first signal and a second
signal, respectively.
[0394] In another illustrative aspect, the first signal and the
second signal can be quantified, and compared to each other to
determine if there is a reduction in the intensity of the second
signal compared to the first signal. In these embodiments, a
reduction in the intensity of the second signal compared to the
first signal can indicate that the patient will benefit from
continuation of treatment with an anti-inflammatory drug. The
patient may then be treated with the anti-inflammatory drug, or
treatment with the anti-inflammatory drug may be continued, if the
patient was already being treated with the anti-inflammatory
drug.
[0395] In one embodiment, the first signal is obtained by
administering the folate-imaging agent conjugate prior to
administration of the anti-inflammatory drug to obtain a "control"
level of signal intensity prior to treatment with the
anti-inflammatory drug. In another embodiment, the first signal is
obtained by administering the folate-imaging agent conjugate on the
same day as treatment with the anti-inflammatory drug is initiated
to obtain a "control" level of signal intensity. The second signal
can be obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug to
determine the level of signal intensity after treatment with the
anti-inflammatory drug for a period of time. Without being bound by
theory, a reduction in the intensity of the second signal compared
to the first signal may reflect a reduction in the number of
inflammatory cells capable of binding folate-imaging agent
conjugates, such as activated macrophages, at the site of
inflammation, inactivation of inflammatory cells at the site of
inflammation, a reduction in the capacity of inflammatory cells to
bind folate-imaging agent conjugates, a reduction in accumulation
of folate-imaging agent conjugates in inflammatory cells at the
site of inflammation, or any other response caused by the
anti-inflammatory drug that reduces the signal intensity produced
by folate-imaging agent conjugates at a site of inflammation.
[0396] In one of the above described embodiments, the second signal
is obtained by administering the folate-imaging agent conjugate
subsequent to the administration of the anti-inflammatory drug. In
one illustrative embodiment, the second signal is obtained by
administering the folate-imaging agent conjugate within about 21
days after administration of the anti-inflammatory drug is
initiated. In another embodiment, the second signal is obtained by
administering the folate-imaging agent conjugate on any one of the
days within about 21 days after the administration of the
anti-inflammatory drug is initiated. In another aspect, the second
signal is obtained by administering the folate-imaging agent
conjugate within about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8
weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12
weeks after the administration of the anti-inflammatory drug is
initiated. In another illustrative embodiment, the second signal is
obtained by administering the folate-imaging agent conjugate on any
one of the days within about 12 weeks after the administration of
the anti-inflammatory drug is initiated. In another embodiment, the
second signal is obtained by administering the folate-imaging agent
conjugate on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day
8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16,
day 17, day 18, day 19, day 20, day 21, day 22, day 23, or day 24
after the administration of the anti-inflammatory drug is
initiated. The day the administration of the anti-inflammatory drug
is initiated is day 0. Additional signals may be obtained by using
a third, fourth, etc. administration step of the folate-imaging
agent conjugate. Any additional administrations of the
folate-imaging agent conjugate may be done at any of the times
described in this paragraph.
[0397] As described herein, the first signal is obtained by
administering the folate-imaging agent conjugate prior to
administration of the anti-inflammatory drug, or on the same day as
treatment with the anti-inflammatory drug is initiated, to obtain a
"control" level of signal intensity prior to treatment with the
anti-inflammatory drug. The second signal can be obtained by
administering the folate-imaging agent conjugate subsequent to the
administration of the anti-inflammatory drug to determine the level
of signal intensity after treatment with the anti-inflammatory drug
for a period of time. The first signal and the second signal can be
quantified, and compared to each other to determine if there is a
reduction in the intensity of the second signal compared to the
first signal. In these embodiments, a reduction in the intensity of
the second signal compared to the first signal can indicate that
the patient will benefit from continued treatment with an
anti-inflammatory drug, or will not benefit from continuation of
treatment with the anti-inflammatory drug. If it is determined that
the patient will not benefit from treatment with a particular
anti-inflammatory drug, a different anti-inflammatory drug can be
used. The methods and uses described herein can be used to
determine whether the new anti-inflammatory drug will benefit the
patient.
[0398] In various embodiments, the second signal can be reduced by
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, or about 100% compared to the
first signal. For any of these embodiments, the reduction may
indicate that the patient should continue to be treated with the
anti-inflammatory drug.
[0399] In an alternate embodiment, the methods and uses described
herein can be used to predict the response of a patient to an
anti-inflammatory drug by making a determination whether the
patient is expected to benefit from initiation of treatment, rather
than continued treatment, with an anti-inflammatory drug, or
whether the patient is not expected to benefit from initiation of
treatment of treatment with an anti-inflammatory drug. In such
embodiments, folate-imaging agent conjugates are administered, in
the absence of treatment with an anti-inflammatory drug, and the
intensity of the signal obtained is used to determine whether the
patient is expected to benefit from initiation of treatment with an
anti-inflammatory drug, or is not expected to benefit from
initiation of treatment of treatment with an anti-inflammatory
drug.
[0400] The methods and uses described herein are applicable to any
anti-inflammatory drugs used to treat a patient with an
inflammatory disease. Anti-inflammatory drugs include, but are not
limited to, non-steroidal anti-inflammatory drugs (NSAID),
analgesics, glucocorticoids, anti-rheumatic drugs, dihydrofolate
reductase inhibitors, TNF-.alpha. inhibitors, biologic response
modifiers, hormonal agents, and combinations thereof. Other
exemplary anti-inflammatory drugs include corticosteroids,
hydrocortisone, prednisolone, prednisone, allopurinol, aspirin,
indomethacin, phenylbutazone, etanercept, infliximab, adalimumab,
rituximab, abatacept, anakinra, efalizumab, methotrexate,
dexamethasone, naproxen, and combinations thereof.
