U.S. patent application number 16/682200 was filed with the patent office on 2020-05-21 for vitamin receptor drug delivery conjugates for treating inflammation.
The applicant listed for this patent is Endocyte, Inc.. Invention is credited to Paul Joseph Kleindl, Christopher Paul Leamon, Yingjuan June Lu, Hari Krishna R. Santhapuram, Iontcho Radoslavov Vlahov, Kevin Yu Wang, Fei You.
Application Number | 20200155555 16/682200 |
Document ID | / |
Family ID | 54354404 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200155555 |
Kind Code |
A1 |
Leamon; Christopher Paul ;
et al. |
May 21, 2020 |
VITAMIN RECEPTOR DRUG DELIVERY CONJUGATES FOR TREATING
INFLAMMATION
Abstract
Described herein are compositions, methods, compounds,
conjugates, and kits for use in targeted drug delivery using drug
delivery conjugates containing hydrophilic spacer linkers for use
in treating disease states caused by pathogenic cell populations,
such as inflammatory cells.
Inventors: |
Leamon; Christopher Paul;
(West Lafayette, IN) ; Vlahov; Iontcho Radoslavov;
(West Lafayette, IN) ; Lu; Yingjuan June; (West
Lafayette, IN) ; Wang; Kevin Yu; (Zionsville, IN)
; You; Fei; (West Lafayette, IN) ; Kleindl; Paul
Joseph; (Lebanon, IN) ; Santhapuram; Hari Krishna
R.; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Endocyte, Inc. |
West Lafayette |
IN |
US |
|
|
Family ID: |
54354404 |
Appl. No.: |
16/682200 |
Filed: |
November 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15811600 |
Nov 13, 2017 |
10500204 |
|
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16682200 |
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|
14671790 |
Mar 27, 2015 |
9877965 |
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|
15811600 |
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|
13518291 |
Jun 21, 2012 |
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PCT/US2010/061897 |
Dec 22, 2010 |
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14671790 |
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12666712 |
Dec 24, 2009 |
9138484 |
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PCT/US08/68093 |
Jun 25, 2008 |
|
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|
14671790 |
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61391230 |
Oct 8, 2010 |
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61386785 |
Sep 27, 2010 |
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61374830 |
Aug 18, 2010 |
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61351032 |
Jun 3, 2010 |
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61291103 |
Dec 30, 2009 |
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61289952 |
Dec 23, 2009 |
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61036186 |
Mar 13, 2008 |
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60946092 |
Jun 25, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/551 20170801;
C07K 7/06 20130101; A61P 9/10 20180101; A61K 47/545 20170801; A61K
31/519 20130101; A61K 31/436 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 47/55 20060101 A61K047/55; A61K 47/54 20060101
A61K047/54; C07K 7/06 20060101 C07K007/06; A61K 31/436 20060101
A61K031/436 |
Claims
1.-2. (canceled)
3. A process for preparing a conjugate of the formula B-L-A
comprising coupling a compound of the formula ##STR00065## wherein
AA is an amino acid; R.sup.1 is cysteine side chain, .beta.-methyl
cysteine side chain, or .beta.,.beta.-dimethyl cysteine side chain;
n is an integer from 0 to 100; and -(AA).sub.n- comprises at least
one fragment of the formula ##STR00066## wherein R is selected from
the group consisting of H, alkyl, cycloalkyl, and arylalkyl; m is
an integer from 1 to about 3; n1 is an integer from 1 to about 6; p
is an integer from 1 to about 5; and (*) indicates the point of
attachment to the rest of the compound, with a compound of the
formula ##STR00067## wherein A is an anti-inflammatory agent, in
the presence of a base.
4. The process of claim 3, wherein the base is NaHCO.sub.3 or
DIPEA.
5. The process of claim 3 or 4, wherein the step of coupling is
carried out in the presence of DMSO.
6. The process of any one of claims 3 to 5, wherein the step of
coupling is carried out in the presence of a buffer.
7. The process of claim 6, wherein the buffer is a phosphate
buffer.
8. The process of any one of claims 3 to 7, wherein the compound of
the formula ##STR00068## is of the formula ##STR00069##
9. The process of any one of claims 3 to 7, wherein the compound of
the formula ##STR00070## is of the formula ##STR00071##
10. The process of any one of claims 3 to 9, wherein n is from 3 to
7.
11. The process of any one of claims 3 to 10, wherein the compound
of the formula ##STR00072## is of the formula ##STR00073##
##STR00074## ##STR00075##
12. The process of any one of claims 3 to 10, further comprising
(a) contacting H-cysteine-(4-methoxytrityl)-2-chlorotrityl-resin in
the presence of DIPEA and PyBOP with a compound of the formula
##STR00076## (b) contacting the product of step (a) with
Fmoc-Glu(OtBu)-OH; (c) contacting the product of step (b) with a
compound of the formula ##STR00077## (d) contacting the product of
step (c) with Fmoc-Glu-OtBu; (e) contacting the product of step (d)
with N.sup.10TFA-Pteroic Acid; (f) contacting the product of step
(e) with 2% hydrazine in DMF; and (g) contacting the product of
step (f) with a mixture of TFA, H.sub.2O, triisopropylsilane, and
ethanedithiol.
13. The process of any one of claims 3 to 10, further comprising
(a) contacting H-cysteine-(4-methoxytrityl)-2-chlorotrityl-resin in
the presence of DIPEA and PyBOP with a compound of the formula
##STR00078## (b) contacting the product of step (a) with
Fmoc-Glu(OtBu)-OH; (c) contacting the product of step (b) with a
compound of the formula ##STR00079## (d) contacting the product of
step (c) with Fmoc-Glu(OtBu)-OH; (e) contacting the product of step
(d) with a compound of the formula ##STR00080## (f) contacting the
product of step (e) with Fmoc-Glu-OtBu; (g) contacting the product
of step (f) with N.sup.10TFA-Pteroic Acid; (h) contacting the
product of step (g) with 2% hydrazine in DMF; and (i) contacting
the product of step (h) with a mixture of TFA, H.sub.2O,
triisopropylsilane, and ethanedithiol.
14. The process of any one of claims 3 to 10, further comprising
(a) contacting H-cysteine-(4-methoxytrityl)-2-chlorotrityl-resin in
the presence of DIPEA and PyBOP with a compound of the formula
##STR00081## (b) contacting the product of step (a) with
Fmoc-Glu(OtBu)-OH; (c) contacting the product of step (b) with a
compound of the formula ##STR00082## (d) contacting the product of
step (c) with a compound of the formula ##STR00083## (e) contacting
the product of step (d) with Fmoc-Glu(OtBu)-OH; (f) contacting the
product of step (e) with a compound of the formula ##STR00084## (g)
contacting the product of step (f) with Fmoc-Glu-OtBu; (h)
contacting the product of step (g) with N.sup.10TFA-Pteroic Acid;
(i) contacting the product of step (i) with 2% hydrazine in DMF;
and (j) contacting the product of step (i) with a mixture of TFA,
H.sub.2O, triisopropylsilane, and ethanedithiol.
15. The process of any one of claims 3 to 10, further comprising
(a) contacting H-cysteine-(4-methoxytrityl)-2-chlorotrityl-resin in
the presence of DIPEA and PyBOP with a compound of the formula
##STR00085## (b) contacting the product of step (a) with
Fmoc-Glu(OtBu)-OH; (c) contacting the product of step (b) with a
compound of the formula ##STR00086## (d) contacting the product of
step (c) with Fmoc-Arg(Pbf)-OH; (e) contacting the product of step
(d) with a compound of the formula ##STR00087## (f) contacting the
product of step (e) with Fmoc-Glu(OtBu)-OH; (g) contacting the
product of step (f) with a compound of the formula ##STR00088## (h)
contacting the product of step (g) with Fmoc-Glu-OtBu; (i)
contacting the product of step (h) with N.sup.10TFA-Pteroic Acid;
(j) contacting the product of step (i) with 2% hydrazine in DMF;
and (k) contacting the product of step (j) with a mixture of TFA,
H.sub.2O, triisopropylsilane, and ethanedithiol.
16. A compound of the formula ##STR00089## ##STR00090##
##STR00091## or a salt thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/811,600, filed Nov. 13, 2017, which is a continuation of
U.S. application Ser. No. 14/671,790, filed Mar. 27, 2015, which is
continuation-in part of U.S. application Ser. No. 13/518,291, filed
Jun. 21, 2012, which is a U.S. national application under 35 U.S.C.
.sctn. 371(b) of International Application Serial No.
PCT/US2010/061897 filed Dec. 22, 2010, and claims priority under 35
USC .sctn. 119(e) to U.S. Provisional Application Ser. No.
61/289,952, filed on Dec. 23, 2009, U.S. Provisional Application
Ser. No. 61/291,103 filed on Dec. 30, 2009, U.S. Provisional
Application Ser. No. 61/351,032, filed on Jun. 3, 2010, U.S.
Provisional Application Ser. No. 61/374,830, filed on Aug. 18,
2010, U.S. Provisional Application Ser. No. 61/386,785, filed on
Sep. 27, 2010, and U.S. Provisional Application Ser. No.
61/391,230, filed on Oct. 8, 2010, and Ser. No. 14/671,790, filed
Mar. 27, 2015, continuation-in-part of U.S. application Ser. No.
12/666,712, filed Dec. 24, 2009, which is a U.S. national
application under 35 U.S.C. .sctn. 371(b) of International
Application Serial No. PCT/US2008/068093 filed Jun. 25, 2008, and
claims priority under 35 USC .sctn. 119(e) to U.S. Provisional
Application Ser. No. 61/036,186, filed on Mar. 13, 2008, and U.S.
Provisional Application Ser. No. 60/946,092 filed on Jun. 25, 2007,
the entire disclosures of each of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to compositions and methods
for use in targeted drug delivery. More particularly, the invention
is directed to cell-surface receptor binding drug delivery
conjugates containing hydrophilic spacer linkers for use in
treating disease states caused by pathogenic cell populations and
to methods and pharmaceutical compositions that use and include
such conjugates.
BACKGROUND
[0003] The mammalian immune system provides a means for the
recognition and elimination of foreign pathogens. While the immune
system normally provides a line of defense against foreign
pathogens, there are many instances where the immune response
itself is involved in the progression of disease. Exemplary of
diseases caused or worsened by the host's own immune response are
autoimmune diseases and other diseases in which the immune response
contributes to pathogenesis. For example, macrophages are generally
the first cells to encounter foreign pathogens, and accordingly,
they play an important role in the immune response, but activated
macrophages can also contribute to the pathophysiology of disease
in some instances.
[0004] The folate receptor is a 38 KD GPI-anchored protein that
binds the vitamin folic acid with high affinity (<1 nM).
Following receptor binding, rapid endocytosis delivers the vitamin
into the cell, where it is unloaded in an endosomal compartment at
low pH. Importantly, covalent conjugation of small molecules,
proteins, and even liposomes to folic acid does not block the
vitamin's ability to bind the folate receptor, and therefore,
folate-drug conjugates can readily be delivered to and can enter
cells by receptor-mediated endocytosis.
[0005] Because most cells use an unrelated reduced folate carrier
to acquire the necessary folic acid, expression of the folate
receptor is restricted to a few cell types. With the exception of
kidney, choroid plexus, and placenta, normal tissues express low or
nondetectable levels of the folate receptor. It has been reported
that the folate receptor .beta., the nonepithelial isoform of the
folate receptor, is expressed on activated (but not resting)
synovial macrophages. Thus, folate receptors are expressed on a
subset of macrophages (i.e., activated macrophages). Folate
receptors of the .beta. isoform are also found on activated
monocytes.
[0006] Accordingly, the present invention relates to the
development of vitamin-targeted therapeutics, such as
folate-targeted therapeutics, to treat inflammation. The folate
conjugates described herein can be used to treat inflammatory
diseases by targeting inflammatory cells that overexpress the
folate receptor.
SUMMARY OF THE INVENTION
[0007] It has been discovered that therapeutic agents, diagnostic
agents, and imaging agents may be conjugated to other compounds to
control or alter their behavior, biodistribution, metabolism,
and/or clearance in vivo. In one illustrative embodiment of the
invention, conjugates of compounds are described that include a
hydrophilic spacer linker. In one aspect, conjugates of compounds
are described that include both a hydrophilic spacer linker and a
targeting ligand. Illustrative of such conjugates are compounds of
the following formula described herein
B-L-A
wherein B is a receptor binding ligand that binds to a target cell
receptor, L is a linker that comprises one or more hydrophilic
spacer linkers, and A is a therapeutic agent (e.g. a drug) that is
desirably delivered to the cell.
[0008] In one variation, the linker L does not include a releasable
linker. In another variation, the linker L includes a releasable
linker. In another embodiment, at least one of the hydrophilic
spacer linkers is formed from or includes at least one
carbohydrate. In one variation, the carbohydrate forms part of the
linker chain connecting B and A. In another variation, the
carbohydrate forms part of a side chain attached to the linker
chain connecting B and A. In one variation, the linker is a
polyvalent linker. In another variation, the linker is a bivalent
linker.
[0009] It is appreciated that in each of the above embodiments,
more than one receptor binding ligand B may be attached to the
linkers described herein. It is further appreciated that more than
one therapeutic agent A may be attached to the linkers described
herein. Such multi-ligand and/or multi-drug conjugates are also
described herein, where the linker (L) comprises a hydrophilic
spacer linker.
[0010] In another embodiment, compounds are described herein that
have reduced uptake by the liver and are less likely to be cleared
by the liver. In one aspect, such compounds are preferentially
cleared by the renal processes as compared to hepatic
processes.
[0011] The therapeutic agent or therapeutic agents A include
therapeutic drugs and any other compound that is desirably or
advantageously delivered to a cell by targeting a cell receptor.
Illustrative drugs include cytotoxic drugs, anti-inflammatory
agents, and the like.
[0012] In the embodiments of compounds, compositions, and methods
described herein, the cells that may be targeted with the
therapeutic agents A include cells that cause inflammation, such as
activated monocytes, activated macrophages, and other inflammatory
cells. The targeting of the cell is accomplished by the appropriate
selection of a receptor binding ligand B. It is appreciated that
selective or specific targeting of a cell in vivo may be
accomplished by selecting a receptor that is preferentially
expressed or overexpressed by the target cell. Illustratively, the
target cell preferentially expresses or overexpresses a vitamin
receptor, such as a folate receptor.
[0013] In another embodiment, the conjugates described herein are
included in pharmaceutical compositions in amounts effective to
treat disease states associated with pathogenic populations of
cells, such as cells associated with inflammation.
[0014] In another embodiment, the conjugates described herein, and
pharmaceutical compositions containing them are used in methods for
treating diseases and disease states associated with pathogenic
populations of cells, such as cells associated with
inflammation.
[0015] In another embodiment, a method for treating a patient with
an inflammatory disease, the method comprising the step of
administering to the patient a composition comprising a drug
delivery conjugate of the formula
BL(A.sup.1)(A.sup.2).sub.m
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0016] m is 0 or 1;
[0017] B is a folate;
[0018] L is a linker that comprises one or more hydrophilic spacer
linkers;
[0019] A.sup.1 is an antifolate; and
[0020] A.sup.2 has the formula
##STR00001##
wherein
[0021] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0022] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0023] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0024] In another embodiment, a pharmaceutical composition
comprising a drug delivery conjugate of the formula
BL(A.sup.1)(A.sup.2).sub.m
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0025] m, B, L, A.sup.1, A.sup.2, Y.sup.A, R.sup.A, R.sup.B,
R.sup.C, and R.sup.D are as described herein.
[0026] In any of the preceding embodiments, the antifolate can be
aminopterin, or an analog, derivative, or conjugate thereof.
[0027] In another embodiment, a method for treating a patient with
an inflammatory disease, the method comprising the step of
administering to the patient a composition comprising a drug
delivery conjugate of the formula
B-L-A.sup.3
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0028] B is a folate;
[0029] L is a linker that comprises one or more hydrophilic spacer
linkers; and
[0030] A.sup.3 has the formula
##STR00002##
wherein
[0031] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0032] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0033] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0034] In another embodiment, a pharmaceutical composition
comprising a drug delivery conjugate of the formula
B-L-A.sup.3
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0035] m, B, L, A.sup.3, Y.sup.A, R.sup.A, R.sup.B, R.sup.C, and
R.sup.D are as described herein.
[0036] In another embodiment, a kit comprising a sterile vial, the
composition of any one of the preceding embodiments, and
instructions for use describing use of the composition for treating
a patient with an inflammatory disease is described.
[0037] In another embodiment, is described a kit comprising a
sterile vial, a composition comprising the compound as a
lyophilized solid of any one of the preceding embodiments, and
instructions describing use of the composition for treating a
patient with an inflammatory disease, wherein the vial is an amber
glass vial with a rubber stopper and an aluminum tear-off seal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1. Measurement of DHFR activity in the lysate of
RAW264.7 cells after no treatment, treatment with EC0746 (1 hour
treatment followed by 24 hours in treatment-free medium), treatment
with EC0746 plus excess folic acid (EC0746/FA, 1 hour treatment
followed by 24 hours in treatment-free medium), treatment with
aminopterin (AMT, 24 hour treatment), treatment with methotrexate
(MXT, 24 hour treatment), and treatment with folic acid (FA, 10
.mu.M).
[0039] FIG. 2A--Viability of RAW264.7 cells, measured using the XTT
assay, treated with EC0746 (EC.sub.50 0.33 nM, 2 hour treatment
followed by 72 hours in treatment-free medium) and treated with
EC0746 and excess folic acid (EC0746/FA, 2 hour treatment followed
by 72 hours in treatment-free medium). FIG. 2B--Inhibition of
LPS-stimulated TNF-.alpha. production in RAW264.7 cells treated
with EC0746 (EC.sub.50 1.43 nM, 2 hour treatment followed by 72
hours in treatment-free medium) and treated with EC0746 and excess
folic acid (EC0746/FA, 2 hour treatment followed by 72 hours in
treatment-free medium).
[0040] FIG. 3. Inhibition of LPS-stimulated cytokine production
(LPS (5 ug/mL), IFN-.gamma. (100 ng/mL) for 24 hours) in
thioglycolate-elicited macrophages. Compounds at 100 nM with
100.times.s FA (2 hour treatment followed by 72 hours in fresh
medium, unstimulated cell; stimulated cells without treatment
(LPS/IFN-.gamma.), treatment with excess folic acid, treatment with
EC0746 (EC0746), treatment with EC0746 and excess folic acid
(EC0746/FA), treatment with methotrexate, and treatment with
aminopterin. Relative folate receptor binding affinities (folic
acid defined as 1.00) EC0746, 0.50; aminopterin, 0.004; and
methotrexate, 0.018.
[0041] FIG. 4A--Arthritis scores for a) untreated animals with
adjuvant induced arthritis and b) animals treated with EC0746 (500
nmol/kg). FIG. 4B--Percentage change in body weight measured for a)
untreated animals with adjuvant induced arthritis and b) animals
treated using EC0746 (500 nmol/kg).
[0042] FIG. 5A--Paw weights measured after the end of the treatment
period for healthy animals, untreated animals with adjuvant induced
arthritis and b) animals treated with EC0746 (500 nmol/kg). FIG.
5B--Spleen weights measured after the end of the treatment period
for healthy animals, untreated animals with adjuvant induced
arthritis and b) animals treated with EC0746 (500 nmol/kg).
[0043] FIG. 6A--Photographs of the hind paws of a healthy control
animal, an untreated animal with adjuvant induced arthritis, and an
animal treated with EC0746 (500 nmol/kg); FIG. 6B--X-rays of the
hind paws of a healthy control animal, an untreated animal with
adjuvant induced arthritis, and an animal treated with EC0746 (500
nmol/kg).
[0044] FIG. 7A--Average arthritis scores measured during the
treatment period for a) untreated animals with adjuvant induced
arthritis, b) animals treated bi-weekly with EC0746, 300 nmole/kg;
c) with methotrexate, 300 nmole/kg; and d) healthy untreated
animals. Treatments administered on the days indicated with the
arrows. FIG. 7B--Percentage weight change measured during the
treatment period for a) untreated animals with adjuvant induced
arthritis, b) animals treated bi-weekly with EC0746, 300 nmole/kg;
c) animals treated bi-weekly with methotrexate, 300 nmole/kg; and
d) healthy untreated animals.
[0045] FIG. 8A--Paw weights measured at the end of the treatment
period for healthy untreated animals, untreated arthritic animals,
animals treated bi-weekly with EC0746 (300 nmole/kg), and animals
treated bi-weekly with methotrexate (300 nmole/kg). FIG. 8B--Spleen
weights measured at the end of the treatment period for healthy
untreated animals, untreated arthritic animals, animals treated
bi-weekly with EC0746 (300 nmole/kg), and animals treated bi-weekly
with methotrexate (300 nmole/kg).
[0046] FIG. 9. Viability of RAW264.7 macrophage cells treated in
media containing the test compound or compounds for 2 hours at the
concentration shown in the graph, followed by 72 hours in fresh
medium without the test compound(s); a) EC0746; b) EC0746+excess
folic acid; c) EC0808 (a D-aminopterin diastereoisomer of EC0746);
and EC0808+excess folic acid.
[0047] FIG. 10. Arthritis score measured for a) untreated arthritic
animals; b) animals treated bi-weekly with methotrexate (500
nmole/kg), and c) animals treated bi-weekly with EC0808 (500
nmole/kg).
[0048] FIG. 11A--Arthritis score measured for a) untreated
arthritic animals; b) animals treated bi-weekly with EC0746 (500
nmole/kg), c) animals treated bi-weekly with EC0746 (500
nmole/kg)+300-fold excess of Re-EC0589), and d) healthy untreated
animals. FIG. 11B--The percentage body weight change measured for
the same animals used to obtain the arthritis scores in FIG. 11A.
Percentage weight change measured for a) untreated arthritic
animals; b) animals treated bi-weekly with EC0746 (500 nmole/kg),
c) animals treated bi-weekly with EC0746 (500 nmole/kg)+300-fold
excess of Re-EC0589), and d) healthy untreated animals.
[0049] FIG. 11C--Arthritis score measured for a) healthy animals,
b) untreated arthritic animals; c) animals treated bi-weekly with
EC0746 (250 nmole/kg), d) animals treated bi-weekly with EC0746
(250 nmole/kg)+500-fold excess of EC0923), and e) animals treated
biweekly with EC0923 alone.
[0050] FIG. 12A--Arthritis score measured for a) untreated
arthritic animals; b) animals treated bi-weekly with methotrexate
(500 nmole/kg), c) animals treated bi-weekly with methotrexate (500
nmole/kg)+300-fold excess of Re-EC20), and d) arthritic animals
treated with Re-EC20. FIG. 12B--The percentage body weight change
measured for the same animals used to obtain the arthritis scores
in FIG. 12A. The percentage weight change measured for a) Untreated
arthritic animals; b) animals treated bi-weekly with methotrexate
(500 nmole/kg), c) animals treated bi-weekly with methotrexate (500
nmole/kg)+300-fold excess of Re-EC20), and d) arthritic animals
treated with Re-EC20.
[0051] FIG. 13A--The average arthritis score measured using a
two-dosage level treatment schedule, a 500 nmole/kg dose was
administered on days 10 and 15 post-arthritis induction and a 100
nmole/kg dose was administered on day 11-14 and 16-19. a) untreated
arthritic animals (collagen-induced arthritis, rats induced
treatment with 500 .mu.g Type II collagen/Freund's complete
adjuvant at day 0 followed by 500 .mu.g Type II collagen/Freund's
incomplete adjuvant at day 7, b) animals treated with EC0746, and
c) animals treated with methotrexate. FIG. 13B--The percentage
weight change measured for the animals used to obtain the arthritis
scores shown in FIG. 13A. a) Untreated arthritic animals, b)
animals treated with EC0746, and c) animals treated with
methotrexate.
[0052] FIG. 14A--Plasma concentrations measured after a single
subcutaneous dose of EC0746 (500 nmole/kg). a) EC0746, b) EC0470
(aminopterin gamma-hydrazide), and c) aminopterin. FIG. 14B--The
C.sub.max for EC0746 of 321 nmole/L is reached at 30 minutes post
injection. The C.sub.max for free drug (aminopterin+aminopterin
hydrazide) of 34 nmole/L is reached at 60 minutes. C.sub.max for
free drug is 9.6% of the total dose. The Area Under Curve value for
EC0746 is 32.5 nmole-min/mL, the Area Under Curve value for free
drug is 7.3 nmole-min/mL (18% of the total).
[0053] FIG. 15. Plasma concentrations measured after a single
subcutaneous dose of aminopterin (500 nmole/kg). The maximum plasma
concentration was measured at 30 minutes post administration.
[0054] FIG. 16A--Percentage body weight change measured for animals
after subcutaneous administration of the indicated dose of
aminopterin biweekly for 2 weeks a) control, 0 nmole/kg, b) 100
nmole/kg, and c) 50 nmole/kg. FIG. 16B--Percentage body weight
change measured for animals after subcutaneous administration of
the indicated dose of treatment compound biweekly for 2 weeks a)
control, no treatment, b) 100 nmole/kg aminopterin, and c) 50
nmole/kg aminopterin, d) 500 nmole/kg EC0746, or e) 2000 nmole/kg
EC0746.
[0055] FIG. 17A--Viability of RAW264.7 cells, measured using the
XTT assay, treated with EC0932 (2 hour treatment followed by 72
hours in treatment-free medium) and treated with EC0932 and excess
folic acid (EC0932/FA, 2 hour treatment followed by 72 hours in
treatment-free medium). FIG. 17B--Inhibition of LPS-stimulated
TNF-.alpha. production in RAW264.7 cells treated with EC0932 (2
hour treatment followed by 72 hours in treatment-free medium) and
treated with EC0932 and excess folic acid (EC0932/FA, 2 hour
treatment followed by 72 hours in treatment-free medium).
