U.S. patent application number 11/481264 was filed with the patent office on 2007-01-11 for imaging and therapeutic method using monocytes.
Invention is credited to Andrew R. Hilgenbrink, Philip S. Low.
Application Number | 20070009434 11/481264 |
Document ID | / |
Family ID | 37478921 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009434 |
Kind Code |
A1 |
Low; Philip S. ; et
al. |
January 11, 2007 |
Imaging and therapeutic method using monocytes
Abstract
The invention relates to a method of treating or diagnosing a
disease state mediated by monocytes. The method utilizes a
composition comprising a conjugate or complex of the general
formula A.sub.b-X wherein the group A.sub.b comprises a ligand that
binds to monocytes, and when the conjugate is being used for
treatment of the disease state, the group X comprises an immunogen,
a cytotoxin, or a compound capable of altering monocyte function,
and when the conjugate is being used for diagnosing the disease
state, the group X comprises an imaging agent.
Inventors: |
Low; Philip S.; (West
Lafayette, IN) ; Hilgenbrink; Andrew R.; (Lafayette,
IN) |
Correspondence
Address: |
BARNES & THORNBURG LLP
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Family ID: |
37478921 |
Appl. No.: |
11/481264 |
Filed: |
July 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60696740 |
Jul 5, 2005 |
|
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60801636 |
May 18, 2006 |
|
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Current U.S.
Class: |
424/1.49 ;
424/85.1; 424/9.6; 530/388.22; 530/391.1 |
Current CPC
Class: |
A61K 38/47 20130101;
A61P 13/12 20180101; A61P 37/00 20180101; A61P 37/06 20180101; A61K
47/64 20170801; A61P 17/00 20180101; A61P 19/02 20180101; A61P
25/00 20180101; A61K 38/19 20130101; A61P 1/04 20180101; A61P 29/00
20180101; A61K 47/545 20170801; A61K 49/0021 20130101; A61K 47/6911
20170801; A61K 49/0043 20130101; A61K 51/044 20130101; A61K 49/0041
20130101; A61P 11/00 20180101; A61K 47/551 20170801; A61K 49/0052
20130101; C09B 11/24 20130101; A61K 51/0459 20130101 |
Class at
Publication: |
424/001.49 ;
424/009.6; 530/391.1; 530/388.22; 424/085.1 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 49/00 20060101 A61K049/00; C07K 16/46 20060101
C07K016/46; C07K 16/28 20060101 C07K016/28; A61K 38/19 20060101
A61K038/19 |
Claims
1. A method for diagnosing a disease state mediated by monocytes,
said method comprising the steps of: isolating monocytes from a
patient suffering from a monocyte mediated disease state;
contacting the monocytes with a composition comprising a conjugate
or complex of the general formula A.sub.b-X where the group A.sub.b
comprises a ligand that binds to monocytes and the group X
comprises an imaging agent; and quantifying the percentage of
monocytes that expresses a receptor for the ligand.
2. The method of claim 1 wherein A.sub.b comprises a folate
receptor binding ligand.
3. The method of claim 1 wherein A.sub.b comprises a
monocyte-binding antibody or antibody fragment.
4. The method of claim 1 wherein the imaging agent comprises a
metal chelating moiety.
5. The method of claim 4 wherein the imaging agent further
comprises a metal cation.
6. The method of claim 5 wherein the metal cation is a
radionuclide.
7. The method of claim 1 wherein the imaging agent comprises a
radionuclide.
8. The method of claim 7 wherein the radionuclide is selected from
the group consisting of technetium, gallium, indium, and a positron
emitting radionuclide.
9. The method of claim 1 wherein the imaging agent comprises a
chromophore.
10. The method of claim 9 wherein the chromophore comprises a
compound selected from the group consisting of fluorescein, Oregon
Green, rhodamine, phycoerythrin, Texas Red, and AlexaFluor 488.
11. The method of claim 1 wherein the patient is suffering from a
disease state selected from the group consisting of rheumatoid
arthritis, osteoarthritis, ulcerative colitis, Crohn's disease,
inflammatory lesions, infections of the skin, osteomyelitis, organ
transplant rejection, pulmonary fibrosis, sarcoidosis, systemic
sclerosis, lupus erythematosus, glomerulonephritis, inflammations
of the skin, and any chronic inflammation.
12. A method for treating a disease state mediated by monocytes,
said method comprising the steps of: administering to a patient
suffering from a monocyte-mediated disease state an effective
amount of a composition comprising a conjugate or complex of the
general formula A.sub.b-X where the group A.sub.b comprises a
ligand that binds to monocytes and the group X comprises an
immunogen, a cytotoxin, or a compound capable of altering monocyte
function; and eliminating the monocyte-mediated disease state.
13. The method of claim 12 wherein A.sub.b comprises a folate
receptor binding ligand.
14. The method of claim 12 wherein A.sub.b comprises a
monocyte-binding antibody or antibody fragment.
15. The method of claim 12 wherein the group X comprises an
immunogen.
16. The method of claim 13 wherein the group X comprises an
immunogen.
17. The method of claim 12 wherein the group X comprises a
cytotoxin.
18. The method of claim 17 wherein the group X further comprises a
liposome.
19. The method of claim 13 wherein the group X comprises a
cytotoxin.
20. The method of claim 19 wherein the group X further comprises a
liposome.
21. The method of claim 12 wherein X comprises a compound capable
of altering monocyte function.
22. The method of claim 21 wherein the compound capable of altering
monocyte function is a cytokine.
23. The method of claim 12 wherein the patient is suffering from a
disease state selected from the group consisting of rheumatoid
arthritis, osteoarthritis, ulcerative colitis, Crohn's disease,
inflammatory lesions, infections of the skin, osteomyelitis, organ
transplant rejection, pulmonary fibrosis, sarcoidosis, systemic
sclerosis, lupus erythematosus, glomerulonephritis, inflammations
of the skin, and any chronic inflammation.
24. The method of claim 13 wherein X comprises a compound capable
of altering monocyte function.
25. The method of claim 24 wherein the compound capable of altering
monocyte function is a cytokine.
26. The method of claim 13 wherein the patient is suffering from a
disease state selected from the group consisting of rheumatoid
arthritis, osteoarthritis, ulcerative colitis, Crohn's disease,
inflammatory lesions, infections of the skin, osteomyelitis, organ
transplant rejection, pulmonary fibrosis, sarcoidosis, systemic
sclerosis, lupus erythematosus, glomerulonephritis, inflammations
of the skin, and any chronic inflammation.
27. A method for diagnosing a disease state mediated by monocytes,
the method comprising the steps of administering parenterally to a
patient a composition comprising a conjugate or complex of the
general formula A.sub.b-X where the group A.sub.b comprises a
ligand that binds to monocytes and the group X comprises an imaging
agent, and quantifying the percentage of monocytes that expresses a
receptor for the ligand.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/696,740,
filed on Jul. 5, 2005, and to U.S. Provisional Application Ser. No.
60/801,636, filed on May 18, 2006, each incorporated by reference
herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for treating and
diagnosing disease states mediated by monocytes. More particularly,
ligands that bind to monocytes are complexed with an imaging agent
for use in diagnosis or to an immunogen, a cytotoxin, or an agent
for altering monocyte function for use in the treatment of
monocyte-mediated disease.
BACKGROUND
[0003] The mammalian immune system provides a means for the
recognition and elimination of foreign pathogens. While the immune
system normally provides a line of defense against foreign
pathogens, there are many instances where the immune response
itself is involved in the progression of disease. Exemplary of
diseases caused or worsened by the host's own immune response are
autoimmune diseases and other diseases in which the immune response
contributes to pathogenesis. For example, macrophages are generally
the first cells to encounter foreign pathogens, and accordingly,
they play an important role in the immune response, but activated
macrophages can also contribute to the pathophysiology of disease
in some instances.
