U.S. patent application number 12/527316 was filed with the patent office on 2010-04-29 for methods and compositions for treating and diagnosing kidney disease.
This patent application is currently assigned to ENDOCYTE, INC.. Invention is credited to Christopher Paul Leamon, Iontcho Radoslavov Vlahov.
Application Number | 20100104626 12/527316 |
Document ID | / |
Family ID | 39690842 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100104626 |
Kind Code |
A1 |
Leamon; Christopher Paul ;
et al. |
April 29, 2010 |
METHODS AND COMPOSITIONS FOR TREATING AND DIAGNOSING KIDNEY
DISEASE
Abstract
The invention relates to a method for diagnosing a kidney
disease state. The method comprises the steps of administering to a
patient a composition comprising a conjugate or complex of the
general formula V-L-D where the group V comprises a vitamin
receptor binding ligand that binds to kidney proximal tubule cells
and the group D comprises a diagnostic marker, and diagnosing the
kidney disease state. The invention also relates to a method for
treating a kidney disease state. The method comprises the steps of
administering to a patient suffering from the disease state an
effective amount of a composition comprising a conjugate or complex
of the general formula V-L-D where the group V comprises a vitamin
receptor binding ligand that binds to kidney proximal tubule cells
and the group D comprises an antigen, a cytotoxin, or a cell growth
inhibitor, and eliminating the disease state.
Inventors: |
Leamon; Christopher Paul;
(West Lafayette, IN) ; Vlahov; Iontcho Radoslavov;
(West Lafayette, IN) |
Correspondence
Address: |
BARNES & THORNBURG LLP
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Assignee: |
ENDOCYTE, INC.
West Lafayette
IN
|
Family ID: |
39690842 |
Appl. No.: |
12/527316 |
Filed: |
February 16, 2008 |
PCT Filed: |
February 16, 2008 |
PCT NO: |
PCT/US08/54189 |
371 Date: |
December 2, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60901778 |
Feb 16, 2007 |
|
|
|
Current U.S.
Class: |
424/450 ;
514/249; 514/291; 544/258 |
Current CPC
Class: |
A61K 49/0002 20130101;
A61K 51/04 20130101; A61P 13/12 20180101; A61K 51/00 20130101; C07D
475/04 20130101; A61K 47/552 20170801; C07D 519/00 20130101; C07K
7/02 20130101; A61K 47/551 20170801 |
Class at
Publication: |
424/450 ;
514/291; 514/249; 544/258 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/436 20060101 A61K031/436; A61K 31/4985
20060101 A61K031/4985; C07D 475/04 20060101 C07D475/04 |
Claims
1. A method for diagnosing a kidney disease state, said method
comprising the steps of: administering to a patient a composition
comprising a conjugate or complex of the general formula V-L-D
where the group V comprises a vitamin receptor binding ligand that
binds to kidney cells and the group D comprises a diagnostic
marker; and diagnosing the kidney disease state.
2-11. (canceled)
12. A method for treating a kidney disease state, said method
comprising the steps of: administering to a patient suffering from
the disease state an effective amount of a composition comprising a
conjugate or complex of the general formula V-L-D where the group V
comprises a vitamin receptor binding ligand that binds to kidney
cells and the group D comprises an antigen, a cytotoxin, or a cell
growth inhibitor; and eliminating the disease state.
13. The method of claim 12 wherein V comprises a folate.
14. (canceled)
15. The method of claim 12 wherein the group D comprises an
antigen.
16. The method of claim 13 wherein the group D comprises an
antigen.
17. The method of claim 12 wherein the group D comprises a
cytotoxin.
18. The method of claim 17 wherein the group D further comprises a
liposome.
19. The method of claim 13 wherein the group D comprises a
cytotoxin.
20. The method of claim 19 wherein the group D further comprises a
liposome.
21. The method of claim 12 wherein the group D comprises a cell
growth inhibitor.
22-23. (canceled)
24. The method of claim 13 wherein the group D comprises a cell
growth inhibitor.
25-26. (canceled)
27. The method of claim 21 wherein the cell growth inhibitor is
rapamycin.
28. The method of claim 24 wherein the cell growth inhibitor is
rapamycin.
29. A compound of the formula V-L-D, wherein V is a folate receptor
binding ligand, L is an optional linker, and D is a cell-growth
inhibitor.
30. The compound of claim 29 wherein V-L-D has the following
formula: ##STR00036## where L is as defined herein, and L is
connected to the rapamycin or analog or derivative thereof at
either of (O*), and the other of (O*) is substituted with R,
wherein R is hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9;
where R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, trifluoromethyl, or F; R.sup.5 and R.sup.6 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms,
(CR.sup.3R.sup.4).sub.fOH, CF.sub.3, F, or CO.sub.2R.sup.11;
R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms,
(CR.sup.3R.sup.4).sub.10H, CF.sub.3, F, or CO.sub.2R.sup.11;
R.sup.8 and R.sup.9 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon
atoms, (CR.sup.3R.sup.4).sub.fOH, CF.sub.3, F, or CO.sub.2R.sup.11;
R.sup.11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10
carbon atoms; b=0-6; d=0-6; and f=0-6.
31. (canceled)
32. The compound of claim 29 wherein the cell growth inhibitor is
an epidermal growth factor receptor kinase inhibitor.
33. The compound of claim 29 wherein the cell growth inhibitor is
an inhibitor of mTOR.
34. The compound of claim 29 wherein the cell growth inhibitor is a
rapamycin.
35. The compound of claim 29 wherein the folate receptor binding
ligand is a folate.
36. The compound of claim 29 wherein the linker is a peptide
comprising one or more amino acids selected from the group
consisting of cysteine, aspartic acid, and arginine, where the
amino acid can be either the D or the L configuration in each
instance.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/901,778, filed on Feb. 16, 2007, the entire
disclosure of which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods and compositions for
treating and diagnosing kidney disease states. More particularly,
ligands that bind to receptors overexpressed on proximal tubule
cells are complexed with a diagnostic marker for use in diagnosis
or to an antigen, a cytotoxin, or a cell growth inhibitor for use
in the treatment of kidney disease states.
BACKGROUND
[0003] Diseases affecting kidney function are prevalent. For
example, polycystic kidney disease (PKD) is a prevalent inherited
disease. Adult PKD is an autosomal dominant disorder affecting
approximately 600,000 people in the United States and 12.5 million
world-wide. Infants can also present with autosomal recessive PKD
which is rapidly developing and which can lead to renal
insufficiency in the neonate. PKD and other kidney disease states
(e.g., Dent's disease and nephrocytinosis) affect and manifest
abnormal growth of kidney proximal tubule cells. PKD results in the
proliferation of kidney epithelial cells and the formation of PKD
renal cysts. The kidneys can become enlarged and symptoms including
pain, bleeding, and kidney stones can occur. Associated problems
include liver cysts, abdominal aneurysm, intracranial aneurysm, and
renal insufficiency. It has been suggested that cellular processes
associated with signal transduction, transcriptional regulation,
and cell-cycle control are involved in cyst formation in PKD.
[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 a
substantial fraction of the vitamins into the cell, where they are
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. 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, and normal
tissues typically express low or nondetectable levels of the folate
receptor. Folate receptors are overexpressed in proximal tubule
cells.
[0005] The invention is based on the manifestation of abnormal
proliferation of kidney proximal tubule cells in PKD and other
kidney disease states that exhibit abnormal proximal tubule cell
proliferation. These kidney disease states can be treated with
ligands that bind to receptors overexpressed on proximal tubule
cells wherein the ligands are complexed with an antigen, a
cytotoxin, or a cell growth inhibitor for use in the treatment of
the kidney disease states. These kidney disease states, including
PKD, can also be diagnosed by using ligands that bind to receptors
overexpressed on proximal tubule cells wherein the ligands are
complexed with a diagnostic marker.
SUMMARY
[0006] In one embodiment, a method for diagnosing a kidney disease
state is provided. The method comprises the steps of administering
to a patient a composition comprising a conjugate or complex of the
general formula V-L-D, where the group V comprises a vitamin
receptor binding ligand that binds to kidney cells and the group D
comprises a diagnostic marker, and diagnosing the kidney disease
state.
[0007] In another embodiment, V comprises a folate receptor binding
ligand or V comprises a folate receptor binding antibody or
antibody fragment. In yet another embodiment, the marker can
comprise a metal chelating moiety, or a fluorescent chromophore. In
another illustrative embodiment, the disease state is selected from
the group consisting of polycystic kidney disease, Dent's disease,
nephrocytinosis, and Heymann nephritis.
[0008] In another embodiment, a method for treating a kidney
disease state is provided. The method comprises the steps of
administering to a patient suffering from the disease state an
effective amount of a composition comprising a conjugate or complex
of the general formula V-L-D where the group V comprises a vitamin
receptor binding ligand that binds to kidney cells and the group D
comprises an antigen, a cytotoxin, or a cell growth inhibitor, and
eliminating the disease state.
[0009] In another embodiment, V comprises a folate receptor binding
ligand or an antibody or antibody fragment that binds to the folate
receptor. In another illustrative aspect, group D comprises an
antigen, a cytotoxin, or a cell growth inhibitor. In yet another
embodiment, the cell growth inhibitor is selected from the group
consisting of epidermal growth factor receptor kinase inhibitors,
inhibitors of the mTOR pathway, DNA alkylators, microtubule
inhibitors, cell cycle inhibitors, and protein synthesis
inhibitors. In another embodiment, the disease state is selected
from the group consisting of polycystic kidney disease, Dent's
disease, nephrocytinosis, and Heymann nephritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows IHC analysis of folate receptor expression in
polycystic kidney disease tissues using a monoclonal antibody
directed to the folate receptor for staining. The upper left panel
shows normal human kidney tissue and the remainder of the panels
show staining of cysts in polycystic kidney disease tissues using
the anti-folate receptor monoclonal antibody.
[0011] FIG. 2 shows IHC analysis of folate receptor expression in
polycystic kidney disease tissues using a polyclonal antibody
directed to the folate receptor for staining. The upper left panel
shows normal mouse kidney tissue and the remainder of the panels
show staining of cysts in polycystic kidney disease tissues using
the anti-folate receptor polyclonal antibody.
[0012] FIG. 3 shows the structure of EC0371, a folate-rapamycin
conjugate.
[0013] FIG. 4 shows an affinity assay comparing the relative
affinities of folic acid (circles; 1.0) and EC0371 (triangles; 0.5)
for the folate receptor.
[0014] FIG. 5 shows the effect of rapamycin and EC0371 on the
viability of KB cells at various free rapamycin and conjugated
rapamycin (EC0371) concentrations. The leftmost panels show
untreated cells. The panels in the second column from the left show
control cells treated with DMSO (diluent). The panels in the third
column from the left show cells treated with 2, 10, or 50 nM
rapamycin. The panels in the rightmost column show cells treated
with 2, 10, or 50 nM EC0371. Neither rapamycin nor EC0371 is
cytotoxic after 24 hours of treatment.