[0056] FIG. 18A--A Western blot showing inhibition of mTOR
signaling in LPS/IFN-.gamma. stimulated RAW264.7 cells. Comparison
between unstimulated cells, untreated stimulated cells, stimulated
cells treated with an mTOR inhibitor (everolimus), stimulated cells
treated with an antifolate (aminopterin), stimulated cells treated
with EC0932, stimulated cells treated with EC0932 plus excess
competitor (EC0823), and stimulated cells treated excess competitor
(EC0823) alone. Treatments with 100 nM compound in cell medium for
2 hours followed by fresh untreated medium. FIG. 18B--measurement
treatment response on pRPS6.
[0057] FIG. 19A--Arthritis score measured for a) untreated animals
with induced arthritis, b) healthy untreated animals, c) animals
treated with EC0932 (250 nmole/kg, biweekly on indicated days), and
d) animals treated with EC0932 and a 500-fold excess of EC0923 (250
nmole/kg, biweekly, on indicated days). FIG. 19B--Percentage change
in body weight measured for the animals used to obtain the
arthritis scores shown in FIG. 19A; a) untreated animals with
induced arthritis, b) healthy untreated animals, c) animals treated
with EC0932 (250 nmole/kg, biweekly on indicated days), and d)
animals treated with EC0932 and a 500-fold excess of EC0923 (250
nmole/kg, biweekly, on indicated days).
[0058] FIG. 20A--Paw weights measured after the completion of the
treatment and observation period used to obtain the data in FIG. 19
for a) untreated animals with induced arthritis, b) healthy
untreated animals, c) animals treated with EC0932 (250 nmole/kg,
biweekly on indicated days), and d) animals treated with EC0932 and
a 500-fold excess of EC0923 (250 nmole/kg, biweekly, on indicated
days). FIG. 20B--Spleen weights measured after the completion of
the treatment and observation period used to obtain the data in
FIG. 20A for a) untreated animals with induced arthritis, b)
healthy untreated animals, c) animals treated with EC0932 (250
nmole/kg, biweekly on indicated days), and d) animals treated with
EC0932 and a 500-fold excess of EC0923 (250 nmole/kg, biweekly, on
indicated days).
[0059] FIG. 21A--Arthritis score measured for a) untreated animals
with induced arthritis, b) healthy untreated animals, c) animals
treated with EC0894 (500 nmole/kg, biweekly), and d) animals
treated with EC0828 (250 nmole/kg, biweekly). FIG. 21B--Percentage
change in body weight measured for the animals used to obtain the
arthritis scores shown in FIG. 21A; a) untreated animals with
induced arthritis, b) healthy untreated animals, c) animals treated
with EC0894 (500 nmole/kg, biweekly), and d) animals treated with
EC0828 (250 nmole/kg, biweekly).
[0060] FIG. 22A--Paw weight measured after completion of the
treatment and observation period used to obtain the data in FIG. 21
for a) untreated animals with induced arthritis, b) healthy
untreated animals, c) animals treated with EC0894 (500 nmole/kg,
biweekly), and d) animals treated with EC0828 (250 nmole/kg,
biweekly). FIG. 22B--Spleen weight measured after completion of the
treatment and observation period used to obtain the data in FIG. 21
for a) untreated animals with induced arthritis, b) healthy
untreated animals, c) animals treated with EC0894 (500 nmole/kg,
biweekly), and d) animals treated with EC0828 (250 nmole/kg,
biweekly).
[0061] FIG. 23A--Arthritis score measured for a) untreated animals
with induced arthritis, b) healthy untreated animals, c) animals
treated with EC0565 (500 nmole/kg, biweekly), and d) animals
treated with EC0828 (250 nmole/kg, biweekly). FIG. 23B--Percentage
change in body weight measured for the animals used to obtain the
arthritis scores shown in FIG. 23A; a) untreated animals with
induced arthritis, b) healthy untreated animals, c) animals treated
with EC0894 (500 nmole/kg, biweekly), and d) animals treated with
EC0828 (250 nmole/kg, biweekly).
[0062] FIG. 24A--Paw weight measured after completion of the
treatment and observation period used to obtain the data in FIG. 23
for a) untreated animals with induced arthritis, b) healthy
untreated animals, c) animals treated with EC0565 (500 nmole/kg,
biweekly), and d) animals treated with EC0828 (250 nmole/kg,
biweekly). FIG. 24B--Spleen weight measured after completion of the
treatment and observation period used to obtain the data in FIG. 23
for a) untreated animals with induced arthritis, b) healthy
untreated animals, c) animals treated with EC0894 (500 nmole/kg,
biweekly), and d) animals treated with EC0828 (250 nmole/kg,
biweekly).
[0063] FIGS. 25A-D--Relative affinities of EC0746 (FIG. 25A),
aminopterin (AMT) (FIG. 25B), methotrexate (MTX) (FIG. 25C), and
EC0932 (FIG. 25D), compared to folic acid (FA), set as 1, to folate
receptors (FR-t) of KB cells. FIG. 25E--In vitro inhibition of DHFR
in FR-positive RAW264.7 cells: Untreated control; EC0746 (2 h pulse
of 100 nm, followed by 22 h "chase" in drug free medium); EC0746
with excess FA (2 h pulse of 100 nm EC0746 with 100-fold excess
folic acid (folate competition), followed by 22 h "chase"); AMT (2
h pulse of 100 nm, followed by 22 h "chase" in drug free medium);
MTX (2 h pulse of 100 nm, followed by 22 h "chase" in drug free
medium); FA alone (2 h pulse of 10 m, followed by 22 h "chase" in
drug free medium).
[0064] FIG. 25F--Relative affinities of EC0746, aminopterin (AMT),
and methotrexate (MTX) compared to folic acid (FA), set as 1, to
folate receptors (FR-P) of CHO cells. a. folic acid (relative
affinity set to 1.0), b. EC0746 (relative affinity 0.270), c.
aminopterin (relative affinity 0.004), and d. methotrexate
(relative affinity 0.005).
[0065] FIG. 26A, B, C, D, E, F, G, H, I. Anti-proliferative Effect
on RAW264.7 cells: FIG. 26A--Viability of RAW264.7 cells, measured
using the XTT assay, LPS (100 ng/mL) added at 4 h before end of
incubation to stimulate cytokine production, treated with EC0746
(EC.sub.50 about 0.3 nM, 2 hour treatment followed by 70 h "chase"
in drug-free medium) and treated with EC0746 and excess folic acid
(EC0746/FA, 2 hour treatment followed by 70 h "chase" in drug-free
medium). FIG. 26B--TNF-.alpha. production from cells treated as in
part a, upon LPS exposure (ED.sub.50 about 1.6 nM). Flow cytometric
analysis (FACS) with propidium iodide (PI) staining of the cell
cycle of the cells of part a for Untreated (FIG. 26C),
EC0746-treated (FIG. 26D) and EC0746/FA-treated cells (FIG. 26E).
FIG. 26F--Cell cycle distribution for Untreated, EC0746 treated and
EC0746/FA treated cells. FIG. 26G--Western blot analysis of
cell-cycle distribution (d) and PCNA expression (e) on whole cell
lysates using a PCNA-specific monoclonal antibody for Untreated
(UTC), EC0746-treated (EC0746-FA) and EC0746/FA (EC0746+FA)-treated
cells.
[0066] FIG. 26H, I--Anti-proliferative Effect on RAW264.7 cells:
FIG. 26H--Viability of RAW264.7 cells, measured using the XTT
assay, LPS (100 ng/mL) added at 4 h before end of incubation to
stimulate cytokine production, 2 hour treatment followed by 70 h
"chase" in drug-free medium). Comparison of EC0746, EC0746 with
excess folic acid (EC0746/FA), aminopterin (AMT), and methotrexate
(MTX).
[0067] FIG. 26I--TNF-.alpha. production from cells treated as in
part a upon LPS exposure. Comparison of EC0746, EC0746 with excess
folic acid (EC0746/FA), aminopterin (AMT), and methotrexate
(MTX).
[0068] FIG. 27A, B, C. EC0746 Modulation of Cytokine Responses in
Thioglycollate-elicited Macrophages (TG-macs). FIG. 27A, B--Rat
cytokine antibody array and plotted results for
cytokines/chemokines (FIG. 27C) for rat TG-macs untreated (LPS) or
with indicated treatment of 100 nM of treatment for 2 h plus a 70 h
chase, and with addition at 24 h prior to end of incubation of LPS
(5 .mu.g/mL) and IFN-.gamma. (100 ng/mL) for EC0746, EC0746 plus
100-fold excess FA (EC0746/FA), folic acid alone (FA), aminopterin
(AMT), and methotrexatre (MTX), respectively, as to IL-10, IL-Ira,
IL-10, MIP-I.alpha., TNF-.alpha., VEGF, CINC-2a/b, CINC-3, sICAM,
LIX, L-selectin, and MIG.
[0069] FIG. 28A, B, C. Amelioration of Systemic Inflammation in
Rats with Adjuvant Induced Arthritis (AIA). FIG. 28A--Arthritis
score with time in days since first treatment; FIG. 28B--% Change
in body weight with time in days since first treatment; and FIG.
28C--% Increase in weight of Paws and Spleen for Healthy, Arthritic
(onset), EC0746-treated (onset), Arthritic (established) and
EC0746-treated (established) AIA rats.
[0070] FIG. 29 A, B, C, D, E, F. Anti-arthritic activity in Rats
with Adjuvant Induced Arthritis (AIA). FIG. 29A--Arthritis score
with time in days since first treatment; FIG. 29B--% Change in body
weight with time in days since first treatment; FIG. 29C--%
Increase in weight of Paws; FIG. 29D--% Increase in weight of
Spleen; FIG. 29E--radiograph of hind paw; and FIG.
29F--Radiographic score for Healthy (where indicated), Arthritic
Control (untreated), and animals treated with EC0746,
EC0746+Competitor, Competitor alone, methotrexate (MTX), or Enbrel
(etanercept) in AIA rats, with EC0923 as the folate-containing
competitor.
[0071] FIG. 30 A, B, C, D. Histology of Rats with Adjuvant Induced
Arthritis (AIA). FIG. 30A--Histology scores for Inflammation,
Pannus, Cartlige damage and Bone Resorption; FIG. 30B--Sum
histology scores; FIG. 30C--Mean paw thickness (.mu.m) and FIG.
30D--images from paws for Healthy (where indicated), Arthritic
Control (untreated), and animals treated with EC0746,
EC0746+Competitor, Competitor alone, methotrexate (MTX), or Enbrel
in AIA rats, with EC0923 as the folate-containing competitor.
[0072] FIG. 31A, B, C. Anti-arthritic activity in Rats with
Adjuvant Induced Arthritis (AIA). FIG. 31A--Arthritis score with
time in days since first treatment; FIG. 31B--% Change in body
weight with time in days since arthritis induction; FIG. 31C--%
Increase in weight of Paws and Spleen for Healthy (where
indicated), Arthritic Control (untreated), and animals treated with
methotrexate (MTX) or MTX+Competitor in AIA rats, with EC0923 as
the folate-containing competitor.
[0073] FIG. 32A, B. Effects of potential EC0746 Metabolites on
RAW264.7 cells. The effects of the potential EC0746 metabolites
aminopterin (AMT) and AMT hydrazide (EC0470) in 72 h incubations of
RAW264.7 macrophages are shown for: FIG. 32A--Cell proliferation in
the XTT assay, and FIG. 32B--LPS-stimulated TNF-.alpha.
production.
[0074] FIG. 33A, B, C. Pharmacokinetics of EC0746 and potential
metabolites aminopterin (AMT) and AMT hydrazide (EC0470) in Rats.
FIG. 33A--Plasma concentrations (nmol/L) of EC0746, AMT and AMT
hydrazide following single subcutaneous EC0746 (500 nmol/kg)
administration. FIG. 33B--Plasma concentrations (nmol/L) of AMT
following single subcutaneous AMT (500 nmol/kg) administration.
FIG. 33C--Pharmacokinetic analysis of the results of FIG. 33A,
B.
[0075] FIG. 34. An animal model for autoimmune disease uveitis.
Rats were immunized with a bovine S antigen peptide emulsified with
Freund's incomplete adjuvant containing M. Tuberculosi and boosted
with pertussis toxin.
[0076] FIG. 35. Uveitis total scores (both eyes) for animals
treated with 500 nmol/kg EC0746 every other day starting on day 7
after EAU induction (open circles) or from untreated animals
(closed circles).
[0077] FIG. 36A, B, C. Representative photographs of rat eyes were
taken on day 15. FIG. 36A--A photograph of an eye from an untreated
animal with experimental autoimmune uveitis (EAU). FIG. 36B--A
photograph of an eye from an animal with EAU treated with EC0746
every other day starting on day 7 after EAU induction. FIG. 36C--A
photograph of an eye from a healthy rat.
[0078] FIG. 37. The effect of EC0746 treatment on protein levels in
the aqueous humor. The protein levels (mg/mL) at day 19 in the
aqueous humor in the anterior portion of the eye are shown for the
left and right eye of each tested animal. Animals 1-5 were
untreated after induction of the EAU. Animals 6-9 were treated with
EC0746 every other day starting on day 7 after EAU induction. On
the far right of the chart, the total of the protein levels in the
aqueous humor samples pooled from both eyes of an untreated, healty
animal.
[0079] FIG. 38A shows that EC0565 induces inhibition of RPS6 in
RAW264.7 cells (1 h pulse/6 h chase), where UTC=Control (untreated
cells); EC0565=(100 nM) and EC0565+x.s. EC17=treatment plus an
excess amount of a non-cytotoxic folate conjugate; FIG. 38B shows
that EC0565 induces inhibition of RPS6 in TG-elicited macrophages
(1 h pulse/6 h chase), in a dose dependent manner, where
UTC=Control (untreated cells); EC0565=treatment (10 nmol, 30 nmol,
100 nmol); EC0565+x.s. folate=treatment (10 nmol, 30 nmol, 100
nmol) plus an excess of a folic acid conjugate (100 .mu.mole);
FAC=treatment with folic acid and 0 nmol of EC0565; and
Everolimus=treatment with unconjugated everolimus (10 nmol, 100
nmol); FIG. 38C shows that EC0565 induces inhibition of RPS6 in
arthitic macrophages (1 h pulse/6 h chase), in a dose dependent
manner, where UTC=Control (untreated cells); EC0565=treatment (1
nmol, 10 nmol and 30 nmol); EC0565+excess folate=treatment (1 nmol,
10 nmol and 30 nmol) plus an excess of a folic acid (100 .mu.mole);
and FAC=treatment with folic acid and 0 nmol of EC0565.
[0080] FIG. 39 compares the arthritis score of animals treated with
biweekly injections of 500 nmol/kg of EC0565 (d) and biweekly
injections of 500 nmol/kg of unconjugated everolimus (c) with
healthy controls (a) and untreated animals (b).
[0081] FIG. 40 shows the percentage weight change observed for the
same animals used to generate the data shown in FIG. 39. (a)
Untreated animals, (b) treated with biweekly injections of 500
nmol/kg of EC0565, (c) untreated healthy control animals, and (d)
treated with biweekly injections of 500 nmol/kg of unconjugated
everolimus.
[0082] FIG. 41 shows the paw weight data (localized disease) for
the animals at the end of the treatment period for each of the
treatment groups described in FIGS. 39 and 40.
[0083] FIG. 42 shows the spleen weight data (systemic disease) for
the animals at the end of the treatment period for each of the
treatment groups described in FIGS. 39 and 40.
[0084] FIG. 43 shows the radiographic analysis of soft tissue and
bone damage in the hind paws of animals treated with EC0565 (500
nmol/kg), Everolimus (500 nmol/kg), methotrexate (MTX, 190
nmol/kg), untreated animals with adjuvant induced arthritis, and
untreated, healthy control animals.
[0085] FIG. 44A compares the arthritis score of animals treated
with biweekly injections of 500 nmol/kg of EC0565 (d) and biweekly
injections of 500 nmol/kg of unconjugated everolimus (c) with
healthy controls (b) and untreated animals (a). FIG. 44B shows the
percentage weight change observed for the the treatment groups
shown in FIG. 44A. FIG. 44C shows the paw weight data (localized
disease) for the animals at the end of the treatment period for
each of the treatment groups described in FIG. 44A, B. FIG. 44D
shows the spleen weight data (systemic disease) for the animals at
the end of the treatment period for each of the treatment groups
described in FIG. 44A, B.
[0086] FIG. 45 shows that EC0565 induces dose-responsive inhibition
of the production of pRPS6 and p70S6K in KB cells (1 h pulse/4 h
chase) using a 30 min camera exposure, where C=Control (untreated
cells); FAC=Folic acid control (100 .mu.M).
[0087] FIG. 46A. Arthritis scores for AIA rats. a) untreated
animals; b) healthy animals; c) EC0565, subcutaneously (s.c.), 500
nmol/kg, tiw; d) everolimus, oral, 500 nmol/kg, tiw; e) etanercept,
s.c. 10 mg/kg, q3d; and e) methotrexate (MTX), oral, 250 nmol/kg,
biw).
[0088] FIG. 46B. Weight change for AIA rats. a) untreated animals;
b) healthy animals; c) EC0565, subcutaneously (s.c.), 500 nmol/kg,
tiw; d) everolimus, oral, 500 nmol/kg, tiw; e) etanercept, s.c. 10
mg/kg, q3d; and e) methotrexate (MTX), oral, 250 nmol/kg, biw).
[0089] FIG. 46C. Paw Weights (a measure of swelling) for AIA rats.
healthy animals; untreated controls; EC0565, subcutaneously (s.c.),
500 nmol/kg, tiw; everolimus, oral, 500 nmol/kg, tiw; etanercept,
s.c. 10 mg/kg, q3d; and methotrexate (MTX), oral, 250 nmol/kg,
biw).
[0090] FIG. 46D. Spleen Weights for AIA rats. Healthy animals;
untreated controls; EC0565, subcutaneously (s.c.), 500 nmol/kg,
tiw; everolimus, oral, 500 nmol/kg, tiw; Enbrel (etanercept), s.c.
10 mg/kg, q3d; and methotrexate (MTX), oral, 250 nmol/kg, biw).
[0091] FIG. 47. Radiographic analysis of hind paws of AIA rats.
Healthy animals; untreated controls; EC0565, subcutaneously (s.c.),
500 nmol/kg, tiw; everolimus, oral, 500 nmol/kg, tiw; Enbrel
(etanercept), s.c. 10 mg/kg, q3d; and methotrexate (MTX), oral, 250
nmol/kg, biw.
[0092] FIG. 48A--Histological study of AIA rats. Scores for
inflammation, pannus formation, cartilage damage, and bone
resorption are shown for untreated animals (untreated control);
everolimus, oral, 500 nmol/kg, tiw; methotrexate (MTX), oral, 250
nmol/kg, biw; EC0565, subcutaneously (s.c.), 500 nmol/kg, tiw; and
etanercept, s.c. 10 mg/kg, q3d. FIG. 48B--The sum of the scores
shown in panel a for each treatment. FIG. 48C--The measured paw
thickness for each treatment shown in FIG. 48A.
[0093] FIG. 49 shows representative photomicrographs (16.times.) of
the ankle closest to the mean summed score for each treatment
group.
[0094] FIG. 50A shows the amount of paw edema for healthy or AIA
rats. Healthy rats, no induces arthritis; arthritis (untreated AIA
rats); treated with EC0565 100 nmol/kg/dose, twice/week; 500
nmol/kg/dose, twice/week; 1000 nmol/kg, twice/week; and 1000
nmol/kg/dose, once/week. FIG. 50B shows the change in spleen weight
of for healthy or AIA rats. Healthy rats, no induces arthritis;
arthritis (untreated AIA rats); treated with EC0565 100
nmol/kg/dose, twice/week; 500 nmol/kg/dose, twice/week; 1000
nmol/kg, twice/week; and 1000 nmol/kg/dose, once/week.
[0095] FIG. 51A shows the average arthritis score for treated rats
with collagen-induced arthritis (CIA). a) untreated CIA animals; b)
ECO565, 1000 nmol/kg/dose, tiw; and c) everolimus, 1000
nmol/kg/dose, tiw.
[0096] FIG. 51B shows the average weight change for animals in
treat as in FIG. 51A. a) untreated CIA animals; b) ECO565, 1000
nmol/kg/dose, tiw; and c) everolimus, 1000 nmol/kg/dose, tiw.
[0097] FIG. 52A shows the plasma concentration of EC0565 and
everolimus over time after a 2 mmol/kg intravenous dose of EC0565.
FIG. 52B shows the plasma concentration of EC0565 and everolimus
over time after a 2 mmol/kg subcutaneous dose of EC0565. FIG. 52C
shows a comparison of the plasma concentration of EC0565 given
subcutaneously or intravenously.
[0098] FIG. 53A shows the effect on Proliferating Cell Nuclear
Antigen (PCNA) in synchronized FR-positive murine macrophage-like
RAW264.7 cells treated with media as measured by Western blot
analysis on whole cell lysates using a monoclonal antibody specific
for PCNA, a) EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM); b) EC0565
(1 nM, 10 nM, 100 nM, and 1000 nM) in the presence of XS EC17 (a
folate receptor binding competitor); c) EC17 alone (1 nM, 10 nM,
100 nM, and 1000 nM); and d) everolimus (1 nM, 10 nM, 100 nM, and
1000 nM).
[0099] FIG. 53B shows the pixel density measurements for the images
shown in FIG. 53A a) EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM); b)
EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM) in the presence of XS
EC17 (a folate receptor binding competitor); c) EC17 alone (1 nM,
10 nM, 100 nM, and 1000 nM); and d) everolimus (1 nM, 10 nM, 100
nM, and 1000 nM).
[0100] FIG. 53C shows a plot of percent control of PCNA data shown
in FIG. 53B for EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM); EC0565
(1 nM, 10 nM, 100 nM, and 1000 nM) in the presence of XS EC17 and
for everolimus (1 nM, 10 nM, 100 nM, and 1000 nM).
DETAILED DESCRIPTION
[0101] Drug delivery conjugates are described herein consisting of
a receptor binding ligand (B), a linker (L) comprising one or more
hydrophilic spacer linkers, and a therapeutic agent (A), e.g. a
drug, that is desirably delivered to a cell. The receptor binding
ligand (B) is covalently attached to the linker (L), and the
therapeutic agent (A), or an analog or derivative thereof, is also
covalently attached to the linker (L). It is to be understood that
the therapeutic agent (A) includes analogs and derivatives thereof
that are attached to the linker (L). The linker (L) comprises one
or more spacer linkers and/or releasable linkers, and combinations
thereof, in any order. In one variation, releasable linkers, and
optional spacer linkers are covalently bonded to each other to form
the linker. In another variation, a releasable linker is directly
attached to the therapeutic agent (A), or analog or derivative
thereof. In another variation, a releasable linker is directly
attached to the receptor binding ligand (B). In another variation,
either or both the receptor binding ligand (B) and the therapeutic
agent (A), or analog or derivative thereof, is attached to a
releasable linker through one or more spacer linkers. In another
variation, each of the receptor binding ligand (B) and the
therapeutic agent (A), or analog or derivative thereof, is attached
to a releasable linker, each of which may be directly attached to
each other, or covalently attached through one or more spacer
linkers.
[0102] From the foregoing, it should be appreciated that the
arrangement of the receptor binding ligand (B), and the therapeutic
agent (A), or analog or derivative thereof, and the various
releasable and optional spacer linkers may be varied widely. In one
aspect, the receptor binding ligand (B), and the therapeutic agent
(A), or analog or derivative thereof, and the various releasable
and optional spacer linkers are attached to each other through
heteroatoms, such as nitrogen, oxygen, sulfur, phosphorus, silicon,
and the like. In variations, the heteroatoms, excluding oxygen, may
be in various states of oxidation, such as N(OH), S(O), S(O).sub.2,
P(O), P(O).sub.2, P(O).sub.3, and the like. In other variation, the
heteroatoms may be grouped to form divalent radicals, such as for
example hydroxylamines, hydrazines, hydrazones, sulfonates,
phosphinates, phosphonates, and the like, including radicals of the
formulae --(NHR.sup.1NHR.sup.2)--, --SO--, --(SO.sub.2)--, and
--N(R.sup.3)O--, wherein R.sup.1, R.sup.2, and R.sup.3 are each
independently selected from hydrogen, alkyl, aryl, arylalkyl,
substituted aryl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, and alkoxyalkyl. In one variation, the linker (L) is a
polyvalent linker. In another variation, more than one receptor
binding ligand (B) is attached to the polyvalent linker. In another
variation, more than one therapeutic agent (A) is attached to the
polyvalent linker. In another variation, more than one receptor
binding ligand (B) and more than one therapeutic agent (A) is
attached to the polyvalent linker.
[0103] In one embodiment, the receptor binding ligand (B) is a
vitamin receptor binding ligand such as a vitamin, or an analog or
a derivative thereof, capable of binding to vitamin receptors. In
another embodiment, the receptor binding ligand (B) is a vitamin,
or analog or derivative thereof, attached to a releasable linker
which is attached to the drug through a linker (L) that is formed
from one or more spacer linkers and/or releasable linkers and/or
hydrophilic spacer linkers. In one variation, both the therapeutic
agent (A) and the vitamin, or analog or derivative thereof, can
each be attached to spacer linkers, where the spacer linkers are
attached to each other through one or more releasable linkers. In
addition, both the therapeutic agent (A) and the vitamin, or analog
or derivative thereof, can each be attached to one or more
releasable linkers, where the releasable linkers are attached to
each other or through a spacer linker. Each of these radicals may
be connected through existing or additional heteroatoms on the
receptor binding ligand (B), therapeutic agent (A), or releasable,
hydrophilic spacer, or additional spacer linker.