[0004] The folate receptor is a 38 KD GPI-anchored protein that
binds the vitamin folic acid with high affinity (<1 nM).
Following receptor binding, rapid endocytosis delivers the vitamin
into the cell, where it is unloaded in an endosomal compartment at
low pH. Importantly, covalent conjugation of small molecules,
proteins, and even liposomes to folic acid does not block the
vitamin's ability to bind the folate receptor, and therefore,
folate-drug conjugates can readily be delivered to and can enter
cells by receptor-mediated endocytosis.
[0005] Because most cells use an unrelated reduced folate carrier
to acquire the necessary folic acid, expression of the folate
receptor is restricted to a few cell types. With the exception of
kidney, choroid plexus, and placenta, normal tissues express low or
nondetectable levels of the folate receptor. However, many
malignant tissues, including ovarian, breast, bronchial, and brain
cancers express significantly elevated levels of the receptor. In
fact, it is estimated that 95% of all ovarian carcinomas
overexpress the folate receptor. It has been reported that the
folate receptor .beta., the nonepithelial isoform of the folate
receptor, is expressed on activated (but not resting) synovial
macrophages. Thus, folate receptors are expressed on a subset of
macrophages (i.e., activated macrophages).
SUMMARY
[0006] It is unknown, however, whether folate receptors are
expressed on monocytes, the precursor cells for macrophages. Thus,
Applicants have undertaken to determine whether folate receptors
are expressed on monocytes and whether monocyte targeting, using a
ligand such as folate, to deliver cytotoxic or other inhibitory
compounds to monocytes, is useful therapeutically. Applicants have
also undertaken to determine whether an imaging agent linked to a
ligand capable of binding to monocytes may be useful for diagnosing
inflammatory pathologies.
[0007] A method is provided for treating and diagnosing disease
states mediated by monocytes. In one embodiment, the monocytes are
activated monocytes. In one embodiment, disease states mediated by
monocytes are treated by delivering an immunogen to the monocytes,
by linking the immunogen to a ligand that binds to monocytes, to
redirect host immune responses to monocytes. In another embodiment,
monocytes can be inactivated or killed by other methods such as by
the delivery to monocytes of cytotoxins or other compounds capable
of altering monocyte function.
[0008] In the embodiment where an immunogen is delivered to
monocytes to inactivate or kill monocytes, ligands that bind to
monocytes are conjugated with an immunogen to redirect host immune
responses to the monocytes, or the ligand is conjugated to a
cytotoxin for killing of monocytes. Ligands that can be used in the
conjugates of the present invention include those that bind to
receptors expressed on monocytes (e.g., activated monocytes), such
as the folate receptor, or ligands such as monoclonal antibodies
directed to cell surface markers expressed on monocytes or other
ligands that bind to activated monocytes. In another embodiment,
ligands that bind to monocytes are conjugated to an imaging agent
and the conjugate is used to diagnose diseases mediated by
monocytes.
[0009] In another embodiment, a method is provided for diagnosing a
disease state mediated by monocytes. The method comprises the steps
of isolating monocytes from a patient suffering from a
monocyte-mediated disease state, contacting the monocytes with a
composition comprising a conjugate or complex of the general
formula A.sub.b-X
[0010] where the group A.sub.b comprises a ligand that binds to
monocytes and the group X comprises an imaging agent, and
quantifying the percentage of monocytes that expresses a receptor
for the ligand. In another embodiment, A.sub.b comprises a folate
receptor binding ligand. In yet another embodiment, A.sub.b
comprises a monocyte-binding antibody or antibody fragment or other
ligands that bind to activated monocytes. In another embodiment,
the imaging agent comprises a metal chelating moiety that binds an
element that is a radionuclide. In still another embodiment, the
imaging agent comprises a chromophore selected from the group
consisting of fluorescein, Oregon Green, rhodamine, phycoerythrin,
Texas Red, and AlexaFluor 488.
[0011] In another embodiment, a method is provided for diagnosing a
disease state mediated by monocytes. The method comprises the steps
of administering parenterally to a patient a composition comprising
a conjugate or complex of the general formula A.sub.b-X where the
group A.sub.b comprises a ligand that binds to monocytes and the
group X comprises an imaging agent, and quantifying the percentage
of monocytes that expresses a receptor for the ligand.
[0012] In another embodiment, a method is provided for treating a
disease state mediated by monocytes. The method comprises the steps
of administering to a patient suffering from a monocyte-mediated
disease state an effective amount of a composition comprising a
conjugate or complex of the general formula A.sub.b-X where the
group A.sub.b comprises a ligand that binds to monocytes and the
group X comprises an immunogen, a cytotoxin, or a compound capable
of altering monocyte function, and eliminating the
monocyte-mediated disease state.
[0013] In yet another embodiment, a compound for diagnosing or
treating a disease state mediated by monocytes is provided. The
compound is selected from the following group of compounds:
##STR1## ##STR2## ##STR3##
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows folate-fluorescein binding to human monocytes
isolated from peripheral blood and left untreated or preincubated
with a 100-fold excess of unlabeled folic acid to compete with
folate-fluorescein for binding.
[0015] FIG. 2 shows folate-fluorescein (folate-FITC e.g.
folate-fluorescein isothiocyanate) binding, quantified by flow
cytometry, to CD11b.sup.+ human monocytes (panel A) and to
CD11b.sup.+ human monocytes preincubated with an excess of
unlabeled folic acid (panel B) to compete with folate-FITC for
binding.
[0016] FIG. 3 shows flow cytometry analysis, using CD11b (A), CD14
(B), CD16 (C), CD69 (D), and HLA-DR (E) antibodies, of CD markers
that are co-expressed with the folate receptor on human
monocytes.
[0017] FIG. 4 shows binding of .sup.3H-folic acid to white blood
cells from humans, dogs, rabbits, rats, mice, or to KB cells. The
cells were either preincubated with a 100-fold excess of unlabeled
folic acid (cross-hatched bars labeled with an "xs") or not
preincubated with excess unlabeled folic acid (solid bars).
[0018] FIG. 5 shows folate-FITC binding, analyzed by flow
cytometry, to peripheral blood monocytes from dogs (panels A and C)
and horses (panels B and D) and competition of binding by unlabeled
folic acid.
[0019] FIG. 6 shows folate-FITC (A-C) or folate-AlexaFluor 488
(D-F) binding, analyzed by flow cytometry, to peripheral blood
monocytes from dogs and competition of binding by unlabeled folic
acid.
[0020] FIG. 7 shows folate-phycoerythrin binding, analyzed by flow
cytometry, to human peripheral blood monocytes and competition by
unlabeled folic acid.
[0021] FIG. 8 shows the percentage of human peripheral blood
monocytes that are folate receptor positive in healthy humans
(squares) and in patients with rheumatoid arthritis (diamonds),
osteoarthritis (upper group of triangles), and fibromyalgia (three
triangles at lowest percentages).
[0022] FIG. 9 shows paw volume over time in rats after arthritis
induction. The rats were treated with folate-flumethasone (50
nmoles/kg/day; squares) or folate-indomethacin (100 (triangles) or
250 (diamonds) nmoles/kg/day) or were untreated (circles).
[0023] FIG. 10 shows the percentage of human peripheral blood
monocytes that are folate receptor positive in patients with
rheumatoid arthritis over the course of therapy.
DETAILED DESCRIPTION
[0024] Methods are provided for treating and diagnosing disease
states mediated (e.g., caused or augmented) by monocytes. Exemplary
disease states include fibromyalgia, rheumatoid arthritis,
osteoarthritis, ulcerative colitis, Crohn's disease, psoriasis,
osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary
fibrosis, sarcoidosis, systemic sclerosis, organ transplant
rejection (GVHD), lupus erythematosus, Sjogren's syndrome,
glomerulonephritis, inflammations of the skin (e.g., psoriasis),
and chronic inflanunations. Such disease states can be diagnosed by
isolating monocytes (e.g., whole blood or peripheral blood
monocytes) from a patient suffering from such disease state,
contacting the monocytes with a composition comprising a conjugate
of the general formula A.sub.b-X wherein the group A.sub.b
comprises a ligand that binds to monocytes, and the group X
comprises an imaging agent, and quantifying the percentage of
monocytes expressing a receptor for the ligand.
