U.S. patent application number 11/807422 was filed with the patent office on 2008-05-22 for copper lowering treatment of inflammatory and fibrotic diseases.
Invention is credited to George J. Brewer.
Application Number | 20080118519 11/807422 |
Document ID | / |
Family ID | 29584487 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080118519 |
Kind Code |
A1 |
Brewer; George J. |
May 22, 2008 |
Copper lowering treatment of inflammatory and fibrotic diseases
Abstract
The present invention relates generally to the field of
prophylaxis and therapy for inflammatory and/or fibrotic diseases
which include responses to injuries. In particular, the present
invention is related to agents that can bind or complex copper such
as thiomolybdate, and to the use of these agents in the prevention
and treatment of inflammatory and/or fibrotic diseases. Exemplary
thiomolybdates include mono-, di-, tri- and tetrathiomolybdate;
these agents are administered to patients to prevent and/or treat
inflammatory and/or fibrotic diseases, such as pulmonary disease
including pulmonary fibrosis and acute respiratory distress
syndrome, liver disease including liver cirrhosis and hepatitis C,
kidney disease including renal interstitial fibrosis, scleroderma,
cystic fibrosis, pancreatic fibrosis, keloid, secondary fibrosis in
the gastrointestinal tract, hypertrophic burn scars, myocardial
fibrosis, Alzheimer's disease, retinal detachment inflammation
and/or fibrosis resulting after surgery, and graft versus host and
host versus graft rejections.
Inventors: |
Brewer; George J.; (Ann
Arbor, MI) |
Correspondence
Address: |
Casimir Jones, S.C.
440 Science Drive, Suite 203
Madison
WI
53711
US
|
Family ID: |
29584487 |
Appl. No.: |
11/807422 |
Filed: |
May 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11057353 |
Feb 14, 2005 |
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11807422 |
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10444204 |
May 23, 2003 |
6855340 |
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11057353 |
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60382993 |
May 24, 2002 |
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Current U.S.
Class: |
424/158.1 ;
424/646 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 29/00 20180101; A61K 33/24 20130101; A61K 33/24 20130101; A61P
13/12 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
424/158.1 ;
424/646 |
International
Class: |
A61K 33/24 20060101
A61K033/24; A61K 39/395 20060101 A61K039/395; A61P 29/00 20060101
A61P029/00 |
Claims
1. A method of treating fibrotic disease in a patient, comprising
administering to the patient having an inflammatory or fibrotic
disease a biologically effective amount of at least a first agent
that binds, chelates or complexes copper.
2-40. (canceled)
41. The method of claim 1, wherein the first agent is a
thiomolybdate.
42. The method of claim 41, wherein the thiomolybdate forms a
thiomolybdate-copper-protein complex in said patient.
43. The method of claim 41, wherein the thiomolybdate is
tetrathiomolybdate.
44. The method of claim 41, wherein the biologically effective
amount of the first agent is between about 20 mg and about 200 mg
per patient.
45. The method of claim 1, wherein administering the first agent
lowers available copper levels.
46. The method of claim 1, wherein administering the first agent
lowers serum ceruloplasmin levels.
47. The method of claim 1, wherein the method further comprises
administering to the patient a therapeutically effective amount of
at least a second agent, where the second agent is chosen from
anti-inflammatory agents, anti-fibrotic agents, and an agent that
binds, chelates or complexes copper.
48. The method of claim 1, wherein the first agent is administered
orally.
49. The method of claim 1, wherein the first agent is administered
by injection.
50. The method of claim 49, where the injection is chosen from
intravascular, intramuscular, or subcutaneous.
51. The method of claim 47, wherein the second agent is chosen from
a steroid, a NSAIDS (non-steroidal anti-inflammatory drugs), a
chemotherapeutic agent as used in some auto-immune diseases, and an
antibody or antisense agent directed to specific cytokines or to
cytokine receptors or to other molecules which enhance inflammation
or fibrosis.
52. The method of claim 46, wherein said ceruloplasmin levels are
lowered to between about 5 to about 15 mg/dl.
53. The method of claim 46, wherein said ceruloplasmin levels are
lowered to between about 10% to about 90% of the ceruloplasmin
level prior to said administering.
54. The method of claim 46, wherein said ceruloplasmin levels are
lowered to approximately 50% of the ceruloplasmin level prior to
said administering.
55. A method of treating inflammatory disease in a patient,
comprising administering to the patient having an inflammatory or
fibrotic disease a biologically effective amount of at least a
first agent that binds, chelates or complexes copper.
56-70. (canceled)
71-78. (canceled)
79-112. (canceled)
113. A method of treating inflammatory or fibrotic disease in a
patient, comprising administering to the patient having an
inflammatory or fibrotic disease a biologically effective amount of
a sustained release formulation of an agent that binds, chelates or
complexes copper.
114-127. (canceled)
128-129. (canceled)
131-132. (canceled)
Description
[0001] The present invention claims priority to U.S. 60/382,993
filed on May 24, 2002, the disclosure of which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
prophylaxis and therapy for inflammatory and fibrotic diseases. In
particular, the present invention is related to agents that can
bind or complex copper, and to the use of these agents in the
prevention and treatment of inflammatory and fibrotic diseases.