[0104] The binding site for the receptor binding ligand (B) can
include receptors for any binding ligand (B), or a derivative or
analog thereof, capable of specifically binding to a receptor
wherein the receptor or other protein is uniquely expressed,
overexpressed, or preferentially expressed by a population of
pathogenic cells. A surface-presented protein uniquely expressed,
overexpressed, or preferentially expressed by the pathogenic cells
is typically a receptor that is either not present or present at
lower concentrations on non-pathogenic cells providing a means for
selective elimination of the pathogenic cells. The drug delivery
conjugates may be capable of high affinity binding to receptors on
activated macrophages, monocytes, or other inflammatory cells. The
high affinity binding can be inherent to the binding ligand or the
binding affinity can be enhanced by the use of a chemically
modified ligand (e.g., an analog or a derivative of a vitamin).
[0105] The drug delivery conjugates described herein can be formed
from, for example, a wide variety of vitamins or receptor-binding
vitamin analogs/derivatives, linkers, and drugs. The drug delivery
conjugates described herein are capable of selectively targeting a
population of pathogenic cells in the host animal due to
preferential expression of a receptor for the binding ligand, such
as a vitamin, accessible for ligand binding, on the pathogenic
cells. Illustrative vitamin moieties that can be used as the
receptor binding ligand (B) include camitine, inositol, lipoic
acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic
acid, riboflavin, thiamine, biotin, vitamin B.sub.12, other water
soluble vitamins, the B vitamins, and the lipid soluble vitamins A,
D, E and K. These vitamins, and their receptor-binding analogs and
derivatives, constitute an illustrative receptor binding ligand (B)
that can be coupled with the therapeutic agent (A) drug by a linker
(L) to form a drug delivery conjugate as described herein.
[0106] The term vitamin is understood to include vitamin analogs
and/or derivatives, unless otherwise indicated. Illustratively,
pteroic acid, which is a derivative of folate, biotin analogs such
as biocytin, biotin sulfoxide, oxybiotin and other biotin
receptor-binding compounds, and the like, are considered to be
vitamins, vitamin analogs, and vitamin derivatives. It should be
appreciated that vitamin analogs or derivatives as described herein
refer to vitamins that incorporate an heteroatom through which the
vitamin analog or derivative is covalently bound to the linker
(L).
[0107] Illustrative vitamin moieties include folic acid, biotin,
riboflavin, thiamine, vitamin B.sub.12, and receptor-binding
analogs and derivatives of these vitamin molecules, and other
related vitamin receptor binding molecules.
[0108] In another embodiment, the cell receptor is a folate
receptor, and the receptor binding ligand (B) is a folate receptor
binding ligand. In another embodiment, B is a folate, such as folic
acid, or an analog or derivative of folic acid that binds to folic
acid receptors. It is to be understood as used herein, that the
term folate is used both individually and collectively to refer to
folic acid itself, and/or to such analogs and derivatives of folic
acid that are capable of binding to folate receptors.
[0109] Illustrative embodiments of folate analogs and/or
derivatives include 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. The dideaza analogs include, for example,
1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of
folate. Other folates useful as complex forming ligands include the
folate receptor-binding analogs aminopterin, amethopterin
(methotrexate), N.sup.10-methylfolate, 2-deamino-hydroxyfolate,
deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed folates, reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein.
[0110] Additional analogs of folic acid that bind to folic acid
receptors are described in U.S. Patent Application Publication Nos.
2005/0227985 and 2004/0242582, the disclosures of which are
incorporated herein by reference. Illustratively, such folate
analogs have the general formula:
##STR00003##
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;
[0111] 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--;
[0112] C.sup.1 and C.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(CH.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;
[0113] 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,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0114] 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; and p, r,
s, and t are each independently either 0 or 1.
[0115] As used herein, it is to be understood that the term folate
refers both individually to folic acid used in forming a drug
delivery conjugate, or alternatively to a folate analog or
derivative thereof that is capable of binding to folate
receptors.
[0116] In one aspect of such folate analogs, when s is 1, t is 0,
and when s is 0, t is 1. In another aspect of such folate analogs,
r is 1, and C.sup.2 of the folate analog is covalently linked to a
naturally occurring amino acid at its alpha-amino group through an
amide bond. Illustrative amino acids include aspartic acid,
glutamic acid, lysine, cysteine, and the like.
[0117] The vitamin can be a folate which includes a nitrogen, and
in this embodiment, the spacer linkers can be alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
wherein each of the spacer linkers is optionally substituted with a
substituent X.sup.1, and the spacer linker is bonded to the folate
nitrogen to form an imide or an alkylamide.
[0118] In the various embodiments described herein, the
substituents X.sup.1 can be alkyl, hydroxyalkyl, amino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, carboxy, carboxyalkyl, guanidinoalkyl, R.sup.4-carbonyl,
R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0119] Illustrative embodiments of vitamin analogs and/or
derivatives also include analogs and derivatives of biotin such as
biocytin, biotin sulfoxide, oxybiotin and other biotin
receptor-binding compounds, and the like. It is appreciated that
analogs and derivatives of the other vitamins described herein are
also contemplated herein. In one embodiment, vitamins that can be
used as the receptor binding ligand (B) in the drug delivery
conjugates described herein include those that bind to vitamin
receptors expressed specifically on activated macrophages or
activated monocytes, such as the folate receptor, which binds
folate, or an analog or derivative thereof as described herein.
[0120] The linker L includes one or more hydrophilic spacer
linkers. Illustrative hydrophilic linkers are described in
WO2009/002993 and U.S. patent application Ser. No. 12/660,712, the
disclosure of which is incorporated by reference herein in its
entirety. In addition, other optional spacer linkers and/or
releasable linkers may be included in L. It is appreciated that
additional spacer linkers may be included when predetermined
lengths are selected for separating receptor binding ligand (B)
from therapeutic agent (A). It is also appreciated that in certain
configurations, releasable linkers may be included. For example, as
described herein in one embodiment, the drug delivery conjugates
may be used to deliver therapeutic agents (A) (e.g. drugs) for
treating inflammation. In such embodiments, it is appreciated that
once delivered, the therapeutic agent (A) is desirably released
from the conjugate. For example, in the configuration where the
receptor binding ligand (B) is folate, or an analog or derivative
thereof, the conjugate may bind to a folate receptor. Once bound,
the conjugate often undergoes the process of endocytosis, and the
conjugate is delivered to the interior of the cell. Cellular
mechanisms may biologically degrade the conjugate to release the
drug "payload" and release the folate compound.
[0121] Accordingly, in other aspects, the conjugates B-L-A
described herein also include the following general formulae:
B-L.sub.S-L.sub.H-A
B-L.sub.H-L.sub.S-A
B-L.sub.S-L.sub.H-L.sub.S-A
B-L.sub.R-L.sub.H-A
B-L.sub.H-L.sub.R-A
B-L.sub.R-L.sub.H-L.sub.R-A
B-L.sub.S-L.sub.R-L.sub.H-A
B-L.sub.R-L.sub.H-L.sub.S-A
B-L.sub.R-L.sub.S-L.sub.H-L.sub.R-A
B-L.sub.H-L.sub.S-L.sub.H-L.sub.R-A
where B, L, and A are as described herein, and L.sub.R is a
releasable linker section, L.sub.S is a spacer linker section, and
L.sub.H is a hydrophilic linker section of linker L. It is to be
understood that the foregoing formulae are merely illustrative, and
that other arrangements of the hydrophilic spacer linker sections,
releasable linker sections, and spacer linker sections are to be
included herein. In addition, it is to be understood that
additional conjugates are contemplated that include a plurality
hydrophilic spacer linkers, and/or a plurality of releasable
linkers, and/or a plurality of spacer linkers.
[0122] It is appreciated that the arrangement and/or orientation of
the various hydrophilic linkers may be in a linear or branched
fashion, or both. For example, the hydrophilic linkers may form the
backbone of the linker (L) forming the conjugate between the folate
and the drug (i.e. therapeutic agent (A)). Alternatively, the
hydrophilic portion of the linker (L) may be pendant to or attached
to the backbone of the chain of atoms connecting the receptor
binding ligand B to the therapeutic agent A. In this latter
arrangement, the hydrophilic portion may be proximal or distal to
the backbone chain of atoms.
[0123] In another embodiment, the linker (L) is more or less
linear, and the hydrophilic groups are arranged largely in a series
to form a chain-like linker in the conjugate. Said another way, the
hydrophilic groups form some or all of the backbone of the linker
(L) in this linear embodiment.
[0124] In another embodiment, the linker (L) is branched with
hydrophilic groups. In this branched embodiment, the hydrophilic
groups may be proximal to the backbone or distal to the backbone.
In each of these arrangements, the linker (L) is more spherical or
cylindrical in shape. In one variation, the linker (L) is shaped
like a bottle-brush. In one aspect, the backbone of the linker (L)
is formed by a linear series of amides, and the hydrophilic portion
of the linker (L) is formed by a parallel arrangement of branching
side chains, such as by connecting monosaccharides, sulfonates, and
the like, and derivatives and analogs thereof.
[0125] It is understood that the linker (L) may be neutral or
ionizable under certain conditions, such as physiological
conditions encountered in vivo. For ionizable linkers, under the
selected conditions, the linker (L) may deprotonate to form a
negative ion, or alternatively become protonated to form a positive
ion. It is appreciated that more than one deprotonation or
protonation event may occur. In addition, it is understood that the
same linker (L) may deprotonate and protonate to form inner salts
or zwitterionic compounds.
[0126] In another embodiment, the hydrophilic spacer linkers are
neutral, i.e. under physiological conditions, the linkers do not
significantly protonate nor deprotonate. In another embodiment, the
hydrophilic spacer linkers may be protonated to carry one or more
positive charges. It is understood that the protonation capability
is condition dependent. In one aspect, the conditions are
physiological conditions, and the linker (L) is protonated in vivo.
In another embodiment, the hydrophilic spacer linkers include both
regions that are neutral and regions that may be protonated to
carry one or more positive charges. In another embodiment, the
hydrophilic spacer linkers include both regions that may be
deprotonated to carry one or more negative charges and regions that
may be protonated to carry one or more positive charges. It is
understood that in this latter embodiment that zwitterions or inner
salts may be formed.
[0127] In one aspect, the regions of the linkers (L) that may be
deprotonated to carry a negative charge include carboxylic acids,
such as aspartic acid, glutamic acid, and longer chain carboxylic
acid groups, and sulfuric acid esters, such as alkyl esters of
sulfuric acid. In another aspect, the regions of the linkers (L)
that may be protonated to carry a positive charge include amino
groups, such as polyaminoalkylenes including ethylene diamines,
propylene diamines, butylene diamines and the like, and/or
heterocycles including pyrollidines, piperidines, piperazines, and
other amino groups, each of which is optionally substituted. In
another embodiment, the regions of the hydrophilic spacer linkers
that are neutral include poly hydroxyl groups, such as sugars,
carbohydrates, saccharides, inositols, and the like, and/or
polyether groups, such as polyoxyalkylene groups including
polyoxyethylene, polyoxypropylene, and the like.
[0128] In one embodiment, the hydrophilic spacer linkers described
herein are formed primarily from carbon, hydrogen, and oxygen, and
have a carbon/oxygen ratio of about 3:1 or less, or of about 2:1 or
less. In one aspect, the hydrophilic linkers described herein
include a plurality of ether functional groups. In another aspect,
the hydrophilic linkers described herein include a plurality of
hydroxyl functional groups. Illustrative fragments that may be used
to form such linkers include polyhydroxyl compounds such as
carbohydrates, polyether compounds such as polyethylene glycol
(PEG) units, and acid groups such as carboxyl and alkyl sulfuric
acids. In one variation, oligoamide spacers, and the like may also
be included in the linker (L).
Illustrative carbohydrate spacers include saccharopeptides as
described herein that include both a peptide feature and sugar
feature; glucuronides, which may be incorporated via [2+3] Huisgen
cyclization, also known as click chemistry; .beta.-alkyl
glycosides, such as of 2-deoxyhexapyranoses (2-deoxyglucose,
2-deoxyglucuronide, and the like), and .beta.-alkyl
mannopyranosides.
[0129] In another illustrative embodiment, the hydrophilic spacer
linkers described herein include a plurality of hydroxyl functional
groups, such as linkers (L) that incorporate monosaccharides,
oligosaccharides, polysaccharides, and the like. It is to be
understood that the polyhydroxyl containing spacer linkers
comprises a plurality of --(CROH)-- groups, where R is hydrogen or
alkyl.
[0130] In another embodiment, the hydrophilic spacer linkers
include one or more of the following fragments:
##STR00004## ##STR00005## ##STR00006##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0131] In another embodiment, the hydrophilic spacer linkers
described herein are formed primarily from carbon, hydrogen, and
nitrogen, and have a carbon/nitrogen ratio of about 3:1 or less, or
of about 2:1 or less. In one aspect, the hydrophilic linkers
described herein include a plurality of amino functional
groups.
[0132] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0133] In each of the foregoing illustrative examples of linkers L,
there are also included in some cases additional spacer linkers
L.sub.S, and/or additional releasable linkers L.sub.R. Those spacer
linker and releasable linkers also may include asymmetric carbon
atoms. It is to be further understood that the stereochemical
configurations shown herein are merely illustrative, and other
stereochemical configurations are contemplated. It is to be further
understood that in the foregoing embodiments, open positions, such
as (*) atoms are locations for attachment of the receptor binding
ligand (B) or the therapeutic agent (A) to be delivered. In
addition, it is to be understood that such attachment of either or
both of B and A may be direct or through an intervening linker (L).
Intervening linkers include other spacer linkers and/or releasable
linkers. Illustrative additional spacer linkers and releasable
linkers that are included in the conjugate described herein are
described in U.S. Pat. No. 7,601,332, the disclosure of which is
incorporated herein by reference.
[0134] In one embodiment, the hydrophilic spacer linker comprises
one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises at least three carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more carbohydrate
containing or polyhydroxyl group containing linkers, and one or
more aspartic acids. In another embodiment, the hydrophilic spacer
linker comprises one or more carbohydrate containing or
polyhydroxyl group containing linkers, and one or more glutamic
acids. In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers, one or more glutamic acids, one or more
aspartic acids, and one or more beta amino alanines. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0135] In another series of variations, in each of the foregoing
embodiments, the hydrophilic spacer linker also includes at least
one arginine.
[0136] Ilustrative spacer linkers include carbonyl, thionocarbonyl,
alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl,
alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl,
carbonyltetrahydrofuranyl,
1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and
1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of said
spacer linkers is optionally substituted with one or more
substituents X.sup.1 as defined herein.
[0137] Illustrative releasable linkers include methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl,
oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl,
iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
herein.
[0138] In any of the embodiments described herein, the substituents
X.sup.2 can be alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl,
amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo,
haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl,
arylalkyl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl
alkanoate, guanidinoalkyl, R.sup.4-carbonyl, R.sup.5-carbonylalkyl,
R.sup.6-acylamino, and R-acylaminoalkyl, wherein R.sup.4 and
R.sup.5 are each independently selected from amino acids, amino
acid derivatives, and peptides, and wherein R.sup.6 and R.sup.7 are
each independently selected from amino acids, amino acid
derivatives, and peptides. In this embodiment the releasable linker
can include nitrogen, and the substituent X.sup.2 and the
releasable linker can form an heterocycle.
[0139] In any of the embodiments described herein, the substituents
X.sup.1 can be alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl,
amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo,
haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl,
arylalkyl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl
alkanoate, guanidinoalkyl, R.sup.4-carbonyl, R.sup.5-carbonylalkyl,
R.sup.6-acylamino, and R.sup.7-acylaminoalkyl, wherein R.sup.4 and
R.sup.5 are each independently selected from the group consisting
of an amino acid, an amino acid derivative, and a peptide, and
wherein R.sup.6 and R.sup.7 are each independently selected from
the group consisting of an amino acid, an amino acid derivative,
and a peptide.
[0140] In any of the embodiments described herein, the substituents
X.sup.1 can be alkyl, hydroxyalkyl, amino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, carboxy, carboxyalkyl, guanidinoalkyl, R.sup.4-carbonyl,
R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0141] The term cycloalkylene as used herein refers to a bivalent
chain of carbon atoms, a portion of which forms a ring, such as
cycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,
3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the
like.
[0142] The term aryl as used herein refers to an aromatic mono or
polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the
like. In addition, aryl may also include heteroaryl.
[0143] The term substituted aryl as used herein refers to the
replacement of one or more hydrogen atoms, generally on carbon,
with a corresponding number of substituents, such as halo, hydroxy,
amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro,
and the like.
[0144] The term heteroaryl as used herein refers to an aromatic
mono or polycyclic ring of carbon atoms and at least one heteroatom
selected from nitrogen, oxygen, and sulfur, such as pyridinyl,
pyrimidinyl, indolyl, benzoxazolyl, and the like.
[0145] The term substituted heteroaryl as used herein refers to the
replacement of one or more hydrogen atoms, generally on carbon,
with a corresponding number of substituents, such as halo, hydroxy,
amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro,
and the like.
[0146] The term optionally substituted as used herein refers to the
replacement of one or more hydrogen atoms, generally on carbon,
with a corresponding number of substituents, such as halo, hydroxy,
amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro,
and the like. In addition, two hydrogens on the same carbon, on
adjacent carbons, or nearby carbons may be replaced with a bivalent
substituent to form the corresponding cyclic structure.
[0147] The term amino acid as used herein refers generally to
aminoalkylcarboxylate, where the alkyl radical is optionally
substituted, such as with alkyl, hydroxy alkyl, sulfhydrylalkyl,
aminoalkyl, carboxyalkyl, and the like, including groups
corresponding to the naturally occurring amino acids, such as
serine, cysteine, methionine, aspartic acid, glutamic acid, and the
like. It is to be understood that such amino acids may be of a
single stereochemistry or a particular mixture of stereochemisties,
including racemic mixtures. In addition, amino acid refers to beta,
gamma, and longer amino acids, such as amino acids of the
formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, omithine, threonine, and the
like. In another illustrative aspect of the vitamin drug delivery
conjugate intermediate described herein, the drug (i.e.,
therapeutic agents (A)), or an analog or a derivative thereof,
includes an alkylthiol nucleophile.
[0148] As used herein the term "antifolate" refers to a compound
that interferes with the metabolism of folic acid and its
derivatives in cellular processes.
[0149] It is to be understood that the above-described terms can be
combined to generate chemically-relevant groups, such as
alkoxyalkyl referring to methyloxymethyl, ethyloxyethyl, and the
like, haloalkoxyalkyl referring to trifluoromethyloxyethyl,
1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like, arylalkyl
referring to benzyl, phenethyl, .alpha.-methylbenzyl, and the like,
and others.
[0150] The term amino acid derivative as used herein refers
generally to an optionally substituted aminoalkylcarboxylate, where
the amino group and/or the carboxylate group are each optionally
substituted, such as with alkyl, carboxylalkyl, alkylamino, and the
like, or optionally protected. In addition, the optionally
substituted intervening divalent alkyl fragment may include
additional groups, such as protecting groups, and the like.
[0151] The term peptide as used herein refers generally to a series
of amino acids and/or amino acid analogs and derivatives covalently
linked one to the other by amide bonds.
[0152] The term "releasable linker" as used herein refers to a
linker (L) that includes at least one bond that can be broken under
physiological conditions (e.g., a pH-labile, acid-labile,
oxidatively-labile, or enzyme-labile bond). It should be
appreciated that such physiological conditions resulting in bond
breaking include standard chemical hydrolysis reactions that occur,
for example, at physiological pH, or as a result of
compartmentalization into a cellular organelle such as an endosome
having a lower pH than cytosolic pH.
[0153] The cleavable bond or bonds may be present in the interior
of a cleavable linker and/or at one or both ends of a cleavable
linker. It is appreciated that the lability of the cleavable bond
may be adjusted by including functional groups or fragments within
the linker L that are able to assist or facilitate such bond
breakage, also termed anchimeric assistance. In addition, it is
appreciated that additional functional groups or fragments may be
included within the linker L that are able to assist or facilitate
additional fragmentation of the conjugates after bond breaking of
the releasable linker. The lability of the cleavable bond can be
adjusted by, for example, substitutional changes at or near the
cleavable bond, such as including alpha branching adjacent to a
cleavable disulfide bond, increasing the hydrophobicity of
substituents on silicon in a moiety having a silicon-oxygen bond
that may be hydrolyzed, homologating alkoxy groups that form part
of a ketal or acetal that may be hydrolyzed, and the like.
[0154] It is understood that a cleavable bond can connect two
adjacent atoms within the releasable linker and/or connect other
linkers (L) or B and/or A, as described herein, at either or both
ends of the releasable linker. In the case where a cleavable bond
connects two adjacent atoms within the releasable linker, following
breakage of the bond, the releasable linker is broken into two or
more fragments. Alternatively, in the case where a cleavable bond
is between the releasable linker and another moiety, such as an
additional heteroatom, additional spacer linker, another releasable
linker, the therapeutic agent A, or analog or derivative thereof,
or the receptor binding ligand B, or analog or derivative thereof,
following breakage of the bond, the releasable linker is separated
from the other moiety.
[0155] It is understood that each of the additional spacer and
releasable linkers are bivalent. It should be further understood
that the connectivity between each of the various additional spacer
and releasable linkers themselves, and between the various
additional spacer and releasable linkers and A and/or B, as defined
herein, may occur at any atom found in the various additional
spacer or releasable linkers.
[0156] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form dithioalkylcarbonylhydrazide, where the hydrazide
forms a hydrazide with the therapeutic agent A, or analog or
derivative thereof.
[0157] In another aspect, the linker (L) comprises a plurality of
additional spacer linkers selected from the group consisting of the
naturally occurring amino acids and stereoisomers thereof.
[0158] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form 2-dithioalkyloxycarbonyl, where the carbonyl forms
a carbonate with the agent A, or analog or derivative thereof.
[0159] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form 2-dithioalkyloxycarbonylhydrazide.
[0160] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form 2-dithioalkylaminocarbonyl, where the carbonyl
forms a carbamate with the therapeutic agent A, or analog or
derivative thereof.
[0161] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form 2-dithioalkylaminocarbonyl, where the carbonyl
forms a carbamate with the therapeutic agent A, or analog or
derivative thereof, and the alkyl is ethyl.
[0162] In another aspect, the linker (L) comprises a releasable
linker, an additional spacer linker, and a releasable linker taken
together to form 2-dithioarylalkyloxycarbonyl, where the carbonyl
forms a carbamate or a carbamoylaziridine with the therapeutic
agent A, or analog or derivative thereof.
[0163] In one aspect, the releasable and spacer linkers may be
arranged in such a way that subsequent to the cleavage of a bond in
the linker (L), released functional groups chemically assist the
breakage or cleavage of additional bonds, also termed anchimeric
assisted cleavage or breakage.
[0164] Illustrative mechanisms for cleavage of the linkers
described herein include the following 1,4 and 1,6 fragmentation
mechanisms
##STR00007##
where X is an exogenous or endogenous nucleophile, glutathione, or
bioreducing agent, and the like, and either of Z or Z' is the
vitamin (e.g. folate), or analog or derivative thereof, or the
drug, or analog or derivative thereof, or a vitamin (e.g. folate)
or drug in conjunction with other portions of the linker (L). It is
to be understood that although the above fragmentation mechanisms
are depicted as concerted mechanisms, any number of discrete steps
may take place to effect the ultimate fragmentation of the linker
(L) to the final products shown. For example, it is appreciated
that the bond cleavage may also occur by acid-catalyzed elimination
of the carbamate moiety, which may be anchimerically assisted by
the stabilization provided by either the aryl group of the beta
sulfur or disulfide illustrated in the above examples. In those
variations of this embodiment, the releasable linker is the
carbamate moiety. Alternatively, the fragmentation may be initiated
by a nucleophilic attack on the disulfide group, causing cleavage
to form a thiolate. The thiolate may intermolecularly displace a
carbonic acid or carbamic acid moiety and form the corresponding
thiacyclopropane. In the case of the benzyl-containing linkers,
following an illustrative breaking of the disulfide bond, the
resulting phenyl thiolate may further fragment to release a
carbonic acid or carbamic acid moiety by forming a resonance
stabilized intermediate. In any of these cases, the releasable
nature of the illustrative linkers described herein may be realized
by whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present.
[0165] It is to be understood that although the above fragmentation
mechanisms are depicted as concerted mechanisms, any number of
discrete steps may take place to effect the ultimate fragmentation
of the linker (L) to the final products shown. Alternatively, the
fragmentation may be initiated by a nucleophilic attack on the
disulfide group, causing cleavage to form a thiolate. The thiolate
may intermolecularly displace a carbonic acid or carbamic acid
moiety and form the corresponding thiacyclopropane. In any of these
cases, the releasable nature of the illustrative linkers (L)
described herein may be realized by whatever mechanism may be
relevant to the chemical, metabolic, physiological, or biological
conditions present. Without being bound by theory, in this
embodiment, acid catalysis, such as in an endosome, may also
initiate the cleavage via protonation of the urethane group. In
addition, acid-catalyzed elimination of the carbamate leads to the
release of CO.sub.2 and the nitrogen-containing moiety attached to
Z, and the formation of a benzyl cation, which may be trapped by
water, or any other Lewis base, as is similarly described
herein.
[0166] In one embodiment, the linkers (L) described herein are
compounds of the following formulae
##STR00008##
where n is an integer selected from 1 to about 4; R.sup.a and
R.sup.b are each independently selected from the group consisting
of hydrogen and alkyl, including lower alkyl such as
C.sub.1-C.sub.4 alkyl that are optionally branched; or R.sup.a and
R.sup.b are taken together with the attached carbon atom to form a
carbocyclic ring; R is an optionally substituted alkyl group, an
optionally substituted acyl group, or a suitably selected nitrogen
protecting group; and (*) indicates points of attachment for the
drug, folate, other linkers (L), or other parts of the
conjugate.
[0167] Another illustrative mechanism involves an arrangement of
the releasable and spacer linkers in such a way that subsequent to
the cleavage of a bond in the linker (L), released functional
groups chemically assist the breakage or cleavage of additional
bonds, also termed anchimeric assisted cleavage or breakage.