[0025] Such disease states can also be diagnosed by administering
parenterally to a patient a composition comprising a conjugate or
complex of the general formula A.sub.b-X where the group A.sub.b
comprises a ligand that binds to monocytes and the group X
comprises an imaging agent, and quantifying the percentage of
monocytes that expresses a receptor for the ligand.
[0026] Monocyte-mediated disease states can be treated in
accordance with the methods disclosed herein by administering an
effective amount of a composition A.sub.b-X wherein A.sub.b
comprises a ligand that binds to monocytes and wherein the group X
comprises an immunogen, a cytotoxin, or a compound capable of
altering monocyte function. Such monocyte targeting conjugates,
when administered to a patient suffering from a monocyte-mediated
disease state, work to concentrate and associate the conjugated
cytotoxin, immunogen, or compound capable of altering monocyte
function with the population of monocytes to kill the monocytes or
alter monocyte 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.
[0027] 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.
[0028] As used herein, the terms "elimination" and "deactivation"
of the monocyte population that expresses the ligand receptor mean
that this monocyte population is killed or is completely or
partially inactivated which reduces the monocyte-mediated
pathogenesis characteristic of the disease state being treated.
[0029] As used herein, "mediated by" in reference to diseases
mediated by monocytes means caused by or augmented by. For example,
monocytes can directly cause disease or monocytes can augment
disease states such as by stimulating other immune cells to secrete
factors that mediate disease states, such as by stimulating T-cells
to secrete TNF-.alpha.. Illustratively, monocytes themselves may
also harbor infections and cause disease and infected monocytes may
cause other immune cells to secrete factors that cause disease such
as TNF-.alpha. secretion by T-cells.
[0030] In one embodiment, monocyte-mediated disease states are
diagnosed in a patient by isolating monocytes from the patient,
contacting the monocytes with a conjugate A.sub.b-X wherein A.sub.b
comprises a ligand that binds to monocytes and X comprises an
imaging agent, and quantifying the percentage of monocytes
expressing the receptor for the ligand. In another embodiment, the
imaging or diagnostic conjugates can be administered to the patient
as a diagnostic composition comprising a conjugate and a
pharmaceutically acceptable carrier and thereafter monocytes can be
collected from the patient to quantify the percentage of monocytes
expressing the receptor for the ligand A.sub.b. In this embodiment,
the composition is typically formulated for parenteral
administration and is administered to the patient in an amount
effective to enable imaging of monocytes. In another embodiment,
disease states can also be diagnosed by administering parenterally
to a patient a composition comprising a conjugate or complex of the
general formula A.sub.b-X where the group A.sub.b comprises a
ligand that binds to monocytes and the group X comprises an imaging
agent, and quantifying the percentage of monocytes that expresses a
receptor for the ligand.
[0031] In one embodiment, for example, the imaging agent (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 imaging agent can comprise a
chromophore such as, for example, fluorescein, rhodamine, Texas
Red, phycoerythrin, Oregon Green, AlexaFluor 488 (Molecular Probes,
Eugene, Oreg.), Cy3, Cy5, Cy7, and the like.
[0032] 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 imaging can be performed by
any suitable imaging method known in the art, such as intravital
imaging.
[0033] The method disclosed herein can be used for both human
clinical medicine and veterinary applications. Thus, the host
animal afflicted with the monocyte-mediated 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 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 inflammatory disease states.
[0034] In the ligand conjugates of the general formula A.sub.b-X,
the group A.sub.b is a ligand that binds to monocytes (e.g.,
activated monocytes) when the conjugates are used to diagnose or
treat disease states. Any of a wide number of monocyte-binding
ligands can be employed. Acceptable ligands include particularly
folate receptor binding ligands, and analogs thereof, and
antibodies or antibody fragments capable of recognizing and binding
to surface moieties expressed or presented on monocytes. In one
embodiment, the monocyte-binding ligand is folic acid, a folic acid
analog or another folate receptor binding molecule. In another
embodiment the monocyte-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
monocytes.
[0035] In one embodiment, the monocyte-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).
[0036] In another embodiment, other vitamins can be used as the
monocyte-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.
[0037] In other embodiments, the monocyte-binding ligand can be any
ligand that binds to a receptor expressed or overexpressed on
activated monocytes including CD40-, CD16-, CD14-, CD11b-, and
CD62-binding ligands, 5-hydroxytryptamine, macropahge inflammatory
protein 1-.alpha., MIP-2, receptor activator of nuclear factor kB
ligand antagonists, monocyte chemotactic protein 1-binding ligands,
chemokine receptor 5-binding ligands, RANTES-binding ligands,
chemokine receptor-binding ligands, and the like.
[0038] The monocyte (e.g., activated monocytes) targeted conjugates
used for diagnosing or treating disease states mediated by
monocytes have the formula A.sub.b-X, wherein A.sub.b is a ligand
capable of binding to monocytes, and the group X comprises an
imaging agent or an immunogen, cytotoxin, or a compound capable of
altering monocyte function. In such conjugates wherein the group
A.sub.b 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 Publication No. 2005/0002942 A1, each incorporated
herein by reference, describe methods and examples for preparing
conjugates useful in accordance with the methods described herein.
The present monocyte-targeted imaging 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.
[0039] In accordance with another embodiment, there is provided a
method of treating disease states mediated by monocytes by
administering to a patient suffering from such disease state an
effective amount of a composition comprising a conjugate of the
general formula A.sub.b-X wherein A.sub.b is as defined above and
the group X comprises a cytotoxin, an immunogen, or a compound
capable of altering monocyte function. In these embodiments, the
monocytes can be activated monocytes and the group A.sub.b 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 are clodronate, anthrax, Pseudomonas
exotoxin, typically modified so that these cytotoxic moieties do
not bind to normal cells, and other toxins or cytotoxic agents
including art-recognized chemotherapeutic agents such as
adrenocorticoids, alkylating agents, antiandrogens, antiestrogens,
androgens, estrogens, antimetabolites such as cytosine arabinoside,
purine analogs, pyrimidine analogs, and methotrexate, busulfan,
carboplatin, chlorambucil, cisplatin and other platinum compounds,
tamoxiphen, taxol, cyclophosphamide, plant alkaloids, prednisone,
hydroxyurea, teniposide, and bleomycin, nitrogen mustards,
nitrosureas, vincristine, vinblastine, MEK kinase inhibitors, MAP
kinase pathway inhibitors, PI-3-kinase inhibitors, mitochondrial
perturbants, NF.kappa.B pathway inhibitors, proteosome inhibitors,
pro-apoptotic agents, glucocorticoids, such as prednisolone,
flumethasone, dexamethasone, and betamethasone, indomethacin,
diclofenac, proteins such as pokeweed, saporin, momordin, and
gelonin, non-steroidal anti-inflammatory drugs (NSAIDs), protein
synthesis inhibitors, didemnin B, verrucarin A, geldanamycin, and
the like. Such toxins or cytotoxic compounds can be directly
conjugated to the monocyte-binding ligand, for example, folate or
another folate receptor-binding ligand, or they can be formulated
in liposomes or other small particles which themselves are targeted
as conjugates of the monocyte-binding ligand typically by covalent
linkages to component phospholipids.
[0040] Similarly, when the group X comprises a compound capable of
altering a monocyte function, for example, a cytokine such as IL-10
or IL-11, the compound can be covalently linked to the targeting
ligand A.sub.b, for example, a folate receptor-binding ligand or a
monocyte-binding antibody or antibody fragment directly, or the
monocyte function altering compound can be encapsulated in a
liposome which is itself targeted to monocytes by pendent monocyte
targeting ligands A.sub.b covalently linked to one or more liposome
components.