[0015] FIG. 6 shows the effects of rapamycin and EC0371 on P-S6
immunostaining in KB cells after 16 hours of incubation with
rapamycin or EC0371. P-S6 is a phosphorylation target of m-TOR and
the antibody used is phospho-specific. The leftmost panels show
untreated cells. The panels in the middle column show cells treated
with 2, 10, or 50 nM rapamycin. The panels in the rightmost column
show cells treated with 2, 10, or 50 nM EC0371. Rapamycin and
EC0371 inhibit P-S6 immunostaining (i.e., phosphorylation of P-S6
through the mTOR pathway).
[0016] FIG. 7 shows an immunoblot using a phospho-specific
antibody. The left panel shows phosphorylation of ribosomal S6 and
S-6 kinase (T389) in untreated cells and cells treated with DMSO
(diluent). The right panel shows that rapamycin (2, 10, and 50 nM)
and EC0371 (folate-rapamyin; 2, 10, and 50 nM) abolish or greatly
reduce phosphorylation of ribosomal S6 and S-6 kinase (T389) which
are phosphorylation targets in the m-TOR pathway.
[0017] FIG. 8 shows the therapeutic effect of EC0371 on the in vivo
development of polycystic kidney disease in the bpk-mutant mouse
model. The leftmost kidney is from a wildtype mouse. The middle
kidney is from a bpk mutant mouse not treated with EC0371. The
rightmost kidney is from a bpk mutant mouse treated with EC0371
showing that EC0371 greatly reduces kidney size.
[0018] FIG. 9 shows the effect on one-kidney weight of EC0371
treatment in multiple bpk mutant mice (rightmost group of symbols).
EC0371-treated bpk mice exhibit a significant decrease in
one-kidney weight as a percentage of total body weight relative to
untreated bpk mice.
[0019] FIG. 10 shows the effect on two-kidney weight of EC0371
treatment in multiple bpk mutant mice (rightmost group of symbols).
EC0371-treated bpk mice exhibit a significant decrease in
two-kidney weight as a percentage of total body weight relative to
untreated bpk mice.
DETAILED DESCRIPTION
[0020] Methods are provided for treating and diagnosing kidney
disease states. Exemplary disease states include PKD, Dent's
disease, nephrocytinosis, Heymann nephritis, and other diseases
manifested by abnormal proliferation of proximal tubule cells of
the kidney. PKD's can include, but are not limited to, autosomal
dominant (adult) polycystic kidney disease and autosomal recessive
(childhood) polycystic kidney disease. These disease states are
characterized by abnormal proliferation of kidney proximal tubule
cells. Such disease states can be diagnosed by contacting kidney
proximal tubule cells with a composition comprising a conjugate of
the general formula V-L-D wherein the group V comprises a ligand
that binds to the kidney proximal tubule cells, and the group D
comprises a diagnostic marker, and diagnosing the disease state.
Such disease states can be treated by contacting kidney proximal
tubule cells with a composition comprising a conjugate of the
general formula V-L-D wherein the group V comprises a ligand that
binds to the kidney proximal tubule cells, and the group D
comprises an antigen, a cytotoxin, or a cell growth inhibitor, and
eliminating the disease state.
[0021] As used herein, the terms "eliminated" and "eliminating" in
reference to the disease state, mean reducing the symptoms or
eliminating the symptoms of the disease state or preventing the
progression or the reoccurrence of disease.
[0022] As used herein, the term "elimination" of the proximal
tubule cell population causing the disease state that expresses the
ligand receptor means that this cell population is killed or is
completely or partially removed or inactivated which reduces the
pathogenic characteristics of the disease state being treated.
[0023] The kidney disease states characterized by abnormal
proliferation of proximal tubule cells can be treated in accordance
with the methods disclosed herein by administering an effective
amount of a composition V-L-D wherein V comprises a ligand that
binds to proximal tubule cells and wherein the group D comprises an
antigen, a cytotoxin, or a cell growth inhibitor. Such targeting
conjugates, when administered to a patient suffering from a kidney
disease state manifested by abnormal proximal tubule cell
proliferation, work to concentrate and associate the conjugated
cytotoxin, antigen, or cell growth inhibitor with the population of
proximal tubule cells to kill the cells or alter cell function. The
conjugate is typically administered parenterally, but can be
delivered by any suitable method of administration (e.g., orally),
as a composition comprising the conjugate and a pharmaceutically
acceptable carrier therefor. Conjugate administration is typically
continued until symptoms of the disease state are reduced or
eliminated, or administration is continued after this time to
prevent progression or reappearance of the disease.
[0024] For diagnosis the typical method of administration of the
conjugates is parenteral administration, but any suitable method
can be used. In this embodiment, kidney disease states can be
diagnosed by administering parenterally to a patient a composition
comprising a conjugate or complex of the general formula V-L-D
where the group V comprises a ligand that binds to proximal tubule
cells and the group D comprises a diagnostic marker, and diagnosing
the disease state.
[0025] In one embodiment, for example, the diagnostic marker (e.g.,
a reporter molecule) can comprise a radiolabeled compound such as a
chelating moiety and an element that is a radionuclide, for example
a metal cation that is a radionuclide. In another embodiment, the
radionuclide is selected from the group consisting of technetium,
gallium, indium, and a positron emitting radionuclide (PET imaging
agent). In another embodiment, the diagnostic marker can comprise a
fluorescent chromophore such as, for example, fluorescein,
rhodamine, Texas Red, phycoerythrin, Oregon Green, AlexaFluor 488
(Molecular Probes, Eugene, Oreg.), Cy3, Cy5, Cy7, and the like.
Imaging agents are described in U.S. Pat. No. 7,128,893 and in U.S.
Patent Publ. No. 20070009434, each incorporated herein by
reference.
[0026] Diagnosis typically occurs before treatment. However, in the
diagnostic methods described herein, the term "diagnosis" can also
mean monitoring of the disease state before, during, or after
treatment to determine the progression of the disease state. The
monitoring can occur before, during, or after treatment, or
combinations thereof, to determine the efficacy of therapy, or to
predict future episodes of disease. The diagnostic method can be
any suitable method known in the art, including imaging methods,
such as intravital imaging.
[0027] The method disclosed herein can be used for both human
clinical medicine and veterinary applications. Thus, the patient or
animal afflicted with the kidney disease state and in need of
diagnosis or therapy can be a human, or in the case of veterinary
applications, can be a laboratory, agricultural, domestic or wild
animal. In embodiments where the conjugates are administered to the
patient or animal, the conjugates can be administered parenterally
to the animal or patient suffering from the kidney disease state,
for example, intradermally, subcutaneously, intramuscularly,
intraperitoneally, or intravenously. Alternatively, the 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, such as a slow pump.
The therapeutic method described herein can be used alone or in
combination with other therapeutic methods recognized for the
treatment of kidney disease states.
[0028] In the ligand conjugates of the general formula V-L-D, the
group V is a ligand that binds to proximal tubule cells when the
conjugates are used to diagnose or treat kidney disease states. Any
of a wide number of binding ligands can be employed. Acceptable
ligands include, for example, folate receptor binding ligands, and
analogs thereof, and antibodies or antibody fragments capable of
recognizing and binding to surface moieties expressed on proximal
tubule cells, in particular when these cells proliferate
abnormally. In one embodiment, the binding ligand is folic acid, a
folic acid analog, or another folate receptor binding molecule. In
another embodiment the binding ligand is a specific monoclonal or
polyclonal antibody or an Fab or an scFv (i.e., a single chain
variable region) fragment of an antibody capable of binding to
receptors overexpressed on proximal tubule cells, for example, when
these cells proliferate abnormally.
[0029] In one embodiment, the binding ligand can be folic acid, a
folic acid analog, or another folate receptor-binding molecule.
Analogs of folate that can be used 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
refers to the art recognized analogs having a carbon atom
substituted for one or two nitrogen atoms in the naturally
occurring folic acid structure. For example, the deaza analogs
include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza
analogs. The dideaza analogs include, for example, 1,5 dideaza,
5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. The foregoing
folic acid analogs are conventionally termed "folates," reflecting
their capacity to bind to folate receptors. Other folate
receptor-binding analogs include 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).
[0030] In another embodiment, other vitamins can be used as the
binding ligand. The vitamins that can be used in accordance with
the methods described herein include niacin, pantothenic acid,
folic acid, riboflavin, thiamine, biotin, vitamin B.sub.12,
vitamins A, D, E and K, other related vitamin molecules, analogs
and derivatives thereof, and combinations thereof.
[0031] In other embodiments, the binding ligand can be any ligand
that binds to a receptor expressed or overexpressed on proximal
tubule cells, in particular when they proliferate abnormally (e.g.,
EGF, KGF, or leptin). In another embodiment, the binding ligand can
be any ligand that binds to a receptor expressed or overexpressed
on proximal tubule cells proliferating abnormally and involved in a
kidney disease state.
[0032] The targeted conjugates used for diagnosing or treating
disease states mediated by proximal tubule cells proliferating
abnormally have the formula V-L-D, wherein V is a ligand capable of
binding to the proximal tubule cells, and the group D comprises a
diagnostic marker or an antigen (such as an immunogen), cytotoxin,
or a cell growth inhibitor. In such conjugates wherein the group V
is folic acid, a folic acid analog, or another folic acid receptor
binding ligand, these conjugates are described in detail in U.S.
Pat. No. 5,688,488, the specification of which is incorporated
herein by reference. That patent, as well as related U.S. Pat. Nos.
5,416,016 and 5,108,921, and related U.S. patent application Ser.
No. 10/765,336, each incorporated herein by reference, describe
methods and examples for preparing conjugates useful in accordance
with the methods described herein. The present targeted diagnostic
and therapeutic agents can be prepared and used following general
protocols described in those earlier patents and patent
applications, and by the protocols described herein.
[0033] In accordance with another embodiment, there is provided a
method of treating kidney disease states by administering to a
patient suffering from such disease state an effective amount of a
composition comprising a conjugate of the general formula V-L-D
wherein V is as defined above and the group D comprises a
cytotoxin, an antigen (i.e., a compound administered to a patient
for the purpose of eliciting an immune response in vivo), or a cell
growth inhibitor. The group V can be any of the ligands described
above. Exemplary of cytotoxic moieties useful for forming
conjugates for use in accordance with the methods described herein
include art-recognized chemotherapeutic agents such as
antimetabolites, methotrexate, busulfan, carboplatin, chlorambucil,
cisplatin and other platinum compounds, plant alkaloids,
hydroxyurea, teniposide, and bleomycin, MEK kinase inhibitors, MAP
kinase pathway inhibitors, PI-3-kinase inhibitors, NF.kappa.B
pathway inhibitors, pro-apoptotic agents, apoptosis-inducing
agents, proteins such as pokeweed, saporin, momordin, and gelonin,
didemnin B, verrucarin A, geldanamycin, toxins, and the like. Such
cytotoxic compounds can be directly conjugated to the targeting
ligand, for example, folate or another folate receptor-binding
ligand, or they can be formulated in liposomes or other small
particles which themselves can be targeted to proximal tubule cells
by pendent targeting ligands V non-covalently or covalently linked
to one or more liposome components.