[0168] In another illustrative embodiment, the linker (L) includes
one or more amino acids. In one variation, the linker (L) includes
a single amino acid. In another variation, the linker (L) includes
a peptide having from 2 to about 50, 2 to about 30, or 2 to about
20 amino acids. In another variation, the linker (L) includes a
peptide having from about 4 to about 8 amino acids. Such amino
acids are illustratively selected from the naturally occurring
amino acids, or stereoisomers thereof. The amino acid may also be
any other amino acid, such as any amino acid having the general
formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, omithine, threonine, and the
like. In one variation, the releasable linker includes at least 2
amino acids selected from asparagine, aspartic acid, cysteine,
glutamic acid, lysine, glutamine, arginine, serine, omithine, and
threonine. In another variation, the releasable linker includes
between 2 and about 5 amino acids selected from asparagine,
aspartic acid, cysteine, glutamic acid, lysine, glutamine,
arginine, serine, omithine, and threonine. In another variation,
the releasable linker includes a tripeptide, tetrapeptide,
pentapeptide, or hexapeptide consisting of amino acids selected
from aspartic acid, cysteine, glutamic acid, lysine, arginine, and
omithine, and combinations thereof.
[0169] In one illustrative embodiment of the invention, a method
for treating a patient with an inflammatory disease, the method
comprising the step of administering to the patient a composition
comprising a drug delivery conjugate of the formula
BL(A.sup.1)(A.sup.2).sub.m
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0170] m is 0 or 1;
[0171] B is a folate;
[0172] L is a linker that comprises one or more hydrophilic spacer
linkers;
[0173] A.sup.1 is an antifolate; and
[0174] A.sup.2 has the formula
##STR00009##
wherein
[0175] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0176] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0177] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0178] In another embodiment, a pharmaceutical composition
comprising a drug delivery conjugate of the formula
BL(A.sup.1)(A.sup.2).sub.m
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0179] m is 0 or 1;
[0180] B is a folate;
[0181] L is a linker that comprises one or more hydrophilic spacer
linkers;
[0182] A.sup.1 is an antifolate; and
[0183] A.sup.2 has the formula
##STR00010##
wherein
[0184] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0185] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0186] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0187] In one aspect, B, L, A.sup.1, and A.sup.2 in the conjugate
BLA.sup.1(A.sup.2).sub.m are connected as shown in the following
formula:
##STR00011##
[0188] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein m is 1
and A.sup.1 and A.sup.2 are each covalently attached to linker L is
described.
[0189] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein L is a
linker of the formula
##STR00012##
[0190] wherein * indicates the point of attachment to the folate;
** indicates the point of attachment to one of A.sup.1 or A.sup.2;
*** indicates the point of attachment to the remaining A.sup.1 or
A.sup.2; F and G are each independently 1, 2, 3 or 4; and W.sup.1
is NH or O is described.
[0191] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
folate is of the formula
##STR00013##
wherein * indicates the point of attachment to the linker;
[0192] 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;
[0193] 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.ident.C--;
[0194] C.sup.1 and C.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(CH.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;
[0195] 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,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0196] 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; and
[0197] p, r, s and t are each independently either 0 or 1 is
described.
[0198] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
antifolate is aminopterin, or an analog, or derivative, thereof is
described.
[0199] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
antifolate is aminopterin hydrazide is described.
[0200] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
folate is of the formula
##STR00014##
wherein * indicates the point of attachment to the linker is
described.
[0201] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein m.sup.1
is 1; R.sup.A and R.sup.B are hydrogen; Y.sup.A is
OCH.sub.2CH.sub.2OR.sup.C; and R.sup.C is a bond connected to L is
described.
[0202] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein F is 2
and G is 1 is described.
[0203] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
drug delivery conjugate is of the formula
##STR00015##
is described.
[0204] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
drug delivery conjugate is of the formula
##STR00016##
[0205] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
drug delivery conjugate is of the formula
##STR00017##
[0206] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
composition further comprises one or more carriers, diluents, or
excipients, or a combination thereof is described.
[0207] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
purity of the drug delivery conjugate is at least 98% is
described.
[0208] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
composition is in a dosage form adapted for parenteral
administration is described.
[0209] In another embodiment, the method of any one of the
preceding embodiments wherein the dose of the drug delivery
conjugate is in the range of 1 to 5 .mu.g/kg is described.
[0210] In another embodiment, the method of any one of the
preceding embodiments wherein the dose of the drug delivery
conjugate is in the range of 1 to 3 .mu.g/kg is described.
[0211] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
disease is selected from the group consisting of arthritis,
including rheumatoid arthritis and osteoarthritis,
glomerulonephritis, proliferative retinopathy, restenosis,
ulcerative colitis, Crohn's disease, fibromyalgia, psoriasis and
other inflammations of the skin, inflammations of the eye,
including uveitis and autoimmune uveitis, osteomyelitis, Sjogren's
syndrome, multiple sclerosis, diabetes, atherosclerosis, pulmonary
fibrosis, lupus erythematosus, sarcoidosis, systemic sclerosis,
organ transplant rejection (GVHD), and chronic inflammations is
described.
[0212] In one embodiment, a compound having the following
formula
##STR00018##
is described.
[0213] In another embodiment, a kit comprising a sterile vial, the
composition or compound of any one of the preceding embodiments,
and instructions for use describing use of the composition for
treating a patient with an inflammatory disease is described.
[0214] In another embodiment, is described a kit comprising a
sterile vial, a composition comprising the compound as a
lyophilized solid of any one of the preceding embodiments, and
instructions describing use of the composition for treating a
patient with an inflammatory disease, wherein the vial is an amber
glass vial with a rubber stopper and an aluminum tear-off seal.
[0215] In another embodiment, the kit of the preceding embodiment
wherein the folate is of the formula
##STR00019##
wherein * indicates the point of attachment to the linker is
described.
[0216] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
antifolate is aminopterin hydrazide is described.
[0217] In another embodiment, the kit of any one of the preceding
embodiments wherein the antifolate is aminopterin hydrazide is
described.
[0218] In another embodiment, the kit of any one of the preceding
embodiments wherein in the conjugate m.sup.1 is 1; R.sup.A and
R.sup.B are hydrogen; Y.sup.A is OCH.sub.2CH.sub.2OR.sup.C; and
R.sup.C is a bond connected to L is described.
[0219] In another embodiment, the kit of any one of the preceding
embodiments wherein in the conjugate F is 2 and G is 1 is
described.
[0220] In another embodiment, the kit of any one of the preceding
embodiments is described wherein the drug delivery conjugate is of
the formula
##STR00020##
[0221] In another embodiment, the kit of any one of the preceding
embodiments is described wherein the drug delivery conjugate is of
the formula
##STR00021##
[0222] In another embodiment, the kit of any one of the preceding
embodiments wherein the drug delivery conjugate is of the
formula
##STR00022##
is described.
[0223] In another embodiment, the kit of any one of the preceding
embodiments wherein the composition or compound is in the form of a
reconstitutable lyophlizate is described.
[0224] In another embodiment, the kit of any one of the preceding
embodiments wherein the dose of the drug delivery conjugate is in
the range of 1 to 5 .mu.g/kg is described.
[0225] In another embodiment, the kit of any one of the preceding
embodiments wherein the dose of the drug delivery conjugate is in
the range of 1 to 3 .mu.g/kg is described.
[0226] In another embodiment, the kit of any one of the preceding
embodiments wherein the purity of the drug delivery conjugate is at
least 98% is described.
[0227] In another embodiment, a kit is described wherein the
compound of any of the preceding compound embodiments is
lyophilized and is in the kit along with a composition for
reconstrituting the lyophilizate.
[0228] In another embodiment, one of A is a derivative or analog of
rapamycin. Illustrative examples of derivatives or analogs of
rapamycin are disclosed in U.S. Pat. Nos. 4,316,885, 4,650,803,
5,100,883, 5,118,677, 5,118,678, 5,120,842, 5,130,307, 5,138,051,
5,151,413, 5,169,851, 5,194,447, 5,221,670, 5,233,036, 5,258,389,
5,260,300, 5,302,584, 5,362,718, 5,378,696, 5,385,908, 5,385,909,
5,385,910, 5,389,639, 5,391,730, 5,463,048, and 5,491,231. The
disclosure of each of the foregoing documents is incorporated by
reference herein in its entirety. In another embodiment one of A is
a derivative of everolimus.
[0229] In one illustrative embodiment of the invention a method for
treating a patient with an inflammatory disease, the method
comprising the step of administering to the patient a composition
comprising a drug delivery conjugate of the formula
B-L-A.sup.3
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0230] B is a folate;
[0231] L is a linker that comprises one or more hydrophilic spacer
linkers; and
[0232] A.sup.3 has the formula
##STR00023##
wherein
[0233] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0234] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0235] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0236] In another embodiment, a pharmaceutical composition
comprising a drug delivery conjugate of the formula
B-L-A.sup.3
or a pharmaceutically acceptable salt, isomer, mixture of isomers,
crystalline form, non crystalline form, hydrate, or solvate
thereof; wherein
[0237] B is a folate;
[0238] L is a linker that comprises one or more hydrophilic spacer
linkers; and
[0239] A.sup.3 has the formula
##STR00024##
wherein
[0240] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0241] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0242] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted is described.
[0243] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein
[0244] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0245] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0246] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, and C(O)R.sup.D,
where R.sup.D is in each instance independently selected from the
group consisting of hydrogen, and alkyl, alkenyl, heteroalkyl,
cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and
heteroarylalkyl, each of which is optionally substituted is
described.
[0247] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein L is a
bivalent linker of the formula
##STR00025##
[0248] wherein * indicates the point of attachment to the folate
and ** indicates the point of attachment to A.sup.3; and F and G
are each independently 1, 2, 3 or 4 is described.
[0249] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
folate is of the formula
##STR00026##
wherein * indicates the point of attachment to the linker;
[0250] 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;
[0251] 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--;
[0252] 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(CH.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;
[0253] 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,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0254] 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; and
[0255] n, p, r, s and t are each independently either 0 or 1 is
described.
[0256] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
folate is of the formula
##STR00027##
wherein * indicates the point of attachment to the linker is
described.
[0257] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein R.sup.A
and R.sup.B are hydrogen; Y.sup.A is OCH.sub.2CH.sub.2OR.sup.C; and
R.sup.C is a bond connected to L is described.
[0258] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein F is 2
and G is 1 is described.
[0259] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
drug delivery conjugate is of the formula
##STR00028##
is described.
[0260] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
composition further comprises one or more carriers, diluents, or
excipients, or a combination thereof is described.
[0261] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
purity of the drug delivery conjugate is at least 98% is
described.
[0262] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
composition is in a dosage form adapted for parenteral
administration is described.
[0263] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
dose of the drug delivery conjugate is in the range of 1 to 5
.mu.g/kg is described.
[0264] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
dose of the drug delivery conjugate is in the range of 1 to 3
.mu.g/kg is described.
[0265] In another embodiment, the method or pharmaceutical
composition of any one of the preceding embodiments wherein the
disease is selected from the group consisting of arthritis,
including rheumatoid arthritis and osteoarthritis,
glomerulonephritis, proliferative retinopathy, restenosis,
ulcerative colitis, Crohn's disease, fibromyalgia, psoriasis and
other inflammations of the skin, osteomyelitis, Sjogren's syndrome,
multiple sclerosis, diabetes, atherosclerosis, pulmonary fibrosis,
lupus erythematosus, sarcoidosis, systemic sclerosis, organ
transplant rejection (GVHD) and chronic inflammations is
described.
[0266] In another embodiment, a kit comprising a sterile vial, the
composition described in any of the preceding embodiments; and
instructions for use describing use of the composition for treating
a patient with an inflammatory disease is described.
[0267] In another embodiment, the kit of any one of the preceding
embodiments wherein the folate is of the formula
##STR00029##
wherein * indicates the point of attachment to the linker is
described.
[0268] In another embodiment, the kit of any one of the preceding
embodiments wherein R.sup.A and R.sup.B are hydrogen; Y.sup.A is
OCH.sub.2CH.sub.2OR.sup.C; and R.sup.C is a bond connected to L is
described.
[0269] In another embodiment, the kit of any one of the preceding
embodiments wherein F is 2 and G is 1 is described.
[0270] In another embodiment, the kit of any one of the preceding
embodiments wherein the drug delivery conjugate is of the
formula
##STR00030##
is described.
[0271] In another embodiment, the kit of any one of the preceding
embodiments wherein the composition is in the form of a
reconstitutable lyophlizate is described.
[0272] In another embodiment, the kit of any one of the preceding
embodiments wherein the dose of the drug delivery conjugate is in
the range of 1 to 5 .mu.g/kg is described.
[0273] In another embodiment, the kit of any one of the preceding
embodiments wherein the dose of the drug delivery conjugate is in
the range of 1 to 3 .mu.g/kg is described.
[0274] In another embodiment, the kit of any one of the preceding
embodiments wherein the purity of the drug delivery conjugate is at
least 98% is described.
[0275] The conjugates or compounds described herein may contain one
or more chiral centers, or may otherwise be capable of existing as
multiple stereoisomers. It is to be understood that in one
embodiment, the invention described herein is not limited to any
particular sterochemical requirement, and that the conjugates,
compounds, and compositions, methods, uses, and medicaments that
include them may be optically pure, or may be any of a variety of
steroisomeric mixtures, including racemic and other mixtures of
enantiomers, other mixtures of diastereomers, and the like. It is
also to be understood that such mixtures of stereoisomers may
include a single stereochemical configuration at one or more chiral
centers, while including mixtures of stereochemical configuration
at one or more other chiral centers.
[0276] Similarly, the compounds or conjugates described herein may
be include geometric centers, such as cis, trans, E, and Z double
bonds. It is to be understood that in another embodiment, the
invention described herein is not limited to any particular
geometric isomer requirement, and that the conjugates, compounds,
and compositions, methods, uses, and medicaments that include them
may be pure, or may be any of a variety of geometic isomer
mixtures. It is also to be understood that such mixtures of
geometric isomers may include a single configuration at one or more
double bonds, while including mixtures of geometry at one or more
other double bonds.
[0277] As used herein, the term "alkyl" includes a chain of carbon
atoms, which is optionally branched. It is to be understood that
alkyl is advantageously of limited length, including
C.sub.1-C.sub.24, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, and C.sub.1-C.sub.4. It is appreciated herein that
shorter alkyl groups add less lipophilicity to the conjugate and
accordingly will have different pharmacokinetic behavior. As used
herein, the term "cycloalkyl" includes a chain of carbon atoms,
which is optionally branched, and where at least a portion of the
chain is cyclic. It is to be understood that a chain forming
cycloalkyl is advantageously of limited length, including
C.sub.3-C.sub.24, C.sub.3-C.sub.12, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, and C.sub.3-C.sub.4. It is appreciated herein that
shorter alkyl groups add less lipophilicity to the conjugate and
accordingly will have different pharmacokinetic behavior.
[0278] As used herein, the term "heteroalkyl" includes a chain of
atoms that includes both carbon and at least one heteroatom, and is
optionally branched. Illustrative heteroatoms include nitrogen,
oxygen, and sulfur. In certain variations, illustrative heteroatoms
also include phosphorus, and selenium. As used herein, the term
"heterocyclyl" including heterocycle includes a chain of atoms that
includes both carbon and at least one heteroatom, and is optionally
branched, where at least a portion of the chain is cyclic.
Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In
certain variations, illustrative heteroatoms also include
phosphorus, and selenium. Illustrative heteocycles include, but are
not limited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl,
piperidinyl, morpholinyl, piperazinyl, homopiperazinyl,
quinuclidinyl, and the like.
[0279] As used herein, the term "amino" includes the group
NH.sub.2, alkylamino, and dialkylamino, where the two alkyl groups
in dialkylamino may be the same or different, i.e. alkylalkylamino.
Illustratively, amino includes methylamino, ethylamino,
dimethylamino, methylethylamino, and the like. In addition, it is
to be understood that when amino modifies or is modified by another
term, such as aminoalkyl, or acylamino, the above variations of the
term amino are included therein. Illustratively, aminoalkyl
includes H.sub.2N-alkyl, methylaminoalkyl, ethylaminoalkyl,
dimethylaminoalkyl, methylethylaminoalkyl, and the like.
Illustratively, acylamino includes acylmethylamino, acylethylamino,
and the like.
[0280] As used herein, the term "optionally substituted amino"
includes derivatives of amino as described herein, such as, but not
limited to, acylamino, urea, and carbamate, and the like.
[0281] As used herein, the term "aryl" includes monocyclic and
polycyclic aromatic carbocyclic and aromatic heterocyclic groups,
each of which may be optionally substituted. As used herein, the
term "heteroaryl" includes aromatic heterocyclic groups, each of
which may be optionally substituted. Illustrative carbocyclic
aromatic groups described herein include, but are not limited to,
phenyl, naphthyl, and the like. Illustrative heterocyclic aromatic
groups include, but are not limited to, pyridinyl, pyrimidinyl,
pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl,
quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl,
isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl,
benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl,
benzisothiazolyl, and the like.
[0282] The term "optionally substituted" as used herein includes
the replacement of hydrogen atoms with other functional groups on
the radical that is optionally substituted. Such other functional
groups illustratively include, but are not limited to, amino,
hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the
like.
[0283] The term "optionally substituted aryl" as used herein
includes the replacement of hydrogen atoms with other functional
groups on the aryl that is optionally substituted. Such other
functional groups illustratively include, but are not limited to,
amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl,
arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives
thereof, carboxylic acids and derivatives thereof, and the
like.
[0284] Illustrative substituents include, but are not limited to, a
radical --(CH.sub.2).sub.mZ, where m is an integer from 0-6 and Z
is selected from halogen, hydroxy, alkanoyloxy, including
C.sub.1-C.sub.6 alkanoyloxy, optionally substituted aroyloxy,
alkyl, including C.sub.1-C.sub.6 alkyl, alkoxy, including
C.sub.1-C.sub.6 alkoxy, cycloalkyl, including C.sub.3-C.sub.8
cycloalkyl, cycloalkoxy, including C.sub.3-C.sub.8 cycloalkoxy,
alkenyl, including C.sub.2-C.sub.6 alkenyl, alkynyl, including
C.sub.2-C.sub.6 alkynyl, haloalkyl, including C.sub.1-C.sub.6
haloalkyl, haloalkoxy, including C.sub.1-C.sub.6 haloalkoxy,
halocycloalkyl, including C.sub.3-C.sub.8 halocycloalkyl,
halocycloalkoxy, including C.sub.3-C.sub.8 halocycloalkoxy, amino,
C.sub.1-C.sub.6 alkylamino, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)amino, alkylcarbonylamino, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylamino, aminoalkyl, C.sub.1-C.sub.6
alkylaminoalkyl, (C.sub.1-C.sub.6 alkyl)(C.sub.1-C.sub.6
alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N--(C.sub.1-C.sub.6
alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z is selected
from --CO.sub.2R.sup.4 and --CONR.sup.5R.sup.6, where R.sup.4,
R.sup.5, and R.sup.6 are each independently selected in each
occurrence from hydrogen, C.sub.1-C.sub.6 alkyl, and
aryl-C.sub.1-C.sub.6 alkyl.
[0285] The term "prodrug" as used herein generally refers to any
conjugate that when administered to a biological system generates a
biologically active conjugate as a result of one or more
spontaneous chemical reaction(s), enzyme-catalyzed chemical
reaction(s), and/or metabolic chemical reaction(s), or a
combination thereof. In vivo, the prodrug is typically acted upon
by an enzyme (such as esterases, amidases, phosphatases, and the
like), simple biological chemistry, or other process in vivo to
liberate or regenerate the more pharmacologically active drug. This
activation may occur through the action of an endogenous host
enzyme or a non-endogenous enzyme that is administered to the host
preceding, following, or during administration of the prodrug.
Additional details of prodrug use are described in U.S. Pat. No.
5,627,165; and Pathalk et al., Enzymic protecting group techniques
in organic synthesis, Stereosel. Biocatal. 775-797 (2000). It is
appreciated that the prodrug is advantageously converted to the
original drug as soon as the goal, such as targeted delivery,
safety, stability, and the like is achieved, followed by the
subsequent rapid elimination of the released remains of the group
forming the prodrug.
[0286] Prodrugs may be prepared from the conjugate described herein
by attaching groups, referred to as prodrug forming groups, that
ultimately cleave in vivo to one or more functional groups present
on the conjugate, such as --OH--, --SH, --CO.sub.2H, --NR.sub.2.
Illustrative prodrugs include but are not limited to carboxylate
esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl,
alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and
amines where the group attached is an acyl group, an
alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrative
esters, also referred to as active esters, include but are not
limited to 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such as
acetoxymethyl, pivaloyloxymethyl, .beta.-acetoxyethyl,
.beta.-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl,
(1-aminoethyl)carbonyloxymethyl, and the like;
alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl,
.alpha.-ethoxycarbonyloxyethyl, .beta.-ethoxycarbonyloxyethyl, and
the like; dialkylaminoalkyl groups, including di-lower alkylamino
alkyl groups, such as dimethylaminomethyl, dimethylaminoethyl,
diethylaminomethyl, diethylaminoethyl, and the like;
2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)
pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and
lactone groups such as phthalidyl, dimethoxyphthalidyl, and the
like.
[0287] Further illustrative prodrugs contain a chemical moiety,
such as an amide or phosphorus group functioning to increase
solubility and/or stability of the conjugates described herein.
Further illustrative prodrugs for amino groups include, but are not
limited to, (C.sub.3-C.sub.20)alkanoyl;
halo-(C.sub.3-C.sub.20)alkanoyl; (C.sub.3-C.sub.20)alkenoyl;
(C.sub.4-C.sub.7)cycloalkanoyl;
(C.sub.3-C.sub.6)-cycloalkyl(C.sub.2-C.sub.16)alkanoyl; optionally
substituted aroyl, such as unsubstituted aroyl or aroyl substituted
by 1 to 3 substituents selected from the group consisting of
halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with one or more of 1 to 3
halogen atoms; optionally substituted
aryl(C.sub.2-C.sub.16)alkanoyl, such as the aryl radical being
unsubstituted or substituted by 1 to 3 substituents selected from
the group consisting of halogen, (C.sub.1-C.sub.3)alkyl and
(C.sub.1-C.sub.3)alkoxy, each of which is optionally further
substituted with 1 to 3 halogen atoms; and optionally substituted
heteroarylalkanoyl having one to three heteroatoms selected from O,
S and N in the heteroaryl moiety and 2 to 10 carbon atoms in the
alkanoyl moiety, such as the heteroaryl radical being unsubstituted
or substituted by 1 to 3 substituents selected from the group
consisting of halogen, cyano, trifluoromethanesulphonyloxy,
(C.sub.1-C.sub.3)alkyl, and (C.sub.1-C.sub.3)alkoxy, each of which
is optionally further substituted with 1 to 3 halogen atoms. The
groups illustrated are exemplary, not exhaustive, and may be
prepared by conventional processes.
[0288] It is understood that the prodrugs themselves may not
possess significant biological activity, but instead undergo one or
more spontaneous chemical reaction(s), enzyme-catalyzed chemical
reaction(s), and/or metabolic chemical reaction(s), or a
combination thereof after administration in vivo to produce the
conjugate described herein that is biologically active or is a
precursor of the biologically active conjugate. However, it is
appreciated that in some cases, the prodrug is biologically active.
It is also appreciated that prodrugs may often serve to improve
drug efficacy or safety through improved oral bioavailability,
pharmacodynamic half-life, and the like. Prodrugs also refer to
derivatives of the conjugates described herein that include groups
that simply mask undesirable drug properties or improve drug
delivery. For example, one or more conjugates described herein may
exhibit an undesirable property that is advantageously blocked or
minimized may become pharmacological, pharmaceutical, or
pharmacokinetic barriers in clinical drug application, such as low
oral drug absorption, lack of site specificity, chemical
instability, toxicity, and poor patient acceptance (bad taste,
odor, pain at injection site, and the like), and others. It is
appreciated herein that a prodrug, or other strategy using
reversible derivatives, can be useful in the optimization of the
clinical application of a drug.
[0289] It is appreciated that such hydrophilic linkers may alter
the stability, metabolism and tissue distribution of the
conjugates. For example, it is understood that in certain
situations, carbohydrate-protein interactions are weaker than
peptide-protein interactions. Thus, it is appreciated that in
various embodiments described herein, the conjugates may lead to
lower binding of serum proteins. These and other physicochemical
differences between the conjugates described herein and others
already reported may include enhanced targeting to target cells and
improved, i.e. more selective or differentially selective
biodistribution profiles. The increased cyctotoxicity may be a
natural consequence of the decreased serum protein binding or the
better or differential biodistribution (i.e. less drug is wasted in
non-specific compartments). This is especially true for the use of
hydrophilic but neutral spacers. Without being bound by theory it
is also suggested that the hydrophilic spacer linkers described
herein may decrease toxicity that might be due at least in part to
non-specific binding interactions.
[0290] In an alternate embodiment, the drug is linked to a
hydrophilic spacer linker, directly or indirectly, to accomplish
the goal of decreasing liver clearance. It has been found herein
that the attachment of hydrophilic groups, either releasable or
not, and more specifically hydrophilic neutral groups, increases
renal-specific delivery.
[0291] It has been observed that liver clearance of folate-drug
conjugates possessing disulfide linkers and peptidic spacers retain
residual and sometimes substantial unfavorable toxicity profiles.
Including the hydrophilic spacers described herein also introduced
vectors for kidney-specific delivery. It is therefore appreciated
that including such linkers in the drug delivery conjugates may
decrease overall liver uptake and consequentially decrease overall
toxicity. Without being bound by theory, it is appreciated that
toxicity at MTD may be caused by non-specific liver clearance,
leading to metabolism and release of free drug into bile and then
the intestine. The local toxicity as well as systemic toxicity (due
to re-absorption) might then occur. By including hydrophilic
linkers in the conjugates described herein, it is believed that
clearance through the kidney may preferentially occur, thus
decreasing and/or avoiding concomitant liver metabolism based
toxicity. Accordingly, measuring total bile clearance of the drug
component from a series of drug-folate conjugates, may be used to
predict which agent would be the least toxic.
