U.S. patent application number 11/823186 was filed with the patent office on 2008-06-12 for treatment and diagnosis of macrophage mediated disease.
Invention is credited to Philip S. Low, Mary Jo Turk.
Application Number | 20080138396 11/823186 |
Document ID | / |
Family ID | 23106203 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138396 |
Kind Code |
A1 |
Low; Philip S. ; et
al. |
June 12, 2008 |
Treatment and diagnosis of macrophage mediated disease
Abstract
The invention relates to a method of treating or
monitoring/diagnosing a disease state mediated by activated
macrophages. The method comprises the step of administering to a
patient suffering from a macrophage 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 capable of binding to activated macrophages, 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 macrophage function, and when the conjugate is being
used for monitoring/diagnosing the disease state, X comprises an
imaging agent. The method is useful for treating a patient
suffering from a disease selected from the group consisting of
rheumatoid arthritis, ulcerative colitis, Crohn's disease,
inflammation, infections, osteomyelitis, atherosclerosis, organ
transplant rejection, pulmonary fibrosis, sarcoidosis, and systemic
sclerosis.
Inventors: |
Low; Philip S.; (West
Lafayette, IN) ; Turk; Mary Jo; (New York,
NY) |
Correspondence
Address: |
BARNES & THORNBURG LLP
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Family ID: |
23106203 |
Appl. No.: |
11/823186 |
Filed: |
June 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10138275 |
May 2, 2002 |
|
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11823186 |
|
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60288208 |
May 2, 2001 |
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Current U.S.
Class: |
424/450 ;
514/171; 514/249 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 47/6849 20170801; A61P 25/00 20180101; A61P 1/04 20180101;
A61P 29/00 20180101; A61P 31/04 20180101; A61P 37/02 20180101; A61K
47/646 20170801; A61P 11/00 20180101; A61K 51/0482 20130101; A61P
9/10 20180101; A61K 51/1027 20130101; A61K 9/127 20130101; A61P
19/04 20180101; A61P 37/00 20180101; A61P 1/00 20180101; A61P 19/02
20180101; A61P 17/00 20180101; A61P 19/00 20180101 |
Class at
Publication: |
424/450 ;
514/249; 514/171 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/4985 20060101 A61K031/4985; A61K 31/56
20060101 A61K031/56; A61P 17/00 20060101 A61P017/00; A61P 19/00
20060101 A61P019/00; A61P 29/00 20060101 A61P029/00 |
Claims
1.-19. (canceled)
20. A method of treating a disease state mediated by activated
macrophages, said method comprising the step of administering to a
patient suffering from the macrophage mediated disease state an
effective amount of a composition comprising a conjugate of the
general formula A.sub.b-X wherein the group A.sub.b comprises
folate, or an analog thereof, wherein folate or the folate analog
binds to the folate receptor and is capable of binding to the
activated macrophages, and wherein the group X comprises a
cytotoxin.
21. The method of claim 20 wherein A.sub.b comprises folate.
22. The method of claim 20 wherein A.sub.b comprises a folate
analog that binds to the folate receptor.
23. The method of claim 20 wherein the group X further comprises a
liposome.
24. The method of claim 20 wherein the patient is suffering from a
disease state selected from the group consisting of arthritis,
ulcerative colitis, Crohn's disease, inflammatory lesions,
infections of the skin, osteomyelitis, organ transplant rejection,
pulmonary fibrosis, sarcoidosis, systemic sclerosis, and any
chronic inflammation.
25. The method of claim 20 further comprising the step of
administering to the patient an adjuvant selected from the group
consisting of TiterMax Gold, Alum, and GPI-100.
26. The method of claim 20 wherein the cytotoxin is selected from
the group consisting of a toxin, an alkylating agent, an
antimetabolite, a steroid, a platinum compound, a nitrosurea, and a
microtubule inhibitor.
27. The method of claim 26 wherein the antimetabolite is an
antifolate.
28. The method of claim 27 wherein the antifolate is
methotrexate.
29. The method of claim 26 wherein the microtubule inhibitor is
selected from the group consisting of vinblastine, vincristine,
taxol, and tamoxiphen.
30. The method of claim 29 wherein the microtubule inhibitor is
vinblastine.
31. The method of claim 29 wherein the microtubule inhibitor is
taxol.
32. The method of claim 26 wherein the steroid is a corticoid.
33. The method of claim 32 wherein the corticoid is an
adrenocorticoid.
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. r 60/288,208, filed
on May 2, 2001.
FIELD OF THE INVENTION
[0002] This invention relates to methods for treating and
monitoring disease states mediated by activated macrophages. More
particularly, ligands that bind to activated macrophages are
complexed with an imaging agent, or an immunogen, a cytotoxin or an
agent for altering macrophage function for administration to a
diseased host for diagnosis and/or treatment of macrophage mediated
disease.
BACKGROUND AND SUMMARY OF THE INVENTION
[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 such as multiple sclerosis, lupus
erythematosus, psoriasis, pulmonary fibrosis, and rheumatoid
arthritis and diseases in which the immune response contributes to
pathogenesis such as atherosclerosis, inflammatory diseases,
osteomyelitis, ulcerative colitis, Crohn's disease, and graft
versus host disease often resulting in organ transplant
rejection.
[0004] Macrophages are generally the first cells to encounter
foreign pathogens, and accordingly, they play an important role in
the immune response. However, activated macrophages can contribute
to the pathophysiology of disease in some instances. Activated
macrophages nonspecifically engulf and kill foreign pathogens
within the macrophage by hydrolytic and oxidative attack resulting
in degradation of the pathogen. Peptides from degraded proteins are
displayed on the macrophage cell surface where they can be
recognized by T cells, and they can directly interact with
antibodies on the B cell surface, resulting in T and B cell
activation and further stimulation of the immune response.
[0005] Rheumatoid arthritis (RA) is a systemic disease
characterized by chronic inflammatory synovitis, usually involving
peripheral joints. The synovial inflammation causes cartilage
deterioration and bone erosion with consequent destruction of joint
integrity. Rheumatoid factors, which are autoantibodies reactive
with the Fc region of IgG, are found in more than two-thirds of
patients with RA indicating that RA has an autoimmune
component.