[0034] In another embodiment, the group D comprises a cell growth
inhibitor, and the inhibitor can be covalently linked to the
targeting ligand V, for example, a folate receptor-binding ligand
or a proximal tubule cell-binding antibody or antibody fragment
(i.e., an antibody to a receptor overexpressed on proximal tubule
cells that are proliferating abnormally). The ligand can be linked
directly, or the ligand can be encapsulated in a liposome which is
itself targeted to the proximal tubule cells by pendent targeting
ligands V covalently or non-covalently linked to one or more
liposome components. Cell growth inhibitors can be selected from
the group consisting of epidermal growth factor receptor kinase
inhibitors and other kinase inhibitors (e.g. rapamycin and other
inhibitors of the mTOR pathway, r-roscovitine and other
cyclin-dependent kinase inhibitors), DNA alkylators (e.g., nitrogen
mustards (e.g., cyclophosphamide), ethyleneamines, alkyl
sulfonates, nitrosoureas, and triazene derivatives), microtubule
inhibitors (e.g., tamoxiphen, paclitaxel, docetaxel (and other
taxols), vincristine, vinblastine, colcemid, and colchicine), cell
cycle inhibitors (e.g., cytosine arabinoside, purine analogs, and
pyrimidine analogs), and protein synthesis inhibitors (e.g.,
proteosome inhibitors). In one embodiment, rapamycin
(RAPAMUNE.RTM., Wyeth Pharmaceuticals, Inc., Madison, N.J.) is the
cell growth inhibitor. Rapamycin is described in Shillingford, et
al., PNAS 103: 5466-5471 (2006), incorporated herein by reference.
In another embodiment, more than one of these drugs can be
conjugated to a ligand, such as folate, to form, for example, a
dual-drug conjugate.
[0035] In another embodiment, conjugates V-L-D where D is an
antigen or a cell growth inhibitor can be administered in
combination with a cytotoxic compound. The cytotoxic compounds
listed above are among the compounds suitable for this purpose.
[0036] In one embodiment, conjugates are described herein, and such
conjugates may be used in the treatment methods described herein.
Illustratively, the conjugates have the general formula
V-L-D
where V is a folate receptor binding ligand, L is an optional
linker, and D is a cell-growth inhibitor, an antigen, or a
cytotoxin.
[0037] In one embodiment, the folate receptor binding ligand is
folate or an analog of folate, or alternatively a derivative of
either folate or an analog thereof. As used herein, the term
"folate" or "folates" may refer to folate itself, or such analogs
and derivatives of folate. However, it is to be understood that
other folate receptor binding ligands in addition to folates are
contemplated herein. Illustratively, such folate receptor binding
ligands include any compound capable or specific or selective
binding to folate receptors, especially those receptors present on
the surface of cells.
[0038] In another embodiment, the optional linker is absent, and
the conjugate is formed by directly attaching the folate receptor
binding ligand to the cell-growth inhibitor, a cytotoxin, or an
antigen. In another embodiment, the optional linker is present and
is a divalent chemical fragment comprising a chain of carbon,
nitrogen, oxygen, silicon, sulfur, and phosphorus. It is to be
understood that the foregoing atoms may be arranged in any
chemically meaningful way. In one variation, peroxide bonds, i.e.
--O--O-- do not form part of the linker. Generally, the linker is
formed from the foregoing atoms by arranging those atoms to form
functional groups, including but not limited to, alkylene,
cycloalkylene, arylene, ether, amino, hydroxylamino, oximino,
hydrazine, hydrazono, thio, disulfide, carbonyl, carboxyl,
carbamoyl, thiocarbonyl, thiocarboxyl, thiocarbamoyl, xanthyl,
silyl, phosphinyl, phosphonyl, phosphate, and like groups that may
be linked together to construct the linker. It is appreciated that
each of these fragments may also be independently substituted.
[0039] In another embodiment, the drug is a cell-growth inhibitor.
Illustrative of such cell-growth inhibitors are epidermal growth
factor (EGF) receptor kinase inhibitors. Further illustrative of
such cell-growth inhibitor are DNA alkylators, microtubule
inhibitors, cell cycle inhibitors, and protein synthesis
inhibitors.
[0040] In another illustrative embodiment, such cell growth
inhibitors are compounds that inhibit the mammalian target of
rapamycin, also referred to as mTOR. mTOR is a serine/threonine
protein kinase that has been reported to regulate cell growth, cell
proliferation, cell motility, cell survival, protein synthesis, and
transcription (see generally, Beevers et al. "Curcumin inhibits the
mammalian target of rapamycin-mediated signaling pathways in cancer
cells," International Journal of Cancer, 119(4):757-64 (2006); Hay
& Sonenberg N "Upstream and downstream of mTOR," Genes &
Development, 18(16): 1926-45 (2004)). mTOR has been shown to
function as the catalytic subunit of two distinct molecular
complexes in cells. mTOR Complex 1 (mTORC1) is composed of mTOR,
regulatory associated protein of mTOR (Raptor), and mammalian
LST8/G-protein fl-subunit like protein (mLST8/G.beta.L). This
complex possesses the classic features of mTOR by functioning as a
nutrient/energy/redox sensor and controlling protein synthesis.
mTOR Complex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive
companion of mTOR (Rictor), G.beta.L, and mammalian
stress-activated protein kinase interacting protein 1 (mSIN1).
mTORC2 has been shown to function as an important regulator of the
cytoskeleton through its stimulation of F-actin stress fibers,
paxillin, RhoA, Rac1, Cdc42, and protein kinase C a (PKC.alpha.).
In addition, mTORC2 has also been reported to be a "PDK2."
[0041] Illustrative of such mTOR inhibitors is rapamycin, and
analogs and derivatives of rapamycin, such as are described in U.S.
Pat. Nos. 7,153,957 (Regioselective synthesis of CCI-779),
7,122,361 (Compositions employing a novel human kinase), 7,105,328
(Methods for screening for compounds that modulate pd-1 signaling),
7,074,804 (CCI-779 Isomer C), 7,060,797 (Composition and method for
treating lupus nephritis), 7,060,709 (Method of treating hepatic
fibrosis), 7,029,674 (Methods for downmodulating immune cells using
an antibody to PD-1), 7,019,014 (Process for producing anticancer
agent LL-D45042), 6,958,153 (Skin penetration enhancing
components), 6,821,731 (Expression analysis of FKBP nucleic acids
and polypeptides useful in the diagnosis of prostate cancer),
6,713,607 (Effector proteins of Rapamycin), 6,680,330 (Rapamycin
dialdehydes), 6,677,357 (Rapamycin 29-enols), 6,670,355 (Method of
treating cardiovascular disease), 6,617,333 (Antineoplastic
combinations), 6,541,612 (Monoclonal antibodies obtained using
rapamycin position 27 conjugates as an immunogen), 6,511,986
(Method of treating estrogen receptor positive carcinoma),
6,440,991 (Ethers of 7-desmethylrapamycin), 6,432,973 (Water
soluble rapamycin esters), 6,399,626 (Hydroxyesters of
7-desmethylrapamycin), and 6,399,625 (1-oxorapamycins), each
incorporated herein by reference.
[0042] In another illustrative embodiment, the linker includes an
amino acid or a peptide from 2 to about 20 amino acids in length.
As used herein, it is to be understood that amino acids are
illustratively selected from the naturally occurring amino acids,
or stereoisomers thereof. In addition, amino acids may be
non-naturally occurring, and have for example 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. It is further appreciated that water
solubilizing amino acids may be included in the linker to
facilitate uptake and transport of the conjugates described herein.
For example, the amino acids may be selected from asparagine,
aspartic acid, cysteine, glutamic acid, lysine, glutamine,
arginine, serine, ornithine, threonine, and the like.
[0043] In another illustrative embodiment, the bivalent linker (L)
comprises one or more spacer linkers, heteroatom linkers, and
releasable (i.e., cleavable) linkers, and combinations thereof, in
any order. The term "releasable linker" as used herein generally
refers to a linker that includes at least one bond that can be
broken under physiological conditions (e.g., a pH-labile,
acid-labile, oxidatively-labile, enzyme-labile bond, and the like).
It is 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.
[0044] It is also understood that a cleavable bond can connect two
adjacent atoms within the releasable linker and/or connect other
linkers or V and/or D, 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
heteroatom linker, a spacer linker, another releasable linker, the
drug, or analog or derivative thereof, or the vitamin, or analog or
derivative thereof, following breakage of the bond, the releasable
linker is separated from the other moiety.
[0045] 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 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.
[0046] In one embodiment, the present invention provides a vitamin
receptor binding drug delivery conjugate. The drug delivery
conjugate consists of a vitamin receptor binding moiety, bivalent
linker (L), and a drug. The vitamin receptor binding moiety is a
vitamin, or an analog or a derivative thereof, capable of binding
to vitamin receptors, and the drug (antigen, cytotoxin, or cell
growth inhibitor) includes analogs or derivatives thereof
exhibiting drug activity. The vitamin, or the analog or the
derivative thereof, is covalently attached to the bivalent linker
(L), and the drug, or the analog or the derivative thereof, is also
covalently attached to the bivalent linker (L). The bivalent linker
(L) comprises one or more spacer linkers, releasable linkers, and
heteroatom linkers, and combinations thereof, in any order. For
example, the heteroatom linker can be nitrogen, and the releasable
linker and the heteroatom linker can be taken together to form a
divalent radical comprising alkyleneaziridin-1-yl,
alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl,
alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl,
sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below. Alternatively, the
heteroatom linkers can be nitrogen, oxygen, sulfur, and the
formulae --(NHR.sup.1NHR.sup.2)--, --SO--, --(SO.sub.2)--, and
--N(R.sup.3)O--, wherein R.sup.1, R.sup.2, and R.sup.3 are each
independently selected from hydrogen, alkyl, aryl, arylalkyl,
substituted aryl, substituted arylalkyl, heteroaryl, substituted
heteroaryl, and alkoxyalkyl. In another embodiment, the heteroatom
linker can be oxygen, the spacer linker can be
1-alkylenesuccinimid-3-yl, optionally substituted with a
substituent X.sup.1, as defined below, and the releasable linkers
can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,
1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein
each of the releasable linkers is optionally substituted with a
substituent X.sup.2, as defined below, and wherein the spacer
linker and the releasable linker are each bonded to the heteroatom
linker to form a succinimid-1-ylalkyl acetal or ketal.
[0047] The spacer linkers can be 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 the
spacer linkers is optionally substituted with a substituent
X.sup.1, as defined below. In this embodiment, the heteroatom
linker can be nitrogen, and the spacer linkers can be
alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer
linkers is optionally substituted with a substituent X.sup.1, as
defined below, and the spacer linker is bonded to the nitrogen to
form an amide. Alternatively, the heteroatom linker can be sulfur,
and the spacer linkers can be alkylene and cycloalkylene, wherein
each of the spacer linkers is optionally substituted with carboxy,
and the spacer linker is bonded to the sulfur to form a thiol. In
another embodiment, the heteroatom linker can be sulfur, and the
spacer linkers can be 1-alkylenesuccinimid-3-yl and
1-(carbonylalkyl)succinimid-3-yl, and the spacer linker is bonded
to the sulfur to form a succinimid-3-ylthiol.