[0292] As described above, the conjugates described herein may be
used to deliver therapeutic agents A (e.g. drugs) to cells in a
selective or specific manner. In one aspect of such delivery,
unwanted clearance mechanisms may also be avoided. It has been
discovered that the hydrophilic spacer linkers described herein
when used to form conjugates of receptor binding ligands B and
therapeutic agents A, can decrease the amount of clearance by the
liver. It has further been discovered that these hydrophilic spacer
linkers tend to favor clearance along renal pathways, such as the
kidney. It has further been discovered that the conjugates
described herein exhibit lower toxicity than the parent therapeutic
agents A by themselves when administered in the same way. Without
being bound by theory, it is suggested that the lower toxicity
arises from the observed decrease in liver clearance mechanism in
favor of renal clearance mechanisms.
[0293] In another embodiment, multi-drug conjugates are described
herein. Several illustrative configurations of such multi-drug
conjugates are contemplated herein, and include the compounds and
configurations described in PCT international publication No. WO
2007/022494, the disclosure of which is incorporated herein by
reference. In one aspect, the linker (L) can be a polyvalent
linker. Illustratively, the polyvalent linkers may connect the
folate receptor binding ligand B to the two or more therapeutic
agents A (e.g. drug) in a variety of structural configurations
[0294] In one illustrative embodiment, one of the therapeutic
agents (e.g. drugs) is aminopterin or aminopterin hydrazide. If an
additional drug is included in the conjugate, it can be a drug of
formula I or a different drug.
##STR00031##
wherein
[0295] Y.sup.A is OR.sup.C or OCH.sub.2CH.sub.2OR.sup.C;
[0296] one of R.sup.A, R.sup.B, or R.sup.C is a bond connected to
L; and
[0297] the other two of R.sup.A, R.sup.B, and R.sup.C are
independently selected in each case from the group consisting of
hydrogen, optionally substituted heteroalkyl, prodrug forming
group, and C(O)R.sup.D, where R.sup.D is in each instance
independently selected from the group consisting of hydrogen, and
alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,
arylalkyl, heteroaryl, and heteroarylalkyl, each of which is
optionally substituted. If the second drug is a drug different than
formula I, the second drug can be selected based on activity
against one or more populations of pathogenic cells, such as
inflammatory cells, with a particular mechanism of action.
Illustrative mechanisms of action include alkylating agents,
microtubule inhibitors, including those that stabilize and/or
destabilize microtubule formation, including beta-tubulin agents,
cyclin dependent kinase (CDK) inhibitors, topoisomerase inhibitors,
protein synthesis inhibitors, protein kinase inhibitors, including
inhibitors of Ras, Raf, PKC, PI3K, and like inhibitors,
transcription inhibitor, antifolates, heat shock protein blockers,
and the like.
[0298] Illustrative alkylating agents include, but are not limited
to, mitomycins CBI, and the like. Illustrative cyclin dependent
kinase (CDK) inhibitors include, but are not limited to, CYC202,
seliciclib, R-roscovitine, AGM-1470, and the like. Illustrative
topoisomerase inhibitors include, but are not limited to,
doxorubicin, other anthracyclines, and the like. Illustrative
protein synthesis inhibitors include, but are not limited to,
bruceantin, and the like. Illustrative protein kinase inhibitors,
including inhibitors of Ras, Raf, PKC, PI3K, and like inhibitors,
include but are not limited to L-779,450, R115777, and the like.
Illustrative transcription inhibitors include, but are not limited
to, .alpha.-amanatin, actinomycin, and the like. Illustrative
antifolates include, but are not limited to, methotrexate,
aminopterin, and the like. Illustrative heat shock protein blockers
include, but are not limited to, geldanamycin, and the like.
[0299] Illustrative microtubule inhibitors, including those that
stabilize and/or destabilize microtubule formation, include
.beta.-tubulin agents, microtubule poisons, and the like.
Illustrative microtubule poisons that bind to selected receptors
include, but are not limited to, inhibitors binding to the vinca
binding site such as arenastatin, dolastatin, halichondrin B,
maytansine, phomopsin A, rhizoxin, ustiloxin, vinblastine,
vincristine, and the like, stabilizers binding to the taxol binding
site such as discodermalide, epothilone, taxol, paclitaxol, and the
like, inhibitors binding to the colchicine binding site such as,
colchicine, combretastatin, curacin A, podophyllotoxin,
steganacine, and the like, and others binding to undefined sites
such as cryptophycin, tubulysins, and the like.
[0300] In one embodiment, the tubulsyin is a naturally occurring
tubulysin. In another embodiment, the tubulsyin is a synthetic or
semi-synthetic tubulysin. Additional tubulysins that may be
included in the conjugates described herein are described in PCT
international application serial No. PCT/US2008/056824, the
disclosure of which is incorporated herein by reference.
[0301] In one aspect of the drug delivery conjugates described
herein, at least one of the drugs is an antifolate. In one
illustrative example, the antifolate is aminopterin. In another
illustrative example, the antifolate is aminopterin hydrazide. In
other embodiments, where a second drug is included, the second drug
can be a DNA alkylation agent. In another embodiment, the second
drug can be a microtubule inhibitor.
[0302] In another embodiment of the drug delivery conjugates
described herein, the second drug is a P-glycoprotein (PGP)
inhibitor.
[0303] In another embodiment of the drug delivery conjugates
described herein, the second drug is a drug having formula I.
[0304] In another embodiment of the drug delivery conjugates
described herein, the second drug is a vinca alkaloid, or an analog
or derivative thereof. Vinca alklaloids described herein include
all members of the vinca indole-dihydroindole family of alkaloids,
such as but not limited to vindesine, vinblastine, vincristine,
catharanthine, vindoline, leurosine, vinorelbine, vinblastinoic
acid, and the like, and analogs and derivatives thereof.
[0305] In another embodiment, methods for treating diseases caused
by or evidenced by pathogenic cell populations, such as
inflammatory cells, are described herein. The drug delivery
conjugates can be used to treat disease states characterized by the
presence of a pathogenic cell population, such as inflammatory
cells, in the host wherein the members of the pathogenic cell
population have an accessible binding site for the folate, or
analog or derivative thereof, wherein the binding site is uniquely
expressed, overexpressed, or preferentially expressed by the
pathogenic cells. The selective elimination of the pathogenic cells
is mediated by the binding of the drug delivery conjugate to a
receptor (e.g., a folate receptor when the conjugate is folate
targeted), which is uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells. A receptor (e.g.,
a folate receptor) uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells is not present or
present at lower concentrations on non-pathogenic cells providing a
means for selective elimination of the pathogenic cells.
[0306] For example, surface-expressed vitamin receptors, such as
the high-affinity folate receptor, are overexpressed activated
macrophages and activated monocytes. Accordingly, the drug delivery
conjugates described herein can be used to treat a variety of
inflammatory cell types that preferentially express folate
receptors, and, thus, have surface accessible binding sites for
ligands, such as folate, or folate analogs or derivatives. In one
aspect, methods are described herein for targeting the conjugates
to maximize targeting of the pathogenic cells for elimination.
[0307] The invention further contemplates the use of combinations
of drug delivery conjugates to maximize targeting of the pathogenic
cells, such as inflammatory cells, for elimination. In accordance
with the invention "elimination", "eliminated", and "eliminating" a
population of cells mean completely eliminating a population of
cells, eliminating some cells, or reducing the symptoms of disease
caused by the cells, such as inflammatory cells.
[0308] The drug delivery conjugates described herein can be used
for both human clinical medicine and veterinary applications. Thus,
the host animal harboring the population of pathogenic cells and
treated with the drug delivery conjugates (e.g., a folate
conjugate) can be human or, in the case of veterinary applications,
can be a laboratory, agricultural, domestic, or wild animal. The
methods described herein can be applied to host animals including,
but not limited 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.
[0309] The methods are applicable to populations of pathogenic
cells that cause inflammation. For example, activated macrophages
or activated monocytes capable of causing a disease state, such as
inflammation, can be eliminated because they uniquely express,
preferentially express, or overexpress folate receptors, or
receptors that bind analogs or derivatives of folate. For example,
the pathogenic cells can be inflammatory cells that are pathogenic
under some circumstances such as cells of the immune system that
are responsible for graft versus host disease, but not pathogenic
under other circumstances.
[0310] In one embodiment, the drug delivery conjugates can be
internalized into the targeted pathogenic cells upon binding of the
binding ligand moiety (e.g. folate) to a receptor, transporter, or
other surface-presented protein that specifically binds the ligand
and which is preferentially expressed on the pathogenic cells. Such
internalization can occur, for example, through receptor-mediated
endocytosis. If the drug delivery conjugate contains a releasable
linker, the B moiety and the drug can dissociate intracellularly
and the drug can act on its intracellular target.
[0311] In an alternate embodiment, the B (e.g. folate) can bind to
the pathogenic cell placing the drug in close association with the
surface of the pathogenic cell. The drug can then be released by
cleavage of the releasable linker. For example, the drug can be
released by a protein disulfide isomerase if the releasable linker
is a disulfide group. The drug can then be taken up by the
pathogenic cell to which the drug delivery conjugate is bound, or
the drug can be taken up by another pathogenic cell in close
proximity thereto. Alternatively, the drug could be released by a
protein disulfide isomerase inside the cell where the releasable
linker is a disulfide group. The drug may also be released by a
hydrolytic mechanism, such as acid-catalyzed hydrolysis, as
described above for certain beta elimination mechanisms, or by an
anchimerically assisted cleavage through an oxonium ion or
lactonium ion producing mechanism. The selection of the releasable
linker or linkers will dictate the mechanism by which the drug is
released from the conjugate. It is appreciated that such a
selection can be pre-defined by the conditions wherein the drug
conjugate will be used. Alternatively, the drug delivery conjugates
can be internalized into the targeted cells upon binding, and the
receptor binding ligand (B) and the drug can remain associated
intracellularly with the drug exhibiting its effects without
dissociation from the vitamin moiety.
[0312] In still another embodiment where the receptor binding
ligand (B) is a folate, the drug delivery conjugate can act through
a mechanism independent of cellular folate receptors. For example,
the drug delivery conjugates can bind to soluble folate receptors
present in the serum or to serum proteins, such as albumin,
resulting in prolonged circulation of the conjugates relative to
the unconjugated drug, and in increased activity of the conjugates
towards the pathogenic cell population, such as inflammatory cells,
relative to the unconjugated drug.
[0313] In another embodiment, where the linker (L) does not
comprise a releasable linker, and B is folate, the folate moiety of
the drug delivery conjugate can bind to the pathogenic cell placing
the drug on the surface of the pathogenic cell to target the
pathogenic cell for attack by other molecules capable of binding to
the drug. Alternatively, in this embodiment, the drug delivery
conjugates can be internalized into the targeted cells upon
binding, and the vitamin moiety and the drug can remain associated
intracellularly with the drug exhibiting its effects without
dissociation from the folate.
[0314] The drug delivery conjugates described herein can comprise a
receptor binding ligand (B) (e.g. a folate), a linker (L), a drug,
and, optionally, heteroatom linkers to link the receptor binding
ligand (B) and the drug to the linker (L). The linker (L) can
comprise a spacer linker, a releasable (i.e., cleavable) linker,
and an heteroatom linker, or combinations thereof.
[0315] In one embodiment, the drug is aminopterin. In another
embodiment, a second drug may be present. Suitable second drugs can
include, but are not limited to: peptides, oligopeptides,
retro-inverso oligopeptides, proteins, protein analogs in which at
least one non-peptide linkage replaces a peptide linkage,
apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors,
amino acids and their derivatives, receptors and other membrane
proteins; antigens and antibodies thereto; haptens and antibodies
thereto; hormones, lipids, phospholipids, liposomes; toxins;
analgesics; bronchodilators; beta-blockers; antihypertensive
agents; cardiovascular agents including antiarrhythmics, cardiac
glycosides, antianginals and vasodilators; central nervous system
agents including stimulants, psychotropics, antimanics, and
depressants; antihistamines; tranquilizers; anti-depressants; H-2
antagonists; anticonvulsants; antinauseants; prostaglandins and
prostaglandin analogs; muscle relaxants; anti-inflammatory
substances; stimulants; decongestants; antiemetics; diuretics;
antispasmodics; antiasthmatics; cough suppressants; mucolytics;
mineral and nutritional additives; adrenocorticoids and
corticosteroids; alkylating agents; antiandrogens; antiestrogens;
androgens; aclamycin and aclamycin derivatives; estrogens;
antimetabolites such as cytosine arabinoside; purine analogs;
pyrimidine analogs; and methotrexate; busulfan; carboplatin;
chlorambucil; cisplatin and other platinum compounds; tamoxiphen;
taxol; paclitaxel; paclitaxel derivatives; Taxotere.RTM.;
cyclophosphamide; daunomycin; daunorubicin; doxorubicin; rhizoxin;
T2 toxin; plant alkaloids; prednisone; hydroxyurea; teniposide;
mitomycins; discodermolides; microtubule inhibitors; epothilones;
tubulysin; cyclopropyl benz[e]indolone; seco-cyclopropyl
benz[e]indolone; O-Ac-seco-cyclopropyl benz[e]indolone; bleomycin
and any other antibiotic; nitrogen mustards; nitrosureas;
vincristine; vinblastine; analogs and derivative thereof such as
deacetylvinblastine monohydrazide; and other vinca alkaloids;
including those described in PCT international publication No. WO
2007/022493; the disclosure of which is incorporated herein by
reference; colchicine; colchicine derivatives; allocolchicine;
thiocolchicine; trityl cysteine; Halicondrin B; dolastatins such as
dolastatin 10; amanitins such as .alpha.-amanitin; camptothecin;
irinotecan; and other camptothecin derivatives thereof;
maytansines; geldanamycin and geldanamycin derivatives;
estramustine; nocodazole; MAP4; colcemid; inflammatory and
proinflammatory agents; peptide and peptidomimetic signal
transduction inhibitors; and any other art-recognized drug or
toxin.
[0316] In another embodiment, the second drug can be selected from
a vinca alkaloid, such as DAVLBH, a cryptophycin, bortezomib,
thiobortezomib, a tubulysin, aminopterin, rapamycin, paclitaxel,
docetaxel, doxorubicin, daunorubicin, everolimus, .alpha.-amanatin,
verucarin, didemnin B, geldanomycin, purvalanol A, ispinesib,
budesonide, dasatinib, an epothilone, a maytansine, and a tyrosine
kinase inhibitor, including analogs and derivatives of the
foregoing. In one variation, the therapeutic agents (A) (e.g.
drugs) are the same and are antifolate compounds. In one variation,
the therapeutic agents (A) (e.g. drugs) are the same and are
aminopterin hydrazide. In another variation, the therapeutic agents
(A) (e.g. drugs) are different, but at least one of the therapeutic
agents (A) is an antifolate.
[0317] In one embodiment, the drugs for use in the methods
described herein remain stable in serum for at least 4 hours. In
another embodiment the drugs have an IC.sub.50 in the nanomolar
range, and, in another embodiment, the drugs are water soluble. If
the drug is not water soluble, the linker (L) can be derivatized to
enhance water solubility. The term "drug" also means any of the
drug analogs or derivatives described hereinabove. It should be
appreciated that in accordance with this invention, a drug analog
or derivative can mean a drug that incorporates a heteroatom
through which the drug analog or derivative is covalently bound to
the linker (L).
[0318] The drug delivery conjugates can comprise a receptor binding
ligand (B) (e.g. a folate), a linker (L), a drug, and, optionally,
heteroatom linkers to link the receptor binding ligand (B) and the
drug to the linker (L). In one illustrative embodiment, it should
be appreciated that a folate analog or derivative can mean a folate
that incorporates a heteroatom through which the folate analog or
derivative is covalently bound to the linker (L). Thus, in this
illustrative embodiment, the folate can be covalently bound to the
linker (L) through a heteroatom linker, or a vitamin analog or
derivative (i.e., incorporating an heteroatom) can be directly
bound to the linker (L). In similar illustrative embodiments, a
drug analog or derivative is a drug, and a drug analog or
derivative can mean a drug that incorporates an heteroatom through
which the drug analog or derivative is covalently bound to the
linker (L). Thus, in these illustrative aspects, the drug can be
covalently bound to the linker (L) through an heteroatom linker, or
a drug analog or derivative (i.e., incorporating an heteroatom) can
be directly bound to the linker (L). The linker (L) can comprise a
spacer linker, a releasable (i. e., cleavable) linker, and a
heteroatom linker to link the spacer linker to the releasable
linker in conjugates containing both of these types of linkers. The
linker can be a bivalent linker.
[0319] Generally, any manner of forming a conjugate between the
linker (L) and the folate or analog or derivative thereof, between
the linker (L) and the drug, or analog or derivative thereof,
including any intervening heteroatom linkers, can be utilized.
Also, any art-recognized method of forming a conjugate between the
spacer linker, the releasable linker, and the heteroatom linker to
form the bivalent linker can be used. The conjugate can be formed
by direct conjugation of any of these molecules, for example,
through complexation, or through hydrogen, ionic, or covalent
bonds. Covalent bonding can occur, for example, through the
formation of amide, ester, disulfide, or imino bonds between acid,
aldehyde, hydroxy, amino, sulfhydryl, or hydrazo groups.
[0320] In another embodiment, the (L) linker includes a chain of
atoms selected from C, N, O, S, Si, and P that covalently connects
the receptor binding ligand (B), the hydrophilic linker, and/or the
therapeutic agent (A). The linker (L) 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 (L) 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 (L). In addition, it is to be understood that the atoms
forming the linker (L) 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 (L). The linker (L) may be bivalent.
[0321] In another embodiment, pharmaceutical compositions
comprising an amount of a drug delivery conjugate effective to
eliminate a population of pathogenic cells, such as inflammatory
cells, in a host animal when administered in one or more doses are
described. The drug delivery conjugate is preferably administered
to the host animal parenterally, e.g., intradermally,
subcutaneously, intramuscularly, intraperitoneally, intravenously,
or intrathecally. Alternatively, the drug delivery conjugate can be
administered to the host animal by other medically useful
processes, such as orally, and any effective dose and suitable
therapeutic dosage form, including prolonged release dosage forms,
can be used.
[0322] Examples of parenteral dosage forms include aqueous
solutions of the conjugates, in an isotonic saline, 5% glucose or
other well-known pharmaceutically acceptable liquid carriers such
as liquid alcohols, glycols, esters, and amides. The parenteral
dosage form can be in the form of a reconstitutable lyophilizate
comprising the dose of the drug delivery conjugate. In one aspect
of the present embodiment, any of a number of prolonged release
dosage forms known in the art can be administered such as, for
example, the biodegradable carbohydrate matrices described in U.S.
Pat. Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of
which are incorporated herein by reference, or, alternatively, a
slow pump (e.g., an osmotic pump) can be used.
[0323] In one illustrative aspect, at least one additional
composition comprising a therapeutic factor can be administered to
the host in combination or as an adjuvant to the above-detailed
methodology, to enhance the drug delivery conjugate-mediated
elimination of the population of pathogenic cells, or more than one
additional therapeutic factor can be administered. The therapeutic
factor can be selected from a chemotherapeutic agent, or another
therapeutic factor capable of complementing the efficacy of the
administered drug delivery conjugate.
[0324] In one illustrative aspect, therapeutically effective
combinations of these factors can be used. In one embodiment, for
example, therapeutically effective amounts of the therapeutic
factor, for example, in amounts ranging from about 0.1
MIU/m.sup.2/dose/day to about 15 MIU/m.sup.2/dose/day in a multiple
dose daily regimen, or for example, in amounts ranging from about
0.1 MIU/m.sup.2/dose/day to about 7.5 MIU/m.sup.2/dose/day in a
multiple dose daily regimen, can be used along with the drug
delivery conjugates to eliminate, reduce, or neutralize pathogenic
cells in a host animal harboring the pathogenic cells (MIU=million
international units; m.sup.2=approximate body surface area of an
average human).
[0325] Additionally, more than one type of drug delivery conjugate
can be used. Illustratively, for example, the patient can be
treated with conjugates with different vitamins (e.g. folates), but
the same drug in a co-dosing protocol. In other embodiments, the
patient can be treated with conjugates comprising the same receptor
binding ligand (B) (e.g. a folate) linked to different drugs, or
various receptor binding ligands (B) linked to various drugs. In
another illustrative embodiment, drug delivery conjugates with the
same or different vitamins (e.g. folates), and the same or
different drugs comprising multiple vitamins and multiple drugs as
part of the same drug delivery conjugate could be used.
[0326] The unitary daily dosage of the drug delivery conjugate can
vary significantly depending on the host condition, the specific
inflammatory disease state being treated, the molecular weight of
the conjugate, its route of administration and tissue distribution,
and the possibility of co-usage of other therapeutic treatments
such as radiation therapy. The effective amount to be administered
to a patient is based on body surface area, patient weight, and
physician assessment of patient condition. In illustrative
embodiments, effective doses can range, for example, from about 1
ng/kg to about 10 mg/kg, from about 100 ng to about 1 mg, from
about 1 .mu.g/kg to about 500 .mu.g/kg, from about 1 .mu.g/kg to
about 100 .mu.g/kg, from about 1 .mu.g/kg to about 50 .mu.g/kg, and
from about 1 .mu.g/kg to about 10 .mu.g/kg. The reference to kg is
kg of patient body weight.
[0327] In another illustrative aspect, any effective regimen for
administering the drug delivery conjugates can be used. For
example, the drug delivery conjugates can be administered as single
doses, or can be divided and administered as a multiple-dose daily
regimen. In other embodiments, a staggered regimen, for example,
one to three days per week can be used as an alternative to daily
treatment, and such intermittent or staggered daily regimen is
considered to be equivalent to every day treatment and within the
scope of the methods described herein. In one embodiment, the
patient is treated with multiple injections of the drug delivery
conjugate to eliminate the population of pathogenic cells, such as
inflammatory cells. In another embodiment, the patient is injected
multiple times (preferably about 2 up to about 50 times) with the
drug delivery conjugate, for example, at 12-72 hour intervals or at
48-72 hour intervals. In other embodiments, additional injections
of the drug delivery conjugate can be administered to the patient
at an interval of days or months after the initial injections(s)
and the additional injections prevent recurrence of the disease
state caused by the pathogenic cells, such as inflammatory
cells.
[0328] In one embodiment, folates, or analogs or derivatives
thereof that can be used in the drug delivery conjugates include
those that bind to folate receptors expressed specifically on
activated macrophages or activated monocytes. The folate-linked
conjugates, for example, can be used to kill or suppress the
activity of activated macrophages or activated monocytes that cause
disease states in the patient. Such conjugates, when administered
to a patient suffering from inflammation, work to concentrate and
associate the conjugated drug in the population of inflammatory
cells to kill the inflammatory cells or suppress their function.
Elimination, reduction, or deactivation of the inflammatory cell
population works to stop or reduce the pathogenesis characteristic
of the disease state being treated. Exemplary of inflammatory
diseases include arthritis, including rheumatoid arthritis and
osteoarthritis, glomerulonephritis, proliferative retinopathy,
restenosis, ulcerative colitis, Crohn's disease, fibromyalgia,
psoriasis and other inflammations of the skin, inflammations of the
eye, including uveitis and autoimmune uveitis, osteomyelitis,
Sjogren's syndrome, multiple sclerosis, diabetes, atherosclerosis,
pulmonary fibrosis, lupus erythematosus, sarcoidosis, systemic
sclerosis, organ transplant rejection (GVHD) and chronic
inflammations is described Administration of the drug delivery
conjugate is typically continued until symptoms of the disease
state are reduced or eliminated.
[0329] As used herein the term uveitis generally refers to an
intraocular inflammatory disease including iritis, cyclitis,
panuveits, posterior uveitis and anterior uveitis. Iritis is
inflammation of the iris. Cyclitis is inflammation of the ciliary
body. Panuveitis refers to inflammation of the entire uveal
(vascular) layer of the eye. Intermediate uveitis, also called
peripheral uveitis, is centered in the area immediately behind the
iris and lens in the region of the ciliary body and pars plana, and
is also termed "cyclitis" and "pars planitis."
[0330] Autoimmune uveitis may occur as a component of an autoimmune
disorder (such as rheumatoid arthritis, Bechet's disease,
ankylosing spondylitis, sarcoidosis, and the like), as an isolated
immune mediated ocular disorder (such as pars planitis or
iridocyclitis, and the like), as a disease unassociated with known
etiologies, and following certain systemic diseases which cause
antibody-antigen complexes to be deposited in the uveal
tissues.
[0331] Illustratively, the drug delivery conjugates administered to
kill inflammatory cells or suppress their function can be
administered parenterally to the animal or patient suffering from
the disease state, for example, intradermally, subcutaneously,
intramuscularly, intraperitoneally, or intravenously in combination
with a pharmaceutically acceptable carrier. In another embodiment,
the drug delivery conjugates can be administered to the animal or
patient by other medically useful procedures and effective doses
can be administered in standard or prolonged release dosage forms.
In another aspect, the therapeutic method can be used alone or in
combination with other therapeutic methods recognized for treatment
of inflammation.
[0332] In other embodiments of the methods described herein,
pharmaceutically acceptable salts of the conjugates described
herein can be used. Pharmaceutically acceptable salts of the
conjugates described herein include the acid addition and base
salts thereof.
[0333] Suitable acid addition salts are formed from acids which
form non-toxic salts.
[0334] 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.
[0335] 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.
[0336] In various embodiments of the methods described herein, the
conjugates described herein may be administered alone or in
combination with one or more other conjugates described herein or
in combination with one or more other drugs (or as any combination
thereof). In one embodiment, the conjugates described herein may be
administered as a formulation 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 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, 21th
Edition (Lippincott Williams & Wilkins, 2005), incorporated
herein by reference.