[0041] In another embodiment, conjugates A.sub.b-X where X is an
immunogen or a compound capable of altering monocyte function, can
be administered in combination with a cytotoxic compound. The
cytotoxic compounds listed above are among the compounds suitable
for this purpose.
[0042] In another method of treatment embodiment, the group X in
the monocyte targeted conjugate A.sub.b-X, comprises an immunogen,
the ligand-immunogen conjugates being effective to "label" the
population of monocytes responsible for disease pathogenesis in the
patient suffering from the disease for specific elimination by an
endogenous immune response or by co-administered antibodies. The
use of ligand-immunogen conjugates in the method of treatment
described herein works to enhance an immune response-mediated
elimination of the monocyte population that expresses the ligand
receptor. Such elimination can be effected through an endogenous
immune response or by a passive immune response effected by
co-administered antibodies.
[0043] The methods of treatment involving the use of
ligand-immunogen conjugates are described in U.S. Patent
Application Publication Nos. U.S. 2001/0031252 A1 and U.S.
2002/0192157 A1, and PCT Publication No. WO 2004/100983, each
incorporated herein by reference.
[0044] 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 immunogen (e.g., an
antigen or a hapten). It is also contemplated that the endogenous
immune response may employ the secretion of cytokines that regulate
such processes as the multiplication 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.
[0045] 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), 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.
[0046] 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-immunogen conjugate wherein the passively administered
antibodies are directed to the immunogen, 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-immunogen 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-immunogen conjugate.
[0047] The preexisting antibodies, induced antibodies, or passively
administered antibodies will be redirected to the monocytes by
preferential binding of the ligand-immunogen conjugates to the
monocyte cell populations, and such pathogenic cells are killed 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.
[0048] Acceptable immunogens for use in preparing the conjugates
used in the method of treatment described herein are immunogens
that are capable of eliciting antibody production in a host animal
or that have previously elicited antibody production in a host
animal, resulting in a preexisting immunity, or that constitute
part of the innate immune system. Alternatively, antibodies
directed against the immunogen may be administered to the host
animal to establish a passive immunity. Suitable immunogens 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-immunogen conjugates will be used to
redirect a previously acquired humoral or cellular immunity to a
population of monocytes in the host animal for elimination of the
monocytes.
[0049] 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.
[0050] The monocyte-binding ligands and immunogens, cytotoxic
agents, compounds capable of altering monocyte function, or imaging
agents, 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 to the
immunogen, 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 monocyte-binding ligands to immunogens,
cytotoxic agents, compounds capable of altering monocyte function,
or imaging agents are described in U.S. Patent Application
Publication No. 2005/0002942 Al and PCT Publication No. WO
2006/012527, each incorporated herein by reference.
[0051] Alternatively, as mentioned above, the ligand complex can be
one comprising a liposome wherein the targeted entity (that is, the
imaging agent, or the immunogen, cytotoxic agent or monocyte
function-altering agent) is contained within a liposome which is
itself covalently linked to the monocyte-binding ligand. Other
nanoparticles, dendrimers, derivatizable polymers or copolymers
that can be linked to therapeutic or imaging agents useful in the
treatment and diagnosis of monocyte-mediated diseases can also be
used in targeted conjugates.
[0052] 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.
[0053] 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.
[0054] The conjugates used in accordance with the methods described
herein of the formula A.sub.b-X are used in one aspect to formulate
therapeutic or diagnostic compositions, for administration to a
patient, 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 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.
[0055] 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 is treated with multiple injections of the
ligand conjugate wherein the targeted entity is an immunogen or a
cytotoxic agent or a compound capable of altering monocyte function
to eliminate the population of pathogenic monocytes. In one
embodiment, the patient 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 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 known to be disposed to
development of monocyte-mediated disease states. In one embodiment,
more than one type of ligand conjugate can be used, for example,
the host animal may be pre-immunized with fluorescein
isothiocyanate and dinitrophenyl and subsequently treated with
fluorescein isothiocyanate and dinitrophenyl linked to the same or
different monocyte targeting ligands in a co-dosing protocol.
[0056] 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 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, for treatment of inflammations of the
skin.
[0057] In any of the embodiments discussed above, the monocytes can
be activated monocytes or other monocyte populations that cause
disease states. The following examples are illustrative embodiments
only and are not intended to be limiting.
EXAMPLE 1
Materials
[0058] 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 were
purchased from Novabiochem (La Jolla, Calif.).
N.sup.10-trifluoroacetylpteroic acid was purchased from Sigma, St.
Louis, Mo. All anti-mouse and anti-human antibodies were purchased
from Caltag Laboratories, Burlingame, Calif.
Folate-R-Phycoerytherin, Folate-Alexa Fluor 488, Folate-Texas Red,
and Folate-Fluorescein and Folate-cysteine were synthesized as
described. Tritium (.sup.3H)-labeled folic acid was obtained from
American Radiolabeled Chemicals (St. Louis, Mo.).
EXAMPLE 2
Synthesis of Folate-Cysteine
[0059] Standard Fmoc peptide chemistry was used to synthesize
folate-cysteine with the cysteine attached to the y-COOH of folic
acid. The sequence Cys-Glu-Pteroic acid (Folate-Cys) was
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 was 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 was used to precipitate the product, and the precipitant was
collected by centrifugation. The product was washed twice with
diethyl ether and dried under vacuum overnight. To remove the
N.sup.10-trifluoracetyl protecting group, the product was dissolved
in a 10% ammonium hydroxide solution and stirred for 30 min at room
temperature. The solution was kept under a stream of nitrogen the
entire time in order to prevent the cysteine from forming
disulfides. After 30 minutes, hydrochloric acid was added to the
solution until the compound precipitated. The product was collected
by centrifugation and lyophilized. The product was analyzed and
confirmed by mass spectroscopic analysis (MW 544, M.sup.+ 545).
##STR4##
EXAMPLE 3
Synthesis of Folate-Cys-AlexaFluor 488
[0060] AlexaFluor 488 C.sub.5-maleimide (Molecular Probes, Eugene,
Oreg.) was dissolved in dimethyl sulfoxide (DMSO) (0.5 mg in 50
.mu.l DMSO). A 1.5 molar equivalent (0.57 mg) of Folate-Cys was
added to the solution and mixed for 4 hours at room temperature.
Folate-Cys-AlexaFluor 488 (Folate-AlexaFluor) was purified by
reverse-phase HPLC on a C18 column at a flow rate of 1 ml/min. The
mobile phase, consisting of 10 mM NH.sub.4HCO.sub.3 buffer, pH 7.0
(eluent A) and acetonitrile (eluent B), was maintained at a 99:1
A:B ratio for the first minute and then changed to 1:99 A:B in a
linear gradient over the next 29 minutes. Folate-Cys-AlexaFluor 488
eluted at 20 minutes. The product was confirmed by mass
spectroscopy and the biologic activity was confirmed by
fluorescence measurement of its binding to cell surface folate
receptors on folate receptor positive M109 cells in culture.
##STR5##
EXAMPLE 4
Synthesis of Folate-Cys-Texas Red
[0061] Texas Red C.sub.2-maleimide (Molecular Probes, Eugene,
Oreg.) was dissolved in dimethyl sulfoxide (DMSO) (1 mg in 200
.mu.l DMSO). A 1.4 molar equivalent (1 mg) of Folate-Cys was added
to the solution and mixed for 4 hours at room temperature.