[0048] In an alternative to the above-described embodiments, the
heteroatom linker can be nitrogen, and the releasable linker and
the heteroatom linker can be taken together to form a divalent
radical comprising alkyleneaziridin-1-yl,
carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, or
sulfonylalkylaziridin-1-yl, wherein each of the releasable linkers
is optionally substituted with a substituent X.sup.2, as defined
below. In this alternative embodiment, the spacer linkers can be
carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl,
carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, wherein
each of the spacer linkers is optionally substituted with a
substituent X.sup.1, as defined below, and wherein the spacer
linker is bonded to the releasable linker to form an aziridine
amide.
[0049] 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 amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the heteroatom linker can be
nitrogen, and the substituent X.sup.1 and the heteroatom linker can
be taken together with the spacer linker to which they are bound to
form an heterocycle.
[0050] The releasable linkers can be methylene, 1-alkoxyalkylene,
1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
haloalkylenecarbonyl, alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl,
oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl,
iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
below.
[0051] In the preceding embodiment, the heteroatom linker can be
oxygen, and the releasable linkers can be methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the oxygen to
form an acetal or ketal. Alternatively, the heteroatom linker can
be oxygen, and the releasable linker can be methylene, wherein the
methylene is substituted with an optionally-substituted aryl, and
the releasable linker is bonded to the oxygen to form an acetal or
ketal. Further, the heteroatom linker can be oxygen, and the
releasable linker can be sulfonylalkyl, and the releasable linker
is bonded to the oxygen to form an alkylsulfonate.
[0052] In another embodiment of the above releasable linker
embodiment, the heteroatom linker can be nitrogen, and the
releasable linkers can be iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the nitrogen
to form an hydrazone. In an alternate configuration, the hydrazone
may be acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form various acylhydrazone
releasable linkers.
[0053] Alternatively, the heteroatom linker can be oxygen, and the
releasable linkers can be alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below, and the releasable
linker is bonded to the oxygen to form a silanol.
[0054] In the above releasable linker embodiment, the drug can
include a nitrogen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom nitrogen
to form an amide, and also bonded to the drug nitrogen to form an
amide.
[0055] In the above releasable linker embodiment, the drug can
include an oxygen atom, the heteroatom linker can be nitrogen, and
the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom linker
nitrogen to form an amide, and also bonded to the drug oxygen to
form an ester.
[0056] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the heteroatom linker can be
nitrogen, and the substituent X.sup.2 and the heteroatom linker can
be taken together with the releasable linker to which they are
bound to form an heterocycle.
[0057] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0058] The drug can include a nitrogen atom, and the releasable
linker can be haloalkylenecarbonyl, optionally substituted with a
substituent X.sup.2, and the releasable linker is bonded to the
drug nitrogen to form an amide.
[0059] The drug can include an oxygen atom, and the releasable
linker can be haloalkylenecarbonyl, optionally substituted with a
substituent X.sup.2, and the releasable linker is bonded to the
drug oxygen to form an ester.
[0060] The drug can include a double-bonded nitrogen atom, and in
this embodiment, the releasable linkers can be
alkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl,
and the releasable linker can be bonded to the drug nitrogen to
form an hydrazone.
[0061] The drug can include a sulfur atom, and in this embodiment,
the releasable linkers can be alkylenethio and carbonylalkylthio,
and the releasable linker can be bonded to the drug sulfur to form
a disulfide.
[0062] The term "aryl" as used herein refers to an aromatic mono or
polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the
like.
[0063] 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.
[0064] The term "substituted aryl" or "substituted heteroaryl" as
used herein refers to aryl or heteroaryl substituted with one or
more substituents selected, such as halo, hydroxy, amino, alkyl or
dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and the
like.
[0065] In addition, the following linkers are contemplated. It is
understood that these linkers may be combined with each other and
other space, heteroatom and releaseable linkers to prepare the
conjugates described herein. Illustrative linkers, and combinations
of spacer and heteroatom linkers include:
##STR00001## ##STR00002## ##STR00003##
Illustrative linkers, and combinations of releasable and heteroatom
linkers include:
##STR00004## ##STR00005##
[0066] Illustrative folate receptor binding ligands include folic
acid, 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 for this
invention are 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 "folate" or "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. Other suitable ligands capable of
binding to folate receptors to initiate receptor-mediated
endocytotic transport of the complex include anti-idiotypic
antibodies to the folate receptor. An exogenous molecule in complex
with an anti-idiotypic antibody to a folate receptor is used to
trigger transmembrane transport of the complex in accordance with
the present invention.
[0067] Generally, any manner of forming a conjugate between the
bivalent linker (L) and the folate receptor-binding ligand, or
between the bivalent linker (L) and the cell-growth inhibitor,
antigen, or cytotoxin, or analog or derivative thereof, including
any intervening heteroatom linkers, may be used. The conjugate may
be formed by direct conjugation of any of these molecules, for
example, 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, hydrazo, and like groups, such as those
described herein.
[0068] The spacer and/or releasable linker (i.e., cleavable linker)
can be any biocompatible linker. The cleavable linker can be, for
example, a linker susceptible to cleavage under the reducing or
oxidizing conditions present in or on cells, a pH-sensitive linker
that may be an acid-labile or base-labile linker, or a linker that
is cleavable by biochemical or metabolic processes, such as an
enzyme-labile linker. Generally, the spacer and/or releasable
linker comprises about 1 to about 50 atoms in length, more
typically about 2 to about 20 carbon atoms. It is appreciated that
lower molecular weight linkers (i.e., those having an approximate
molecular weight of about 30 to about 300) may be employed.
Precursors to such linkers are selected to have suitably reactive
groups at the points of attachment, such as nucleophilic or
electrophilic functional groups, or both, optionally in a protected
form with a readily cleavable protecting group to facilitate their
use in synthesis of the intermediate species.
[0069] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00006##
wherein:
[0070] R is --O--C.dbd.O.CR.sup.7R.sup.8R.sup.9;
[0071] R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
--(CR.sup.12R.sup.13).sub.fOR.sup.10, --CF.sub.3, --F, or
--CO.sub.2R.sup.10;
[0072] R.sup.10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, triphenylmethyl,
benzyl, alkoxymethyl of 2-7 carbon atoms, chloroethyl, or
tetrahydropyranyl; R.sup.8 and R.sup.9 are taken together to form
X;
[0073] X is 2-phenyl-1,3,2-dioxaborinan-5-yl or
2-phenyl-1,3,2-dioxaborinan-4-yl, wherein the phenyl may be
optionally substituted;
[0074] R.sup.12 and R.sup.13 are each, independently, hydrogen,
alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of
2-7 carbon atoms, trifluoromethyl, or --F;
[0075] f=0-6; and
[0076] L is as defined herein.
[0077] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00007##
wherein R in each instance is the same or different and is
independently selected from the group consisting of alkyl of 1-6
carbon atoms, phenyl and benzyl; and L is as defined herein.
[0078] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00008##
where L is as defined herein, and L is connected to the rapamycin
or analog or derivative thereof at either of (O*), and the other of
(O*) is substituted with R, wherein R is hydrogen or
--(R.sup.a--W--R.sup.b).sub.n--;
[0079] W is a linking group;
[0080] R.sup.a is selected from the group consisting of carbonyl,
--S(O)--, --S(O).sub.2--, --P(O).sub.2--, --P(O)(CH.sub.3)--,
--C(S)--, and --CH.sub.2C(O)--;
[0081] R.sup.b is a selected from the group consisting of carbonyl,
--NH--, --S--, --CH.sub.2--, and --O--; and
[0082] n=1-5.
[0083] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00009##
where L is as defined herein, and L is connected to the rapamycin
or analog or derivative thereof at either of (O*), and the other of
(O*) is substituted with R, wherein R is hydrogen, thioalkyl of 1-6
carbon atoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of 1-6
carbon atoms, dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of
2-12 carbon atoms, hydroxyalkoxyalkyl of 2-12 carbon atoms,
acyloxyalkyl of 3-12 carbon atoms, aminoalkyl of 1-6 carbon atoms,
alkylaminoalkyl of 1-6 carbon atoms per alkyl group,
dialkylaminoalkyl of 1-6 carbon atoms per alkyl group,
alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of
3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkyl
having 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of
4-9 carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, or
dioxolanylallyl.
[0084] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00010##
where L is as defined herein, and L is connected to the rapamycin
or analog or derivative thereof at either of (O*), and the other of
(O*) is substituted with R, wherein R is hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9;
where
[0085] R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, trifluoromethyl, or F; R.sup.5 and R.sup.6 are each,
independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms,
(CR.sup.3R.sup.4).sub.fOR.sup.10, CF.sub.3, F, or CO.sub.2R.sup.11;
R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
carbon atoms, alkynyl of 2-7 carbon atoms,
(CR.sup.3R.sup.4).sub.fOR.sup.10, CF.sub.3, F, or
CO.sub.2R.sup.11;
[0086] R.sup.8 and R.sup.9 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, (CR.sup.3R.sup.4).sub.tOR.sup.10, CF.sub.3, F, or
CO.sub.2R.sup.11;
[0087] R.sup.10 is hydrogen or
COCH.sub.2SCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nOCH.sub.3;
[0088] R.sup.11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenylalkyl of
7-10 carbon atoms;
[0089] b=0-6; d=0-6; f=0-6; and n=5-450.
[0090] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00011##
[0091] where L is as defined herein, and L is connected to the
rapamycin or analog or derivative thereof at either of (O*), and
the other of (O*) is substituted with R, wherein R is hydrogen or
--CO(CR.sup.3R.sup.4).sub.b(CR.sup.5R.sup.6).sub.dCR.sup.7R.sup.8R.sup.9;
where
[0092] R.sup.3 and R.sup.4 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, trifluoromethyl, or F;
[0093] R.sup.5 and R.sup.6 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, (CR.sup.3R.sup.4).sub.fOH, CF.sub.3, F, or
CO.sub.2R.sup.11;
[0094] R.sup.7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
(CR.sup.3R.sup.4).sub.fOH, CF.sub.3, F, or CO.sub.2R.sup.11;
[0095] R.sup.8 and R.sup.9 are each, independently, hydrogen, alkyl
of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, (CR.sup.3R.sup.4).sub.fOH, CF.sub.3, F, or
CO.sub.2R.sup.11;
[0096] R.sup.11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenylalkyl of
7-10 carbon atoms;
b=0-6; d=0-6; and f=0-6.
[0097] In one aspect of each of the foregoing, L includes an amino
acid or a peptide. In another aspect of each of the foregoing, L
includes amino acids selected from cysteine, aspartic acid,
glutamic acid, arginine, and lysine. It is to be understood that
either enantiomer of such amino acids may be included in such
illustrative linkers in each instance. In another aspect of each of
the foregoing, L includes a releasable linker. In one variation,
the releasable linker comprises a disulfide bond. In another
variation, the releasable linker comprises a carbonate.