[0337] In one illustrative aspect, a pharmaceutically acceptable
carrier includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, and combinations thereof, that are
physiologically compatible. In some embodiments, the carrier is
suitable for parenteral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions.
[0338] In various embodiments, liquid formulations may include
suspensions and solutions. Such formulations may comprise a
carrier, for example, water, ethanol, polyethylene glycol,
propylene glycol, methylcellulose or a suitable oil, and one or
more emulsifying agents and/or suspending agents. Liquid
formulations may also be prepared by the reconstitution of a solid,
for example, from a sachet.
[0339] In one embodiment, an aqueous suspension may contain the
conjugates described herein 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.
[0340] In one illustrative embodiment, dispersible powders and
granules suitable for preparation of an aqueous suspension by the
addition of water provide the conjugate in admixture with a
dispersing or wetting agent, suspending agent and one or more
preservatives. Additional excipients, for example, coloring agents,
may also be present.
[0341] 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.
[0342] In other embodiments, isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride can be
included in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, monostearate salts
and gelatin.
[0343] In one aspect, a conjugate as described herein may be
administered directly into the blood stream, into muscle, or into
an internal organ. Suitable routes for such parenteral
administration include intravenous, intraarterial, intraperitoneal,
intrathecal, epidural, intracerebroventricular, intraurethral,
intrastemal, intracranial, intratumoral, intramuscular and
subcutaneous delivery. Suitable means for parenteral administration
include needle (including microneedle) injectors, needle-free
injectors and infusion techniques.
[0344] In one illustrative aspect, parenteral formulations are
typically aqueous solutions which may contain carriers or
excipients such as salts, carbohydrates and buffering agents
(preferably at a pH of from 3 to 9), but, for some applications,
they may be more suitably formulated as a sterile non-aqueous
solution or as a dried form to be used in conjunction with a
suitable vehicle such as sterile, pyrogen-free water. In other
embodiments, any of the liquid formulations described herein may be
adapted for parenteral administration of the conjugates described
herein. The preparation of parenteral formulations under sterile
conditions, for example, by lyophilization under sterile
conditions, may readily be accomplished using standard
pharmaceutical techniques well known to those skilled in the art.
In one embodiment, the solubility of a conjugate used in the
preparation of a parenteral formulation may be increased by the use
of appropriate formulation techniques, such as the incorporation of
solubility-enhancing agents.
[0345] In various embodiments, formulations for parenteral
administration may be formulated to be for immediate and/or
modified release. In one illustrative aspect, conjugates of the
invention may be administered in a time release formulation, for
example in a composition which includes a slow release polymer. The
conjugates can be prepared with carriers that will protect the
conjugates against rapid release, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PGLA). Methods for the preparation of such
formulations are generally known to those skilled in the art. In
another embodiment, the conjugates described herein or compositions
comprising the conjugates may be continuously administered, where
appropriate.
[0346] In one embodiment, sterile injectable solutions can be
prepared by incorporating the conjugate in the required amount in
an appropriate solvent with one or a combination of ingredients
described above, as required, followed by filtered sterilization.
Typically, dispersions are prepared by incorporating the conjugate
into a sterile vehicle which contains a dispersion medium and any
additional ingredients from those described above. In the case of
sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the conjugate plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
[0347] The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable to
high drug concentration. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. In one
embodiment, the proper fluidity can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants.
[0348] In one embodiment, compositions described herein comprise a
drug delivery conjugate having a purity of at least 90%. In another
embodiment, the drug delivery conjugate has a purity of at least
95%. In another embodiment, the drug delivery conjugate has a
purity of at least 96%. In another embodiment, the drug delivery
conjugate has a purity of at least 97%. In another embodiment, the
drug delivery conjugate has a purity of at least 98%. In another
embodiment, the drug delivery conjugate has a purity of at least
99%.
[0349] The drug delivery conjugates described herein can be
prepared by art-recognized synthetic methods. The synthetic methods
are chosen depending upon the selection of the optionally addition
heteroatoms or the heteroatoms that are already present on the
spacer linkers, releasable linkers, the drug, and/or or the
receptor binding ligand (B). In general, the relevant bond forming
reactions are described in Richard C. Larock, "Comprehensive
Organic Transformations, a guide to functional group preparations,"
VCH Publishers, Inc. New York (1989), and in Theodora E. Greene
& Peter G. M. Wuts, "Protective Groups ion Organic Synthesis,"
2d edition, John Wiley & Sons, Inc. New York (1991), the
disclosures of which are incorporated herein by reference.
Additional details for preparing functional groups, including
amides and esters, ketals and acetals, succinimides, silyloxys,
hydrazones, acyl hydrazines, semicarbazones, disulfides,
carbonates, sulfonates, and the like contained in the linker,
including releasable linkers are described in U.S. patent
application publication No. US 2005/0002942 A1, incorporated herein
by reference in its entirety.
[0350] General formation of folate-peptides. The folate-containing
peptidyl fragment Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) can
be prepared by a polymer-supported sequential approach using
standard methods, such as the Fmoc-strategy on an acid-sensitive
Fmoc-AA-Wang resin (1), as shown in Scheme 1.
##STR00032##
[0351] In this illustrative embodiment of the processes described
herein, R.sub.1 is Fmoc, R.sub.2 is the desired
appropriately-protected amino acid side chain, and DIPEA is
diisopropylethylamine. Standard coupling procedures, such as PyBOP
and others described herein or known in the art are used, where the
coupling agent is illustratively applied as the activating reagent
to ensure efficient coupling. Fmoc protecting groups are removed
after each coupling step under standard conditions, such as upon
treatment with piperidine, tetrabutylammonium fluoride (TBAF), and
the like. Appropriately protected amino acid building blocks, such
as Fmoc-Glu-OtBu, N.sup.10-TFA-Pte-OH, and the like, are used, as
described in Scheme 1, and represented in step (b) by Fmoc-AA-OH.
Thus, AA refers to any amino acid starting material that is
appropriatedly protected. It is to be understood that the term
amino acid as used herein is intended to refer to any reagent
having both an amine and a carboxylic acid functional group
separated by one or more carbons, and includes the naturally
occurring alpha and beta amino acids, as well as amino acid
derivatives and analogs of these amino acids. In particular, amino
acids having side chains that are protected, such as protected
serine, threonine, cysteine, aspartate, and the like may also be
used in the folate-peptide synthesis described herein. Further,
gamma, delta, or longer homologous amino acids may also be included
as starting materials in the folate-peptide synthesis described
herein. Further, amino acid analogs having homologous side chains,
or alternate branching structures, such as norleucine, isovaline,
.beta.-methyl threonine, .beta.-methyl cysteine,
.beta.,.beta.-dimethyl cysteine, and the like, may also be included
as starting materials in the folate-peptide synthesis described
herein.
[0352] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-support peptide
2, where n is an integer and may equal 0 to about 100. Following
the last coupling step, the remaining Fmoc group is removed (step
(a)), and the peptide is sequentially coupled to a glutamate
derivative (step (c)), deprotected, and coupled to TFA-protected
pteroic acid (step (d)). Subsequently, the peptide is cleaved from
the polymeric support upon treatment with trifluoroacetic acid,
ethanedithiol, and triisopropylsilane (step (e)). These reaction
conditions result in the simultaneous removal of the t-Bu, t-Boc,
and Trt protecting groups that may form part of the
appropriately-protected amino acid side chain. The TFA protecting
group is removed upon treatment with base (step (f)) to provide the
folate-containing peptidyl fragment 3.
[0353] In each of the foregoing synthetic processes, the
intermediates may be coupled with any additional hydrophilic spacer
linkers, other spacer linkers, releasable linkers, or the
therapeutic agent A. In variations of each of the foregoing
processes, additional hydrophilic spacer linkers, other spacer
linkers, or releasable linkers may be inserted between the receptor
binding ligand B and the indicated hydrophilic spacer linkers. In
addition, it is to be understood that the left-to-right arrangement
of the bivalent hydrophilic spacer linkers is not limiting, and
accordingly, the therapeutic agent A, the receptor binding ligand
B, additional hydrophilic spacer linkers, other spacer linkers,
and/or releasable linkers may be attached to either end of the
hydrophilic spacer linkers described herein.
METHOD EXAMPLES
Example
Adjuvant-Induced Arthritis (AIA) Model
[0354] Female Lewis rats were fed a folate-deficient diet (Harlan
Teklad, Indianapolis, Ind.) for 9-10 days prior to arthritis
induction. The adjuvant-induced arthritis (AIA) was induced by
intradermal inoculation (at the base of tail) of 0.5 mg of
heat-killed Mycobacteria butyricum (BD Diagnostic Systems, Sparks,
Md.) in 100 .mu.L light mineral oil (Sigma). Ten days after
arthritis induction, paw edema in rats was assessed using a
modified arthritis scoring system: 0=no arthritis; 1=swelling in
one type of joint; 2=swelling in two types of joint; 3=swelling in
three types of joint; 4=swelling of the entire paw. A total score
for each rat is calculated by summarizing the scores for each of
the four paws, giving a maximum score of 16 for each rat. On Day 10
post arthritis induction, rats with a total arthritis score of
.gtoreq.2 were removed from the study and the remaining rats were
distributed evenly across the control and treatment groups (n=5 for
all groups except that n=2-3 for healthy controls). All treatments
started on Day 10 unless mentioned otherwise.
Example
EC0746 Demonstrated FR-Mediated Inhibition of DHFR, Viability, and
LPS-Stimulated TNF-.alpha. Production in RAW264.7 Cells
[0355] RAW264.7 cells were treated with vehicle (medium), EC0746
(100 nM) without or with 100-fold excess free folate, aminopterin
(AMT, 100 nM), methotrexate (MTX, 100 nM), or excess free folate
alone (10 .mu.M). After 1 h incubation, the drug-containing media
were replaced with fresh medium and the cells were allowed to
incubate further for 24 h. At the end of incubation, the cells were
lysed and the DHFR activity in cell lysates was measured using a
commercial DHFR assay kit (Sigma-Aldrich, Saint Louis, Mo.). See
FIG. 1.
[0356] For XTT cell viability and TNF-.alpha. inhibition assays.
RAW264.7 cells in 96-well plates were treated with vehicle (culture
medium) or 10-fold serial dilutions of EC0746 without or with
100-fold excess free folate. After 2 h incubation, the
drug-containing media were replaced and the cells were allowed to
incubate further for 70 h. Four hours prior to the end of
incubation, LPS was added to the treated cells at a final
concentration of 100 ng/mL. 100 .mu.L of the culture supernatants
were collected for TNF-.alpha. analysis using a commercial ELISA
kit. See FIG. 2B. The cell viability was assessed by adding XTT
(2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide-
) to the remaining medium for an additional 4 h following the
manufacturer's instructions (Roche Applied Science, Indianapolis,
Ind.) See FIG. 2A. Both results were expressed as % absorbance
(minus background) relative to untreated control in triplicates.
The results demonstrated that EC0746 inhibited the viability of
RAW264.7 cells and the ability of these cells to produce
TNF-.alpha. in response to LPS.
Example
EC0746 Inhibited Lps-Stimulated Cytokine Production from
Thioglycollate-Elicited Macrophages in a Fr-Dependent Manner
[0357] To obtain thioglycollate-elicited macrophages, female Lewis
rats were dosed once intraperitoneally with an aged thioglycollate
medium (20 ml/kg) and euthanized 3 days later. The peritoneal
cavity of the animals was lavaged with 60-70 ml of ice-cold PBS
buffer to collect peritoneal extrudate. Thioglycollate-elicited
macrophages in the peritoneal fluids were obtained after a red cell
lysing step and a 2-hour adherence in cell culture medium
containing 1% heat-inactivated fetal bovine serum.
[0358] Rat thioglycollate-elicited macrophages were treated with
medium only, methotrexate (100 nM), aminopterin (100 nM), EC0746
(100 nM) without or with 100-fold excess free folate (10 .mu.M), or
excess free folate alone (10 .mu.M). The drug-containing media were
removed after 2 h incubation and the cells were allowed to incubate
further for an additional 70 h in fresh medium. Twenty-four hours
prior to the end of incubation, LPS (5 .mu.g/mL) and IFN-.gamma.
(100 ng/mL) were added to the above cells to stimulate cytokine
production. Cytokines (TNF-.alpha., IL-1.alpha., IL-6, IL-10,
MIP-1.alpha., etc.) released into the cell culture medium were
measured using a rat cytokine array assay kit (R&D Systems,
Minneapolis, Minn.). See FIG. 3.
Example
EC0746 Treatment Reduced Local (Paw) and Systemic (Spleen)
Inflammation in Rats with Adjuvant Arthritis
[0359] Rats with adjuvant arthritis were treated subcutaneously
with EC0746 (500 nmol/kg) on days 10, 13, 17, and 20 post arthritis
induction. The animals in the healthy and arthritis control groups
were left untreated. The arthritis scores and animal body weights
were recorded three times a week (see FIG. 4A, B). At the
completion of study (days 24-25), the rats were euthanized by
CO.sub.2 asphyxiation and processed for paw and spleen weights. The
results showed that EC0746-treatment rats had significantly less
arthritis score, paw weight (i.e. paw swelling), and spleen weights
(see FIG. 5A, B). The overall reduction of local and systemic
inflammation in the EC0746-treated rats rendered these animals
better body weight compared to animals in the untreated arthritis
control group.
Example
EC0746 Treatment Prevented Bone Damage in Rats with Adjuvant
Arthritis
[0360] Rats with adjuvant arthritis were treated subcutaneously
with EC0746 (500 nmol/kg) on days 10, 13, 17, and 20 post arthritis
induction. The animals in the healthy and arthritis control groups
were left untreated. At the completion of study, rat hind paws were
photographed and the animals were euthanized by CO.sub.2
asphyxiation. X-ray radiographic images of hind paws were taken
immediately using a Kodak Imaging Station In Vivo FX (Carestream
Molecular Imaging, New Haven, Conn.). The representative X-ray
images of hind paws were shown for a healthy rat and EC0746-treated
or untreated arthritic rats. Compared to severe bone erosion seen
with the untreated arthritis control animal, EC0746 treatment
starting at the on-set of arthritis development (day 10)
effectively halted paw swelling/inflammation and prevented bone
erosion. See FIG. 6A, B.
Example
EC0746 was as Effective as Mtx at Equal Molar Subcutaneous
Doses
[0361] Rats with adjuvant arthritis were treated subcutaneously
with EC0746 (300 nmol/kg) or methotrexate (MTX, 300 nmol/kg) on
days 10, 13, 17, and 20 post arthritis induction. The animals in
the healthy and arthritis control groups were left untreated. The
arthritis score and animal body weight were recorded three times a
week (see FIG. 7A, B). At the completion of study, the rats were
euthanized by CO.sub.2 asphyxiation and processed for paw and
spleen weights. The results showed that biweekly subcutaneously
dosed EC0746 and methotrexate were similarly active in reducing
local (paw) and systemic (spleen) inflammation in these arthritic
animals. See FIG. 8A, B.
Example
Anti-Arthritis Activity of EC0746 (but not MTX) could be Partially
Blocked by a Co-Injected Folate Competitor
[0362] Rats with adjuvant arthritis were treated subcutaneously
with EC0746 (500 nmol/kg) or MTX (500 nmol/kg) in the absence or
presence of 300-fold excess of Re-EC0589 (150 .mu.mol/kg, Rhenium
complex of EC0589) or Re-EC20 (150 .mu.mol/kg, Rhenium complex of
EC20), respectively, on days 10, 13, 17, and 20 post arthritis
induction. The arthritis score and animal body weight were recorded
three times a week. Both EC0589 and Re-EC20 served as a FR-binding
competitor. The animals in the arthritis control group were left
untreated. The results showed that the anti-arthritis activity of
EC0746 but not MTX could be partially blocked by excess amount of a
FR-binding competitor.
##STR00033##
Example
Collagen-Induced Arthritis (CIA) Model
[0363] The collagen-induced arthritis (CIA) was induced in female
Lewis rats on folate-deficient diet (Harlan Teklad, Indianapolis,
Ind.). On Day 0, rats were immunized with 500 .mu.g of bovine
collagen Type II (Chondrex, Redmond, Wash.) formulated with
Freund's complete adjuvant. A booster immunization was given on Day
7 with 250 .mu.g of the bovine collagen formulated with Freund's
incomplete adjuvant. Arthritis disease was assessed by a
qualitative clinical score system described by the manufacturer
(Chondrex, Redmond, Wash.): 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 of wrist,
3=Severe redness and swelling of the entire paw including digits,
and 4=Maximally inflamed limb with involvement of multiple joints.
On Day 10 post first immunization, rats were distributed evenly
(according to the arthritis score) across the control and treatment
groups. The CIA rats were given ten consecutive subcutaneous doses
of EC0746 and methotrexate on days 10-19. For both drugs, an
induction dose (500 nmol/kg) was given on days 10 and 15 and a
maintenance dose (100 nmol/kg) was given on days 11-14 and 16-19.
The animals in the arthritis control group were left untreated. The
arthritis score and animal body weight were recorded five times a
week. The result showed that EC0746 was also effective in rats with
collagen-induced arthritis. See FIG. 13A, B.
Example
EC0746 Plasma Pharmacokinetics after a Single S.C. Dose
[0364] Female Lewis rats with rounded tip jugular vein catheters
(Harlan) were fed regular rodent diet and used in this study. The
animals were given a single subcutaneous dose of EC0746 at 500
nmol/kg. Whole blood samples (300 .mu.l) were collected from the
animals at the following time points: 1 min, 10 min, 30 min, 1 h, 2
h, 3 h, 4 h, and 8 h after injection. The blood samples were placed
into anti-coagulant tubes containing 1.7 mg/mL of K3-EDTA and 0.35
mg/mL of N-Maleoyl-beta-alanine (0.35 mg/mL). Plasma samples were
obtained by centrifugation for 3 min at .about.2,000 g and stored
at -80.degree. C. The amount of EC0746 and its released base drugs
(EC0470 & aminopterin) were determined by HPLC using the EC0746
injection solution as the standard (see FIG. 14). The result showed
that approximately 18% of free drug exposure/release (EC0470 &
aminopterin) was detected in the plasma after a single subcutaneous
dose of EC0746. However, the Tmax of EC0746 was observed at
.about.30 min while EC0470 and aminopterin showed a delayed Tmax at
.about.1 h. See FIG. 14. A similar method was used to determine the
plasma pharmacokinetics of aminopterin (see FIG. 15).
Example
Maximum Tolerated Dose (MTD) of Aminopterin and EC0746
[0365] Healthy rats were administered a subcutaneous injection of
the indicated dose of aminopterin or EC0746 biweekly for 2 weeks;
control animals, no treatment, 100 nmole/kg aminopterin, 50
nmole/kg aminopterin, 500 nmole/kg EC0746, or 2000 nmole/kg EC0746.
The animals were weighed daily. See FIG. 16A, B. A dose of 0.1
.mu.mol/kg of aminopterin in folate deficient rats is above the
MTD; therefore, the projected MTD of EC0746 would be <0.5
.mu.mol/kg based solely on .about.20% free drug release shown in
the previous Example. However, the MTD of EC0746 is actually 2.0
.mu.mol/kg, which is equivalent to 0.4 .mu.mol/kg, or--8.times.
higher than the MTD for free aminopterin. On a molar basis for
total aminopterin, the MTD is 40.times. higher. The therapeutic
index (MTD for healthy animals/ED.sub.50 for treatment of
adjuvant-induced arthristis in female Lewis rats) for EC746 is
about 9.5 (2000/210).
Example
Mechanism of Action: EC0932 as a Folate-Targeted Antifolate
[0366] RAW264.7 cells in 96-well plates were treated with vehicle
(culture medium) or 10-fold serial dilutions of EC0932 without or
with 100-fold excess free folate. The drug-containing medium was
replaced after 2 h treatment and the cells were allowed to incubate
further in standard medium for 70 h. Four hours prior to the end of
incubation, LPS was added to the treated cells at a final
concentration of 100 ng/mL. 100 .mu.L of the culture supernatants
were collected for TNF-.alpha. analysis using a commercial ELISA
kit (see FIG. 17B). The cell viability was assessed by adding XTT
(2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide-
) to the remaining medium for an additional 4 h following the
manufacturer's instructions (Roche Applied Science, Indianapolis,
Ind.). See FIG. 17A. Both results were expressed as % absorbance
(minus background) relative to untreated control in triplicates.
The results demonstrated that EC0932 inhibited the viability of
RAW264.7 cells and their ability to produce TNF-.alpha. in response
to LPS.
Example
Mechanism of Action: EC0932 as a Folate-Targeted mTOR Inhibitor
[0367] RAW264.7 cells were treated with medium only (UTC), 100 nM
everolimus (mTOR inhibitor), 100 nM aminopterin (antifolate), 100
.mu.M EC0823 (C, a FR-binding competitor), or 100 nM EC0932 without
or with 100 .mu.M of EC0823. The drug-containing media were removed
after 2 h and the cells were allowed to incubate for 4 h in fresh
medium. Afterwards, the cell lysates were collected and subjected
to Western blot analysis for phosphorylation of S6 ribosomal
protein (p-RPS6), a downstream target in the mTOR signaling
pathway. The data showed that EC0932 treatment resulted in
down-regulation of p-RPS6 and its inhibitory effect could be
partially blocked by excess EC0823, a FR-binding competitor. See
FIG. 18.
##STR00034##
Example
EC0932 Demonstrates FR-Specific Activity Against Adjuvant
Arthritis
[0368] Rats with adjuvant arthritis were treated subcutaneously
with EC0932 (250 nmol/kg) in the absence or presence of 500-fold
excess of EC0923 (125 .mu.mol/kg) on days 10, 13, 17, and 20 post
arthritis induction. The animals in the healthy and arthritis
control groups were left untreated. The arthritis scores and animal
body weights were recorded three times a week (see FIG. 19A, B). At
the completion of study (day 24), the rats were euthanized by
CO.sub.2 asphyxiation and processed for paw and spleen weights (see
FIG. 20A, B). The results showed that the anti-arthritis activity
of EC0932 was blocked by EC0923, a folate competitor with similar
affinity as free folic acid.
##STR00035##
Example
EC0828 Demonstrates FR-Specific Activity Against Adjuvant
Arthritis
[0369] Using similar methods to those herein described, the effect
of EC0828 on arthritis was measured. See FIGS. 21-24.
Example
Relative Folate Receptor Affinity
[0370] Using displacement of .sup.3H-folic acid from
folate-receptor-.alpha. positive KB cells the relative folate
affinity (FA) for several compounds was measured. Folic acid (1.0),
aminopterin (0.008), methotrexate (0.018), EC0746 (0.5), and EC0932
(0.26). See FIGS. 25A-D.
[0371] Using displacement of .sup.3H-folic acid from
folate-receptor-beta positive CHO-FR-.beta. cells, the relative
folate affinity (FA) for several compounds was measured. Folic acid
(1.0), aminopterin (0.004), methotrexate (0.005), and EC0746
(0.27). See FIG. 25F.
Example
EC0746 is a High-Affinity FR-Specific DHFR Inhibitor
[0372] The FR-binding affinity of EC0746 was directly compared to
that of aminopterin (AMT) and methotrexate (MTX) in a competitive
binding assay using KB cells as the source of FR. .sup.3H-folic
acid was used as the competitive ligand and the relative affinity
of folate itself was set to 1. As shown in FIGS. 25A-D, EC0746
displayed a much higher affinity to KB cells (FR-.alpha. positive)
than AMT and MTX with relative affinity values of 0.50, 0.004, and
0.018 respectively. To demonstrate FR-specific activities in-vitro,
we first tested the ability of EC0746 to inhibit DHFR, an
intracellular target involved in cellular division. FR-positive
RAW264.7 cells were given a 2-h pulse of 100 nM EC0746 without or
with 100-fold excess folic acid (folate competition) followed by a
22-h "chase" in a drug-free medium. As shown in FIG. 25E, EC0746
inhibited DHFR activity in RAW264.7 cells to a similar degree as
AMT and MTX, but excess folic acid completely abolished its
inhibitory effect. Since excess folic acid alone (10 .mu.M) did not
have any impact, our data suggested that EC0746 may be similar to
AMT and MTX with regard to DHFR inhibition but its activity is
FR-specific.
Example
EC0746 Demonstrates an Anti-Proliferative Effect on RAW264.7
Cells
[0373] To study the anti-proliferative effect of EC0746, RAW264.7
cells (.about.40% confluency) were given a 2 h treatment of serial
dilutions of EC0746 without or with excess folic acid and followed
by a 70-h "chase" in drug-free medium. At 4 h before the end of
incubation, LPS (100 ng/ml) was added to the culture medium to
stimulate cytokine production. As determined by the XTT assay (FIG.
26A), EC0746 showed a dose-dependent inhibition of cell
proliferation (ED.sub.50=0.3 nM); however, the maximum effect was
.about.50% when compared to the untreated cells. The EC0746-treated
RAW264.7 cells produced less TNF-.alpha. upon LPS exposure
(ED.sub.50=1.6 nM) (FIG. 26B). The anti-proliferative and anti-TNF
activities of EC0746 were 100% competable with excess folic acid.
Interestingly, RAW264.7 cells that had "survived" the EC0746
treatment at .gtoreq.1 nM concentrations showed no sign of
additional growth when redispersed in fresh medium for 72 h.