[0006] RA is seen throughout the world in as much as 2% of the
population, with 80% of RA patients developing the disease between
the ages of 35 and 50. The clinical manifestations of RA include
pain, swelling, and tenderness in the joints resulting in
limitation of motion, weakness, fatigue, and weight loss. RA is a
systemic disease and, consequently, has extra-articular
manifestations, especially in patients with high titers of
rheumatoid factors. These symptoms include rheumatoid nodules with
an inner zone of necrotic material, a mid-zone of macrophages, and
an outer zone of granulated tissue, muscle atrophy, osteoporosis,
pulmonary fibrosis, and rheumatoid vasculitis which may result in
cutaneous ulceration, digital gangrene, or neurovascular
disease.
[0007] Rheumatoid synovitis, characteristic of RA, results in an
increase in the number of synovial lining cells, hyperplasia and
hypertrophy of the synovial lining cells, microvascular injury,
edema, and infiltration of cells such as T cells, macrophages, and
dendritic cells. The rheumatoid synovium is characterized by the
presence of secreted products of immune cells such as factors
secreted by T lymphocytes including IL-2, IFN-.delta., IL-6, IL-10,
GM-CSF and TGF.alpha. and .beta. and factors secreted by activated
macrophages including IL-1, IL-6, IL-8, IL-10, GM-CSF, macrophage
CSF, and TGF.beta.. The production of these cytokines appears to
account for much of the pathology of RA including inflammation of
the synovium, synovial cell proliferation, cartilage and bone
deterioration, and systemic symptoms of the disease.
[0008] RA may be treated using various therapies including physical
therapy, rest, and splinting. Therapeutic agents are also used for
the treatment of RA including aspirin and nonsteroidal
anti-inflammatory drugs to control local inflammation. However,
these agents have a minimal effect on the progression of the
disease and are associated with toxic side effects.
Disease-modifying anti-rheumatic drugs, such as
.alpha.-penicillamine and sulfasalazine, are also used to treat RA,
but the benefit from these drugs is delayed for weeks or months and
these drugs have toxic side effects. Immunosuppressive and
cytotoxic drugs suppress symptoms of RA in some patients, but are
associated with toxicity. Intra-articular glucocorticoids have also
been used, but provide only transient relief. Accordingly, there is
a need for the development of new therapies with reduced toxicity
that are efficacious for the treatment of RA and other diseases
caused or worsened by activated macrophages.
[0009] The folate receptor (FR) is a 38 KDa 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 alter the
vitamin's ability to bind the folate receptor, and therefore,
folate-drug conjugates can readily enter cells by receptor-mediated
endocytosis.
[0010] Because most cells use an unrelated reduced folate carrier
(RFC) to acquire the necessary folic acid, expression of the folate
receptor is restricted to a few cell types. With the exception of
kidney and placenta, normal tissues express low or nondetectable
levels of FR. 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 recently
been reported that FRB, the nonepithelial isoform of the folate
receptor, is expressed on activated (but not resting) synovial
macrophages. Thus, Applicants have attempted to utilize
folate-linked compounds potentially capable of altering the
function of activated macrophages, to treat macrophage-mediated
disease states. For example, Applicants have found that
folate-linked immunogens can be used to redirect the host immune
response in arthritic animals to activated macrophages at the site
of inflammation to deplete macrophages and reduce arthritic
inflammation.
[0011] Scintigraphic imaging agents are a million times more
sensitive than magnetic resonance imaging (MRI) contrast agents,
and their selectivity can be enhanced by their targeting to
lesion-specific cell markers. Indeed, the radioisotope .sup.99mTc
has been delivered to arthritic tissues using nonspecific IgG,
anti-CD4 antibodies, CD11b/CD14-glycolipopeptide ligands, and
E-selectin binding peptides. Preclinical studies with such
radioimaging agents have clearly emphasized the value of imaging
arthritic tissues in-vivo, however, the selectively of the current
imaging agents is not yet optimal, and none of the present
compounds is targeted exclusively to activated macrophages. In view
of the emergence of folate receptor activity during macrophage
activation, Applicants have undertaken to determine whether a
folate-targeted .sup.99mTc imaging agent might be used to image
arthritic lesions in vivo.
[0012] To determine whether expression of this high affimity FR
might be exploited to selectively target drugs to activated
macrophages at sites of inflammation, folic acid has been
conjugated to a .sup.99mTc chelator, and its distribution evaluated
in both normal and diseased tissues of rats with adjuvant-induced
arthritis. The folate-linked .sup.99mTc chelate complex, termed
EC20, was indeed found to concentrate in the arthritic extremities
of diseased rats, but not in the joints of healthy rats. The
intensity of the gamma scintigraphic images of affected tissues was
found to be greatly reduced in the presence of excess competing
folic acid. Furthermore, liver and spleen of arthritic animals also
showed enhanced uptake of EC20 and increased levels of FR,
confirming that systemic activation of macrophages accompanies
adjuvant-induced arthritis. Depletion of macrophages from arthritic
animals reduced tissue FR content and concomitantly abolished
uptake of EC20. Furthermore, Kupffer cells isolated from rats with
adjuvant-induced arthritis exhibited a significantly higher binding
capacity for folate conjugates than Kupffer cells from healthy
rats. Thus, Applicants have found that EC20 is useful for assaying
the participation of activated macrophages in inflammatory
pathologies such as rheumatoid arthritis.
[0013] The present invention is directed to a method for treating
and monitoring disease states mediated by activated macrophages. In
accordance with one embodiment of the present invention, disease
states mediated by activated macrophages are treated by redirecting
host immune responses to activated macrophages or by altering the
function of activated macrophages or by direct killing of activated
macrophages. In one aspect of the invention, to promote killing of
activated macrophages, ligands that bind specifically to activated
macrophages are conjugated with an immunogen to redirect host
immune responses to the activated macrophage population, or they
are conjugated to a cytotoxin for direct killing of macrophages.
Ligands that can be used in the conjugates of the present invention
include those that bind to receptors expressed specifically on
activated macrophages, such as the folate receptor, or ligands such
as monoclonal antibodies directed to cell surface markers
specifically expressed on activated macrophages. In another aspect
of the invention ligands that bind specifically to activated
macrophages are conjugated with an imaging agent; the conjugate is
administered to a patient for diagnosing and monitoring the
progression of diseases mediated by activated macrophages.