[0098] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00012##
where L is as defined herein, and L is connected to the rapamycin
or analog or derivative thereof at either of (O*). In one aspect, L
includes an amino acid or a peptide. In another aspect, L includes
amino acids selected from cysteine, aspartic acid, glutamic acid,
arginine, and lysine. It is to be understood that either enantiomer
of such amino acids may be included in such illustrative linkers in
each instance. In another aspect, L includes a releasable linker.
In one variation, the releasable linker comprises a disulfide bond.
In another variation, the releasable linker comprises a
carbonate.
[0099] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00013##
where L is as defined herein, and L is connected to the rapamycin
or analog or derivative thereof at either of (O*). In one aspect, L
includes an amino acid or a peptide. In another aspect, L includes
amino acids selected from cysteine, aspartic acid, glutamic acid,
arginine, and lysine. It is to be understood that either enantiomer
of such amino acids may be included in such illustrative linkers in
each instance. In another aspect, L includes a releasable linker.
In one variation, the releasable linker comprises a disulfide bond.
In another variation, the releasable linker comprises a
carbonate.
[0100] In another illustrative embodiment, the conjugate is a
compound of the following formula:
##STR00014##
where L is as defined herein. In one aspect, L includes an amino
acid or a peptide. In another aspect, L includes amino acids
selected from cysteine, aspartic acid, glutamic acid, arginine, and
lysine. It is to be understood that either enantiomer of such amino
acids may be included in such illustrative linkers in each
instance.
[0101] In another illustrative embodiment, the conjugate is a
compound of the following formula (EC0371; see also FIG. 3):
##STR00015##
[0102] The compounds described herein may be prepared by general
organic synthetic reactions, such as those described in U.S. patent
application Ser. No. 10/765,336, the disclosure of which is
incorporated herein by reference.
[0103] Briefly, the following chemical transformations are
described for preparing the compounds described herein.
[0104] General amide and ester formation. For example, where the
heteroatom linker is a nitrogen atom, and the terminal functional
group present on the spacer linker or the releasable linker is a
carbonyl group, the required amide group can be obtained by
coupling reactions or acylation reactions of the corresponding
carboxylic acid or derivative, where L is a suitably-selected
leaving group such as halo, triflate, pentafluorophenoxy,
trimethylsilyloxy, succinimide-N-oxy, and the like, and an amine,
as illustrated in Scheme 1.
##STR00016##
[0105] Coupling reagents include DCC, EDC, RRDQ, CGI, HBTU, TBTU,
HOBT/DCC, HOBT/EDC, BOP-Cl, PyBOP, PyBroP, and the like.
Alternatively, the parent acid can be converted into an activated
carbonyl derivative, such as an acid chloride, a
N-hydroxysuccinimidyl ester, a pentafluorophenyl ester, and the
like. The amide-forming reaction can also be conducted in the
presence of a base, such as triethylamine, diisopropylethylamine,
N,N-dimethyl-4-aminopyridine, and the like. Suitable solvents for
forming amides described herein include CH.sub.2Cl.sub.2,
CHCl.sub.3, THF, DMF, DMSO, acetonitrile, EtOAc, and the like.
Illustratively, the amides can be prepared at temperatures in the
range from about -15.degree. C. to about 80.degree. C., or from
about 0.degree. C. to about 45.degree. C. Amides can be formed
from, for example, nitrogen-containing aziridine rings,
carbohydrates, and .alpha.-halogenated carboxylic acids.
Illustrative carboxylic acid derivatives useful for forming amides
include compounds having the formulae:
##STR00017##
and the like, where n is an integer such as 1, 2, 3, or 4.
[0106] Similarly, where the heteroatom linker is an oxygen atom and
the terminal functional group present on the spacer linker or the
releasable linker is a carbonyl group, the required ester group can
be obtained by coupling reactions of the corresponding carboxylic
acid or derivative, and an alcohol.
[0107] Coupling reagents include DCC, EDC, CDI, BOP, PyBOP,
isopropenyl chloroformate, EEDQ, DEAD, PPh.sub.3, and the like.
Solvents include CH.sub.2Cl.sub.2, CHCl.sub.3, THF, DMF, DMSO,
acetonitrile, EtOAc, and the like. Bases include triethylamine,
diisopropyl-ethylamine, and N,N-dimethyl-4-aminopyridine.
Alternatively, the parent acid can be converted into an activated
carbonyl derivative, such as an acid chloride, a
N-hydroxysuccinimidyl ester, a pentafluorophenyl ester, and the
like.
[0108] General ketal and acetal formation. Furthermore, where the
heteroatom linker is an oxygen atom, and the functional group
present on the spacer linker or the releasable linker is
1-alkoxyalkyl, the required acetal or ketal group can be formed by
ketal and acetal forming reactions of the corresponding alcohol and
an enol ether, as illustrated in Scheme 2.
##STR00018##
[0109] Solvents include alcohols, CH.sub.2Cl.sub.2, CHCl.sub.3,
THF, diethylether, DMF, DMSO, acetonitrile, EtOAc, and the like.
The formation of such acetals and ketals can be accomplished with
an acid catalyst. Where the heteroatom linker comprises two oxygen
atoms, and the releasable linker is methylene, optionally
substituted with a group X.sup.2 as described herein, the required
symmetrical acetal or ketal group can be illustratively formed by
acetal and ketal forming reactions from the corresponding alcohols
and an aldehyde or ketone, as illustrated in Scheme 3.
##STR00019##
[0110] Alternatively, where the methylene is substituted with an
optionally-substituted aryl group, the required acetal or ketal may
be prepared stepwise, where L is a suitably selected leaving group
such as halo, trifluoroacetoxy, triflate, and the like, as
illustrated in Scheme 4. The process illustrated in Scheme 4 is a
conventional preparation, and generally follows the procedure
reviewed by R. R. Schmidt et al., Chem. Rev., 2000, 100, 4423-42,
the disclosure of which is incorporated herein by reference.
##STR00020##
[0111] The resulting arylalkyl ether is treated with an oxidizing
agent, such as DDQ, and the like, to generate an intermediate
oxonium ion that is subsequently treated with another alcohol to
generate the acetal or ketal.
[0112] General succinimide formation. Furthermore, where the
heteroatom linker is, for example, a nitrogen, oxygen, or sulfur
atom, and the functional group present on the spacer linker or the
releasable linker is a succinimide derivative, the resulting
carbon-heteroatom bond can be formed by a Michael addition of the
corresponding amine, alcohol, or thiol, and a maleimide derivative,
where X is the heteroatom linker, as illustrated in Scheme 5.
##STR00021##
[0113] Solvents for performing the Michael addition include THF,
EtOAc, CH.sub.2Cl.sub.2, DMF, DMSO, H.sub.2O and the like. The
formation of such Michael adducts can be accomplished with the
addition of equimolar amounts of bases, such as triethylamine,
Hunig's base or by adjusting the pH of water solutions to 6.0-7.4.
It is appreciated that when the heteroatom linker is an oxygen or
nitrogen atom, reaction conditions may be adjusted to facilitate
the Michael addition, such as, for example, by using higher
reaction temperatures, adding catalysts, using more polar solvents,
such as DMF, DMSO, and the like, and activating the maleimide with
silylating reagents.
[0114] General silyloxy formation. Furthermore, where the
heteroatom linker is an oxygen atom, and the functional group
present on the spacer linker or the releasable linker is a silyl
derivative, the required silyloxy group may be formed by reacting
the corresponding silyl derivative, and an alcohol, where L is a
suitably selected leaving group such as halo, trifluoroacetoxy,
triflate, and the like, as illustrated in Scheme 6.
##STR00022##
[0115] Silyl derivatives include properly functionalized silyl
derivatives such as vinylsulfonoalkyl diaryl, or diaryl, or alkyl
aryl silyl chloride. Instead of a vinylsulfonoalkyl group, a
.beta.-chloroethylsulfonoalkyl precursor may be used. Any aprotic
and anhydrous solvent and any nitrogen-containing base may serve as
a reaction medium. The temperature range employed in this
transformation may vary between -78.degree. C. and 80.degree.
C.
[0116] General hydrazone formation. Furthermore, where the
heteroatom linker is a nitrogen atom, and the functional group
present on the spacer linker or the releasable linker is an iminyl
derivative, the required hydrazone group can be formed by reacting
the corresponding aldehyde or ketone, and a hydrazine or
acylhydrazine derivative, as illustrated in Scheme 7, equations (1)
and (2) respectively.
##STR00023##
[0117] Solvents that can be used include THF, EtOAc,
CH.sub.2Cl.sub.2, CHCl.sub.3, CCl.sub.4, DMF, DMSO, MeOH and the
like. The temperature range employed in this transformation may
vary between 0.degree. C. and 80.degree. C. Any acidic catalyst
such as a mineral acid, H.sub.3CCOOH, F.sub.3CCOOH,
p-TsOH.H.sub.2O, pyridinium p-toluene sulfonate, and the like can
be used. In the case of the acylhydrazone in equation (2), the
acylhydrazone may be prepared by initially acylating hydrazine with
a suitable carboxylic acid or derivative, as generally described
above in Scheme 1, and subsequently reacting the acylhydrazide with
the corresponding aldehyde or ketone to form the acylhydrazone.
Alternatively, the hydrazone functionality may be initially formed
by reacting hydrazine with the corresponding aldehyde or ketone.
The resulting hydrazone may subsequently be acylated with a
suitable carboxylic acid or derivative, as generally described
above in Scheme 1.
[0118] General disulfide formation. Furthermore, where the
heteroatom linker is a sulfur atom, and the functional group
present on the releasable linker is an alkylenethiol derivative,
the required disulfide group can be formed by reacting the
corresponding alkyl or aryl sulfonylthioalkyl derivative, or the
corresponding heteroaryldithioalkyl derivative such as a
pyridin-2-yldithioalkyl derivative, and the like, with an
alkylenethiol derivative, as illustrated in Scheme 8.
##STR00024##
[0119] Solvents that can be used are THF, EtOAc, CH.sub.2Cl.sub.2,
CHCl.sub.3, CCl.sub.4, DMF, DMSO, and the like. The temperature
range employed in this transformation may vary between 0.degree. C.
and 80.degree. C. The required alkyl or aryl sulfonylthioalkyl
derivative may be prepared using art-recognized protocols, and also
according to the method of Ranasinghe and Fuchs, Synth. Commun.
18(3), 227-32 (1988), the disclosure of which is incorporated
herein by reference. Other methods of preparing unsymmetrical
dialkyl disulfides are based on a transthiolation of unsymmetrical
heteroaryl-alkyl disulfides, such as 2-thiopyridinyl,
3-nitro-2-thiopyridinyl, and like disulfides, with alkyl thiol, as
described in WO 88/01622, European Patent Application No.
0116208A1, and U.S. Pat. No. 4,691,024, the disclosures of which
are incorporated herein by reference.