[0401] In the various embodiments described herein, the methods or
uses described herein are applicable to inflammatory diseases,
including, but not limited to, inflammatory diseases selected from
the group consisting of arthritis, osteoarthritis, rheumatoid
arthritis, atherosclerosis, psoriasis, ischemia/reperfusion injury,
pulmonary fibrosis, organ transplant rejection, ulcerative colitis,
impact trauma, osteomyelitis, multiple sclerosis, scleroderma,
Crohn's disease, Sjogren's syndrome, glomerulonephritis, systemic
sclerosis, sarcoidosis, an inflammatory lesion, and chronic
inflammation.
[0402] The methods and uses described herein, can be used 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 and uses described herein can be applied to humans,
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.
[0403] As used herein, the term "folate-imaging agent conjugate"
for the methods and uses described herein, means a folate ligand
linked to an imaging agent. Thus, the folate-imaging agent
conjugate comprises a folate ligand linked to an imaging agent. The
folate-imaging agent conjugates described below provide a means for
detecting a signal in a patient treated with a folate-imaging agent
conjugate.
[0404] In various embodiments, the folate ligand linked to the
imaging agent in the folate-imaging agent conjugate can include,
but is not limited to, folate, 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 to form folate-imaging agent conjugates for use in
the methods and uses described herein 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 "folates" bind to folate
receptors.
[0405] Additional folate ligands that can be used in the
folate-imaging agent conjugates have the general formula:
##STR00045##
[0406] wherein X and Y are each-independently selected from the
group consisting of halo, R.sup.2, OR.sup.2, SR.sup.3, and
NR.sup.4R.sup.5;
[0407] U, V, and W represent divalent moieties each independently
selected from the group consisting of --(R.sup.6a)C.dbd., --N.dbd.,
--(R.sup.6a)C(R.sup.7a)--, and --N(R.sup.4a)--; Q is selected from
the group consisting of C and CH; T is selected from the group
consisting of S, O, N, and --C.dbd.C--;
[0408] A.sup.1 and A.sup.2 are each independently selected from the
group consisting of oxygen, sulfur, --C(Z)--, --C(Z)O--, --OC(Z)--,
--N(R.sup.4b)--, --C(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)--,
--OC(Z)N(R.sup.4b)--, --N(R.sup.4b) C(Z)O--,
--N(R.sup.4b)C(Z)N(R.sup.5b)--, --S(O)--, --S(O).sub.2--,
--N(R.sup.4a)S(O).sub.2--, --C(R.sup.6b)(R.sup.7b)--,
--N(C.ident.CH)--, --N(C H.sub.2C.ident.CH)--, C.sub.1-C.sub.12
alkylene, and C.sub.1-C.sub.12 alkyeneoxy, where Z is oxygen or
sulfur;
[0409] R.sup.1 is selected from the group consisting of hydrogen,
halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; R.sup.2,
R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.5, R.sup.5b, R.sup.6b,
and R.sup.7b are each independently selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl,
C.sub.1-C.sub.12 alkenyl, C.sub.1-C.sub.12 alkynyl,
alkoxy)carbonyl, and (C.sub.1-C.sub.12 alkylamino)carbonyl;
[0410] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6 and R.sup.7 are taken together
to form a carbonyl group; R.sup.6a and R.sup.7a are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; or R.sup.6a
and R.sup.7a are taken together to form a carbonyl group;
[0411] L is a divalent linker as described herein;
[0412] n, p, r, s and t are each independently either 0 or 1;
and
[0413] wherein * indicates the attachment point to the rest of the
conjugate.
[0414] It is appreciated that the forgoing folate ligands can be
present in one or more tautomeric forms.
[0415] In various embodiments, the imaging agent portion of the
folate-imaging agent conjugate can be any imaging agent useful for
medical imaging. For example the imaging agent can be a radioactive
imaging agent or a chromophore, and the chromophore can be a
fluorophore. In one illustrative embodiment, the fluorophore is
selected from the group consisting of fluorescein, rhodamine, Texas
Red, phycoerythrin, Oregon Green, AlexaFluor 488 (Molecular Probes,
Eugene, Oreg.), Cy3, Cy5, and Cy7.
[0416] In another aspect, the fluorophore is a fluorescent agent
selected from Oregon Green fluorescent agents, including but not
limited to Oregon Green 488, Oregon Green 514, and the like,
AlexaFluor fluorescent agents, including but not limited to
AlexaFluor 488, AlexaFluor 647, and the like, fluorescein, and
related analogs, rhodamine fluorescent agents, including but not
limited to tetramethylrhodamine, and the like, DyLight fluorescent
agents, including but not limited to DyLight 680, and the like, CW
800, Texas Red, phycoerythrin, and others. In another embodiment,
the fluorophore is selected from a fluorescein, a rhodamine, a
phycoerythrin, a long wavelength fluorescent dye, and a cyanine.
Illustrative fluorophores are shown in the following illustrative
general structures:
##STR00046##
where X is oxygen, nitrogen, or sulfur, and where X is attached to
linker L; Y is OR.sup.a, NR.sup.a.sub.2, or NR.sup.a.sub.3.sup.+;
and Y' is O, NR.sup.a, or NR.sup.a.sub.2.sup.+; where each R is
independently selected in each instance from H, fluoro, sulfonic
acid, sulfonate, and salts thereof, and the like; and R.sup.a is
hydrogen or alkyl, and, in another embodiment,
##STR00047##
where X is oxygen, nitrogen, or sulfur, and where X is attached to
linker L; and each R is independently selected in each instance
from H, alkyl, heteroalkyl, and the like; and n is an integer from
0 to about 4.