[0014] In one embodiment, a method of treating or
monitoring/diagnosing a disease state mediated by activated
macrophages is provided. The method comprises the step of
administering to a patient suffering from a macrophage 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 capable of binding to activated
macrophages, 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 macrophage function, and when the
conjugate is being used for monitoring/diagnosing the disease
state, X comprises an imaging agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows folate-targeted imaging of arthritic rats
(whole body scintigraphic images).
[0016] FIG. 2 shows folate-targeted imaging of arthritic rats
(assessment of liver, spleen and kidney by scintigraphy).
[0017] FIG. 3 shows effects of macrophage depletion (black bars
represent arthritic rats, light gray bars represent healthy rats,
and dark gray bars represent arthritic rats depleted of macrophages
by clodronate treatment).
[0018] FIG. 4 shows folate receptor-mediated uptake of EC20 in
arthritic tissues (light gray bars represent biodistribution of
EC20 in the presence of a 500-fold excess of free folic acid, black
bars represent biodistribution of EC20 in the absence of a 500-fold
excess of free folic acid, dark gray bars represent the
biodistribution of the same complex lacking a folate moiety
(EC28)).
[0019] FIG. 5 shows folate receptor expression in various tissues
of arthritic rats (black bars represent arthritic rats, light gray
bars represent healthy rats, and dark gray bars represent arthritic
rats depleted of macrophages by clodronate treatment).
[0020] FIG. 6 shows expression of a functional folate receptor on
liver macrophages of arthritic rats.
[0021] FIG. 7 shows increased uptake of a folate-targeted imaging
agent in a patient with an inflamed joint.
[0022] FIG. 8 shows a chemical structure representing the
folate-linked chelator EC20.
[0023] FIG. 9 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis (diamonds represent folate-FITC
(left foot), squares represent PBS (left foot), triangles represent
folate-FITC (right foot), and X's represent PBS (right foot)).
[0024] FIG. 10 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis (light gray diamonds represent
administration of folate-FITC, dark gray squares represent
administration of methotrexate, dark gray triangles represent
administration of saline, and black lines represent animals without
arthritis). The bottom four lines represent the uninjected paw.
[0025] FIG. 11 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis in the right (uninjected) paw
(light gray diamonds represent administration of folate-FITC at
3000 nmoles/kg, light gray squares represent administration of
folate-FITC at 600 nmoles/kg, light gray triangles represent
administration of folate-FITC at 120 nmoles/kg, light gray X's
represent administration of methotrexate, dark gray circles
represent administration in 3 doses (as in Example 14), light gray
circles represent administration of saline, and black squares
represent animals without arthritis).
[0026] FIG. 12 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis (black diamonds=FF, gray
triangles=FF+low, vertical hash marks=FF+high, gray squares=early
MTX, gray X's=late MTX, thick black lines=arthritis, gray
circles=no arthritis, black circles=FF+mid, horizontal hash
marks=late FFMTX).
[0027] FIG. 13 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis (black diamonds represent
animals with no arthritis, dark gray squares represent animals with
arthritis, light gray triangles represent administration of
folate-FITC, light gray circles represent administration of
methotrexate, and light gray diamonds represent administration of
the combination of folate-FITC and methotrexate).
[0028] FIG. 14 demonstrates immunotherapy mediated protection
against adjuvant-induced arthritis (black diamonds=FF10, gray
squares=FF2, gray triangles=FF, gray X's =IL10, black X's=IL2, open
circles=PBS).
DETAILED DESCRIPTION OF THE INVENTION
[0029] Methods are provided in accordance with the present
invention for either treating or monitoring/diagnosing a disease
state mediated by activated macrophages. Exemplary of diseases
known to be mediated by activated macrophages include rheumatoid
arthritis, ulcerative colitis, Crohn's disease, psoriasis,
osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary
fibrosis, sarcoidosis, systemic sclerosis, organ transplant
rejection (GVHD) and chronic inflammations. Such disease states can
be monitored by first 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 the group
A.sub.b comprises a ligand capable of binding to activated
macrophages, and the group X comprises an imaging agent and
thereafter scanning the patient with an imaging device capable of
detecting the imaging agent. Macrophage mediated disease states can
be treated in accordance with this invention by administering an
effective amount of a composition of the above formula wherein
A.sub.b comprises a ligand capable of binding to an activated
macrophage and wherein the group X comprises an immunogen, a
cytotoxin, or a cytokine capable of altering macrophage function.
Such macrophage targeting conjugates, when administered to a
patient suffering from an activated macrophage mediated disease
state, work to concentrate and associate the conjugated cytotoxin,
immunogen, or cytokine with the population of activated macrophages
to kill the activated macrophages or alter macrophage function.
Elimination or deactivation of the activated macrophage population
works to stop or reduce the activated macrophage mediated
pathogenesis characteristic of the disease state being treated. The
conjugate is typically administered parenterally 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.
[0030] In one embodiment of the invention activated macrophage
mediated disease states are monitored or diagnosed in a patient by
administering a conjugate A.sub.b-X wherein A.sub.b comprises a
ligand capable of binding to activated macrophages and X comprises
an imaging agent and thereafter scanning the patient with an
imaging device capable of detecting localized concentration of the
imaging agent. The imaging or diagnostic conjugates are, similar to
those therapeutic conjugates outlined above, typically administered
as a diagnostic composition comprising a conjugate and a
pharmaceutically acceptable carrier. The composition is typically
formulated for parenteral administration and is administered to the
patient in an amount effective to enable imaging of the locale of
activated macrophage populations. The nature of the imaging agent
component of the conjugate is dictated by the imaging methodology.
Thus, for example, the imaging agent can comprise a chelating
moiety and a metal cation, for example, a radionuclide or a nuclear
resonance imaging contrast agent, such as gadolinium. Typically the
activated macrophage targeted imaging agent is administered to a
patient, and following a period of time to allow delivery and
concentration of the imaging agent in the activated macrophage cell
populations, the patient is subjected to the imaging procedure and
imaging is enabled by the targeted imaging agent.