[0120] General carbonate formation. Furthermore, where the
heteroatom linker is an oxygen atom, and the functional group
present on the spacer linker or the releasable linker is an
alkoxycarbonyl derivative, the required carbonate group can be
formed by reacting the corresponding hydroxy-substituted compound
with an activated alkoxycarbonyl derivative where L is a suitable
leaving group, as illustrated in Scheme 9.
##STR00025##
[0121] Solvents that can be used are THF, EtOAc, CH.sub.2Cl.sub.2,
CHCl.sub.3, CCl.sub.4, DMF, DMSO, and the like. The temperature
range employed in this transformation may vary between 0.degree. C.
and 80.degree. C. Any basic catalyst such as an inorganic base, an
amine base, a polymer bound base, and the like can be used to
facilitate the reaction.
[0122] General semicarbazone formation. Furthermore, where the
heteroatom linker is a nitrogen atom, and the functional group
present on one spacer linker or the releasable linker is an iminyl
derivative, and the functional group present on the other spacer
linker or the other releasable linker is an alkylamino or
arylaminocarbonyl derivative, the required semicarbazone group can
be formed by reacting the corresponding aldehyde or ketone, and a
semicarbazide derivative, as illustrated in Scheme 10.
##STR00026##
[0123] Solvents that can be used are THF, EtOAc, CH.sub.2Cl.sub.2,
CHCl.sub.3, CCl.sub.4, DMF, DMSO, MeOH and the like. The
temperature range employed in this transformation may vary between
0.degree. C. and 80.degree. C. Any acidic catalyst such as a
mineral acid, H.sub.3CCOOH, F.sub.3CCOOH, p-TsOH.H.sub.2O,
pyridinium p-toluene sulfonate, and the like can be used. In
addition, in forming the semicarbazone, the hydrazone functionality
may be initially formed by reacting hydrazine with the
corresponding aldehyde or ketone. The resulting hydrazone may
subsequently by acylated with an isocyanate or a carbamoyl
derivative, such as a carbamoyl halide, to form the semicarbazone.
Alternatively, the corresponding semicarbazide may be formed by
reacting hydrazine with an isocyanate or carbamoyl derivative, such
as a carbamoyl halide to form a semicarbazide. Subsequently, the
semicarbazide may be reacted with the corresponding aldehyde or
ketone to form the semicarbazone.
[0124] General sulfonate formation. Furthermore, where the
heteroatom linker is an oxygen atom, and the functional group
present on the spacer linker or the releasable linker is sulfonyl
derivative, the required sulfonate group can be formed by reacting
the corresponding hydroxy-substituted compound with an activated
sulfonyl derivative where L is a suitable leaving group such as
halo, and the like, as illustrated in Scheme 11.
##STR00027##
[0125] Solvents that can be used are THF, EtOAc, CH.sub.2Cl.sub.2,
CHCl.sub.3, CCl.sub.4, and the like. The temperature range employed
in this transformation may vary between 0.degree. C. and 80.degree.
C. Any basic catalyst such as an inorganic base, an amine base, a
polymer bound base, and the like can be used to facilitate the
reaction.
[0126] General formation of folate-peptides. The folate-containing
peptidyl fragment Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) is
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 12.
##STR00028##
[0127] 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 12, and represented in step (b) by Fmoc-AA-OH.
Thus, AA refers to any amino acid starting material that is
appropriately 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.
[0128] 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 (O) to provide the
folate-containing peptidyl fragment 3.
[0129] In another method of treatment embodiment, the group D in
the targeted conjugate V-L-D, comprises an antigen (i.e., a
compound that is administered for the purpose of eliciting an
immune response in vivo), the ligand-antigen conjugates being
effective to "label" the population of proximal tubule cells
responsible for disease pathogenesis in the patient suffering from
the kidney disease for specific elimination by an endogenous immune
response or by co-administered antibodies. The use of
ligand-antigen conjugates in the method of treatment described
herein works to enhance an immune response-mediated elimination of
the proximal tubule cells proliferating abnormally that overexpress
the ligand receptor. Such elimination can be effected through an
endogenous immune response or by a passive immune response effected
by co-administered antibodies.
[0130] The methods of treatment involving the use of ligand-antigen
conjugates are described in U.S. patent application Ser. Nos.
09/822,379, 10/138,275, and PCT Application Serial No.
PCT/US04/014097, each incorporated herein by reference.
[0131] The endogenous immune response can include a humoral
response, a cell-mediated immune response, and any other immune
response endogenous to the host animal, including
complement-mediated cell lysis, antibody-dependent cell-mediated
cytotoxicity (ADCC), antibody opsonization leading to phagocytosis,
clustering of receptors upon antibody binding resulting in
signaling of apoptosis, antiproliferation, or differentiation, and
direct immune cell recognition of the delivered antigen (e.g., a
hapten). It is also contemplated that the endogenous immune
response may employ the secretion of cytokines that regulate such
processes as the multiplication, differentiation, and migration of
immune cells. The endogenous immune response may include the
participation of such immune cell types as B cells, T cells,
including helper and cytotoxic T cells, macrophages, natural killer
cells, neutrophils, LAK cells, and the like.
[0132] The humoral response can be a response induced by such
processes as normally scheduled vaccination, or active immunization
with a natural antigen or an unnatural antigen or hapten, e.g.,
fluorescein isothiocyanate (FITC) or dinitrophenyl (DNP), with the
unnatural antigen inducing a novel immunity. Active immunization
involves multiple injections of the unnatural antigen or hapten
scheduled outside of a normal vaccination regimen to induce the
novel immunity. The humoral response may also result from an innate
immunity where the host animal has a natural preexisting immunity,
such as an immunity to .alpha.-galactosyl groups.
[0133] Alternatively, a passive immunity may be established by
administering antibodies to the host animal such as natural
antibodies collected from serum or monoclonal antibodies that may
or may not be genetically engineered antibodies, including
humanized antibodies. The utilization of a particular amount of an
antibody reagent to develop a passive immunity, and the use of a
ligand-antigen conjugate wherein the passively administered
antibodies are directed to the antigen, would provide the advantage
of a standard set of reagents to be used in cases where a patient's
preexisting antibody titer to potential antigens is not
therapeutically useful. The passively administered antibodies may
be "co-administered" with the ligand-antigen conjugate, and
co-administration is defined as administration of antibodies at a
time prior to, at the same time as, or at a time following
administration of the ligand-antigen conjugate.
[0134] The preexisting antibodies, induced antibodies, or passively
administered antibodies will be redirected to the proximal tubule
cells proliferating abnormally by binding of the ligand-antigen
conjugates to the proximal tubule cell populations overexpressing
the receptor for the ligand, and such pathogenic cells are killed
or eliminated or reduced in number by complement-mediated lysis,
ADCC, antibody-dependent phagocytosis, or antibody clustering of
receptors. The cytotoxic process may also involve other types of
immune responses, such as cell-mediated immunity.
[0135] Acceptable antigens for use in preparing the conjugates used
in the method of treatment described herein are antigens that are
capable of eliciting antibody production in a patient or animal or
that have previously elicited antibody production in a patient or
animal, resulting in a preexisting immunity, or that constitute
part of the innate immune system. Alternatively, antibodies
directed against the antigen may be administered to the patient or
animal to establish a passive immunity. Suitable antigens for use
in the invention include antigens or antigenic peptides against
which a preexisting immunity has developed via normally scheduled
vaccinations or prior natural exposure to such agents such as polio
virus, tetanus, typhus, rubella, measles, mumps, pertussis,
tuberculosis and influenza antigens, and .alpha.-galactosyl groups.
In such cases, the ligand-antigen conjugates will be used to
redirect a previously acquired humoral or cellular immunity to a
population of proximal tubule cells proliferating abnormally in the
patient or animal for elimination of the proximal tubule cells or
reduction in number or inactivation, completely or partially.
[0136] Other suitable immunogens include antigens or antigenic
peptides to which the host animal has developed a novel immunity
through immunization against an unnatural antigen or hapten, for
example, fluorescein isothiocyanate (FITC) or dinitrophenyl, and
antigens against which an innate immunity exists, for example,
super antigens and muramyl dipeptide.
[0137] The proximal tubule cell-binding ligands and antigens,
cytotoxic agents, and cell growth inhibitors, or diagnostic
markers, as the case may be, in forming conjugates for use in
accordance with the methods described herein can be conjugated by
using any art-recognized method for forming a complex. This can
include covalent, ionic, or hydrogen bonding of the ligand V to the
group D compound, either directly or indirectly via a linking group
such as a divalent linker. The conjugate is typically formed by
covalent bonding of the ligand to the targeted entity through the
formation of amide, ester or imino bonds between acid, aldehyde,
hydroxy, amino, or hydrazo groups on the respective components of
the complex or, for example, by the formation of disulfide bonds.
Methods of linking binding ligands to antigens, cytotoxic agents,
or cell growth inhibitors, or diagnostic markers are described in
U.S. patent application Ser. Nos. 10/765,336 and 60/590,580, each
incorporated herein by reference.
[0138] Alternatively, as mentioned above, the ligand complex can be
one comprising a liposome wherein the targeted entity (that is, the
diagnostic marker, or the antigen, cytotoxic agent or cell growth
inhibitor) is contained within a liposome which is itself
covalently linked to the binding ligand. Other nanoparticles,
dendrimers, derivatizable polymers or copolymers that can be linked
to therapeutic or diagnostic markers useful in the treatment and
diagnosis of kidney disease states can also be used in targeted
conjugates.
[0139] In one embodiment of the invention the ligand is folic acid,
an analog of folic acid, or any other folate receptor binding
molecule, and the folate ligand is conjugated to the targeted
entity by a procedure that utilizes trifluoroacetic anhydride to
prepare .gamma.-esters of folic acid via a pteroyl azide
intermediate. This procedure results in the synthesis of a folate
ligand, conjugated to the targeted entity only through the
.gamma.-carboxy group of the glutamic acid groups of folate.
Alternatively, folic acid analogs can be coupled through the
.alpha.-carboxy moiety of the glutamic acid group or both the
.alpha. and .gamma. carboxylic acid entities.
[0140] The therapeutic methods described herein can be used to slow
the progress of disease completely or partially. Alternatively, the
therapeutic methods described herein can eliminate or prevent
reoccurrence of the disease state.
[0141] The conjugates used in accordance with the methods described
herein of the formula V-L-D are used in one aspect to formulate
therapeutic or diagnostic compositions, for administration to a
patient or animal, wherein the compositions comprise effective
amounts of the conjugate and an acceptable carrier therefor.
Typically such compositions are formulated for parenteral use. The
amount of the conjugate effective for use in accordance with the
methods described herein depends on many parameters, including the
nature of the disease being treated or diagnosed, the molecular
weight of the conjugate, its route of administration and its tissue
distribution, and the possibility of co-usage of other therapeutic
or diagnostic agents. The effective amount to be administered to a
patient or animal is typically based on body surface area, patient
weight and physician assessment of patient condition. An effective
amount can range from about to 1 ng/kg to about 1 mg/kg, more
typically from about 1 .mu.g/kg to about 500 .mu.g/kg, and most
typically from about 1 .mu.g/kg to about 100 .mu.g/kg.