[0417] In another embodiment, the fluorophore has the formula
##STR00048##
or a salt thereof wherein
[0418] W is a bond or
##STR00049##
where x is an integer from about 1 to about 4;
[0419] W.sup.A is a double bond or
##STR00050##
where y is an integer from about 1 to about 4;
[0420] R.sup.C and R.sup.D are independently hydrogen or alkyl, or
R.sup.C and R.sup.D and the atoms to which they are attached form a
cycloalkene;
[0421] T is hydrogen, fluoro, chloro, or hydroxy, alkyl,
heteroalkyl, alkoxy, aryl, heteroaryl, aryloxy, or heteroaryloxy,
each of which is optionally substituted, or T is O**, S**,
R.sup.17N**, where ** represents the attachment point to the rest
of the conjugate;
[0422] R.sup.8 represents from 1 to 3 substituents independently
selected in each instance from the group consisting of alkyl,
heteroalkyl, alkoxy, alkylhydroxy, fluoro, sulfonic acid, or a salt
thereof, alkylsulfonic acid, or a salt thereof, or an amine, and an
alkylamine; or R.sup.8 represents 2 to 3 substituents where at
least two substituents are on adjacent carbons and together with
the atoms to which they are attached form an optionally substituted
fused aromatic ring, and the other substituent, if present, is
alkyl, heteroalkyl, alkoxy, alkylhydroxy, fluoro, sulfonic acid, or
a salt thereof, alkyl sulfonic acid, or a salt thereof, or an
amine, and an alkylamine;
[0423] R.sup.11 represents from 1 to 3 substituents independently
selected in each instance from the group consisting of alkyl,
heteroalkyl, alkoxy, alkylhydroxy, fluoro, sulfonic acid, or a salt
thereof, alkylsulfonic acid, or a salt thereof, or an amine, and an
alkylamine; or R.sup.11 represents 2 to 3 substituents where at
least two substituents are on adjacent carbons and together with
the atoms to which they are attached form a fused aromatic ring,
and the other substituent, if present, is alkyl, heteroalkyl,
alkoxy, alkylhydroxy, fluoro, sulfonic acid, or a salt thereof,
alkylsulfonic acid, or a salt thereof, or an amine, and an
alkylamine;
[0424] L.sub.1 is an alkylene linked via a divalent linker to a
folate;
[0425] Het is selected from the group consisting of
##STR00051##
where * is the attachment point to W;
[0426] R.sup.9, R.sup.10, R.sup.12, R.sup.13, R.sup.14, and
R.sup.17 are in each instance independently selected from the group
consisting of alkyl, heteroalkyl, hydroxyalkyl, alkylamine,
aminoalkyl, thioalkyl, alkylsulfonic acid, or salt thereof, and
alkylcarboxylic acid, or a salt thereof;
[0427] R.sup.15 represents from 0 to 3 substituents selected from
the group consisting of fluoro, alkyl, alkoxy, sulfonic acid, or a
salt thereof, and heteroalkyl; and
[0428] R.sup.16 is selected from the group consisting of alkyl,
heteroalkyl, hydroxyalkyl, alkylamine, aminoalkyl, thioalkyl,
alkylsulfonic acid, or salt thereof, and alkylcarboxylic acid, or a
salt thereof; or R.sup.16 is alkylene*** where *** represents the
attachment point to the rest of the conjugate via a divalent
linker. In other embodiments, x is an integer from 0 to 4. In other
embodiments, y is an integer from 0 to 4.
[0429] In another embodiment, the imaging agent portion of the
folate-imaging agent conjugate can be a radioactive imaging agent.
For example, the folate-imaging agent conjugate can be a compound
of either of the following formulas where the imaging agent is a
metal chelator, and wherein M is a radionuclide:
##STR00052##
[0430] In these embodiments, the radionuclide can be selected from
the group consisting of an isotope of gallium, an isotope of
indium, an isotope of copper, an isotope of technetium, and an
isotope of rhenium. In another embodiment, the radionuclide can be
an isotope of technetium. In yet another embodiment, the
radionuclide can be .sup.99m-technetium.
[0431] In another embodiment, the imaging agent is
technetium-99m-labeled EC20 (.sup.99mTc-EC20). .sup.99mTc-EC20 has
been developed and provides for detection of tissues or cells
expressing folate receptors capable of binding folate.
.sup.99mTc-EC20 has the formula
##STR00053##
The term EC20 can be used to identify the non-radioactive reagent
lacking a radionuclide. EC20 has the formula:
##STR00054##
In the context of administration to patients for detecting and
assessing tissues and cells expressing folate receptors capable of
binding folate, ".sup.99mTc-EC20" is used herein to denote the
radioactive drug substance, or a pharmaceutically acceptable salt
thereof. It will be appreciated that .sup.99mTc-EC20 may be present
in solution or suspension in an ionized form, including a
deprotonated form. "EC20" is used herein to denote the
non-radioactive reagent lacking a radionuclide, or a
pharmaceutically acceptable salt of EC20. It will be appreciated
that EC20 may be present in solution or suspension in an ionized
form, including a deprotonated form.
[0432] In yet another embodiment, the folate-imaging agent
conjugate has the formula:
##STR00055##
or a pharmaceutically acceptable salt of any of these
compounds.
[0433] In another embodiment, the folate imaging agent conjugate
has the formula:
##STR00056##
[0434] wherein F is a folate ligand, L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; Ar is an aryl group,
including heteroaryl groups, that includes one or more substituents
R.sup.f comprising a radiophore or a precursor to a radiophore.
[0435] In another embodiment, the folate-imaging agent conjugate
has the formula:
##STR00057##
[0436] wherein F is a folate ligand; L is an optional bivalent
linker; n is an integer selected from 1 to about 100, or from 1 to
about 20, or n is 1, 2, 3, 4, 5, 6, 7, or 8; R.sup.f is as
described below; and m is an integer selected from 1 to about
3.
[0437] In another embodiment of the folate-imaging agent conjugate,
has the formula:
##STR00058##
wherein L is an optional bivalent linker; n is an integer selected
from 1 to about 100; R.sup.f is as defined in the various
embodiments herein; and m is an integer selected from 1 to about
3.
[0438] In each of the embodiments described above with R.sup.f, a
suitable radiophore may be prepared using the fluorine isotope
.sup.18F. Other useful positron-emitting isotopes may also be
employed, such as .sup.34Cl, .sup.45Ti, .sup.51Mn, .sup.61Cu,
.sup.63Zn, .sup.82Rb, .sup.68Ga .sup.66Ga, .sup.11C, .sup.13N,
.sup.15O, and .sup.18F. In one illustrative embodiment, the
radioisotope is selected from .sup.34Cl, .sup.64Cu, .sup.68Ga,
.sup.66Ga, or .sup.18F. In one aspect R.sup.f is .sup.18F, nitro,
or --N(CH.sub.3).sub.3.sup.+.