Folate-Cys-Texas Red (Folate-Texas Red) was purified by
reverse-phase HPLC on a C18 column at a flow rate of 1 ml/min. The
mobile phase, consisting of 10 mM NH.sub.4HCO.sub.3 buffer, pH 7.0
(eluent A) and acetonitrile (eluent B), was maintained at a 99:1
A:B ratio for the first five minutes and then changed to 70:30 A:B
in a linear gradient over the next 30 minutes followed by a 1:99
A:B linear gradient over the last 15 minutes. Folate-Cys-Texas Red
eluted as two isomer peaks at 44.5 and 45.8 minutes. The product
was confirmed by mass spectroscopy and the biologic activity was
confirmed by fluorescence measurement of its binding to cell
surface folate receptors on folate receptor positive M109 cells in
culture. ##STR6##
EXAMPLE 5
Synthesis of Folate-Oregon Green 514
[0062] Standard Fmoc peptide chemistry was used to synthesize a
folate peptide linked to Oregon Green (Molecular Probes, Eugene,
Oreg.) attached to the .gamma.-COOH of folic acid. The sequence
Lys-Glu-Pteroic acid (Folate-Cys) was 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 followed by
a N.sup.10-trifluoroacetylpteroic acid was linked to a
Fmoc-protected lysine wang resin containing a 4-methyltrityl
protecting group on the .epsilon.-amine. The methoxytrityl
protecting group on the .epsilon.-amine of lysine was removed with
1% trifluoroacetic acid in dichloromethane to allow attachment of
Oregon Green (Folate-Oregon Green). A 1.5 molar equivalent of
Oregon Green carboxylic acid, succinimidyl ester was reacted
overnight with the peptide and then washed thoroughly from the
peptide resin beads. The Folate-Oregon Green was then cleaved from
the resin with a 95% trifluoroacetic acid-2.5% water-2.5%
triisopropylsilane solution. Diethyl ether was used to precipitate
the product, and the precipitant was collected by centrifugation.
The product was washed twice with diethyl ether and dried under
vacuum overnight. To remove the N.sup.10-trifluoracetyl protecting
group, the product was dissolved in a 10% ammonium hydroxide
solution and stirred for 30 min at room temperature. The product
was precipitated with combined isopropanol and ether, and the
precipitant was collected by centrifugation. ##STR7##
EXAMPLE 6
Synthesis of Folate-R-Phycoerythrin
[0063] Folate-phycoerythrin was 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 was added to a solution of R-phycoerythrin
pyridyldisulfide (Sigma, St. Louis, Mo.) in phosphate buffered
saline (PBS), pH 7.4. The solution was allowed to react overnight
at 4.degree. C. and the labeled protein (Mr .about.260 kDa) was
purified by gel filtration chromatography using a G-15 desalting
column. The folate labeling was 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 were
intensely fluorescent, while the sample in the presence of excess
folic acid showed little cellular fluorescence.
EXAMPLE 7
Synthesis of Folate-Fluorescein
[0064] Folate-FITC was synthesized as described by Kennedy, M. D.
et al. in Pharmaceutical Research, Vol. 20(5); 2003. ##STR8##
EXAMPLE 8
Synthesis of Folate-D-R-D-D-C--Prednisolone
[0065] Standard Fmoc peptide chemistry was used to synthesize
folate-aspartate-arginine-aspartate-aspartate-cysteine
(Folate-Asp-Arg-Asp-Asp-Cys, Folate-D-R-D-D-C) with the amino acid
spacer attached to the .gamma.-COOH of folic acid. The sequence
Cys-Asp-Asp-Arg-Asp-Glu-Pteroic acid (Folate-Asp-Arg-Asp-Asp-Cys)
was 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.
Fmoc-D-Asp(OtBu)-OH was linked to a trityl-protected Cys linked to
a 2-Chlorotrityl resin. A second Fmoc-D-Asp(OtBu)-OH followed by
Fmoc-Arg(Pbf)-OH, Fmoc-D-Asp(OtBu)-OH and Fmoc-Glu-OtBu were added
successively to the resin. N.sup.10-trifluoroacetylpteroic acid was
then attached to the .gamma.-COOH of Glu. The
Folate-Asp-Arg-Asp-Asp-Cys was cleaved from the resin using a 92.5%
trifluoroacetic acid-2.5% water-2.5% triisopropylsilane-2.5%
ethanedithio solution. Diethyl ether was used to precipitate the
product, and the precipitant was collected by centrifugation. The
product was washed twice with diethyl ether and dried under vacuum
overnight. To remove the N.sup.10-trifluoracetyl protecting group,
the product was dissolved in a 10% ammonium hydroxide solution and
stirred for 30 min at room temperature. The solution was kept under
a stream of nitrogen the entire time in order to prevent the
cysteine from forming disulfides. After 30 minutes, hydrochloric
acid was added to the solution until the compound precipitated. The
product was collected by centrifugation and lyophilized. The
product was analyzed and confirmed by mass spectroscopic analysis
(MW 1046). ##STR9##
EXAMPLE 9
Synthesis of Folate-Indomethacin
[0066] ##STR10##
[0067] 2-(2-Pyridyldithio)ethanol was synthesized by dissolving 1.5
equivalents of Aldrithiol (Sigma, St. Louis, Mo.) with 6
equivalents of 4-dimethylaminopyridine (DMAP) in dichloromethane
(DCM). The solution was purged with nitrogen and 1 equivalent of
mercaptoethanol was added dropwise to the Aldrithiol solution over
the course of 15 minutes. The reaction proceeded at room
temperature for 30 minutes at which time no odor of mercaptoethanol
remained. The reaction was diluted 100-fold with DCM and 5 g of
activated carbon was added per gram of Aldrithiol. The reaction
mixture was filtered and the solvent removed. The mixture was
resuspended in 70:30 (Petroleum ether:Ethylacetate (EtOAc)) and
purified by flash chromatography on a 60 .ANG. silica gel column.
The product was monitored by thin layer chromatography and
collected.
[0068] Folate-indomethacin was synthesized following a modified
method published by Kalgutkar et al. in the Journal of Med. Chem.
2000, 43; 2860-2870 where the anti-inflammatory (indomethacin) was
linked through an ester bond with the 2-(2-Pyridyldithio)ethanol.
Briefly, 1 equivalent of indomethacin was dissolved in DCM along
with 0.08 equivalents DMAP, 1.1 equivalents 2-(2-Pyridyldithio)
ethanol and 1.1 equivalents 1,3-dicyclohexyl-carbodiimide. The
reaction proceeded at room temperature for 5 hours. The reaction
was purified by chromatography on silica gel (EtOAc:hexanes,
20:80). One equivalent of the purified compound was dissolved in
DMSO and to it were added 1.5 equivalents of the
folate-Asp-Arg-Asp-Asp-Cys peptide. The resulting solution was
reacted for 3 hours at room temperature followed by purification
using a HPLC reverse-phase C18 column at a flow rate of 1 ml/min.
The mobile phase, consisting of 10 mM NH.sub.4HCO.sub.3 buffer, pH
7.0 (eluent A) and acetonitrile (eluent B), was maintained at a
99:1 A:B ratio for the first five minutes and then changed to 70:30
A:B in a linear gradient over the next 30 minutes. The recovered
final product was confirmed by mass spectrometry.
EXAMPLE 10
Synthesis of Folate-Diclofenac
[0069] ##STR11##
[0070] Folate-diclofenac was synthesized by the method described in
Example 9 except that diclofenac was used in place of indomethicin.
In various embodiments, n=1, 2, or 3, and where n is illustratively
2.
EXAMPLE 11
Synthesis of Folate-Cys-Prednisolone
[0071] The folate glucocorticoid conjugate of prednisolone was
prepared as follows. A 1.1 molar equivalent of prednisone was
dissolved in tetrahydrofuran (THF). In a separate vial, a 0.7 molar
equivalent of dimethylaminopyridine, 1 molar equivalent of
tri(hydroxyethyl)amine and 1 molar equivalent of the linker
(synthesis described in PCT Publication No. WO 2006/012527,
incorporated herein by reference) were dissolved in
dichloromethane. An approximately equal volume of both solutions
were combined, mixed and reacted at room temperature for 4 hours.