Because DHFR is an S-phase enzyme that increases during the S-phase
of mitosis, RAW264.7 cells pre-treated with EC0746 were stained
with propidium iodide and analyzed for the status of cell cycle and
the expression of proliferating cell nuclear antigen (PCNA). For
flow cytometric analysis (FACS) of the cell cycle, the cells were
recovered, fixed in 70% ethanol/PBS solution, and resuspended in
FACS buffer (PBS supplemented with 1% BSA). After a brief treatment
with RNase A (Roche Molecular Biochemicals), the cells were stained
with 50 .mu.g/ml of propidium iodide (Invitrogen, Carlsbad, Calif.)
before FACS analysis. Western blot analysis was carried out on
whole cell lysates using a monoclonal antibody specific for PCNA
(PC10, Cell Signaling, Danvers, Mass.). After incubation with a
peroxidase-conjugated secondary antibody, the signals were
visualized using SuperSignal West Pico Chemiluminescent Substrate
system (ThermoScientific, Waltham, Mass.) following the
manufacturer's instructions. The images were acquired using a G:BOX
Chemi HR 16 gel imaging system (Syngene, Frederick, Md.). As shown
in FIGS. 26C-F, EC0746 treated RAW264.7 cells showed an increase in
number of cells in the S-phase, but the effect was again competable
by excess folic acid. Western blot analysis indicated no change in
PCNA expression in EC0746-treated and untreated cells (FIG. 26G).
Taken together, these data demonstrated that EC0746 completely
halted the proliferation of RAW264.7 cells in a FR-dependent
manner, but did not appear to kill them; instead, the cells were
arrested at the S-phase of the cell cycle.
Example
EC0746 Modulates Cytokine Responses in Rat Thioglycollate-Elicited
Macrophages
[0374] Because rat TG-macs are responsive to inflammation stimuli
in-vitro, we examined the ability of EC0746 to block cytokine
response after exposure to LPS and IFN-.gamma., two signals
required for full activation of macrophages. Thus, rat TG-macs were
treated with 100 nM of EC0746 following our standard condition of 2
h pulse plus a 70-h chase and without or with folate competition.
At 24 h prior to the end of incubation, LPS (5 .mu.g/mL) and
IFN-.gamma. (100 ng/mL) were added to the culture medium to
stimulate the release of cytokines, chemokines, and other
inflammatory mediators. As detected with a rat cytokine antibody
array (FIG. 27), EC0746 inhibited a range of cytokines/chemokines,
11 of which showed a significant FR-specific inhibition (P<0.05,
EC0746 versus EC0746 plus folic acid), including IL-1.beta.,
IL-1.gamma.a, MIP-1.alpha., TNF-.alpha., VEGF, CINCs, sICAM, LIX,
L-selectin, and MIG. These data also indicated that (i) the levels
of FRs on rat TG-macs were sufficient for EC0746 to take a remedial
effect on cytokine responses associated with macrophage activation
and (ii) the observed anti-inflammatory action of EC0746 can be
independent of macrophage proliferation.
Example
EC0746 Ameliorates Local and Systemic Inflammation in Rats with
Adjuvant Arthritis
[0375] The rat adjuvant induced arthritis (AIA) model resembles the
characteristics of RA in humans and has been widely used to study
the impact of novel anti-inflammatory agents. In this study, AIA
rats with early-onset or established disease (mean arthritis scores
of 0 and 2, respectively) were given a biweekly regimen of EC0746
(s.c., 500 nmol/kg/dose) for 2-week starting on day 10-13 after
arthritis induction. Multiple study endpoints were taken to assess
the efficacy including (i) arthritis score and body weight obtained
during the course of therapy and (ii) paw and spleen weights
collected 4 days after the last dose. As measured by the
semiquantitative visual scoring system, EC0746 was found to be very
effective in rats with low arthritis at the first day of treatment
(FIG. 28A). In rats with more established AIA, the same treatment
improved, although to a lesser degree, the overall severity of the
joint disease. While the untreated arthritic controls lost
approximately 14-16% of their original weight, the animals treated
with EC0746 from the early-onset maintained a good body weight
(FIG. 28B). On the other hand, EC0746 did not stop the animals with
more established arthritis at the time of treatment from losing
weight, but to a lesser extent in relation to the untreated
arthritic controls. When the percent increase in paw/spleen weights
was evaluated (FIG. 28C), EC0746 treatment resulted in
approximately 10-(paw) and 3-fold (spleen) improvements in rats
with low arthritis, and the corresponding improvements in rats with
more established arthritis were at .about.2.5- and 2-fold,
respectively (see P values in the figure legend).
[0376] To further investigate dose-response relationship and
schedule-dependency, EC0746 was administered to rats with
developing AIA (day 10) at 25-500 nmol/kg/dose (biweekly) or 1000
nmol/kg/dose (once weekly). As summarized in Table 1, below,
biweekly EC0746 dosed for 2 weeks displayed a linear dose response
in inhibiting paw edema from 25 to 250 nmol/kg/dose with an
R-squared value of 0.997, and no statistical differences between
250 and 500 nmol/kg/dose were seen. When dosed once weekly for 2
weeks, EC0746 at 1000 nmol/kg/dose remained to be highly effective,
but this schedule did not control the fast progressing AIA to the
same degree as the biweekly regimen at 250-500 nmol/kg/dose.
Overall, EC0746 was found very effective against AIA and capable of
controlling local (joints) and systemic (spleen) inflammation and
halting the progression of arthritis.
TABLE-US-00001 TABLE 1 % Inhibition % Dose in paw Weight Treatment
(nmol/kg) Frequency edema.sup.1 Splenomagly.sup.2 loss.sup.3
Control -- -- 0 117 .+-. 22 -16 .+-. 1 EC0746 25* biw 0 .+-. 25* 73
.+-. 8 -17 .+-. 1 (sc) 100* biw 35 .+-. 11* 52 .+-. 13 -14 .+-. 1
250* biw 91 .+-. 4* 25 .+-. 6 -0.5 .+-. 3 500 biw 91 .+-. 9 37 .+-.
7 0.4 .+-. 4 1000 qw 72 .+-. 12 39 .+-. 5 -7 .+-. 3 MTX 250 biw 70
.+-. 5 42 .+-. 17 -14 .+-. 2 (oral) 1650 qw 47 .+-. 10.sup..dagger.
-- -10 .+-. 2 MTX (sc) 250 biw 78 .+-. 10 24 .+-. 3 -2 .+-. 4 500
biw 88 .+-. 7 20 .+-. 4 -2 .+-. 7 Enbrel 10 q3d 46 .+-. 9 42 .+-. 7
-15 .+-. 2 (sc) mg/kg .sup.1%Inhibition in paw edema is calculated
based on paw weight on Day 24: 100.times. (Arthritic control -
Treated) / (Arthritic control - Healthy). .sup.2Splenomagly is
defined as % increase in spleen weight relative to the spleen
weights of healthy rats. .sup.3On Day 24 relative to body weight on
the first day of treatment. * Linear regression analysis showed an
R-squared value of 0.997. .sup..dagger. Calculated based on
arthritis scores on Day 24 (paw weights were not obtained).
Example
EC0746 Anti-Arthritic Activity was FR-Mediated and Different from
MTX
[0377] RA patients receiving MTX are frequently given folate
supplementation to reduce its side effects. We found simply mixing
AMT with free folic acid (1:1) to match the dose of EC0746 (500
nmol/kg/dose, biweekly) was 100% lethal to AIA rats. Thus, the
anti-arthritic effect of EC0746 in AIA rats was not due to the
apparent "folate supplementation of AMT". Given the difference in
FR-binding affinity, we suspected that EC0746 and its active
comparator MTX would have a different mechanism of action in the
AIA model where FR-positive macrophages are abundant. To verify
this theory, we undertook in-vivo folate competition studies using
EC0923, a water-soluble folate-containing competitor (relative
affinity value of 0.84 on KB cells), as a FR-blocking agent in the
AIA rats. All test compounds (EC0746, 250 nmol/kg/dose; MTX, 250
nmol/kg/dose; EC0923, 125 .mu.mol/kg/dose) were administered
biweekly for 2 weeks. EC0923 was pre- and co-administered at a
total of 500-fold excess of EC0746 and MTX.
[0378] As shown in FIG. 29, EC0746 alone was very effective, but
its activity could be nearly completely blocked by the presence of
excess EC0923. This included all parameters assessed: arthritis
score (FIG. 29A), change in body weight (FIG. 29B), and percent
increases in paw and spleen weights (FIG. 29C, D) (see P values in
the figure legend). EC0923 treatment alone did not have any impact
on the development or severity of the disease compared to the
untreated arthritic controls (FIG. 29). Radiographic analysis (hind
paws) confirmed severe periarticular soft tissue swelling, joint
space narrowing, bone erosion, periostal new bone formation, and
osteolysis in arthritic control animals and the animals treated
with EC0923 (FIG. 29E, F). There were minimum radiographic changes
seen in EC0746-treated animals, but the animals treated with EC0746
plus EC0923 showed a significant joint damage (P<0.05).
Histologically, animals treated with EC0923 alone were also similar
to untreated arthritic controls in all parameters assessed (i.e.
ankle inflammation, bone resorption, pannus formation, and
cartilage damage) (FIG. 30A). As shown in FIG. 30B, the mean sum of
histology scores (out of a maximum score of 20) was .about.15.3 and
14.2 for the arthritic control and EC0923-treated animals,
respectively. The corresponding dorsal to ventral paw thickness was
.about.9.8 mm and .about.9.4 mm, compared .about.5.8 mm for the
healthy animals (FIG. 30C). In contrast, 3 of 5 animals treated
with EC0746 had no lesions (FIG. 30D), resulting in 88-100%
decreases in individually scored parameters (FIG. 30A) and an
overall 94% decrease in summed scores when compared to untreated
arthritic controls (FIG. 30B). The dorsal to ventral thickness of
EC0746-treated paws was also significantly decreased by 94% (FIG.
30C). The animals treated with EC0746 plus the 500-fold excess
EC0923 did have significantly decreased inflammation scores (24%),
but all other scored parameters were non-significantly decreased
(9-21%, FIG. 30A, B). The dorsal to ventral paw thickness in
EC0746/EC0923-treated paws was also significantly decreased by 22%
from that of the arthritic control animals (FIG. 30C), suggesting a
small non FR-targeted effect. On an equimolar basis, s.c. MTX
treatment (250 nmol/kg/dose, 4 dose.times.2 weeks) also
significantly improved the development and severity of arthritis
(Table 1, above, FIG. 31). However, the anti-arthritic activity of
MTX was not significantly blocked by excess EC0923 in arthritis
score (FIG. 31A) and percent increases in paw and spleen weights
(FIG. 31B). The animals treated with MTX plus the folate competitor
did actually lose more body weight compared to the animals treated
with MTX (FIG. 31C). Taken together, our data suggested that EC0746
and MTX are different from each other in terms of treating active
inflammation via FR-targeted and non-targeted mechanisms of action,
respectively.
Example
EC0746 is More Efficacious than Oral Methotrexate and Subcutaneous
Etanercept
[0379] Since methotrexate (MTX) and etanercept are part of the
current standard of care for RA, we compared EC0746 against both
drugs in rats with adjuvant arthritis using clinically relevant
dosing routes. The animals were treated with subcutaneous EC0746
(250 nmo/kg) and oral MTX (250 nmol/kg) on days 10, 13, 17, and 20
post arthritis induction. Etanercept was given subcutaneously at 10
mg/kg once every 3 days starting on day 10. At the completion of
any study (day 24), rats were euthanized by CO.sub.2 asphyxiation
and processed for paw weight (cut at the hairline) and spleen
weight. The differences in total paw weight between arthritic rats
after treatment and that of healthy rats from the same experiment
were used to verify the extent of paw edema. The removed hind paws
were immersion-fixed in 10% buffered formalin and subjected to
radiographic and/or histopathological analyses. In some cases,
X-ray radiographic images of the arthritic hind paws were taken
using a Kodak Imaging Station In Vivo FX system (Carestream
Molecular Imaging, New Haven, Conn.).
[0380] As shown in FIG. 29 and Table 1, above, subcutaneously
administered EC0746 (250 nmol/kg/dose, 4 doses.times.2 weeks) was
found more efficacious than oral MTX on an equimolar basis in most
clinical and radiographic parameters assessed: arthritis score
(FIG. 29A), change in body weight (FIG. 29B), percent inhibition in
paw edema (Table 1, above, calculated from FIG. 29C) and
radiographic score (FIG. 29E, F). Although EC0746 and MTX-treated
arthritic animals had significantly decreased spleen weight
compared to arthritic controls, they were not statistically
different from each other (FIG. 29D, Table 1, above). In the same
study, EC0746 was found more effective than etanercept in all
parameters assessed except for the spleen. Histological grading of
arthritis compared to the untreated controls showed oral
MTX-treated animals had significant reductions (66-84%) in all
scored parameters (i.e. ankle inflammation, bone resorption, pannus
formation, and cartilage damage (FIG. 30A). There was a 73%
significant decrease in the summed histological score (FIG. 30B).
While etanercept was not as effective as oral MTX, animals treated
with etanercept also had significant reductions in inflammation
(42%) and bone resorption (55%), which contributed to a significant
43% decrease in the summed score (FIG. 30A, B). The dorsal to
ventral paw thicknesses in both MTX and etanercept-treated rats
were significantly decreased by 63% and 40%, respectively (FIG.
30C). Overall, EC0746 consistently outperformed both oral MTX and
s.c. etanercept in all histological parameters assessed, with
further decreases in the summed scores and dorsal to ventral paw
thicknesses.
Example
EC0746 is Less Toxic than Aminopterin and Methotrexate in
Folate-Deficient Rats
[0381] Since the toxicity of antifolates can be easily masked by
rodent diets enriched with folate, healthy rats on a
folate-deficient diet (Harlan) were used to determine the
maximum-tolerated-dose levels (MTD) of EC0746, AMT and MTX. The
animals were given biweekly injections of EC0746, AMT, and MTX for
two weeks. The MTD dose was defined as the dose that had caused at
least 13-14% weight loss combined with clinical signs of stress and
at least one animal in the >MTD dose group needed to be
euthanized. Standard hematologic and blood chemistry parameters
were examined as needed along with histopathology. The MTDs of
EC0746, MTX and AMT were determined to be 2000, 1000, and 50
nmol/kg, respectively. At above the MTD dose level, the main
toxicities of EC0746 were similar to those of AMT and MTX,
including manifested gastrointestinal distress (diarrhea), swollen
muzzle, immunosuppression (bone marrow, thymus), low
white-blood-cell count, low platelet count, and infections. While
immunosuppression is the dose-limiting toxicity of all three of
these compounds, EC0746 at its MTD dose in rats showed less
gastrointestinal-associated toxicities than AMT and MTX. Overall,
EC0746 was approximately 40-fold less toxic than AMT and 2-fold
less toxic than MTX on an equimolar basis in these folate-deficient
animals; however, its toxicity profile at above MTD levels was not
dissimilar from that of AMT and MTX.
Example
EC0746 Pharmacokinetics in Rats
[0382] EC0746 is bioavailable after subcutaneous administration in
rats and has a serum protein binding of .about.46%. Both AMT and
AMT hydrazide are anticipated metabolites because EC0746 contains a
hydrazide/disulfide-based releasable linker. Notably, AMT hydrazide
and AMT are equally potent on RAW264.7 cells by inhibiting cell
proliferation (FIG. 32A) and LPS-stimulated TNF-.alpha. production
(FIG. 32B). Thus, the plasma concentrations of EC0746 and two
primary metabolites, AMT and AMT hydrazide, were determined by
LC/MS/MS after a single subcutaneous EC0746 administration. As
shown in FIG. 33A, subcutaneously administered EC0746 (500 nmol/kg)
reached the blood stream within minutes, peaked around 10-30 min,
and maintained a plateau until 60 minutes. EC0746 was cleared
rapidly from the blood with an elimination half-life of .about.35
min. Interestingly, the peak appearances of AMT and AMT hydrazide
in the plasma were nearly superimposable in the EC0746-dosed rats
with a 30-min delay from the EC0746 C.sub.max. For comparison, the
pharmacokinetics of subcutaneously dosed AMT (500 nmol/kg) was also
examined (FIG. 33B). The AMT C.sub.max was more similar to that of
EC0746 than to those of EC0746-derived AMT/AMT hydrazide seen in
FIG. 33A. However, the elimination half-life of subcutaneously
administered free AMT was .about.140 min, more similar to that of
AMT (.about.117 min) and AMT hydrazide (.about.187 min) released
from EC0746. Based on area-under-the-curve, .about.18% of active
drug exposure/release (AMT plus AMT hydrazide) was detected in the
plasma over 8 h collection period in the EC0746 dosed animals (FIG.
33C).
Example
Animal Experimental Autoimmune Uveitis Model
[0383] Experimental autoimmune uveitis (EAU) was induced in female
Lewis rats maintained on a folate-deficient diet (Harlan Teklad,
Indianapolis, Ind.). On Day 0, the animals were immunized
subcutaneously with 25 .mu.g of bovine S--Ag PDSAg peptide
formulated with Freund's incomplete adjuvant containing 0.5 mg of
M. Tuberculosis H37Ra. Purified pertussis toxin (PT) was given at a
dosage of 1 .mu.g per animal on the same day via intraperitoneal
injection. The severity of uveitis in each eye was assessed by a
qualitative visual score system: 0=No disease, eye is translucent
and reflects light (red reflex); 0.5 (trace)=Dilated blood vessels
in the iris, 1=Engorged blood vessels in iris, abnormal pupil
contraction; 2=Hazy anterior chamber, decreased red reflex;
3=Moderately opaque anterior chamber, but pupil still visible, dull
red reflex; and 4=Opaque anterior chamber and obscured pupil, red
reflex absent, proptosis. This assessment yields a maximum uveitis
score of 8 per animal. FIG. 34 shows images the eyes of an animal
(upper right) with severe uveitis on its right eye (bottom) and a
healthy eye (upper right).
Example
EC0746 Treatment Effectively Reduced EAU Inflammation
[0384] Animals treated according to the preceding method to induce
EAU were randomized and distributed into two groups: (1) the
untreated experimental autoimmune uveitis control group and (2) the
EC0746 treated experimental autoimmune uveitis group. The animals
in the experimental autoimmune uveitis control group were
untreated. The animals in the EC0746 treatment group were given
subcutaneous doses of EC0746 at a dosage of 500 nmol/kg every other
day starting on day 7 after EAU induction. The uveitis score and
animal body weight were recorded for each animal on days 7-9 and
11-15, see FIG. 35 (the uveitis score, calculated as described in
the preceding example, is shown). On day 19, the animals were
euthanized and the aqueous humor samples were collected from the
anterior chamber for total protein analysis (see FIG. 37).
Increased protein levels in aqueous humor are symptomatic of ocular
inflammation.
Example
Adjuvant-Induced Arthritis (AIA) Model
[0385] Female Lewis rats were fed a folate-deficient diet (Harlan
Teklad, Indianapolis, Ind.) for 9-10 days prior to arthritis
induction. The adjuvant-induced arthritis (AIA) was induced by
intradermal inoculation (at the base of tail) of 0.5 mg of
heat-killed Mycobacteria butyricum (BD Diagnostic Systems, Sparks,
Md.) in 100 .mu.L light mineral oil (Sigma). Ten days after
arthritis induction, paw edema in rats was assessed using a
modified arthritis scoring system: 0=no arthritis; 1=swelling in
one type of joint; 2=swelling in two types of joint; 3=swelling in
three types of joint; 4=swelling of the entire paw. A total score
for each rat is calculated by summarizing the scores for each of
the four paws, giving a maximum score of 16 for each rat. On Day 10
post arthritis induction, rats with a total arthritis score of
.gtoreq.2 were removed from the study and the remaining rats were
distributed evenly across the control and treatment groups (n=5 for
all groups except that n=2-3 for healthy controls). All treatments
started on Day 10 unless mentioned otherwise.
Example
EC0565 Mediated Fr-Specific Inhibition of mTOR Signaling in
Macrophages
[0386] To examine the targeting effect of EC0565 on FR-positive
macrophages, RAW264.7, thioglycolate-elicited macrophages
(TG-macs), and arthritic macrophages from AIA rats (AIA-macs) were
treated with medium only (UTC), everolimus (10 and 100 nM), EC0565
(1, 10, 30, and 100 nM), or EC0565 (1, 10, 30, and 100 nM) plus 100
.mu.M excess of a folate competitor (EC17 or free folate). The
drug-containing media were removed after 1 h and the cells were
allowed to incubate from 6 h in fresh medium. Afterwards, the cell
lysates were collected and subjected to Western blot analysis for
phosphorylation of S6 ribosomal protein (p-RPS6), a downstream
target in the mTOR signaling pathway.
[0387] EC0565 treatment resulted in down-regulation of p-RPS6 at
nanomolar concentrations in all macrophages tested (see FIGS.
38A-C). EC0565 appeared to be less potent than everolimus (see FIG.
38B), but its inhibitory effect was dose dependent (see FIG. 38C)
and mediated by the FR (see FIGS. 38A-C). The presence of excess
EC17 (see FIG. 38A, a folate-containing ligand) or free folic acid
(see FIG. 38B, C) reversed the effect EC0565 on these cells. More
importantly, despite the lower FR expression in TG-macs and
AIA-macs than in RAW264.7 cells, these results suggested that the
amount of FRs on these ex-vivo isolated macrophages were sufficient
to deliver a FR-specific target inhibition of the mTOR-signaling
pathway.
Example
Subcutaneously Dosed EC0565 and Everolimus were Similarly
Active
[0388] AIA rats were treated subcutaneously (twice a week) with
EC0565 (500 nmol/kg) and everolimus (500 nmol/kg) on days 10, 13,
17, and 20. The animals in the healthy and arthritis control groups
were left untreated. The arthritis scores (see FIG. 39) and animal
body weights (see FIG. 40) were recorded three times a week. At the
completion of study (day 24), the rats were euthanized by CO.sub.2
asphyxiation and processed for paw and spleen weights (see FIGS. 41
and 42, respectively). Without being bound be theory, it is
believed that, given its low water solubility, the bioavailability
of everolimus after subcutaneous administration was lower than that
of EC0565. In this study, EC0565 was shown to be as active or more
active as everolimus against adjuvant-induced arthritis.
Example
Radiographic Analysis Confirmed Less Tissue/Bone Damage in
EC0565-Treated Arthritic Paws
[0389] AIA rats were treated subcutaneously (twice a week) with
EC0565 (500 nmol/kg), everolimus (500 nmol/kg), and methotrexate
(190 nmol/kg) on days 10, 13, 17, and 20. The animals in the
healthy and arthritis control groups were left untreated. At the
completion of study (day 24), the rats were euthanized by CO.sub.2
asphyxiation and the hind paws were fixed in 10% PBS-buffered
formalin and subjected to radiographic analysis. All radiographs
were evaluated by a board-certified radiologist without knowledge
of the assignment of treatment groups. The following radiographic
changes were graded numerically according to severity: increased
soft tissue volume (0-4), narrowing or widening of joint spaces
(0-5), subchondral erosion (0-3), periosteal reaction (0-4),
osteolysis (0-4), subluxation (0-3) and degenerative joint changes
(0-3). The maximum possible score per foot was 26. In this study,
EC0565-treated rats showed minimal tissue and bone damage in their
hind paws when compared to arthritis control, everolimus-treated,
and methotrexate-treated animals (see FIG. 43).
Example
Anti-Arthritis Activity of EC0565 could be Partially Blocked by A
Folate Competitor
[0390] The AIA rats were treated subcutaneously with EC0565 (500
nmol/kg) in the absence (c) or presence (d) of 500-fold excess of
EC0923 (250 .mu.mol/kg) on days 10, 13, and 17. The animals in the
healthy (b) and arthritis control (a) groups were left untreated.
The arthritis scores (see FIG. 44A) and animal body weights (see
FIG. 44B) were recorded three times a week. At the completion of
study (day 24), the rats were euthanized by CO.sub.2 asphyxiation
and processed for paw (see FIG. 44C) and spleen (see FIG. 44D)
weights. In this study, the anti-arthritis activity of EC0565 was
partially blocked by EC0923, a folate competitor with similar
affinity as free folic acid.
Example
mTOR Knockdown
[0391] Western Blot Analysis. The data shown in FIG. 45 indicate
that EC0565 (folate-sugar-everolimus) can cause a dose-dependent,
and specific knockdown of the downstream targets of mTOR
(intracellular target for everolimus). Without being bound by
theory, in it believed that folate delivers everolimus inside the
cell where everolimus inhibits mTOR, which is the mammalian target
of rapamycin and a ser/thr kinase. Inhibition of mTOR's downstream
targets (P70 S6-kinase and Ribosomal S6) results, as shown on the
Western blot.
Example
Comparison of EC0565 with Oral Everolimus and Subcutaneous
Etanercept in AIA Rats
[0392] Methotrexate (MTX) and etanercept (Enbrel.RTM.) are part of
the current standard of care for RA. Treatment with EC0565 was
compared to treatment with its base drug everolimus, MTX, and
etanercept in rats with adjuvant arthritis using clinically
relevant dosing routes. AIA rats were treated 3 times a week with
subcutaneous EC0565 (500 nmo/kg) and oral everolimus (500 nmol/kg)
on days 10, 12, 14, 17, 19, and 21 post arthritis induction.
Biweekly oral MTX (250 nmol/kg) was given on days 10, 13, 17, and
20. Etanercept was given subcutaneously at 10 mg/kg once every 3
days starting on day 10. At the completion of each study (day 24),
rats were euthanized by CO.sub.2 asphyxiation and processed for paw
weight (cut at the hairline) and spleen weight. The removed hind
paws were immersion-fixed in 10% buffered formalin and subjected to
radiographic and histopathological analyses.
[0393] As shown in FIGS. 46A-46D, subcutaneously administered
EC0565 (500 nmol/kg/dose, 6 doses in 2 weeks) was more efficacious
than oral everolimus on an equimolar basis in the following
clinical parameters assessed: arthritis score, percent change in
body weight, and paw weight. EC0565 and everolimus-treated
arthritic animals had significantly decreased spleen weight
compared to arthritic controls. The difference between treatment
with EC0565 and treatment with erverolimus on spleen weight was not
statistically different. EC0565 was found to be more effective than
oral MTX and subcutaneous etanercept in all parameters assessed
except for the effect on spleen weight.
[0394] Radiographic analysis of hind paws FIG. 47 revealed that
EC0565-treated rats had minimal tissue and bone damage in their
hind paws when compared to arthritis control, oral
everolimus-treated, etanercept-treated, and oral MTX treated
animals.