[0031] The method of the present invention can be used for both
human clinical medicine and veterinary applications. Thus, the host
animals afflicted with the activated macrophage mediated disease
state can be humans, or in the case of veterinary applications,
they can be laboratory, agricultural, domestic or wild animals. The
conjugates administered in accordance with the methods of this
invention are preferably 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
of the present invention may be used alone or in combination with
other therapeutic methods recognized for the treatment of
macrophage mediated disease states.
[0032] In the ligand conjugates of the general formula A.sub.b-X in
accordance with the present invention, the group A.sub.b is a
ligand capable of binding to activated macrophages. Any of a wide
number of macrophage binding moieties can be employed. Acceptable
ligands include particularly folate receptor binding ligands and
antibodies or antibody fragments capable of recognizing and
specifically binding to surface moieties uniquely or preferentially
expressed or presented in/on activated macrophages. In one
embodiment the activated macrophage binding ligand is folic acid, a
folic acid analog or other folate receptor binding molecules.
Activated macrophages express a 38 LD GPI-anchored folate receptor
that binds folate and folate-derivatized compounds with
subnanomolar affinity (i.e., <1 nM). In another embodiment the
activated macrophage binding ligand is a specific monoclonal or
polyclonal antibody or Fab or scFv (i.e., a single chain variable
region) fragments of antibodies capable of specific binding to
activated macrophages.
[0033] The activated macrophage targeted conjugates used for
diagnosing and monitoring disease states mediated by activated
macrophages in accordance with this invention are formed to target
and, thus, to concentrate an imaging agent at the site of activated
macrophage populations in the diseased patient. In such conjugates
of the formula A.sub.b-X, A.sub.b is a ligand capable of binding to
activated macrophages and the group X comprises an imaging agent.
In one embodiment the imaging agent comprises a chelating agent and
a metal cation, typically either a radionuclide or a nuclear
magnetic resonance imaging enhancer or contrast agent, such as
gadolinium. Such conjugates wherein the group A.sub.b is folic
acid, a folic acid analog, or another folic acid receptor binding
ligand 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,
each incorporated herein by reference, describe methods and
examples for preparing chelate conjugates useful in accordance with
the present invention. The present macrophage targeted imaging
agents can be prepared and used following general protocols
described in those earlier patents. The present diagnostic method,
however, is based in part on the discovery that folate targeted
conjugates can be used to concentrate conjugated imaging entities
in and at activated macrophage populations enabling monitoring and
diagnosis of disease states characterized by concentration of
activated macrophages at the site of disease.
[0034] In accordance with one embodiment of the present invention
there is provided a method of treating disease states mediated by
activated macrophages 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 macrophage function. Exemplary of
cytotoxic moieties useful for forming conjugates for use in
accordance with the present method include 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, inflammatory and
proinflammatory agents, and the like. Such toxins or cytotoxic
components can be directly conjugated to the activated macrophage
binding moiety, for example, folate or other folate receptor
binding ligands, or they can be formulated in liposomes which
themselves are targeted as conjugates of macrophage binding
entities typically by covalent linkages to component phospholipids.
Similarly, when the group X comprises a compound capable of
altering a macrophage function, for example, a cytokine such as
IL-10 or IL-11, the cytokine can be covalently linked to the
targeting moiety A.sub.b, for example, a folate receptor binding
ligand or an antibody or antibody fragment directly, or the
macrophage function altering cytokine can be encapsulated in a
liposome which is itself targeted to activated macrophages by
pendent macrophage targeting entities A.sub.b covalently linked to
one or more phospholipid liposome components.
[0035] In another embodiment the ligand-immunogen conjugates can be
administered in combination with a cytotoxic compound. The
compounds listed in the preceding paragraph are among the compounds
suitable for this purpose.
[0036] In another method of treatment embodiment of the present
invention the group X in the activated macrophage targeted
conjugate A.sub.b-X, comprises an immunogen, the ligand-immunogen
conjugates being effective to "label" the population of activated
macrophages 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 in
accordance with this invention works to enhance an immune
response-mediated elimination of the activated macrophage
population. Such can be effected through an endogenous immune
response or by a passive immune response effected by
co-administered antibodies. The endogenous immune response may
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/hapten. It
is also contemplated that the endogenous immune response will
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.
[0037] In another embodiment, the ligand-immunogen conjugate can be
internalized and the immunogen can be degraded and presented on the
macrophage cell surface for recognition by immune cells to elicit
an immune response directed against macrophages presenting the
degraded immunogen.
[0038] The humoral response may 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 a-galactosyl groups. 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 other 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. It is
contemplated that the preexisting antibodies, induced antibodies,
or passively administered antibodies will be redirected to the
activated macrophages by preferential binding of the
ligand-immunogen conjugates to the activated macrophage 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, as well as secondary responses that arise when the
attracted antigen-presenting cells phagocytose the activated
macrophages and present antigens of such cells to the immune system
for elimination of other activated macrophages presenting such
antigens.
[0039] Acceptable immunogens for use in preparing the conjugates
used in the method of treatment of the present invention 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 activated macrophages in
the host animal for elimination of such cells. 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. It is also contemplated that MHC I restricted peptides
could be linked to the ligand for use in redirecting cellular
immunity to macrophages and eliciting T cell killing of
macrophages.
[0040] The macrophage binding ligands and immunogens, cytotoxic
agents, cytokines or imaging agents, as the case may be in forming
conjugates for use in accordance with the present invention, may be
conjugated by using any art-recognized method for form 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. 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
macrophage function altering agent) is contained within a liposome
which is itself covalently linked to the activated macrophage
binding ligand.
[0041] 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 a and
.gamma. carboxylic acid entities.
[0042] The conjugates used in accordance with this invention of the
formula A.sub.b-X are used in one aspect of this invention to
formulate therapeutic or diagnostic compositions comprising
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 invention 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.
[0043] When used for monitoring or diagnosis, imaging procedures
are typically carried out about 1 to about 6 hours post
administration of the activated macrophage targeted imaging
agent.
[0044] 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 for the purpose of defining
this invention such an intermittent or staggered daily regimen is
considered to be equivalent to every day treatment and within the
scope of this invention. In one embodiment of the invention the
patient is treated with multiple injections of the ligand conjugate
wherein the targeted entity is an immunogen or a cytotoxic agent to
eliminate the population of pathogenic activated macrophages. In
one embodiment, the patient is treated, for example, injected
multiple times with the ligand conjugate at, for example, at 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 activated macrophage mediated disease states. In
one embodiment of the invention 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 activated macrophage
targeting ligands in a co-dosing protocol.