[0439] In another embodiment, the folate ligand portion of the
folate-imaging agent conjugate comprises a compound of the
formula:
##STR00059##
[0440] In yet another embodiment, the folate-imaging agent
conjugate has the formula:
##STR00060##
[0441] wherein:
[0442] X is an amino acid or a derivative thereof, and
[0443] Y is a dye that has a fluorescence excitation and emission
spectra in the near infrared range, and said compound maintains or
enhances the fluorescence of Y.
[0444] The amino acid X may be selected from the group consisting
of tyrosine, cysteine, lysine, a derivative of tyrosine, a
derivative of cysteine and a derivative of lysine. In a particular
embodiment, the amino acid X is tyrosine, and in another
embodiment, the amino acid X is a derivative of tyrosine selected
from the group consisting of:
##STR00061##
and racemic mixtures thereof.
[0445] In another embodiment, the dye Y may have the formula:
##STR00062##
[0446] wherein:
[0447] X' is independently selected from the group consisting of O,
S, N and C, and
[0448] R' is independently selected from the group consisting of
CH2 and CH2CH2. In some embodiments, the dye Y is selected from the
group consisting of LS288, IR800, SP054, S0121, KODAK IRD28, S2076,
S0456 and derivatives thereof.
[0449] In another embodiment, the folate-imaging agent conjugate
has the formula:
##STR00063##
wherein W, X, Y, Z each are H, Na.sup.+, K.sup.+ or
NH.sub.4.sup.+.
[0450] The folate ligand and the imaging agent in the
folate-imaging agent conjugates, may be linked in any chemically
relevant way. In one embodiment, the linker includes a chain of
atoms selected from C, N, O, S, Si, and P that covalently connects
the folate ligand to the imaging agent. The linker may have a wide
variety of lengths, such as in the range from about 2 to about 100
atoms. 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, 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 to form
divalent cyclic structures that form the linker, including cyclo
alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes,
and the like in the linker. The linker may be bivalent. In another
embodiment, there is no linker in the folate-imaging agent
conjugate, rather the folate ligand and the imaging agent are
directly linked.
[0451] In other embodiments of the methods and uses described
herein, pharmaceutically acceptable salts of the folate-imaging
agent conjugates described herein are contemplated.
Pharmaceutically acceptable salts of the folate-imaging agent
conjugates described herein include the acid addition and base
salts thereof.
[0452] Suitable acid addition salts are formed from acids which
form non-toxic salts. Illustrative examples include the acetate,
aspartate, benzoate, besylate, bicarbonate/carbonate,
bisulphate/sulphate, borate, camsylate, citrate, edisylate,
esylate, formate, fumarate, gluceptate, gluconate, glucuronate,
hexafluorophosphate, hibenzate, hydrochloride/chloride,
hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate,
malate, maleate, malonate, mesylate, methylsulphate, naphthylate,
2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate,
pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,
saccharate, stearate, succinate, tartrate, tosylate and
trifluoroacetate salts.
[0453] Suitable base salts of the conjugates described herein are
formed from bases which form non-toxic salts. Illustrative examples
include the arginine, benzathine, calcium, choline, diethylamine,
diolamine, glycine, lysine, magnesium, meglumine, olamine,
potassium, sodium, tromethamine and zinc salts. Hemi-salts of acids
and bases may also be formed, for example, hemi-sulphate and
hemi-calcium salts.
[0454] In one embodiment, the folate-imaging agent conjugates
described herein may be administered as a composition in
association with one or more pharmaceutically acceptable carriers.
The carriers can be excipients. 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 folate-imaging agent
conjugates 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.
[0455] In one illustrative aspect, a pharmaceutically acceptable
carrier can include any 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 the compositions for use in the
methods and uses described herein. Other possible carriers include,
for example, water, ethanol, polyethylene glycol, propylene glycol,
methylcellulose, or a suitable oil, and one or more emulsifying
agents and/or suspending agents. In another embodiment, the
pharmaceutically acceptable carrier is a liquid carrier and the
liquid carrier can be selected from the group consisting of saline,
glucose, alcohols, glycols, esters, amides, and a combination
thereof. In another embodiment, the folate-imaging agent conjugate
is in a sterile, pyrogen-free, aqueous solution.
[0456] In one embodiment, an aqueous suspension may contain the
active materials in admixture with appropriate excipients. Such
excipients are suspending agents, for example, sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents which may be a naturally-occurring phosphatide, for
example, lecithin; a condensation product of an alkylene oxide with
a fatty acid, for example, polyoxyethylene stearate; a condensation
product of ethylene oxide with a long chain aliphatic alcohol, for
example, heptadecaethyleneoxycetanol; a condensation product of
ethylene oxide with a partial ester derived from fatty acids and a
hexitol such as polyoxyethylene sorbitol monooleate; or a
condensation product of ethylene oxide with a partial ester derived
from fatty acids and hexitol anhydrides, for example,
polyoxyethylene sorbitan monooleate. The aqueous suspensions may
also contain one or more preservatives, for example, ascorbic acid,
ethyl, n-propyl, or p-hydroxybenzoate; or one or more coloring
agents.
[0457] Suitable emulsifying agents may be naturally-occurring gums,
for example, gum acacia or gum tragacanth; naturally-occurring
phosphatides, for example, soybean lecithin; and esters including
partial esters derived from fatty acids and hexitol anhydrides, for
example, sorbitan mono-oleate, and condensation products of the
said partial esters with ethylene oxide, for example,
polyoxyethylene sorbitan monooleate. In other embodiments, isotonic
agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride can be included in the composition. In
another embodiment, solubility enhancing agents may be used.
[0458] In one aspect, the folate-imaging agent conjugates can be
administered parenterally. Suitable routes for such parenteral
administration include intravenous, intraarterial, intraperitoneal,
intrathecal, epidural, intracerebroventricular, intraurethral,
intrasternal, intracranial, intramuscular and subcutaneous
delivery. Suitable means for parenteral administration include
needle (including microneedle) injectors, needle-free injectors and
infusion techniques.