[0395] Histological grading of arthritis compared to the untreated
controls showed oral everolimus-treated animals had significant
reductions (44-61%) in all scored parameters (i.e. ankle
inflammation, bone resorption, pannus formation, and cartilage
damage) (FIG. 48A). There was a 52% significant decrease in the
summed histological score (FIG. 48B). Dorsal to ventral paw
thickness was significantly decreased by 44% (FIG. 48C). While
etanercept was not as effective as oral MTX, animals treated with
etanercept also had significant reductions in inflammation (42%)
and bone resorption (55%), which contributed to a significant 43%
decrease in the summed score (FIG. 48B). The dorsal to ventral paw
thicknesses in both MTX and etanercept-treated rats were
significantly decreased by 63% and 40%, respectively (FIG. 48C).
Animals treated with EC0565 had significant 88-100% decreases in
all scored parameters (FIG. 48A), with an overall 94% significant
decrease in summed scores (FIG. 48B). Dorsal to ventral paw
thickness was significantly decreased by 94% (FIG. 48C). Overall,
EC0565 consistently outperformed everolimus, MTX, and etanercept in
all histological parameters assessed with further decreases in the
summed scores and dorsal to ventral paw thicknesses. The
representative photomicrographs (16.times.) of the ankle closest to
the mean summed score for each group are shown in FIG. 49.
Example 2
EC0565 Anti-Arthritic Activity is Dose and Schedule-Dependent
[0396] To further investigate dose-response relationship and
schedule-dependency, EC0565 was administered to rats with
developing AIA (day 10) at 100, 500, and 1000 nmol/kg/dose
(biweekly) or 1000 nmol/kg/dose (once weekly). At completion of the
study (day 24), rats were euthanized by CO.sub.2 asphyxiation and
processed for paw weight (cut at the hairline) and spleen weight.
The differences in total paw weight between arthritic rats after
treatment and that of healthy rats were used as a measure of the
extent of paw edema (FIG. 50A). Similarly, the differences in
spleen weight between arthritic rats after treatment and that of
healthy rats were used as a measure of the extent of splenomegaly
(FIG. 50B). As shown in FIG. 50A, EC0565 dosed for 2 weeks
displayed a linear dose response in inhibiting paw edema from 100
to 1000 nmol/kg/dose with an R-squared value of 0.862. As shown in
FIG. 50B, biweekly EC0565 treatment displayed a linear dose
response in inhibiting splenomegaly from 0 to 500 nmol/kg/dose with
an R-squared value of 0.909 and no statistical difference between
500 and 1000 nmol/kg was seen. When dosed once weekly for 2 weeks,
EC0565 at 1000 nmol/kg/dose remained highly effective, but this
schedule did not control the fast progressing AIA to the same
degree as the biweekly regimen at 500-1000 nmol/kg/dose (FIG. 50A,
B). Overall, EC0565 was found very effective against AIA and
capable of controlling local (joints) and systemic (spleen)
inflammation and halting the progression of arthritis.
Example 3
Collagen-Induced Arthritis (CIA) Model
[0397] Collagen-induced arthritis (CIA) was induced in female Lewis
rats on folate-deficient diet (Harlan Teklad, Indianapolis, Ind.).
On Day 0, rats were immunized with 500 .mu.g of bovine collagen
Type II (Chondrex, Redmond, Wash.) formulated with Freund's
complete adjuvant. A booster immunization was given on day 7 with
250 .mu.g of the bovine collagen formulated with Freund's
incomplete adjuvant. Arthritis disease was assessed by a
qualitative clinical score system described by the manufacturer
(Chondrex, Redmond, Wash.): 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 of wrist,
3=Severe redness and swelling of the entire paw including digits,
and 4=Maximally inflamed limb with involvement of multiple joints.
On Day 10 post first immunization, rats were distributed evenly
(according to the arthritis score) across the control and treatment
groups. The CIA rats were given subcutaneous doses of EC0565 and
everolimus at 1000 nmol/kg 3 times a week. The animals in the
arthritis control group were left untreated. The arthritis score
and animal body weight were recorded daily during weekdays. As
shown in FIGS. 51A and 51B, EC0565 was also effective in rats with
collagen-induced arthritis.
Example 4
EC0565 Shows Higher Water Solubility and Bioavailability than
Everolimus in Rats
[0398] Everolimus, the base drug of EC0565 has poor water
solubility (1-10 .mu.M) and low and variable oral bioavailability
(.about.12% in rats, Journal of Pharmacokinetics and
Pharmacodynamics, Vol. 34, No. 3, June 2007). These limitations
render formulation of this drug difficult and contribute to a
relatively narrow therapeutic index. In contrast, EC0565 displays a
improved water solubility at >1 mM in phosphate-buffered saline
(pH 7.4). The bioavailability of EC0565 after subcutaneous
injection was measured. Female Lewis rats with rounded tip jugular
vein catheters (Harlan) were fed regular rodent diet. The treated
animals were given a single intravenous or subcutaneous dose of
EC0565 at 2 .mu.mol/kg. For intravenous administration (FIG. 52A),
whole blood samples (300 .mu.l) were collected from the animals at
1 min, 3 min, 7 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h post
injection. For subcutaneous administration (FIG. 52B), whole blood
samples (300 .mu.l) were collected from the animals at 1 min, 10
min, 30 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 12 h post injection. The
blood samples were placed into anti-coagulant tubes containing 1.7
mg/mL of K3-EDTA and 0.35 mg/mL of N-maleoyl-beta-alanine (0.35
mg/mL). Plasma samples were obtained by centrifugation for 3 min at
.about.2,000 g and stored at -80.degree. C. The amount of EC0565
and its released base drug (everolimus) were determined by HPLC
using the EC0565 injection solution as the standard. The results
(based on the area under the curve) showed that approximately 18%
and 17% of free drug exposure/release were detected in the plasma
after a single intravenous or subcutaneous dose of EC0565,
respectively (FIG. 52A, B). The Tmax for EC0565 and everolimus
after subcutaneous injection were observed at .about.1 h (FIG.
52B). Based on the area under the curve, the bioavailablity of
EC0565 after subcutaneous administration (compared to intravenous
administration) was calculated to be .about.128% (FIG. 52C).
Example 5
EC0565 Inhibits Proliferating Cell Nuclear Antigen IN RAW264.7
CELLS
[0399] Proliferating Cell Nuclear Antigen (PCNA) is a cell-cycle
regulated nuclear protein that is often used to evaluate cellular
proliferative activity. To study anti-proliferative effect of
EC0565, FR-positive murine macrophage-like RAW264.7 cells (serum
deprived for 36 h for synchonization) were given a 2 h treatment of
EC0565 (1, 10, 100, and 1000 nM) without or with 1000.times. excess
of EC17 (as a folate competitor) followed by a 48-h chase in
drug-free medium. For comparison, the cells were also treated for
48 h with everolimus (1, 10, 100, and 1000 nM). All media contained
1% DMSO due to the low water solubility of everolimus. Western blot
analysis was carried out on whole cell lysates using a monoclonal
antibody specific for PCNA (PC10, Cell Signaling, Danvers, Mass.).
After incubation with a peroxidase-conjugated secondary antibody,
the signals were visualized using SuperSignal West Pico
Chemiluminescent Substrate system (ThermoScientific, Waltham,
Mass.) following the manufacturer's instructions. The images were
acquired using a G:BOX Chemi HR 16 gel imaging system (Syngene,
Frederick, Md.). As shown in FIG. 53A, both everolimus and EC0565
inhibited PCNA activities in the synchronized RAW264.7 cells. The
inhibitory activity of EC0565 at 1 nM was 100% blocked by the
presence of excess EC17, while EC17 alone was benign (FIGS. 53B-C).
As the EC0565 concentration was increased from 10, 100, to 1000 nM,
EC0565 showed both FR-specific and non FR-specific anti-PCNA
effects. This data indicates that EC0565 reduces PCNA activity in
RAW264.7 cells in a FR-dependent manner, especially at lower
concentrations.
COMPOUND EXAMPLES
##STR00036##
[0400] Example. (3,4), (5,6)-Bisacetonide-D-Gluconic Acid Methyl
Ester
[0401] In a dry 250 mL round bottom flask, under argon
.delta.-gluconolactone (4.14 g, 23.24 mmol) was suspended in
acetone-methanol (50 mL). To this suspension dimethoxypropane
(17.15 mL, 139.44 mmol) followed by catalytic amount of
p-toulenesulfonic acid (200 mg) were added. This solution was
stirred at room temperature for 16 h. TLC (50% EtOAc in petroleum
ether) showed that all of the starting material had been consumed
and product had been formed. Acetone-methanol was removed under
reduced pressure. The residue of the reaction was dissolved in
EtOAc and washed with water. The organic layer was washed with
brine, dried over Na.sub.2SO.sub.4, and concentrated to dryness.
This material was then loaded onto a SiO.sub.2 column and
chromatographed (30% EtOAc in petroleum ether) to yield pure (3,4),
(5,6)-bisacetonide-D-gluconic acid methyl ester (3.8 g, 56%) and
regio-isomer (2,3), (5,6)-bisacetonide-D-gluconic acid methyl ester
(0.71 g, 10%). .sup.1H NMR data was in accordance with the required
products. C.sub.13H.sub.22O.sub.7; MW 290.31; Exact Mass:
290.14.
##STR00037##
Example. (3,4), (5,6)-Bisacetonide-D-Gluconic Amide
[0402] 20 g of the methyl ester was dissolved in 100 mL methanol,
cooled the high-pressure reaction vessel with dry ice/acetone,
charged with 100 mL liquid ammonia, warmed up to room temperature
and heated to 160.degree. C./850 PSI for 2 hours. The reaction
vessel was cooled to room temperature and released the pressure.
Evaporation of the solvent gave brownish syrup, and minimum amount
of isopropyl alcohol was added to make the homogeneous solution
with reflux. The solution was cooled to -20.degree. C. and the
resulting solid was filtered to give 8.3 g of solid. The mother
liquid was evaporated, and to the resulting residue, ether was
added and refluxed until homogeneous solution was achieved. The
solution was then cooled to -20.degree. C. and the resulting solid
was filtered to give 4.0 g product. The solid was combined and
recrystallized in isopropyl alcohol to give 11.2 g (59%) of the
white amide product. C.sub.12H.sub.21NO.sub.6; MW 275.30; Exact
Mass: 275.14.
##STR00038##
Example. (3,4), (5,6)-Bisacetonide-1-Deoxy-1-Amino-D-Glucitol
[0403] In a dry 100 mL round bottom flask, under argon, LiAlH.sub.4
(450 mg, 11.86 momol)) was dissolved in THF (10 mL) and cooled to
0.degree. C. To this suspension (3,4),
(5,6)-bisacetonide-D-gluconic amide (1.09 g, 3.96 mmol) in THF (30
mL) was added very slowly over 15 min. This mixture was refluxed
for 5 h. TLC (10% MeOH in methylene chloride) showed that all of
the starting material had been consumed and product had been
formed. The reaction mixture was cooled to room temperature, and
then cooled to ice-bath temperature, diluted with diethyl ether (40
mL), slowly added 0.5 mL of water, 0.5 mL of 15% aq. NaOH, and then
added 1.5 mL of water. The reaction mixture was warmed to room
temperature and stirred for 30 min. MgSO.sub.4 was added and
stirred for additional 15 min and filtered. The organic layer was
concentrated to dryness to yield (3,4),
(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol. .sup.1H NMR data was
in accordance with the product. C.sub.12H.sub.23NO.sub.5; MW
261.31; Exact Mass: 261.16.
##STR00039##
Example. EC0475
[0404] O-Allyl protected Fmoc-Glu (2.17 g, 1 eq), PyBOP (2.88 g, 1
eq), and DIPEA (1.83 mL, 2 eq) were added to a solution of
(3,4),(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol (1.4 g, 5.3
mmol) in dry DMF (6 mL) and the mixture was stirred at RT under Ar
for 2 h. The solution was diluted with EtOAc (50 mL), washed with
brine (10 mL.times.3), organic layer separated, dried (MgSO.sub.4),
filtered and concentrated to give a residue, which was purified by
a flash column (silica gel, 60% EtOAc/petro-ether) to afford 1.72 g
(50%) allyl-protected EC0475 as a solid. Pd(Ph.sub.3).sub.4 (300
mg, 0.1 eq) was added to a solution of allyl-protected EC0475 (1.72
g, 2.81 mmol) in NMM/AcOH/CHCl.sub.3 (2 mL/4 mL/74 mL). The
resulting yellow solution was stirred at RT under Ar for 1 h, to
which was added a second portion of Pd(Ph.sub.3).sub.4 (300 mg, 0.1
eq). After stirring for an additional 1 h, the mixture was washed
with 1 N HCl (50 mL.times.3) and brine (50 mL), organic layer
separated, dried (MgSO.sub.4), filtered, and concentrated to give a
yellow foamy solid, which was subject to chromatography (silica
gel, 1% MeOH/CHCl.sub.3 followed by 3.5% MeOH/CHCl.sub.3) to give
1.3 g (81%) EC0475 as a solid material. MW 612.67; Exact Mass:
612.27.
##STR00040##
Example. Tetra-Saccharoglutamate-Bis-tGlu-Folate Spacer EC0491
[0405] EC0491 was synthesized by SPPS in eight steps according to
the general peptide synthesis procedure described herein starting
from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following
SPPS reagents:
TABLE-US-00002 Reagents Mmol equivalent MW Amount
H-Cys(4-methoxytrityl)- 0.1 0.167 g 2-chlorotrityl-Resin (loading
0.56 mmol/g) EC0475 0.13 1.3 612.67 0.080 g Fmoc-Glu(OtBu)-OH 0.2 2
425.5 0.085 g EC0475 0.13 1.3 612.67 0.080 g EC0475 0.13 1.3 612.67
0.080 g Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 g EC0475 0.13 1.3
612.67 0.080 g Fmoc-Glu-OtBu 0.2 2 425.5 0.085 g
N.sup.10TFA-Pteroic Acid.cndot.TFA 0.2 2 408 0.105 g (dissolve in
10 ml DMSO) DIPEA 0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2 520
0.104 g
[0406] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step: Column:
Waters NovaPak C18 300.times.19 mm; Buffer A=10 mM ammonium
acetate, pH 5; B=ACN; Method: 100% A for 5 min then 0% B to 20% B
in 20 minutes at 26 ml/min; yield .about.100 mg, 51%.
C.sub.76H.sub.118N.sub.18O.sub.41S; MW 1971.91; Exact Mass:
1970.74.
##STR00041##
Example
[0407] EC0479 was synthesized by SPPS according to the general
peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00003 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)- 0.094 0.16 g 2-chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.4 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
2.0 425.47 0.080 g EC0475 0.13 1.4 612.67 0.082 g Fmoc-Arg(Pbf)-OH
0.19 2.0 648.77 0.12 g EC0475 0.13 1.4 612.67 0.082 g
Fmoc-Glu(OtBu)-OH 0.19 2.0 425.47 0.080 g EC0475 0.13 1.4 612.67
0.082 g Fmoc-Glu-OtBu 0.19 2.0 425.47 0.080 g N.sup.10TFA-Pteroic
Acid 0.16 1.7 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq
41 .mu.L or of AA 49 .mu.L PyBOP 1.0 eq 122 mg or of AA 147 mg
[0408] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0409] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent for 15 min with argon bubbling, drain, wash the
resin once with cleavage reagent, and combine the solution. Rotavap
until 5 ml remains and precipitate in diethyl ether (35 mL).
Centrifuge, wash with diethyl ether, and dry. The crude solid was
purified by HPLC.
[0410] HPLC Purification step. Column: Waters Atlantis Prep T3 10
.mu.m OBD 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 20 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give .about.70 mg EC0479 (35% yield). .sup.1H NMR and LC/MS were
consistent with the product. MW 2128.10; Exact Mass: 2126.84.
##STR00042##
[0411] EC0488. This compound was prepared by SPPS according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00004 Reagents mmol equivalent MW amount
H-Cys(4-methoxytrityl)- 0.10 0.17 g 2-chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH
0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g
Fmoc-Glu-OtBu 0.19 1.9 425.47 0.080 g N.sup.10TFA-Pteroic Acid 0.16
1.6 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq of AA
PyBOP 1.0 eq of AA
[0412] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0413] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3.times. (10 min, 5 min, 5 min) with argon
bubbling, drain, wash the resin once with cleavage reagent, and
combine the solution. Rotavap until 5 ml remains and precipitate in
diethyl ether (35 mL). Centrifuge, wash with diethyl ether, and
dry. About half of the crude solid (.about.100 mg) was purified by
HPLC.
[0414] HPLC Purification step. Column: Waters Xterra Prep MS C18 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 25 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give 43 mg EC0488 (51% yield). .sup.1H NMR and LC/MS (exact mass
1678.62) were consistent with the product. MW 1679.63; Exact Mass:
1678.62.
##STR00043##
EC0536 Conjugate Intermediate
##STR00044##
[0416] EC0632 Conjugate intermediate. C52H72N14O28S, MW 1373.27,
Exact Mass: 1372.44, prepared from the corresponding tert-butyl
protected carboxylates.
##STR00045##
[0417] EC0669 Conjugate intermediate. C49H71N13O24S, MW 1258.23,
Exact Mass: 1257.45
##STR00046##
Example. Synthesis of Coupling Reagent EC0311
[0418] DIPEA (0.60 mL) was added to a suspension of
HOBt-OCO.sub.2--(CH.sub.2).sub.2-SS-2-pyridine HCl (685 mg, 91%) in
anhydrous DCM (5.0 mL) at 0.degree. C., stirred under argon for 2
minutes, and to which was added anhydrous hydrazine (0.10 mL). The
reaction mixture was stirred under argon at 0.degree. C. for 10
minutes and room temperature for an additional 30 minutes,
filtered, and the filtrate was purified by flash chromatography
(silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil
(371 mg), solidified upon standing.
##STR00047##
[0419] EC0593 Multidrug intermediate for two drugs.
C68H103N17O35S2, MW 1782.77, Exact Mass: 1781.62
##STR00048##
[0420] EC0613 Multidrug intermediate for three drugs.
C90H140N22O47S4, MW 2410.45, Exact Mass: 2408.81
##STR00049##
[0421] EC0542 Optionally selective multidrug intermediate for two
drugs. C85H118N18O36S2, C, 50.24; H, 5.85; N, 12.41; O, 28.34; S,
3.16, MW 2032.08, Exact Mass: 2030.74
##STR00050##
[0422] EC0559 Optionally selective multidrug intermediate for two
drugs. C90H121N19O36S3, MW 2141.22, Exact Mass: 2139.74
##STR00051##
[0423] EC0682 Optionally selective multidrug intermediate for two
drugs. C95H132N20O42S2, MW 2290.30, Exact Mass: 2288.82
##STR00052##
[0424] EC0646 Conjugate of Aminopterin and intermediate for
multidrug conjugate. C106H140N26O41S3, MW 2530.59, Exact Mass:
2528.88
Example
##STR00053##
[0426] EC0746 Conjugate of aminopterin. C87H122N26O40S2; C, 46.73;
H, 5.50; N, 16.29; O, 28.62; S, 2.87; MW 2236.180, Exact Mass:
2234.775.
##STR00054##
[0427] Reaction of mixed carbonate 101 with t-butyl-carbazate in
the presence of diisopropylethylamine (DIPEA) gave the
corresponding t-butyl-carbazate 102. Trifluoroacetic acid (TFA)
mediated Boc deprotection of 102 in the presence of
triisopropylsilane (TIPS) resulted in pyridyldisulfanylethyl
carbazate 103 as a TFA salt. Coupling of carbazate 103 with
protected glutamic acid 104, using
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBop) and DIPEA, yielded glutamic acid derivative 105.
4-dimethylaminopyridine (DMAP) mediated Fmoc deprotection of 105
followed by in situ coupling with commercially available
4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic
acid 106 using PyBop and hydroxybenzotriazol (HOBt) resulted in
protected aminopterin hydrazide 107. Treatment of 107 with TFA
removed the t-butyl moiety to yield pyridinedisulfanyl-activated
aminopterin hydrazide 108. .sup.1H NMR (DMSO-d.sub.6 &
D.sub.2O) .delta. 8.82 (s, 1H), 8.44 (d, J=4.7 Hz, 1H), 7.80 (m,
2H), 7.71 (d, J=8.8 Hz, 2H), 7.24 (t, J=4.6 Hz, 1H), 6.74 (d, J=8.8
Hz, 2H), 4.60 (s, 2H), 4.32 (dd, J=5.0 Hz, 1H), 4.20 (t, J=6.0 Hz,
2H), 3.07 (t, J=6.0 Hz, 2H), 2.22 (t, J=7.8 Hz, 2H), 2.15-1.94 (m,
2H). ESI-MS: (M+H).sup.+=Calculated 668.2; found 668.2. Treatment
of a suspension of EC0488 in phosphate buffer under argon with
NaHCO.sub.3 resulted in a clear yellow solution. A
dimethylsulfoxide (DMSO) solution of 108 was added to this mixture
at once under vigorous stirring to yield EC0746. .sup.1H NMR
(DMSO-d.sub.6 & D.sub.2O) .delta. 8.67 (s, 1H) 8.60 (s, 1H),
7.62 (d, J=9 Hz, 2H), 7.59 (d, J=9 Hz, 2H), 6.71 (d, J=8.7 Hz, 2H),
6.62 (d, J=8.1 Hz, 2H), 4.47 (m, 4H), 4.26-4.04 (m, 10H), 3.70-3.30
(m, 22H), 3.30-3.10 (m, 6H), 3.10-2.76 (m, 9H), 2.40-2.04 (m, 15H),
2.04-1.60 (m, 4H). ESI-MS: [(M+2H).sup.2+]/2=Calculated 1119.09;
found 1119.10.
[0428] EC0808 is an isomer of EC0746 having the opposite
configuration at the stereogenic carbon indicated with the
arrow.
##STR00055##
[0429] EC0932, everolimus-aminopterin hydrazide conjugate
C149H218N30O57S4; C, 51.58; H, 6.33; N, 12.11; O, 26.28; S, 3.70;
MW 3469.752; Exact Mass: 3467.396.
##STR00056##
[0430] EC0828 everolimus-aminopterin hydrazide conjugate.
C149H217N29O58S4; C, 51.56; H, 6.30; N, 11.70; O, 26.74; S, 3.70;
MW: 3470.737; Exact Mass: 3468.381.
##STR00057##
Example. Everolimus (2'-pyridyldisulfanyl)ethyl carbonate
(EC0564)
[0431] In a 10 mL round bottom flask, under argon atmosphere,
everolimus (130 mg, 0.136 mmol) was dissolved in 2.0 mL of
CH.sub.2Cl.sub.2.
2-[Benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl]-pyridine
(104.4 mg, 0.271 mmol) followed by DMAP (49.85 mg, 0.41 mmol) were
added. The reaction mixture was stirred for 30 min. Progress of the
reaction was monitored by analytical HPLC (0.1% TFA in water,
pH=2.0 and acetonitrile). The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with sat. NH.sub.4Cl. The organic layer
was dried over Na.sub.2SO.sub.4 and concentrated to yield
everolimus (2'-pyridyldisulfanyl)ethyl carbonate, EC0564.
Example. Everolimus-EC0488 Conjugate (EC0565)
[0432] In a 25 mL round bottom flask, folate linker (EC0488, 104
mg, 0.06 mmol) was dissolved in 2.0 mL of DMSO, and 0.13 mL of
DIPEA (20 equiv) were added. The everolimus carbonate derivative
(EC0564, 38 mg, 1.0 eq) in 1.0 mL of DMSO was added quickly to the
above solution. The resulting clear solution was stirred under
argon. Progress of the reaction was monitored by analytical HPLC
(20 mM NH.sub.4OAc buffer, pH=5.0 and acetonitrile). After 20 min,
reaction mixture was injected on a prep-HPLC. HPLC purification
conditions--column: Waters X-Bridge Prep MS Cis 10 .mu.m
19.times.100 mm; solvent A: 20 mM ammonium acetate, pH 5; solvent
B: acetonitrile; method: 5 min 10% B to 25 min 80% B 25 mL/min.
Fractions containing EC0565 were collected and freeze-dried to
afford 68 mg (50% yield, over 2 steps from everolimus) of fluffy
yellow solid. C.sub.121H.sub.183N.sub.17O.sub.50S.sub.2; C, 53.04;
H, 6.73; N, 8.69; O, 29.20; S, 2.34; MW 2739.96; Exact Mass:
2738.17.
##STR00058##
[0433] EC0606 Conjugate of Everolimus and intermediate for
multidrug conjugate.
[0434] C141H203N19O52S3, C, 54.76; H, 6.62; N, 8.61; O, 26.90; S,
3.11, MW 3092.42, Exact Mass: 3090.30
##STR00059##
[0435] EC0634 Intermediate for optional non-targeted delivery.
C63H95N9O30S2, MW 1522.60, Exact Mass: 1521.56
##STR00060##
[0436] EC0586 Intermediate for optional non-target delivery.
C48H83N9O30S, MW 1298.28, Exact Mass: 1297.50
##STR00061##
[0437] EC0539 Conjugate of lysine analog of aminopterin.
##STR00062##
[0438] EC0544 Conjugate of cysteine analog of aminopterin.
C83H116N24O37S2, C, 47.33H, 5.55; N, 15.96; O, 28.11; S, 3.05, MW
2106.08, Exact Mass: 2104.74
##STR00063##
[0439] EC0551 Conjugate of aminopterin. C86H120N24O39S2, C, 47.42;
H, 5.55; N, 15.43; O, 28.65; S, 2.94, MW 2178.14, Exact Mass:
2176.76
##STR00064##
[0440] EC0647 Bis aminopterin conjugate. C110H147N33O45S4, MW,
2779.80, Exact Mass: 2777.9112, m/z: 2778.91 (100.0%), 2777.91
(74.4%), 2779.92 (62.2%)
* * * * *