[0142] Any effective regimen for administering the ligand
conjugates can be used. For example, the ligand conjugates can be
administered as single doses, or they can be divided and
administered as a multiple-dose daily regimen. Further, a staggered
regimen, for example, one to three days per week can be used as an
alternative to daily treatment, and such an intermittent or
staggered daily regimen is considered to be equivalent to every day
treatment and within the scope of this disclosure. In one
embodiment, the patient or animal is treated with multiple
injections of the ligand conjugate wherein the targeted entity is
an antigen or a cytotoxic agent or a cell growth inhibitor to
eliminate the population of pathogenic proximal tubule cells. In
one embodiment, the patient or animal is treated, for example,
injected multiple times with the ligand conjugate at, for example,
12-72 hour intervals or at 48-72 hour intervals. Additional
injections of the ligand conjugate can be administered to the
patient or animal at intervals of days or months after the initial
injections, and the additional injections prevent recurrence of
disease. Alternatively, the ligand conjugates may be administered
prophylactically to prevent the occurrence of disease in patients
or animals known to be disposed to development of kidney disease
states. In one embodiment, more than one type of ligand conjugate
can be used, for example, the patient or animal may be
pre-immunized with fluorescein isothiocyanate and dinitrophenyl and
subsequently treated with fluorescein isothiocyanate and
dinitrophenyl linked to the same or different targeting ligands in
a co-dosing protocol.
[0143] The ligand conjugates are administered in one aspect
parenterally and most typically by intraperitoneal injections,
subcutaneous injections, intramuscular injections, intravenous
injections, intradermal injections, or intrathecal injections. The
ligand conjugates can also be delivered to a patient or animal
using an osmotic pump. Examples of parenteral dosage forms include
aqueous solutions of the conjugate, for example, a solution in
isotonic saline, 5% glucose or other well-known pharmaceutically
acceptable liquid carriers such as alcohols, glycols, esters and
amides. The parenteral compositions for use in accordance with this
invention can be in the form of a reconstitutable lyophilizate
comprising the one or more doses of the ligand conjugate. In
another aspect, the ligand conjugates can be formulated as one of
any of a number of prolonged release dosage forms known in the art
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. The
ligand conjugates can also be administered topically such as in an
ointment or a lotion, for example, or in a patch form.
[0144] The following examples are illustrative embodiments only and
are not intended to be limiting.
Example 1
Materials
[0145] Fmoc-protected amino acid derivatives, trityl-protected
cysteine 2-chlorotrityl resin (H-Cys(Trt)-2-ClTrt resin
#04-12-2811), Fmoc-lysine(4-methyltrityl) wang resin,
2-(1H-benzotriaxol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphage (HBTU) and N-hydroxybenzotriazole can be
purchased from Novabiochem (La Jolla, Calif.).
N.sup.10-trifluoroacetylpteroic acid can be purchased from Sigma,
St. Louis, Mo.
Example 2
Synthesis of Folate-Cysteine
[0146] Standard Fmoc peptide chemistry can be used to synthesize
folate-cysteine with the cysteine attached to the .gamma.-COOH of
folic acid. The sequence Cys-Glu-Pteroic acid (Folate-Cys) will be
constructed by Fmoc chemistry with HBTU and N-hydroxybenzotriazole
as the activating agents along with diisopropyethylamine as the
base and 20% piperidine in dimethylformamide (DMF) for deprotection
of the Fmoc groups. An .alpha.-t-Boc-protected
N-.alpha.-Fmoc-L-glutamic acid will be linked to a trityl-protected
Cys linked to a 2-Chlorotrityl resin.
N.sup.10-trifluoroacetylpteroic acid was then attached to the
.gamma.-COOH of Glu. The Folate-Cys was cleaved from the resin
using a 92.5% trifluoroacetic acid-2.5% water-2.5%
triisopropylsilane-2.5% ethanedithio solution. Diethyl ether will
be used to precipitate the product, and the precipitant was
collected by centrifugation. The product will be washed twice with
diethyl ether and dried under vacuum overnight. To remove the
N.sup.10-trifluoracetyl protecting group, the product will be
dissolved in a 10% ammonium hydroxide solution and stirred for 30
min at room temperature. The solution will be kept under a stream
of nitrogen the entire time in order to prevent the cysteine from
forming disulfides. After 30 minutes, hydrochloric acid will be
added to the solution until the compound precipitates. The product
will be collected by centrifugation and lyophilized. The product
will be analyzed and confirmed by mass spectroscopic analysis.
##STR00029##
Example 3
Synthesis of Folate-R-Phycoerythrin
[0147] Folate-phycoerythrin will be synthesized by following a
procedure published by Kennedy M. D. et al. in Pharmaceutical
Research, Vol. 20(5); 2003. Briefly, a 10-fold excess of
folate-cysteine will be added to a solution of R-phycoerythrin
pyridyldisulfide (Sigma, St. Louis, Mo.) in phosphate buffered
saline (PBS), pH 7.4. The solution will be allowed to react
overnight at 4.degree. C. and the labeled protein (Mr .about.260
kDa) will be purified by gel filtration chromatography using a G-15
desalting column. The folate labeling will be confirmed by
fluorescence microscopy of M109 cells incubated with
folate-phycoerythrin in the presence and absence of 100-fold excess
of folic acid. After a 1-h incubation and 3 cells washes with PBS,
the treated cells will be intensely fluorescent, while the sample
in the presence of excess folic acid will show little cellular
fluorescence.
Example 4
Synthesis of Folate-Fluorescein
[0148] Folate-FITC will be synthesized as described by Kennedy, M.
D. et al. in Pharmaceutical Research, Vol. 20(5); 2003.
##STR00030##
Example 5
Liposome Preparation
[0149] Liposomes will be prepared following methods by Leamon et
al. in Bioconjugate Chemistry 2003, 14, 738-747. Briefly, lipids
and cholesterol will be purchased from Avanti Polar Lipids
(Alabaster, Ala.). Folate-targeted liposomes will consist of 40
mole % cholesterol, either 4 mole % or 6 mole % polyethyleneglycol
(Mr.about.2000)-derivatized phosphatidylethanolamine (PEG2000-PE,
Nektar AL, Huntsville, Ala.), either 0.03 mole % or 0.1 mole %
folate-cysteine-PEG3400-PE and the remaining mole % will be
composed of egg phosphatidylcholine. Non-targeted liposomes will be
prepared identically with the absence of
folate-cysteine-PEG3400-PE. Lipids in chloroform will be dried to a
thin film by rotary evaporation and then rehydrated in PBS
containing the drug. Rehydration will be accomplished by vigorous
vortexing followed by 10 cycles of freezing and thawing. Liposomes
will be extruded 10 times through a 50 nm pore size polycarbonate
membrane using a high-pressure extruder (Lipex Biomembranes,
Vancouver, Canada).
Example 6
Synthesis of Folate-Saporin
[0150] The protein saporin will be purchased from Sigma (St. Louis,
Mo.). Folate-saporin will be prepared following folate-protein
conjugation methods published by Leamon and Low in The Journal of
Biological Chemistry 1992, 267(35); 24966-24971. Briefly, folic
acid will be dissolved in DMSO and incubated with a 5 fold molar
excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 30
minutes at room temperature. The saporin will be dissolved in 100
mM KH.sub.2PO.sub.4, 100 mM boric acid, pH 8.5. A 10-fold molar
excess of the "activated" vitamin will be added to the protein
solution and the labeling reaction was allowed to proceed for 4
hours. Unreacted material will be separated from the labeled
protein using a Sephadex G-25 column equilibrated in
phosphate-buffered saline, pH 7.4.
Example 7
Synthesis of Folate-Peptides
[0151] Generally, the reagents shown in the following table were
used in the preparation of this example and other examples:
TABLE-US-00001 Reagent (mmol) equivalents Amount
H-Cys(4-methoxytrityl)-2- 0.56 1 1.0 g chlorotrityl-Resin (loading
0.56 mmol/g) Fmoc-.beta.-aminoalanine(NH- 1.12 2 0.653 g MTT)-OH
Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g
Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Glu-OtBu 1.12 2 0.477 g
N.sup.10TFA-Pteroic Acid 0.70 1.25 0.286 g (dissolve in 10 ml DMSO)
DIPEA 2.24 4 0.390 mL PyBOP 1.12 2 0.583 g
[0152] The coupling step was performed as follows: 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 6
coupling steps. At the end wash the resin with 2% hydrazine in DMF
3.times. (5 min) to cleave TFA protecting group on Pteroic
acid.
[0153] Cleave the peptide analog from the resin using the following
reagent, 92.5% (50 ml) TFA, 2.5% (1.34 ml) H.sub.2O, 2.5% (1.34 ml)
Triisopropylsilane, 2.5% (1.34 ml) ethanedithiol, the cleavage step
was performed as follows: Add 25 ml cleavage reagent and bubble for
1.5 hr, drain, and wash 3.times. with remaining reagent. Evaporate
to about 5 mL and precipitate in ethyl ether. Centrifuge and dry.
Purification was performed as follows: Column--Waters NovaPak
C.sub.18 300.times.19 mm; Buffer A=10 mM Ammonium Acetate, pH 5;
B=CAN; 1% B to 20% B in 40 minutes at 15 ml/min, to 350 mg (64%);
HPLC-RT 10.307 min., 100% pure, .sup.1H HMR spectrum consistent
with the assigned structure, and MS (ES-): 1624.8, 1463.2, 1462.3,
977.1, 976.2, 975.1, 974.1, 486.8, 477.8.
Example 8
Synthesis of Folate-.gamma.-Asp-Arg-Asp-Asp-Cys
##STR00031##
[0155] According to the general procedure of the prior example and
Scheme 12, Wang resin bound 4-methoxytrityl (MTT)-protected
Cys-NH.sub.2 was reacted according to the following sequence: 1) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 2) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.
Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.
Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.
Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6)
N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, and Pbf
protecting groups were removed with TFA/H.sub.2O/TIPS/EDT
(92.5:2.5:2.5:2.5), and the TFA protecting group was removed with
aqueous NH.sub.4OH at pH=9.3. Selected .sup.1H NMR (D.sub.2O)
.delta. (ppm) 8.68 (s, 1H, FA H-7), 7.57 (d, 2H, J=8.4 Hz, FA H-12
&16), 6.67 (d, 2H, J=9 Hz, FA H-13 & 15), 4.40-4.75 (m,
5H), 4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H), 2.55-2.95 (m, 8H),
2.42 (m, 2H), 2.00-2.30 (m, 2H), 1.55-1.90 (m, 2H), 1.48 (m, 2H);
MS (ESI, m+H.sup.+) 1046.
Example 9
Synthesis of
Folate-.gamma.-Asp-Asp-Asp-(.beta.-NH.sub.2-Ala)-Cys
##STR00032##
[0157] According to the general procedure of the prior example and
Scheme 12, Wang resin bound 4-methoxytrityl (MTT)-protected
Cys-NH.sub.2 was reacted according to the following sequence: 1) a.