[0459] 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 (e.g., an
aqueous solution).
[0460] The preparation of parenteral formulations under sterile
conditions may readily be accomplished using standard
pharmaceutical techniques well-known to those skilled in the art.
In one embodiment, the folate-imaging agent conjugate can be
present in the form of a reconstituted lyophilizate.
[0461] Any effective regimen for administering the folate-imaging
agent conjugate can be used. For example, the folate-imaging agent
conjugate can be administered as single doses, or can be divided
and administered as a multiple-dose regimen. The effective amount
to be administered to a patient is based on body surface area, and
mass. 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).
[0462] The folate-imaging agent conjugate can 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.
[0463] In another embodiment, the folate-imaging agent conjugate
can 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 folate-imaging agent
conjugate can 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.
[0464] The folate-imaging agent conjugates 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 folate-imaging agent conjugates 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.
[0465] It is appreciated that the folate-imaging agent conjugates
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 folate-imaging agent conjugates
described herein may exist in multiple crystalline or amorphous
forms before reconstitution and administration to the patient. In
another embodiment, the folate-imaging agent conjugate is provided
in a sterile container or package.
[0466] In another embodiment, the folate-imaging agent conjugates
having a radioactive imaging agent, such as .sup.99mTc-EC20, have a
radiochemical 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%. As
used herein, purity determinations may be based on weight
percentage, mole percentage, and the like.
[0467] In addition, purity determinations for the folate-imaging
agent conjugates may be based on the absence or substantial absence
of certain predetermined components, such as, but not limited to,
folic acid, oxidation products, disulfide components not containing
a folate ligand, and the like. In those instances, purity
measurements, including weight percentage and mole percentage
measurements, are related to the components of the solution
exclusive of the solvent. The folate-imaging agent conjugates may
have 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%.
[0468] The purity of the folate-imaging agent conjugates 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.
[0469] Any type of medical imaging procedure known in the art can
be used to detect the signal produced in the patient by the
folate-imaging agent conjugate. Examples of medical imaging
procedures include, but are not limited to, the use of radioactive
imaging agents in imaging procedures, the use of fluorescent
imaging agents or other types of dyes in medical imaging, positron
emission tomography (PET), magnetic resonance imaging, computed
tomography, scintigraphic imaging, SPECT, SPECT/CT, planar imaging,
optical imaging, ultrasound, and the like. The images can be
quantitated by any method known in the art for quantitation of
medical images, including but not limited to, densitometry,
quantitative PET imaging, and the like.
[0470] In one embodiment specific for .sup.99mTc-EC20, for the
imaging procedure the patient is injected with 0.5 mg of unlabeled
folic acid, followed within 1 to 3 minutes by a 1 to 2 mL injection
of 0.1 mg of EC20 labeled with 20 to 25 mCi of .sup.99mTc and
imaging is performed 1 to 2 hours later. In this embodiment, the
imaging methods can be selected from the group consisting of
planar, SPECT, and SPECT/CT imaging.
[0471] In any embodiment described herein, unlabeled folic acid can
be administered to the patient, and, in such embodiments, the
unlabeled folic acid can be administered to the patient prior to
administration of the folate-imaging agent conjugate, or within one
hour of administration of the folate-imaging agent conjugate.
[0472] In another embodiment, the methods and uses 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.
Example 1
Animal Models
[0473] All animal procedures were approved by the Purdue Animal
Care and Use Committee in accordance with guidelines from the
National Institutes of Health. Mice were maintained in a
temperature and humidity controlled room on a 12-h dark-light cycle
with food and water available ad libitum.
Example 2
Collagen-Induced Arthritis
[0474] Collagen-induced arthritis (CIA) was initiated using
established methods on 6-7 week old female DBA/1 mice (Jackson
Laboratories) maintained on folate-deficient diet (Harlan-Teklad).
Briefly, mice were immunized at the base of the tail with 100 .mu.g
bovine type II collagen emulsified in complete Freund's adjuvant
(Chondrex, Inc., Redmond, Wash., USA). Mice were then boosted 21
days later with a similar injection of 100 .mu.g bovine type II
collagen emulsified in incomplete Freund's adjuvant. After four
days, onset of arthritis was synchronized in all mice with an
intraperitoneal injection of 25 .mu.g lipopolysaccharide (LPS)
dissolved in saline. Three days later mice were distributed equally
across control and treatment groups (n=5). Healthy mice and disease
control mice received daily intraperitoneal injections of 100 .mu.L
saline. Diseased mice to be tested for response to therapy received
intraperitoneal injections of methotrexate (9 mg/kg, every 3 days),
dexamethasone (0.5 mg/kg, daily), etanercept (300 .mu.g, daily), or
abatacept (300 .mu.g, daily), or an oral gavage with naproxen (50
mg/kg, 5.times. week). Arthritis scores were assessed every other
day by researchers blinded to the various treatment groups, using
the following scoring system: 0=normal; 1=mild, but definite
redness and swelling of the ankle or wrist, or apparent redness and
swelling limited to individual digits, regardless of the number of
affected digits; 2=moderate redness and swelling of ankle or wrist;
3=severe redness and swelling of the entire paw including digits;
4=maximally inflamed limb with involvement of multiple joints. A
total score for each mouse was calculated by summing the scores for
each of the four paws, allowing a maximum possible score of 16 per
animal. Paw thickness was measured with calipers every other day
starting on the first day of treatment. On day 3 of treatment, mice
were anesthetized with isoflurane and imaged with .sup.99mTc-EC20
as described below. Mice were again imaged and then euthanized by
CO.sub.2 asphyxiation on day 11 of treatment.
Example 3
Ulcerative Colitis
[0475] Ulcerative colitis was induced as previously described in S.