The reaction was monitored by thin layer chromatography using
40:10:1 (Dichloromethane:Acetonitrile:Methanol). The product had an
R.sub.f=0.52. The product was purified on a silica column (Silica
32-63, 60 .ANG.) using the same ratio of solvents. The recovered
product was dried in preparation for conjugation to a
folate-peptide. The derivatized glucocorticoid was dissolved in
DMSO, to which was added a 1.5 molar equivalent of either the
folate-cys or folate-Asp-Arg-Asp-Asp-Cys peptide. The resulting
solution was reacted for 3 hours at room temperature followed by
purification using a HPLC reverse-phase C18 column at a flow rate
of 1 ml/min. The mobile phase, consisting of 10 mM
NH.sub.4HCO.sub.3 buffer, pH 7.0 (eluent A) and acetonitrile
(eluent B), was maintained at a 99:1 A:B ratio for the first minute
and then changed to 1:99 A:B in a linear gradient over the next 39
minutes. The folate-glucocorticoid conjugate eluted at
approximately 26 minutes. The recovered final product was confirmed
by mass spectrometry. ##STR12##
EXAMPLE 12
Synthesis of Folate-Cys-Dexamethasone
[0072] ##STR13##
[0073] Folate-cys-dexamethasone was synthesized by a procedure
similar to that described in Example 11 except that the
glucocorticoid was dexamethasone.
EXAMPLE 13
Synthesis of Folate-Cys-Flumethasone
[0074] ##STR14##
[0075] Folate-cys-flumethasone was synthesized by a procedure
similar to that described in Example 11 except that the
glucocorticoid was flumethasone.
EXAMPLE 14
Isolation of Peripheral Blood Mononuclear Cells (PBMC)
[0076] An 8-10 ml sample of whole blood was collected in EDTA
anticoagulant tubes. PBMCs were isolated from the blood samples
using Ficoll-Paque Plus (Amersham Biosciences, Piscataway, N.J.)
and by following the manufacture's provided protocol. Briefly, the
blood sample was diluted 50:50 with a balanced salt solution
(described below). 8mL of Ficoll-Paque Plus was added to a 50 ml
conical centrifuge tube. The diluted blood sample (approximately
16-20 ml) was layered on top of the Ficoll gradient. The sample was
centrifuged at 400.times.g for 30 minutes at room temperature.
Following centrifugation, the plasma layer (top clear layer) was
removed using a pipette leaving the lymphocyte/monocyte layer
undisturbed. The hazy cell layer immediately below the plasma layer
was removed, being careful to remove the entire cell interface but
a minimum amount of the Ficoll layer. The isolated cells were put
into a sterile 50 ml conical centrifuge tube and diluted 3-fold
(vol/vol) using the balanced salt solution. The resulting cell
solution was gently mixed and centrifuged at 100.times.g for 10
minutes at room temperature to pellet the cells. The supernatant
was removed and the cells were resuspended in folate deficient RPMI
1640 medium supplemented with 10% heat-inactivated FBS, penicillin
(100 IU/ml) and streptomycin (100 .mu.g/ml). Cells were seeded in
microcentrifuge tubes or microscopy chambers as dictated by the
experiment.
EXAMPLE 15
Balanced Salt Solution
[0077] Balanced Salt Solution Preparation (As prepared by Amersham
Biosciences) TABLE-US-00001 Solution A Concentration. (g/L)
Anhydrous D-glucose 0.1 percent 1.0 CaCl.sub.2 .times. 2H.sub.2O
5.0 .times. 10.sup.-5M 0.0074 MgCl.sub.2 .times. 6H.sub.2O 9.8
.times. 10.sup.-4M 0.1992 KCl 5.4 .times. 10.sup.-3M 0.4026 TRIS
0.145 M 17.565
[0078] Dissolve in approximately 950 ml distilled water and add 10
N HCl until pH is 7.6 before adjusting the volume to 1 L.
TABLE-US-00002 Solution B Concentration (g/L) NaCl 0.14M 8.19
To prepare the balanced salt solution mix 1 volume Solution A with
9 volumes Solution B.
EXAMPLE 16
Ligand Binding
[0079] All binding experiments were conducted on ice or in a
4.degree. C. cold room unless indicated otherwise. Folate conjugate
and .sup.3H-folic acid binding studies were performed by incubating
cells with a 100 nM concentration of the indicated folate dye
conjugate for 45 minutes. Competition samples were prepared by
pre-incubating the appropriate samples with a 100-fold excess
concentration of folic acid (10 .mu.M) five minutes prior to adding
the folate dye conjugate. An acidic wash to strip cell-surface
bound folate conjugates was performed on indicated samples by
washing the cell sample with a 150 mM NaCl solution adjusted to pH
3.5 with acetic acid. All antibody labeling was optimized by
titration. Optimal labeling was most often achieved with a
1/1000-1/10,000 dilution of the manufacture's stock antibody
solution. After cells were labeled with folate dye conjugates
and/or antibodies, the samples were washed twice with PBS to remove
non-specific binding. Analysis of folate conjugate binding and/or
antibody binding was analyzed by confocal microscopy or by flow
cytometry (FCS Calibur, BD, Franklin Lakes, N.J.). After washing
.sup.3H-folic acid samples to remove non-specific binding, cells
were dissolved in 0.25M NaOH and radioactivity was counted on a
scintillation counter.
EXAMPLE 17
Synthesis of Folate Resonance Energy Tranfer Reporter
[0080] Compound 1 was prepared by following standard Fmoc chemistry
on an acid-sensitive trityl resin loaded with Fmoc-L-Cys (Trt)-OH,
as described previously (adapted to the shown peptide sequence).
The crude compound 1 was purified by HPLC using a VYDAC protein and
peptide C18 column. The HPLC-purified 1 was then reacted with
tetraethylrhodamine methanethiosulfonate (Molecular Probes, Eugene,
Oreg.) in DMSO to afford compound 2, in the presence of
diisopropylethylamine (DIPEA). The desired product was isolated
from the reaction mixture by preparative HPLC as described above.
The final conjugation was performed by mixing excess DIPEA with 2
(in DMSO) followed by addition of BODIPY FL NHS ester (Molecular
Probes, Eugene, Oreg.). Compound 3 was then isolated from this
reaction mixture by preparative HPLC. ##STR15##
EXAMPLE 18
Laser Imaging
[0081] Fluorescence resonance energy transfer (FRET) imaging of
monocytes to determine uptake of folate-linked imaging agents will
be carried out using a confocal microscopy. An Olympus IX-70
inverted microscopy (Olympus, USA) equipped with an Olympus FW300
scanning box and an Olympus 60X/1.2 NA water objective will be used
to image the cells. Separate excitation lines and emission filters
will be used for each fluorochrome (BODIPY FL, 488 nm (excitation)
and 520/40 nm (emission); rhodamine, 543 nm (excitation) and 600/70
nm (emission)). Two laser sources with 543 nm (He--Ne) and 488 nm
(Argon) wavelength can be used to excite BODIPY FL and rhodamine
separately to obtain two color images when needed. Confocal images
can be acquired with a size of 512.times.512 pixels at 2.7 second
scan time and images can be processed using FluoView (Olympus)
software.
EXAMPLE 19
Liposome Preparation
[0082] Liposomes were prepared following methods by Leamon et al.
in Bioconjugate Chemistry 2003, 14, 738-747. Briefly, lipids and
cholesterol were purchased from Avanti Polar Lipids (Alabaster,
Ala.). Folate-targeted liposomes consisted of 40 mole %
cholesterol, either 4 mole % or 6 mole % polyethyleneglycol
(Mr.about.2000)-derivatized phosphatidylethanolamine (PEG2000-PE,
Nektar Ala., Huntsville, Ala.), either 0.03 mole % or 0.1 mole %
folate-cysteine-PEG3400-PE and the remaining mole % was composed of
egg phosphatidylcholine. Non-targeted liposomes were prepared
identically with the absence of folate-cysteine-PEG3400-PE. Lipids
in chloroform were dried to a thin film by rotary evaporation and
then rehydrated in PBS containing the drug. Rehydration was
accomplished by vigorous vortexing followed by 10 cycles of
freezing and thawing. Liposomes were then extruded 10 times through
a 50 nm pore size polycarbonate membrane using a high-pressure
extruder (Lipex Biomembranes, Vancouver, Canada).