Fmoc-.beta.-aminoalanine(NH-MTT)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 3) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 5) a. Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20%
Piperidine/DMF; 6) N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The
MTT, tBu, and TFA protecting groups were removed with a. 2%
hydrazine/DMF; b. TFA/H.sub.2O/TIPS/EDT (92.5:2.5:2.5:2.5).
##STR00033##
Example 10
Synthesis of Folate-.alpha.-Asp-Arg-Asp-Asp-Cys
[0158] According to the general procedure of the prior example and
Scheme 12, Wang resin bound MTT-protected Cys-NH.sub.2 was reacted
according to the following sequence: 1) a. Fmoc-Asp(OtBu)-OH,
PyBOP, DIPEA; b. 20% Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH,
PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a. Fmoc-Arg(Pbf)-OH, PyBOP,
DIPEA; b. 20% Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP,
DIPEA; b. 20% Piperidine/DMF; 5) a. Fmoc-Glu(.gamma.-OtBu)-OH,
PyBOP, DIPEA; b. 20% Piperidine/DMF; 6) N.sup.10-TFA-pteroic acid,
PyBOP, DIPEA. The MTT, tBu, and Pbf protecting groups were removed
with TFA/H.sub.2O/TIPS/EDT (92.5:2.5:2.5:2.5), and the TFA
protecting group was removed with aqueous NH.sub.4OH at pH=9.3. The
.sup.1H NMR spectrum was consistent with the assigned
structure.
##STR00034##
Example 11
Synthesis of Folate-.gamma.-D-Asp-D-Arg-D-Asp-D-Asp-D-Cys
[0159] According to the general procedure of the prior example and
Scheme 12, Wang resin bound MTT-protected D-Cys-NH.sub.2 was
reacted according to the following sequence: 1) a.
Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 2) a.
Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.
Fmoc-D-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.
Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.
Fmoc-D-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6)
N.sup.10-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, and Pbf
protecting groups were removed with TFA/H.sub.2O/TIPS/EDT
(92.5:2.5:2.5:2.5), and the TFA protecting group was removed with
aqueous NH.sub.4OH at pH=9.3. The .sup.1H NMR spectrum was
consistent with the assigned structure.
Example 12
Synthesis of Folate-Rapamycin (EC0371)
##STR00035##
[0161] This example was prepared according to the prior scheme and
the processes described herein.
Example 13
Animal Models
[0162] To test the ligand-cytotoxin, ligand-antigen, and
ligand-cell growth inhibitor conjugates in animal models of PKD,
well-established animal models will be used. Those animal models
are described in Shillingford, et al., PNAS 103: 5466-5471 (2006),
Piontek, et al., J. Am. Soc. Nephrol. vol. 15: 3035-3043 (2004),
Brown, et al., Kidney Int. vol. 63: 1220-1229 (2003), and Nauta, et
al., Pediatr. Nephrol. vol. 7: 163-172 (1993), incorporated herein
by reference.
Example 14
Immunofluorescence
[0163] The ability of folate-conjugated rapamycin to inhibit the
mTOR pathway was tested in KB cells using immunostaining for P-S6
as a marker (see FIG. 6). The immunostaining procedure was
performed according to the following protocol:
[0164] 1. Aspirate media from cells and immediately add 1 ml/well
of 10% neutral-buffered formalin (NBF) to each well.
[0165] 2. Fix cells for 15 minutes at room temp with gentle orbital
shaking.
[0166] 3. Aspirate NBF from cells and wash briefly with 2 changes
(1 ml/well) of 1.times.PBS.
[0167] 4. Aspirate PBS and add 1 ml of quench solution and quench
for 10 minutes at room temperature with gentle orbital shaking.
[0168] 5. Aspirate quench and wash briefly with 2 changes (1
ml/well) of 1.times.PBS.
[0169] 6. Aspirate PBS and add 1 ml/well cell
block/permeabilization (CBP) solution and incubate for 30 minutes
at 37.degree. C.
[0170] 7. Prepare P-S6 (S235/6)/(3-tubulin antibody solution by
diluting antibody 1:200 in CBP solution (for 12 coverslips make
1194 ul CBP+6 ul P-S6 and .beta.-tubulin). Mix thoroughly.
[0171] 8. Remove the lid from the cell culture dish and place in a
humidified chamber. Cut parafilm to the size of the lid and press
firmly on to the lid.
[0172] 9. Using a pair of needle-nose tweezers transfer a
coverslip, cell-side up, to its corresponding well position on the
lid. Pipet 150 .mu.l of P-S6/.beta.-tubulin antibody solution onto
the coverslip. Repeat for all remaining coverslips.
[0173] 10. Close the lid and incubate overnight at 4.degree. C.
[0174] 11. The following day, make 100 ml cell wash (CW)
solution.
[0175] 12. Pipet 1 ml CW solution into each well of a fresh 12-well
plate. Using two pairs of tweezers, one placed on the back of the
coverslip, carefully pick up the coverslip and place back in the
corresponding well.
[0176] 13. Incubate for 5 minutes with gentle orbital shaking.
Aspirate and repeat wash 2.times..
[0177] 14. During washes, dilute fluorescent-conjugated anti-rabbit
FITC and anti-mouse TXR secondary antibodies 1:200 in CBP.
Centrifuge 10 minutes at 4.degree. C., 13,000 rpm, to remove
aggregates.
[0178] 15. After washing, repeat steps 8 and 9 with diluted
fluorescent-conjugated secondary antibody solution. Incubate for 1
hour at 37.degree. C.
[0179] 16. Repeat steps 12 and 13.
[0180] 17. Rinse 1.times. with 1.times.PBS.
[0181] 18. Aspirate and wash 2.times.3 minutes with
1.times.PBS+0.1% Triton-X 100 with gentle orbital shaking.
[0182] 19. Aspirate and rinse 2.times. with 1.times.PBS.
[0183] 20. Aspirate and add 1 ml of 10% NBF to post-fix secondary
antibodies. Incubate for 10 minutes at room temperature with gentle
orbital shaking.
[0184] 21. Aspirate and wash 1.times.5 minutes with
1.times.PBS.
[0185] 22. Aspirate and add 1 ml of 1.times.PBS+DAPI (1 mg/ml
stock, 1:50,000 dilution). Incubate for 5-10 minutes.
[0186] 23. Thaw Prolong Gold mounting medium. Dispense two drops on
a slide. Using needle-nose tweezers remove individual coverslips,
wipe excess solution from backside and place on top of mounting
medium, cell-side down. Gently squeeze out any air bubbles with the
opposite end of the tweezers.
[0187] 25. Allow mounting medium to harden for at least 1 hour,
preferably overnight. View slides under a suitable microscope
equipped for fluorescence. Store slides at -20.degree. C.
Example 15
Folate Receptor Immunohistochemistry
[0188] Immunohistochemistry was performed as described in PCT Publ.
No. WO/2006/105141, incorporated herein by reference. As shown in
FIGS. 1 and 2, monoclonal and polyclonal antibodies to the folate
receptor stain cysts in polycystic kidney disease tissues
indicating folate receptor overexpression in the cells that form
PKD cysts (see also Table 1 below).
TABLE-US-00002 TABLE 1 Polycystic Kidney Tissue from Mice and
Humans Polycystic Kidney Disease IHC Results Specimen ID 3+ 2+ 1+ 0
PKD PKD Case 7 10% 30% 40% 20% PKD Case 8 0% 30% 60% 10% PKD Case 9
0% 10% 20% 70% PKD Case 10 0% 0% 40% 60% Control N1 10% 20% 30% 40%
Kidney N3 Tissue was not normal kidney N4 30% 30% 30% 10% Serous
ITOC02407A 0% 20% 50% 30% OVCA ITOC02463A 10% 20% 10% 60%
ITOC02556A 0% 0% 0% 100% PKD ORPK666B 10% 10% 50% 30% BPK6468D 0%
10% 20% 70% BPK6467B 0% 20% 40% 40% Control ORPK665B 0% 0% 40% 60%
Kidney ORPK667B 0% 5% 80% 15% BPK6466B 0% 5% 75% 20% BPK6469B 0% 0%
0% 100%
Example 16
Relative Affinity Assay
[0189] Binding assays were run to determine the relative affinities
of EC0371 and folic acid at the folate receptor. KB cells were
incubated for 1 hour at 37.degree. C. with 100 nM .sup.3H-folic
acid in the presence and absence of increasing competitor
concentrations. As shown in FIG. 4 (error bars represent 1 standard
deviation (n=3)), the relative affinity of EC0371 at the folate
receptor is 0.5 compared to a relative affinity of 1.0 for folic
acid.
Example 17
Cell Viability
[0190] Cell viability was examined in KB cells following incubation
for 16 hours in Rapamycin (2, 10, and 50 nM), EC0371 (2, 10, and 50
nM), DMSO (diluent), and media alone (FIG. 5). At 24 hours, neither
rapamycin nor EC0371 was found to be cytotoxic at any of the
concentrations tested.
Example 18
P-S6 and P-S6K Immunoblots
[0191] Folate-rapamycin was found to be highly effective in
inhibiting mTOR in cultured cells. Folate receptor-positive KB
cells were treated with either unconjugated rapamycin (2, 10, or 50
nM) or folate-rapamycin (2, 10, or 50 nM) for 16 hours. The
activity of mTOR was determined by immunoblotting using
phospho-specific antibodies against P-S6 and P-S6K (FIG. 7).
Example 19
Therapeutic Effects of EC0371 In Vivo
[0192] The therapeutic effect of EC0371 (folate-conjugated
rapamycin) was tested on in vivo development of polycystic kidney
disease in the bpk-mutant mouse model. Bpk-mutant mice develop
polycystic kidney disease (PKD) starting at embryogenesis due to a
point mutation in the gene encoding bicaudal C. All nephron
segments are affected, and most bpk-mutant mice die between
postnatal days 24-30 due to severely enlarged cystic kidneys and
renal failure.
[0193] All mice were genotyped by PCR prior to treatment. Wildtype
(Wt) and bpk-mutant (bpk) mice were then segregated into the
following three groups: no treatment (n=5 Wt, 2 bpk); vehicle
treatment (n=5 Wt, 3 bpk); and EC0371 treatment (n=4 Wt, 4
bpk).
[0194] EC0371 was prepared by reconstitution in sterile PBS to a
concentration of 1 mM, then diluted 1:5 for a final concentration
of 0.2 mM (2 nmol/.mu.l). Mice were injected (i.p.) daily with
either EC0371 (3 .mu.mol/kg), vehicle (PBS), or received no
injection, from postnatal day 7 to day 21. On day 21, whole body
weight was recorded and blood was collected. Mice were then
sacrificed and the kidneys, liver, spleen, and thymus were removed
and weighed. EC0371 treatment of bpk-mutant mice was found to
significantly improve the PKD phenotype as measured by kidney size
(FIG. 8), and proportion of kidney(s) to whole body weight (FIGS. 9
and 10).
* * * * *