Wirtz, C. Neufert, B. Weigmann, M. F. Neurath, Chemically induced
mouse models of intestinal inflammation. Nat Protoc. 2, 541-546
(2007), incorporated herein by reference. Seven week old Balb/c
mice (Harlan Laboratories) maintained on a folate-deficient diet
were administered 5% dextran sodium sulfate (DSS) in their drinking
water. Mice were divided into treatment groups (n=10 per group).
Healthy mice and disease control mice received 100 .mu.L saline
daily by oral gavage. Diseased mice were treated daily with
cimetidine (100 mg/kg) or sulphasalazine (150 mg/kg) by oral
gavage. Healthy mice were maintained on normal water and similarly
treated with saline. Disease symptoms were assessed daily and
quantitated by adding the scores from each of the following tests:
Weight loss: 0=no weight loss, 1=1-5% weight loss, 2=6-10% weight
loss, 3=11-15% weight loss, 4=>15% weight loss; Stool
appearance: 0=normal, 1=loose feces, 2=diarrhea; Hematochezia
(blood in stool): 0=no blood, 1=positive via guaiac paper,
2=visually bloody; Overall appearance: 0=normal, 1=ruffled
fur/altered gait, 2=lethargic, moribund. On day 4 of therapy, half
of each treatment group (n=5) received an intraperitoneal injection
of .sup.99mTc-EC20. After 4 hours, the injected mice were
euthanized and the bladder and kidneys were removed to reduce
background radiation associated with .sup.99mTc-EC20 undergoing
excretion. Mice were then imaged to assess uptake of
.sup.99mTc-EC20 in the colon, and the colons were removed and
measured with calipers to evaluate colon shortening as a measure of
disease severity. On day 8, the remaining mice were injected with
.sup.99mTc-EC20 and analyzed similarly.
Example 4
Atherosclerosis
[0476] Five week old male ApoE-/- mice were purchased from Jackson
Laboratories and placed on an adjusted calorie diet (42% from fat,
Harlan Laboratories). Healthy control mice (C57BL/6) were similarly
maintained on normal chow. Mice were divided into therapy groups
(n=5). Healthy mice and disease control mice received 100 .mu.L
saline daily by oral gavage. Diseased mice were treated daily with
valsartan (1 mg/kg), or fluvastatin (3 mg/kg) by oral gavage. After
three weeks of treatment, mice were imaged with 300 .mu.Ci
.sup.99mTc-EC20. Mice were again imaged with .sup.99mTc-EC20 after
12 weeks of therapy and then euthanized by CO.sub.2 asphyxiation.
Aortas were dissected and H&E and oil red 0 staining was
performed.
Example 5
Pulmonary Fibrosis
[0477] Pulmonary fibrosis was induced in mice as previously
described in B. B. Moore, C. M. Hogaboam, Murine models of
pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 294,
L152-160 (2008), incorporated herein by reference. Briefly, 6 week
old female C57BL/6 mice (Harlan Laboratories) maintained on a
folate-deficient diet were anaesthetized with isoflurane, and 50
.mu.L of bleomycin (2 U/kg body weight) dissolved in saline was
intratracheally instilled into each mouse. Healthy control mice
were similarly intratracheally instilled with 50 .mu.L saline. Mice
were then separated into treatment groups (n=5). Healthy mice and
disease control mice received daily intraperitoneal injections of
100 .mu.L saline. Diseased mice were injected every day
intraperitoneally with dexamethasone (0.5 mg/kg) or etanercept (300
.mu.g). Mice were imaged with .sup.99mTc-EC20 after 6 days of
treatment and again after 15 days of treatment. Mice were then
euthanized by CO.sub.2 asphyxiation and bronchoalveolar lavage
fluid was collected and cells were counted using a Beckman Coulter
Z.TM. Series COULTER COUNTER.RTM. Cell and Particle Counter. The
left lung was fixed in formalin and submitted to the Purdue
Histology & Phenotyping Laboratory for H&E staining and the
right lung was used for analysis of hydroxyproline content using a
hydroxyproline assay kit from Sigma-Aldrich (St. Louis, Mo.).
Example 6
[0478] Preparation of .sup.99mTc-EC20 and imaging of sites of
macrophage accumulation in inflamed mice .sup.99mTc-EC20 was
prepared as previously described in C. P. Leamon, M. A. Parker, I.
R. Vlahov, L. Xu, J. A. Reddy, M. Vetzel, N. Douglas, Synthesis and
Biological Evaluation of EC20: A New Folate-Derived,
.sup.99mTc-Based Radiopharmaceutical. Bioconjugate Chem. 13,
1200-1210 (2002), incorporated herein by reference. A preparation
method for .sup.99mTc-EC20 is also described in U.S. Pat. No.
7,128,893, incorporated herein by reference. Briefly, 1 mL of 15
mCi/mL sodium pertechnetate (Cardinal Health) was added to 0.1 mg
of EC20 (a gift from Endocyte, Inc.) and heated at 100.degree. C.
for 18 min. After diluting with saline, 100 .mu.L .sup.99mTc-EC20
(150 .mu.Ci (.about.75 nmol of EC20 per kilogram for CIA,
ulcerative colitis and pulmonary fibrosis mice) or 300 .mu.Ci
(.about.150 nmol of EC20 per kilogram for atherosclerosis)) was
injected intraperitoneally into the desired mice, and unbound
.sup.99mTc-EC20 was allowed to clear from tissues for 4 h. Mice
were then either anesthetized with 3% isoflurane (CIA,
atherosclerosis, and pulmonary fibrosis mice) or euthanized
(ulcerative colitis mice) prior to image acquisition using a Kodak
Image Station operated with Kodak molecular imaging software
(version 4.5; Carestream Molecular Imaging). Radioimages were
acquired for 2 minutes using a radioisotopic phosphor screen, no
illumination source, 4.times.4 binning, focal plane=5, FOV=160, and
f-stop=0. White light images were acquired for 0.175 seconds with
white light transillumination source, no binning, focal plane=5,
FOV=160, and f-stop=11.