EXAMPLE 20
Synthesis of Folate-Pokeweed
[0083] Pokeweed antiviral protein was purchased from Worthington
Biochemical Corporation (Lakewood, N.J.).
N-succinimidyl-3-[2-pyridyldithio]propionate (SPDP; Pierce,
Rockford, Ill.) was dissolved in dimethylformamide (9.6 mM). While
on ice, a 5 fold molar excess of SPDP (.about.170 nmoles) was added
to the pokeweed solution (1 mg/ml PBS, MW.about.29,000). The
resulting solution was gently mixed and allowed to react for 30
minutes at room temperature. The non-conjugated SPDP was removed
using a centrifuge molecular weight concentrator (MWCO 10,000)
(Millipore, Billerica, Mass.). The resulting protein solution was
resuspended in PBS containing 10 mM EDTA to a final volume of 1 mL.
Approximately a 60 fold molar excess of folate-Asp-Arg-Asp-Asp-Cys
peptide (2000 nmoles) was added to the protein solution and allowed
to react for 1 hour. The non-reacted folate-Asp-Arg-Asp-Asp-Cys
peptide was removed using the centrifuge concentrators as
previously described. The protein was washed twice by resuspending
the protein in PBS and repeating the protein concentration by
centrifugation.
EXAMPLE 21
Synthesis of Folate-Saporin
[0084] The protein saporin was purchased from Sigma (St. Louis,
Mo.). Folate-saporin was prepared following folate-protein
conjugation methods published by Learnon and Low in The Journal of
Biological Chemistry 1992, 267(35); 24966-24971. Briefly, folic
acid was 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 was 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 was added to the protein solution and
the labeling reaction was allowed to proceed for 4 hours. Unreacted
material was separated from the labeled protein using a Sephadex
G-25 column equilibrated in phosphate-buffered saline, pH 7.4.
EXAMPLE 22
Synthesis of Folate-Momordin and Folate-Gelonin
[0085] The proteins momordin and gelonin were purchased from Sigma
(St. Louis, Mo.). Folate-cys pyridyldisulfide was prepared by
reacting folate-cys with Aldrithiol (Sigma, St. Louis, Mo.). Both
proteins were dissolved in 0.1M HEPPS buffer, pH 8.2. A 6-fold
molar excess of Trouts reagent (Aldrich St. Louis, Mo.) dissolved
in DMSO (16 mM) was added to each protein solution. The solutions
were allowed to react for 1 hour at room temperature. Unreacted
material was separated from the protein using a Sephadex G-25
column equilibrated in 0.1M phosphate buffer, pH 7.0. Ellmans test
for the presence of free thios were positive for both proteins.
While the protein solution was on ice, a 5-fold molar excess of
folate-cys pyridyldisulfide dissolved in DMSO was added. The
resulting solution was warmed up to room temperature and reacted
for 30 minutes. Unreacted material was separated from the labeled
protein using a Sephadex G-25 column equilibrated in
phosphate-buffered saline, pH 7.4.
EXAMPLE 23
Preparation of Folate-Targeted Clodronate or Prednisolone Phosphate
Liposomes
[0086] Liposomes were prepared following methods by Leamon et al.
in Bioconjugate Chemistry 2003, 14; 738-747. Briefly, lipids and
cholesterol were purchased from Avanti Polar Lipids (Alabaster,
Ala.). Folate-targeted liposomes consisted of 40 mole %
cholesterol, 5 mole % polyethyleneglycol
(Mr.about.2000)-derivatized phosphatidylethanolamine (PEG2000-PE,
Nektar Ala., Huntsville, Ala.), 0.03 mole %
folate-cysteine-PEG3400-PE and 54.97 mole % egg
phosphatidylcholine. Lipids in chloroform were dried to a thin film
by rotary evaporation and then rehydrated in PBS containing either
clodronate (250 mg/ml) or prednisolone phosphate (100 mg/ml).
Rehydration was accomplished by vigorous vortexing followed by 10
cycles of freezing and thawing. Liposomes were then extruded 10
times through a 50 nm pore size polycarbonate membrane using a
high-pressure extruder (Lipex Biomembranes, Vancouver, Canada). The
liposomes were separated from unencapsulated clodronate or
prednisolone phosphate by passage through a CL4B size exclusion
column (Sigma, St. Louis, Mo.) in PBS. Average particle size was
between 70 and 100 nm.
EXAMPLE 24
Folate-FITC Binding to Human Monocytes
[0087] Folate-FITC binding to human monocytes and to human
monocytes preincubated with a 100-fold excess of unlabeled folic
acid was measured. Peripheral blood monocytes were isolated as
described in Examples 14 and 15 and folate-FITC binding and flow
cytometry were performed as described in Example 16. As shown in
FIG. 1, folate-FITC bound to human peripheral blood monocytes in
the absence of unlabeled folic acid and binding was competed in the
presence of a 100-fold excess of unlabeled folic acid.
EXAMPLE 25
Folate-FITC Binding to CD11b.sup.+ Human Monocytes
[0088] Folate-FITC binding to CD11b.sup.+ human monocytes and to
CD11b.sup.+ human monocytes preincubated with a 100-fold excess of
unlabeled folic acid was quantified. Peripheral blood monocytes
were isolated as described in Examples 14 and 15 and folate-FITC
binding and flow cytometry were performed as described in Example
16. As shown in FIG. 2, folate-FITC bound to 45.9% of human
peripheral blood monocytes in the absence of unlabeled folic acid
and to 2% of human peripheral blood monocytes in the presence of a
100-fold excess of unlabeled folic acid.
EXAMPLE 26
Binding to Human Monocytes of Folate-FITC and Antibodies to CD
Markers
[0089] Folate-FITC binding and binding of antibodies to CD11b,
CD14, CD16, CD69, and HLA-DR markers on human monocytes was
quantified. Peripheral blood monocytes were isolated as described
in Examples 14 and 15 and folate-FITC and antibody binding and flow
cytometry were performed as described in Example 16. As shown in
FIG. 3, CD11b, CD14, CD16, CD69, and HLA-DR markers are
co-expressed with the folate receptor on human peripheral blood
monocytes. It has been reported that CD14- and CD16-expressing
monocytes are a population of proinflammatory monocytes (Weber et
al., J. Leuk. Biol., 67:699-704 (2000) and Ziegler-Heitbrock, J.
Leuk. Biol., 67:603-606 (2000)) suggesting that the
folate-receptor-expressing monocytes (about 2% of total circulating
white blood cells) are proinflammatory monocytes.
EXAMPLE 27
Binding of .sup.3H-Folic Acid to White Blood Cells
[0090] .sup.3H-Folic acid binding to white blood cells was
quantified as described in Example 16. White blood cells were
preincubated with a 100-fold excess of unlabeled folic acid for the
samples labeled "xs." As shown in FIG. 4, folate receptors are
detectable on white blood cells from dogs and mice and on KB
cells.
EXAMPLE 28
Folate-FITC Binding to Peripheral Blood Monocytes from Dogs and
Horses
[0091] Folate-FITC binding to peripheral blood monocytes from dogs
and horses was quantified for monocytes preincubated or not
preincubated with a 100-fold excess of unlabeled folic acid.
Peripheral blood monocytes were isolated as described in Examples
14 and 15 and folate-FITC binding and flow cytometry were performed
as described in Example 16. As shown in FIG. 5, folate receptors
were detectable on peripheral blood monocytes of both dogs and
horses.