[0045] The ligand conjugates are administered in accordance with
this invention parenterally and most typically by intraperitoneal
injections, subcutaneous injections, intramuscular injections,
intravenous 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, 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 of the
invention, 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.
EXAMPLE 1
Materials
[0046] EC20 (a folate-linked chelator .sup.99mTc), EC28 (the same
.sup.99mTc chelate complex without folate), and folate-fluorescein
isothiocyanate (folate-FITC) were gifts from Endocyte, Inc. (West
Lafayette, Ind.). Heat-killed Mycoplasma butericum was purchased
from BD Biosciences (Sparks, Md.). Folic acid, light mineral oil,
clodronate, collagenase-A, and streptavidin-R-phycoerythrin were
obtained from Sigma Chemical Co. (St. Louis, Mo.), and Dubelco's
Modified Eagle Medium (DMEM) was from Gibco-BRL (Gathersberg, Md.).
.sup.3H-folic acid was obtained from American Radiolabeled
Chemicals, Inc. (St. Louis, Mo.) and Microcon.RTM.-30 membranes
were purchased from Millipore Corp. (Bedford, Mass.). RK-4-biotin
and ED2-R-phycoerythrin antibodies were acquired from Bachem
Biosciences, Inc. (Philadelphia, Pa.) and Accurate Chemical and
Scientific Corp. (Westbury, N.Y.), respectively.
EXAMPLE 2
Animal Model of Arthritis
[0047] Arthritis was induced in 150-200 g female Lewis rats
(Charles River Laboratories, Inc., Wilmington, Mass.), n=4/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. Disease was allowed to progress for 21 days, and animals
were weighed on a daily basis to ensure the status of their health.
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 folate-FITC in order to lower
serum folate levels to physiologically relevant concentrations.
Control rats were also maintained on a folate-deficient diet but
not induced to develop arthritis.
EXAMPLE 3
Elimination of Endogenous Macrophages
[0048] Evaluation of macrophage independent uptake of the
folate-linked imaging agent was accomplished by killing endogenous
macrophages with liposomal clodronate. Liposomes were formed by
rehydrating a thin film of egg phosphatidylcholine (60 mole %) and
cholesterol (40 mole %) in an isotonic clodronate solution (250
mg/ml). Small unilamellar vesicles were then generated by extrusion
of the liposomes ten times through a 100 nm polycarbonate membrane
using a 10 ml thermobarrel extruder (Lipex Biomembranes, Vancouver,
Canada). Unencapsulated clodronate was removed by dialysis through
a Spectrapor 300,000 M.sub.r-cutoff cellulose acetate membrane
(Spectrum Laboratories, Rancho Domingues, Calif.), and the
clodronate concentration in the retained liposomes was determined
as described in J. Microencapsul. 3(2) 109-14 (1986). Seventeen
days following induction of the arthritis and three days prior to
administration of the imaging agent (EC20), rats destined for
macrophage depletion received a single intraperitoneal injection of
clodronate liposomes containing 20 mg clodronate.
EXAMPLE 4
Scintigraphy and Biodistribution Analysis
[0049] Twelve hours prior to administration of imaging agent, all
animals received 5 ml of normal saline subcutaneously to ensure
proper excretion of unbound imaging agent. Twenty-one days
following induction of arthritis, rats (n=3 per group) were
injected intraperitoneally with 500 .mu.Ci (2.3 nmoles/rat) of
either EC20 (folate +chelator), EC20+500-fold molar excess folic
acid, or EC28 (no folate moiety). Four hours later, rats underwent
either nuclear scintigraphic imaging or biodistribution
analysis.
[0050] For scintigraphy, rats were anesthetized with ketamine and
xylazine, and positioned in ventral recumbency on the image
acquisition surface. Image acquisition was performed for one minute
at a count rate of 50-75,000 counts per minute using a Technicare
Omega 500 Sigma 410 Radioisotope Gamma Camera. Following
acquisition of whole body images, radiation of the upper body
(above the stifles) was blocked using 1/8'' lead plates, and images
of the posterior limbs were obtained. All data were analyzed using
a Medasys.TM. MS-DOS-based computer equipped with Medasys.TM.
Pinnacle software.
[0051] For biodistribution analysis, rats were euthanized by
intraperitoneal injection of nebutal or pentobarbitol sodium.
Liver, spleen, heart, lungs, intestine, and kidneys were then
harvested and radiation in each tissue was determined by counting
in a gamma counter (Packard BioScience Co., Meridian, Conn.).
EXAMPLE 5
Assay of Tissue Folate Receptor Levels
[0052] Folate receptor levels in each tissue were determined
follows. Briefly, tissues were homogenized and cell membranes were
isolated by centrifugation. Membrane proteins were solubilized
overnight, transferred into a Microcon.RTM.-30 filtration device,
and incubated with 50 nM .sup.3H-folic acid. A duplicate of each
sample, used to determine non-specific binding, was also exposed to
50 nM .sup.3H-folic acid, but in the presence of 1000-fold excess
unlabeled folic acid. After unbound .sup.3H-folic acid was washed
through the membrane, membrane protein with bound .sup.3H-folic
acid was recovered and counted in a scintillation counter (Packard
BioScience Co.) to determine the number of active folate receptors
per gram of tissue.
EXAMPLE 6
Identification of the Folate Receptor Expressing Cell Type In
Liver
[0053] Arthritic and healthy rats were first anesthetized with
ketamine and xylazine, and then a midline incision was made,
starting in the lower abdomen and extending through the thoracic
cavity. A 24-gauge catheter was inserted into the hepatic vein, and
a 24-gauge needle was inserted in the cardiac left ventricle to
serve as an outlet for the perfusion fluid. Rats were then perfused
by delivery of normal saline, followed by collagenase A solution
(0.05% in Gey's balanced salt solution) through the catheter. Each
solution was perfused for two minutes at a rate of 20 ml/minute.