Example 7
Imaging with .sup.99mTc-EC20 Predicts Response to Treatment in a
Murine Model of Rheumatoid Arthritis
[0479] Mice were induced to develop collagen-induced arthritis
(CIA, a well-established model of rheumatoid arthritis) and
subjected 28 days later to treatment with methotrexate,
dexamethasone or saline (disease control). Three days after
initiation of therapy, the mice were injected with 150 .mu.Ci of
.sup.99mTc-EC20 and subjected to their first planned radioimage
analysis. As seen in FIG. 1A, mice induced to develop CIA but not
treated with any anti-inflammatory drug exhibited the accumulation
of .sup.99mTc-EC20 in their inflamed appendages, confirming
involvement of folate receptor-positive activated macrophages in
the autoimmune disease. Inflamed mice injected with either
methotrexate or dexamethasone displayed markedly less
.sup.99mTc-EC20 in the affected appendages, despite revealing no
reduction in disease symptoms (arthritis score, paw swelling) at
this early time point (FIGS. 1 B & C). Moreover, after 11 days
of continuous therapy, when the treated mice had finally responded
to their respective therapies and joint inflammation had
substantially resolved (FIGS. 1 B & C), images of the same mice
(FIG. 7) confirmed the significantly reduced uptake of
.sup.99mTc-EC20 in the treated mice relative to disease control
mice. These data show that uptake of .sup.99mTc-EC20 in the
inflamed joints of CIA mice soon after initiation of therapy can be
used to predict an eventual response of the mice to both
methotrexate and dexamethasone.
[0480] To explore whether the ability of .sup.99mTc-EC20 to predict
response to methotrexate and dexamethasone might also extend to
biologic therapies, the above study was repeated, only etanercept
and abatacept were substituted for methotrexate and dexamethasone.
As seen in FIG. 2A, mice treated with the aforementioned biologics
also showed significantly less uptake of .sup.99mTc-EC20 on day 3
of therapy than mice in the disease control group, even though no
significant difference in clinical symptoms was again measurable at
this early time point (FIGS. 2 B & C). Moreover, analysis of
disease symptoms on day 11 of therapy revealed that the treated
mice did indeed eventually respond to etanercept and abatacept with
significantly reduced inflammation. Radioimages of the mice on day
11 (FIG. 8) demonstrated a similar .sup.99mTc-EC20 biodistribution
to the radioimages on day 3. These data show that images acquired
with .sup.99mTc-EC20 early in the course of therapy can also
predict the response of CIA mice to a biologic therapy.
[0481] In order to evaluate whether imaging with .sup.99mTc-EC20
might also be useful for identifying patients that will eventually
fail to respond to a particular therapy, CIA mice were treated with
naproxen, an anti-inflammatory drug (NSAID) used to treat
rheumatoid arthritis in humans, but previously found to have little
efficacy in the CIA model of arthritis in mice. Importantly,
.sup.99mTc-EC20 images on day 3 of therapy revealed no significant
difference in uptake between naproxen-treated and disease control
groups, showing that mice unable to respond to therapy also fail to
show a decrease in .sup.99mTc-EC20 accumulation (FIG. 3A).
Following 11 days of naproxen therapy, there was still no
significant difference in arthritis scores or paw thickness between
treated and disease control mice (FIGS. 3 B & C). These data
show that .sup.99mTc-EC20 images acquired before changes in disease
symptoms occur can accurately identify mice that will eventually
fail to respond to therapy.
Example 8
Imaging with .sup.99mTc-EC20 Predicts Response to Therapy in a
Murine Model of Ulcerative Colitis
[0482] Mice (n=10/group) were administered 5% dextran sulfate
sodium (DSS) in water to induce ulcerative colitis and then treated
daily with cimetidine, sulphasalazine or saline (disease control).
Then on day 4, half of the mice (n=5/group) were injected with 150
.mu.Ci .sup.99mTc-EC20 and euthanized in preparation for subsequent
imaging of their colons. As seen in FIG. 4A, cimetidine- and
sulphasalazine-treated mice showed significantly less
.sup.99mTc-EC20 uptake than saline-treated mice, even though there
was no significant difference in their clinical scores or colon
lengths at this early time point (FIGS. 4 B & C). More
importantly, after 8 days of continuous therapy, treated mice were
found to have significantly lower clinical scores and reduced colon
shortening when compared to disease control mice (FIGS. 4 B &
C). And as before, radioimages remained essentially unchanged from
those collected at the earlier time point (FIG. 9). Taken together,
these data show that imaging with .sup.99mTc-EC20 shortly after the
initiation of therapy can also predict response to treatment in a
murine model of ulcerative colitis.
Example 9
Imaging with .sup.99mTc-EC20 Predicts Response to Therapy in a
Murine Model of Atherosclerosis
[0483] The ability to predict response to therapy might be most
useful when applied to treatment of inflammatory/autoimmune
diseases that are either intrinsically difficult to monitor or slow
to respond to therapeutic intervention. Because atherosclerosis
suffers from both disadvantages due to its inaccessible location
and slow rate of progression, .sup.99mTc-EC20 was analyzed to
determine if it might be effective in predicting response to
therapy in a common murine model of heart disease. For this
purpose, five week old ApoE-/- mice were fed a high fat diet and
treated daily with valsartan or fluvastatin, both of which have
been shown to reduce plaque formation in murine models of
atherosclerosis. After 3 weeks of daily treatment, when no
morphological symptoms of heart disease could be detected, mice
were anesthetized and imaged with .sup.99mTc-EC20. As seen in FIG.
5A, ApoE-/- mice treated with either valsartan or fluvastatin
displayed significantly less .sup.99mTc-EC20 uptake in their chest
cavities than mice treated with saline. Moreover, after 12 weeks of
daily therapy, the valsartan- and fluvastatin-treated groups showed
significantly reduced aortic wall thickening than their
saline-treated counterparts (FIG. 5B). Radioimages on week 12
revealed a similar uptake pattern to images obtained on week 3
(FIG. 10). These data show that imaging with .sup.99mTc-EC20
shortly after the initiation of therapy can also predict response
to treatment in a murine model of atherosclerosis.