EXAMPLE 29
Folate-FITC or Folate-AlexaFluor 488 Binding to Peripheral Blood
Monocytes from Dogs
[0092] Folate-FITC binding or folate-AlexaFluor 488 binding to
peripheral blood monocytes from dogs was quantified for monocytes
preincubated or not preincubated with a 100-fold excess of
unlabeled folic acid. Peripheral blood monocytes were isolated as
described in Examples 14 and 15 and folate-FITC and
folate-AlexaFluor 488 binding and flow cytometry were performed as
described in Example 16. As shown in FIG. 6, folate receptors were
detectable on peripheral blood monocytes of dogs using either
folate-FITC or folate-AlexaFluor 488.
EXAMPLE 30
Folate-Phycoerythrin Binding to Human Peripheral Blood
Monocytes
[0093] Folate-phycoerythrin binding to human peripheral blood
monocytes was quantified for monocytes preincubated or not
preincubated with a 100-fold excess of unlabeled folic acid.
Peripheral blood monocytes were isolated as described in Examples
14 and 15 and folate-phycoerythrin binding and flow cytometry were
performed as described in Example 16. As shown in FIG. 7, folate
receptors were detectable on human peripheral blood monocytes using
folate-phycoerythrin.
EXAMPLE 31
Folate-FITC Binding to Peripheral Blood Monocytes from Healthy
Humans and Patients with Arthritis or Fibromyalgia
[0094] Folate-FITC binding to peripheral blood monocytes from
healthy humans (squares) and from patients with rheumatoid
arthritis (diamonds), osteoarthritis (upper group of triangles),
and fibromyalgia (three triangles at lowest percentages) was
quantified. Peripheral blood monocytes were isolated as described
in Examples 14 and 15 and folate-FITC binding and flow cytometry
were performed as described in Example 16. As shown in FIG. 8,
folate receptors were detectable on peripheral blood monocytes of
humans using folate-FITC. In this assay, patients with fibromyalgia
appear to have lower percentages of folate-receptor expressing
monocytes in peripheral blood than healthy individuals. The
difference may be due to differentiation of monocytes into
macrophages and to the egress of activated macrophages from the
circulation and localization of activated macrophages to sites of
inflammation. Regardless of the reason for this difference, the
results in FIG. 8 suggest that folate-imaging agent conjugates may
be useful in diagnosing monocyte-mediated disease states, and that
one such monocyte-mediated disease state may be fibromyalgia.
EXAMPLE 32
Animal Model of Arthritis
[0095] Arthritis was induced in 150-200 g female Lewis rats
(Harlan, Indianapolis, Ind.), n=2-5/dose group. Briefly, 0.5 mg of
heat-killed Mycoplasma butericum, suspended in mineral oil (5
mg/ml), was injected on day 0 into the left hind foot of rats
following anesthesia with ketamine and xylazine. All treated
animals developed arthritis, as evidenced by dramatic swelling in
the injected paw, progressive swelling in all noninjected limbs due
to the systemic progression of arthritis, and radiographic analysis
of affected limbs. All rats were maintained on a folate-deficient
diet (DYETS, Inc., Bethlehem, Pa.) for 3 weeks prior to
administration of therapeutic agents in order to lower serum folate
levels to physiologically relevant concentrations. Control rats
were also maintained on a folate-deficient diet but were not
induced to develop arthritis.
EXAMPLE 33
Effect of Therapeutic Agents on Adjuvant-Induced Arthritis
[0096] The protocol described in Example 32 for arthritis induction
was followed. The efficacy of folate-flumethasone (50
nmoles/kg/day) and folate-indomethacin (100 or 250 nmoles/kg/day)
against adjuvant-induced arthritis in rats was investigated. Rats
were injected intraperitoneally with either saline (control rats)
or folate-flumethasone (50 nmoles/kg/day) or folate-indomethacin
(100 or 250 nmoles/kg/day) starting at day 4. Calipers were used to
measure left foot dimensions on the days indicated in FIG. 9. The
sudden increase in swelling of the adjuvant-injected foot is due to
influx of neutrophils which have no folate receptors. Consequently,
the therapy has no impact on this phase of paw swelling. However,
the data in FIG. 9 suggests that after about 7 days
folate-flumethasone and folate-indomethacin have potent therapeutic
effects in this adjuvant-induced arthritis model by eliminating or
inactivating monocytes as a result of binding and internalization
by monocytes of folate-flumethasone or folate-indomethacin.
EXAMPLE 34
Folate-FITC Binding to Peripheral Blood Monocytes from Patients
with Arthritis
[0097] Folate-FITC binding to peripheral blood monocytes from
patients with rheumatoid arthritis was quantified. Peripheral blood
monocytes were isolated as described in Examples 14 and 15 and
folate-FITC binding and flow cytometry were performed as described
in Example 16. As shown in FIG. 10, folate receptors were
detectable on peripheral blood monocytes of humans by using
folate-FITC. Patient #1 (x-axis shows patient #) was treated with
Enbrel/methotrexate, patient #2 was treated with methotrexate,
patient #3 was treated with Medrol, patient #4 was treated with
Methotrexate/Azulfidine/Plaquenil, Tbuprofen, prednisone, patient
#5 was treated with Methotrexate/Azulfidine/Plaquenil, Celebrex,
Medrol, patient #6 was treated with
Methotrexate/Azulfidine/Plaquenil, Celebrex, prednisone, and
patient #7 was treated with Plaquenil, Arava. In this assay, the
percentage of folate-receptor expressing monocytes in peripheral
blood of patients with arthritis decreased over the course of
arthritis therapy. The results in FIG. 10 indicate that folate
receptor-expressing monocytes contribute to the pathogenesis of
arthritis.
[0098] The foregoing exemplified embodiments are intended to be
illustrative of the invention described herein, and should not be
construed as limiting. It is to be understood that several
variations of those embodiments are contemplated, and are intended
to be included herein.
[0099] Illustratively, in each of Examples 2 through 13, a wide
variety of folate analogs and derivatives may be substituted for
folate itself in forming the folate linker conjugates. Those
analogs and derivatives, or protected forms thereof, may be
included in the synthetic protocols described herein. In addition,
structural modifications of the linker portion of the conjugates is
contemplated herein. For example, a number of amino acid
substitutions may be made to the linker portion of the conjugate,
including but not limited to naturally occurring amino acids, as
well as those available from conventional synthetic methods. In one
aspect, beta, gamma, and longer chain amino acids may be used in
place of one or more alpha amino acids. In another aspect, the
stereochemistry of the chiral centers found in such molecules may
be selected to form various mixture of optical purity of the entire
molecule, or only of a subset of the chiral centers present. In
another aspect, the length of the peptide chain included in the
linker may be shortened or lengthened, either by changing the
number of amino acids included therein, or by including more or
fewer beta, gamma, or longer chain amino acids. In another aspect,
the selection of amino acid side chains in the peptide portion may
be made to increase or decrease the relative hydrophilicity of the
linker portion specifically, or of the overall molecule
generally.
[0100] Similarly, the length and shape of other chemical fragments
of the linkers described herein may be modified. In one aspect,
where the linker includes an alkylene chain, such as is found in
Examples 3, 4, and 7, the alkylene may be longer or shorter, or may
include branched groups, or include a cyclic portion, which may be
in line or spiro relative to the alkylene chain. In another aspect,
where the linker includes a beta thiol releasable fragment, such as
the thioethyloxy bivalent fragment in Examples 8 through 13, it is
appreciated that other intervening groups connecting the thiol end
to the hydroxy or carbonate end may be used in place of the
ethylene bridge, such as but not limited to optionally substituted
benzyl groups, where the hydroxy end is connected at the benzyl
carbon and the thiol end is connected through the ortho or para
phenyl position, and vice versa.
[0101] In another illustrative embodiment, structural modifications
may be made to the linker to include additional releasable linkers,
such as those described in U.S. Patent Application Publication No.
2005/0002942.
* * * * *