Immediately after perfusion, livers were removed and the membranous
outer tissue was dissected away. The remaining gelatinous tissue
was suspended in collagenase-A solution (0.025% in DMEM) and
incubated at 37.degree. C. for two hours in the presence of 1 .mu.M
folate-FITC or 1 .mu.M folate-FITC+1 mM folic acid. Cells were then
washed three times to removed unbound folate-FITC and immediately
prepared for flow cytometry.
EXAMPLE 7
Flow Cytometry Sample Preparation and Analysis
[0054] Liver cell preparations, which had been exposed to
folate-FITC, were treated for 10 mm at 4.degree. C. with ammonium
chloride lysis buffer (150 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 1 mM
EDTA, pH 7.4) to lyse red blood cells. Following three washes with
phosphate buffered saline, the remaining cells were incubated for 1
h at 4.degree. C. with either ED2 R-Phycoerythrin-labeled mouse
anti-rat macrophage antibody, or RK-4 biotin-labeled mouse-anti rat
granulocyte antibody. Cells were again washed two times, and those
that had received the biotinylated primary antibody were further
incubated with streptavidin-R-Phycoerythrin for 30 minutes.
Following two final washes, cells were examined for FITC and
phycoerythrin dual color staining on a FACScan Coulter XL flow
cytometer.
EXAMPLE 8
Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis
[0055] The protocol described in Example 2 for arthritis induction
was followed. The efficacy of a folate-FITC conjugate
(folate-fluorescein isothiocyanate conjugate) against
adjuvant-induced arthritis in rats was investigated. Each rat used
in the experiment was immunized at the base of the tail with
FITC-KLH (150 .mu.g) to induce antibodies against FITC on days -38
and -10 before administration of Mycoplasma butericum (adjuvant) to
induce arthritis. The immunization of FITC-KLH was done in
combination with an adjuvant (i.e., such as TiterMax Gold (150
.mu.g), Alum (150 .mu.g), or GPI-100 (150 .mu.g) which are all
adjuvants to induce antibodies against FITC as opposed to the
adjuvant used to induce arthritis). The immunized animals were then
injected on day 0 in the left foot pad with 0.5 mg of heat-killed
Mycoplasma butyricum (adjuvant) to initiate development of
arthritis. Then on days 1, 2, 3, 9, 11, and 14, post-adjuvant
(Mycoplasma butyricum) injection, the rats were injected
intraperitoneally with either saline (control rats) or 2000
nmoles/kg of folate-FITC (FF). Calipers were used to measure left
and right foot dimensions daily. With reference to FIG. 9, those
measurements are plotted for both the adjuvant-injected feet (top
two curves) and the non-treated feet (bottom two curves). A sudden
increase in swelling of the adjuvant-injected feet is due to influx
of neutrophils which have no folate receptors. Consequently, the
immunotherapy has no impact on this phase of paw swelling. However,
after about 10 days, activated macrophages invade both injected
feet and uninjected feet, causing bone degradation and further
inflammation. These activated macrophages have functional folate
receptors, and, as shown, they are eliminated or reduced by binding
folate-hapten conjugates such as folate-FITC.
EXAMPLE 9
Folate-Targeted Imaging of Arthritic Rats
[0056] The protocols described in Examples 2 and 4 were followed.
As noted above, activated but not resting macrophages express a
receptor for the vitamin folic acid. To determine whether folate
might be exploited to target .sup.99mTc to sites of arthritic
inflammation, EC20, a folate-linked chelator of .sup.99mTc (see
FIG. 8) was administered intraperitoneally to rats (n=5/group) and
scintigraphic images were acquired with a gamma camera. Due to the
rapid clearance of EC20, excellent contrast was obtained by at
least four hours post-administration (FIG. 1). Importantly, whole
body uptake was significantly more intense in arthritic rats
compared to healthy rats, and this uptake was greatly reduced when
EC20 was administered together with a saturating dose of free folic
acid. This suggests that uptake by all tissues is primarily
determined by a folic-specific receptor.
[0057] Intense organ uptake of EC20 prevented visualization of
limbs in whole body images of the arthritic rats. However, images
of posterior limbs could be easily acquired when mid and upper body
radiation was shielded. With such shielding, arthritic limbs
displayed much greater EC20 uptake than healthy extremities, and
this uptake was completely eliminated in the presence of excess
free folic acid (FIG. 1). Furthermore, the left rear foot of the
arthritic animals, where inflammation was most severe, displayed
greater uptake than the less severely affected right rear foot
(FIG. 1).
[0058] From the whole body images, it could be concluded that
abdominal organs were responsible for a majority of EC20 uptake in
the arthritic animals. To confirm this assessment, liver, spleen
and kidney were removed and imaged separately (FIG. 2). Livers of
arthritic rats demonstrated the highest uptake of EC20, while
livers of healthy rats displayed minimal uptake. Only those spleens
taken from arthritic rats could be visualized. Free folic acid
completely blocked EC20 uptake in liver and spleen, however, the
free vitamin only partially decreased uptake by the kidney.
EXAMPLE 10
Effects Of Macrophaze Depletion
[0059] The protocols described in Examples 2, 3, and 4 were
followed, except that 0.25 mCi of EC20 was administered. In order
to determine whether macrophages might be responsible for the
uptake of EC20, resident macrophages were systemically eliminated
from arthritic rats using a liposomal clodronate preparation (n=3
rats/group). By four days after clodronate treatment, evaluation of
paw size revealed that clodronate-treated rats were significantly
less inflamed than untreated rats (data not shown). To determine
whether macrophage elimination would influence uptake of the
folate-linked imaging agent, EC20 biodistribution analysis was then
performed on the clodronate-treated rats and compared to the same
analysis of both healthy rats and arthritic rats not treated with
clodronate. As shown in FIG. 3 (black bars represent arthritic
rats, light gray bars represent healthy rats, and dark gray bars
represent arthritic rats depleted of macrophages by clodronate
treatment), depletion of macrophages decreased liver uptake of
EC20.about.20-fold in arthritic rats, while retention in the spleen
and intestine was reduced by a factor of three. In most tissues,
clodronate treatment depressed EC20 uptake even below those levels
observed in healthy rats, confirming the hypothesis that activated
macrophages account for most of EC20 retention in normal tissues.
In contrast, kidney uptake of EC20 was elevated in rats depleted of
macrophages, most likely because the decreased internalization of
EC20 by activated macrophages rendered more EC20 available for
binding to kidney folate receptors.