Example 10
Imaging with .sup.99mTc-EC20 Predicts Response to Treatment in a
Murine Model of Pulmonary Fibrosis
[0484] Because the folate receptor-positive activated macrophages
that accumulate in murine models of rheumatoid arthritis and
ulcerative colitis consist predominantly of classically activated
(M1) macrophages, .sup.99mTc-EC20 imaging was analyzed to determine
if it might also prove useful in predicting response to therapy in
an autoimmune disease mediated primarily by alternatively activated
(M2) macrophages. Since pulmonary fibrosis is thought to be
primarily driven by M2 macrophages, C57BL/6 mice were induced to
develop an acute form of lung fibrosis by intratracheal
instillation of bleomycin. Immediately following disease induction,
mice were treated daily with intraperitoneal injections of
dexamethasone, etanercept, or saline, and then imaged with
.sup.99mTc-EC20 six days later. As shown in FIG. 6A, dexamethasone-
and etanercept-treated mice displayed decreased accumulation of
.sup.99mTc-EC20 compared to their saline-treated disease controls.
Moreover, on day 15 of treatment, when the mice were again imaged
with .sup.99mTc-EC20 (FIG. 11), dexamethasone- and
etanercept-treated mice exhibited decreased total cell counts in
their bronchoalveolar lavage fluids, as well as reduced
hydroxyproline contents in their resected lung tissues compared to
saline-treated controls (FIGS. 6B & C). H&E analysis of the
lungs confirmed the reduced fibrosis in mice treated with
dexamethasone or etanercept (FIG. 6D). Taken together, these
results indicate that .sup.99mTC-EC20 imaging can successfully
predict response to treatment in an inflammatory disease mediated
by alternatively activated macrophages, such as for pulmonary
fibrosis.
Example 11
Treated Mice have Reduced Macrophage Accumulation in a Murine Model
of Ulcerative Colitis
[0485] Ulcerative colitis was induced as described. Seven week old
Balb/c mice (Harlan Laboratories) maintained on a folate-deficient
diet were administered 5% dextran sodium sulfate (DSS) in their
drinking water. Healthy control mice were maintained on normal
water. Mice were divided into treatment groups (n=3 per group).
Healthy mice and disease control mice received 100 .mu.L saline
daily by oral gavage. Diseased mice were treated daily with
cimetidine (100 mg/kg) or sulphasalazine (150 mg/kg) by oral
gavage. After 4 days of treatment, mice were euthanized and large
intestines were removed. Equal weights of the large intestines were
digested and made into single cell suspensions. Cells were included
with mouse macrophage marker F480-PE and analyzed by flow
cytometry. Treated mice were found to have reduced macrophage
accumulation compared to the disease control group (FIG. 12).
Example 12
Compound Preparation Method
##STR00064##
[0487] Preparation of Pte-L-Try-S0456 (the final compound as shown
above). Fmoc-Tyr(.sup.tBu)-Wang Resin was combined with a mixture
of piperidine, dichloromethane (DCM), and dimethylformamide (DMF)
in a solid phase peptide synthesis vessel. A solution of
N.sup.10-(trifluoroacetyl)pteroic acid,
(2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (HATU), Hunig's base (di-iso-propylethylamine)
and dimethylformamide (DMF) was added to the resin mixture. The
resin was washed alternately with dimethylformamide (DMF) and
isopropyl alcohol (IPA) and (DCM). The resin product was dried
under argon. The TFA-Pte-L-Tyr compound was cleaved from the resin
with TFA:H.sub.2O:TIPS (95:2.5:2.5). The cleaved product was dried
under vacuum conditions. The resulting TFA-Pte-L-Tyr solid was
suspended in water with 50456 fluorescent dye (CAS 162093-39-2).
The mixture was treated with aqueous sodium hydroxide (NaOH) and
heated to 100.degree. C. for 30 min. After the mixture was cooled
to room temperature it was added with stiffing to acetone to
precipitate the Pte-L-Try-S0456. The solid was washed with acetone
followed by drying under vacuum conditions. Further purification of
the solid product could be accomplished by dissolving the solid in
water, filtering the solution, and diluting the solution with
i-PrOH. The precipitated solid is collected by filtration, washed
sequentially with i-PrOH and acetone, and dried under vacuum
conditions.
Example 13
Predicting Response to Treatment in Mice with Arthritis
[0488] Collagen-induced arthritis (CIA) was initiated as described
above. The mice were treated with etanercept or dexamethasone. On
day 3, mice were given an intraperitoneal (i.p.) injection of 10
nmol OTL-38 dye. After 4 hours, mice were anesthetized with
isofluorane and imaged on the IVIS Lumina. On day 11, mice were
given an i.p. injection of 10 nmol OTL-38 dye. After 4 hours, mice
were euthanized and imaged on the IVIS Lumina. Results are shown in
FIGS. 13-19.
Example 14
Predicting Response to Treatment in Mice with Ulcerative
Colitis
[0489] Ulcerative colitis was induced in mice as described above.
On day 4, mice were given an intravenous (i.v.) injection of 10
nmol OTL-38 dye. Mice were euthanized after 2 hours. The bladder
was emptied and kidneys were removed. On day 10, mice were given an
i.v. injection of 10 nmol OTL dye. Mice were euthanized after 2
hours. The bladder was emptied and the kidneys were removed.
Results are shown in FIGS. 20-27.
Example 15
Predicting Response to Treatment in Mice with Atherosclerosis
[0490] Atherosclerosis was initiated as described above. The mice
were treated with valsartan or fluvastatin. On day 21, mice were
given an intraperitoneal (i.p.) injection of 10 nmol OTL-38 dye.
After 4 hours, mice were anesthetized with isofluorane and imaged.
Results are shown in FIG. 28.
Example 16
Predicting Response to Treatment in Mice with Pulmonary
Fibrosis
[0491] Pulmonary fibrosis was initiated as described above. The
mice were treated with etanercept or dexamethasone. On day 6, mice
were given an intraperitoneal (i.p.) injection of 10 nmol OTL-38
dye. After 4 hours, mice were anesthetized with isofluorane and
imaged. Results are shown in FIG. 29.
* * * * *