EXAMPLE 11
Folate Receptor-Mediated Uptake of EC20 in Arthritic Tissues
[0060] The protocols described in Examples 2 and 4 were followed.
Two additional biodistribution studies were conducted to confirm
that EC20 uptake by tissues of arthritic rats is mediated by the
folate receptor (n=3 rats/group). First, the biodistribution of
EC20 was examined in the presence (light gray bars) and absence
(black bars) of a 500-fold excess of free folic acid. As seen in
FIG. 4, almost complete elimination of EC20 uptake was observed in
all tissues except kidney, indicating that binding was indeed
mediated by a folate receptor. In fact, excess folic acid
competitively reduced EC20 retention in liver, spleen, heart, lung,
intestine and blood to near background levels (FIG. 4). Second, to
confirm the role of folate in EC20-mediated targeting of the
chelated .sup.99mTc, the biodistribution of the same complex
lacking a folate moiety (EC28) was also examined (dark gray bars).
As also displayed in FIG. 4, uptake of EC28 was negligible in all
tissues except kidney, where retention of the non-targeted complex
was similar to that of EC20 in the presence of competing folic
acid.
EXAMPLE 12
Folate Receptor Expression in Various Tissues of Arthritic Rats
[0061] The protocols described in Examples 2, 4, and 5 were used.
The above results suggest that the folate receptor is responsible
for tissue uptake of EC20. In order to confirm this, Applicants
attempted to directly quantitate the folate binding protein in
various rat tissues. Active folate receptor could be detected in
each of the major organs examined, and FR levels were significantly
increased in arthritic rats (FIG. 5; black bars=arthritic rats;
light gray bars=healthy rats). Further, FR content correlated well
with uptake of EC20 seen in the biodistribution studies. In fact,
the FR assay revealed roughly equivalent levels of receptor in
arthritic liver and spleen, in accordance with the similar uptake
of EC20 by the same organs (FIG. 4). Significantly, systemic
elimination of macrophages by clodronate treatment (dark gray bars)
lowered folate receptor levels in all arthritic tissues (FIG. 5),
also in good agreement with the EC20 biodistribution analysis.
Finally, the FR assay confirmed that neither induction of arthritis
nor clodronate treatment alters the levels of FR in kidney or
heart, where FR is not thought to be associated with activated
macrophages.
EXAMPLE 13
Expression of a Functional Folate Receptor on Liver Macrophages of
Arthritic Rats
[0062] The protocols described in Examples 2, 4, 6 and 7 were used.
To further confirm that the elevated uptake of EC20 in livers of
arthritic rats is due to a macrophage population, livers were
resected from collagenase-perfused rats and their disaggregated
cells examined for folate conjugate uptake, using folate-FITC as a
fluorescent marker for FR expression. By also labeling the same
liver cell suspension with an antibody specific for rat liver
macrophages, it was possible to demonstrate that macrophages are
indeed the cell type that expresses elevated levels of folate
receptor in arthritic animals (FIG. 6). Thus, flow cytometric
analysis revealed that 70% of the liver macrophages of arthritic
rats bound folate-FITC compared to only 30% of the liver
macrophages of healthy rats (FIG. 6). Further, the FITC intensity
of the arthritic macrophages was higher than that of macrophages
from healthy livers. Since binding of folate-FITC was suppressed in
the presence of an excess of free folic acid (1 mM), we concluded
that uptake of the folate conjugate by liver macrophages was
mediated by the folate receptor.
[0063] Using an antibody specific for granulocytes, we also
examined whether tissue infiltrating neutrophils might take up
folate conjugates. Although very few neutrophils were found in the
liver, those that were detected exhibited no binding capacity for
folate-FITC (data not shown). Mac-l+peripheral blood cells were
also tested and similarly found to have no binding affinity for the
folate-conjugate (data not shown). In fact, no peripheral blood
cells sorted positive for FITC fluorescence, suggesting that only
resident tissue macrophages (and clearly only a subpopulation of
those) express FR in the liver.
[0064] Finally, to begin to explore whether activated macrophages
might be targeted with folate-linked drugs in human patients, we
obtained permission to examine the whole body images of the 28
suspected ovarian cancer patients enrolled in a recently completed
clinical trial of the gamma imaging agent, .sup.111 In-DTPA-Folate.
As shown in FIG. 7, one patient displayed significant imaging agent
uptake in the right knee, but not the left knee. Importantly, no
other patients demonstrated any measurable joint uptake. Upon
request, the attending physician contacted the anonymous patient
and inquired whether she had been experiencing any chronic joint
discomfort. The physician responded that the patient reported
arthritis in the right knee.
Discussion
[0065] Activated macrophages are thought to be intimately involved
in the pathogenesis of rheumatoid arthritis. Activated macrophages
directly destroy joint tissue by secreting metalloproteinases and
attracting/activating other immune cells by releasing cytokines.
The quantitation of activated macrophages in joint tissues might be
of diagnostic value, since activated macrophage content correlates
well with articular destruction and poor disease prognosis in
humans.
[0066] Gamma camera scintigraphy of rats receiving EC20
demonstrated that arthritic appendages are indeed illuminated by
folate-targeted .sup.99mTc. In contrast, the legs and feet of
healthy rats could not be visualized, demonstrating the selectivity
of the imaging agent for arthritis applications. Although the
intensities of internal organs also increased in adjuvant-induced
arthritis, interference from such tissues did not appear to
compromise the methodology, since gamma radiation from internal,
organs could be easily screened. The fact that excellent contrast
can be obtained within one to two hours of EC20 injection further
shows that imaging agent administration, gamma camera scintigraphy,
and image analysis can be completed during the same
examination.
[0067] Systemic activation of macrophages has been documented in
rats with adjuvant-induced arthritis. Thus, it was important to
establish the specific participation of macrophages in the elevated
uptake of EC20, since a folate-targeted imaging agent had never
previously been examined in arthritic animals. Three experiments
were conducted for this purpose. First, clodronate-loaded liposomes
were employed to systemically deplete macrophages from the treated
rats. Not only were the resulting tissue FR levels greatly reduced,
but uptake of EC20 in the macrophage-rich organs was also nearly
eliminated, suggesting that resident macrophages can indeed account
for both FR expression and EC20 retention in the RES organs.
Second, liver cells were disaggregated by collagenase treatment and
individual cells were evaluated for folate conjugate uptake. As
noted in FIG. 6, the vast majority of cells testing positive for
folate conjugate uptake also sorted positive for the macrophage
marker, ED2, confirming that FR is indeed present on the
macrophages. Finally, because other immune and myelocytic cells are
known to be elevated in tissues of rats with adjuvant-induced
arthritis, it was conceivable that still another extravasating
blood cell type might be involved in the uptake of EC20. However,
neither liver-infiltrating granulocytes nor any blood cell in
circulation displayed any capacity to bind folate-FITC. Thus,
activated macrophages would seem to be the predominant cell type
internalizing folate conjugates in the organs of arthritic
rats.
[0068] It was surprising to find that up to 30% of the liver
macrophages in healthy rats also expressed the folate receptor
(FIG. 6). Since a functional folate receptor is not found on
resting synovial macrophages, it is tempting to speculate that the
folate-FITC binding fraction in the healthy rats might also
constitute an activated population. Two observations may support
this conjecture. First, activated macrophages are also found in
healthy tissues following exposure to immune stimulants such as
foreign antigens. Given the role of the liver in clearing foreign
substances from the body, a low level of resident macrophage
activation does not seem unreasonable. Second, the folate-FITC (and
EC20) binding population of liver cells increased significantly
upon induction of localized inflammation and systemic macrophage
activation.
[0069] With the ability to exploit folate to deliver attached
molecules to activated macrophages now established, folate-linked
imaging agents will allow the early development or continued
progression of rheumatoid arthritis to be assessed. Since graft
versus host disease, multiple sclerosis, Crohn's disease,
ulcerative colitis, psoriasis, osteomyelitis, and even
atherosclerosis may also be caused/aggravated by activated
macrophages, it is possible that the diagnosis/evaluation of these
diseases could be aided by a folate-linked imaging/contrast agent.
The avid folate conjugate uptake by activated macrophages in both
arthritic joints and liver indicates that macrophages can be
effectively targeted regardless of their anatomical location.
EXAMPLE 14
[0070] Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis For the assay shown in FIG. 10, the protocol described in
Example 8 was followed except that 3000 nmoles/kg of folate-FITC
was administered per day (3 doses on days 1, 2, and 3) and
folate-FITC was delivered using an osmotic pump implanted into the
peritoneal cavity of the rat. Methotrexate (MTX) was administered
at a dose of 0.15 mg by intraperitoneal injection one time per day
on days 1, 8, and 15 after adjuvant administration. MTX was used in
place of folate-FITC for animals treated with MTX. The results for
both the left (injected) and right (uninjected) paw are shown. The
results show that folate-FITC (FF) inhibits adjuvant-induced
arthritis as well as MTX.
EXAMPLE 15
P Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis
[0071] The protocol described in Example 14 was followed except
that only the right paw volume was measured and folate-FITC (FF)
was administered at doses of 3000, 600, and 120 nmoles/kg (FIG.
11). Also, FF was administered at 3000 nmoles/kg in either three
doses as in Example 14 (indicated as "3 doses" in FIG. 11) or on
days 1, 2, 3, 9, 11, and 14 as described in Example 8 (indicated as
"FF3000" in FIG. 11). The results show that FF inhibits
adjuvant-induced arthritis in the right paw of the arthritic rats
(inflammation presumably appears in the uninjected right paw due to
the systemic progression of arthritis), and that prolonged
treatment with FF is more effective than 3 initial doses for
treatment of adjuvant-induced arthritis.
EXAMPLE 16
Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis
[0072] The protocol described in Example 8 was followed except MTX
was used at a dose of 0.15 mg (FF+low, early MTX, late MTX and
lateFFMTX) to treat some animals in place of FF (see FIG. 12).
Other animals were treated with 0.75 mg of MTX (FF+mid) or 1.5 mg
of MTX (FF+high). For "early MTX" treatments, the rats were
injected with MTX on days 1, 8, and 15 after arthritis induction.
For "late MTX" treatments, the rats were injected with MTX on days
8 and 15 after adjuvant administration. All measurements were of
the uninjected right paw. The results show that folate-FITC (FF) in
combination with MTX (early or late treatments and a low, high, or
middle dose of MTX) inhibits adjuvant-induced arthritis better than
FF alone.
EXAMPLE 17
Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis
[0073] For the results shown in FIG. 13, the protocol described in
Example 8 was followed except that some animals were treated with
MTX alone (0.15 mg) on days 1, 8, and 15 after arthritis induction
or were treated with MTX (0.15 mg; days 1, 8, and 15) in
combination with FF as described in Example 14. The results show
that the combination of FF and MTX inhibits adjuvant-induced
arthritis to a greater extent than MTX or FF alone.
EXAMPLE 18
Immunotherapy Mediated Protection Against Adjuvant-Induced
Arthritis
[0074] The protocol described in Example 8 was followed except that
IL-10 (10,000 U; FF10) or IL-2 (3 .mu.g/kg; FF2) was administered
along with the treatments with FF (i.e., the cytokines were
administered by intraperitoneal injections on days 1, 2, 3, 9, 11,
and 14 after adjuvant administration; see FIG. 14). The
measurements made were measurements of the right, noninjected paw.
The results show that either IL-10 or IL-2 prevent the inhibition
of adjuvant-induced arthritis resulting from treatment with FF. All
of the above immunotherapy results taken together indicate that
folate-linked agents which are cytotoxic for macrophages can be
used to treat macrophage-mediated disease states.
EXAMPLE 19
Folate-Targeted Imaging of Arthritic Rats
[0075] For the assay shown in FIG. 15, the protocols were as
described in Examples 2 and 4 except that some animals were treated
with FF (2000 nmoles/kg; days 1, 2, 3, 9, 11, and 14) or MTX (0.15
mg; days 1, 8, and 15) as described in Examples 8 and 14,
respectively. The results show that FF or MTX prevent EC20 uptake
in all organs examined except the kidney. It is likely that EC20
uptake is reduced in most organs making more EC20 available for
excretion through the kidney accounting for the increase in EC20
detected in kidney tissues.
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