BACKGROUND
[0003] Many diseases begin with inflammation, which if excessive,
may overwhelm and kill the patient, or if the patient survives,
often leads to a disabling fibrosis, which ultimately may also kill
the patient.
[0004] A classic example is ARDS (Acute Respiratory Distress
Syndrome) which may be initiated by any one of several lung
injuries (smoke inhalation, near drowning, some infections, etc.).
About one third of these patients die, overwhelmed by inflammatory
processes in the lung. In those patients that don't die, there is a
high risk of developing interstitial pulmonary fibrosis, which is
itself often a progressive and fatal disease. A similar pattern is
seen in inflammatory diseases of the liver (such as hepatitis C)
which lead to cirrhosis, in inflammatory diseases of the kidney,
such as glomerulonephritis, which lead to glomerulosclerosis and
renal interstitial fibrosis, and in diseases of the skin, leading
to systemic sclerosis (scleroderma). Other diseases with similar
patterns include pancreatic fibrosis, bowel inflammations leading
to fibrosis and obstruction, and acute burn multiple organ
damage.
[0005] Recently, evidence has been accumulating that the common
condition of inflammation may also underlie many other chronic and
debilitating diseases, such as Alzheimer's, heart disease,
osteoporosis and diabetes, and that drugs that fight inflammation
may have a role in preventing or delaying those diseases, or at
least slowing them down. Typically, anti-inflammatory drugs belong
to the class of drugs known as nonsteroidal anti-inflammatory drugs
(NSAIDS), which include ibuprofen and naproxen, aspirin, and
prescription drugs known as cox-2-inhibitors, including celecoxib
(Celebrex) and rofecoxib (Vioxx) as well as diclofenac (Voltaren),
indomethacin (Indocin), and other less commonly used drugs.
However, patients at risk for these common and chronic diseases are
not encouraged to take anti-inflammatory drugs, other than aspirin,
at least in part because regular use of these drugs is not safe for
everyone. Side effects can include stomachache or nausea in up to
20 percent of patients, and stomach or intestinal ulcers and
bleeding in 2 percent to 4 percent of those who take the drugs for
a year, especially for people over 60. The stomach bleeding can
occur with little warning, and it can be fatal. Even low doses of
aspirin can cause stomach bleeding in some people, and it can also
cause a slight increase in the risk of a less common type of
stroke, also brought on by cerebral hemorrhage. Anti-inflammatory
drugs may make kidney disease worse, and cox-2 drugs have been
suggested to cause an increase in the risk of heart attack.
[0006] Currently there is no effective therapy for these
inflammatory and/or fibrotic diseases. Moreover, anti-inflammatory
drugs possess high levels of risk, especially with prolonged use.
Thus, what is needed is an effective therapy for inflammatory
and/or fibrotic disease; preferably, such therapy is also safe,
especially for long-term use.
SUMMARY OF THE INVENTION
[0007] The present invention provides an effective and safe therapy
for inflammatory and fibrotic diseases, which include response to
injury. This is accomplished by treating patients suffering from
such diseases with agents which reduce the level of endogenous
copper; in some embodiments, this is accomplished by treating
patients with agents which complex or bind copper, which for some
agents may result in the formation of a tripartite
agent-copper-protein complex, thus decreasing endogenous copper
levels. Effective agents include but are not limited to copper
binding or complexing thiomolybdates. The compositions and methods
of the present invention result in effective therapy without the
side effects and risks associated with anti-inflammatory drugs, and
without the side effects and risks associated with other copper
reducing agents.
[0008] The present invention provides a method of treating
inflammatory or fibrotic disease in a patient, comprising
administering to the patient having an inflammatory or fibrotic
disease a biologically effective amount of at least a first agent
that binds or complexes copper. In some embodiments, the first
agent is a thiomolybdate; in other embodiments, the thiomolybdate
forms a thiomolybdate-copper-protein complex in a patient. In yet
other embodiments, the thiomolybdate is tetrathiomolybdate. In some
embodiments, the patient is a human; in other embodiments, the
patient is a non-human animal. In some embodiments, the
biologically effective amount of the first agent is between about
20 mg and about 200 mg per patient. In some embodiments,
administering the first agent lowers endogenous copper levels; in
other embodiments, administering the first agent lowers serum
ceruloplasmin levels. In some embodiments, the first agent is
administered orally. In other embodiments, the first agent is
administered by injection; in further embodiments, the injection is
chosen from intravascular, intramuscular, or subcutaneous
injection.
[0009] The present invention also provides a method of treating
inflammatory and/or fibrotic disease in a patient, comprising
administering to the patient having an inflammatory and/or fibrotic
disease a therapeutically effective amount of at least a first
agent that binds or complexes copper. In some embodiments, the
first agent is a thiomolybdate. In further embodiments, the
thiomolybdate forms a thiomolybdate-copper-protein complex in a
patient. In yet other embodiments, the thiomolybdate is
tetrathiomolybdate. In some embodiments, the patient is a human; in
other embodiments, the patient is a non-human animal. In some
embodiments, the therapeutically effective amount of the first
agent is between about 20 mg and 200 mg per patient administered
over a therapeutically effective time or period. In other
embodiments, the therapeutically effective amount of the first
agent is between about 20 mg and 200 mg per patient per day. In
some embodiments, administering the first agent lowers endogenous
copper levels; in further embodiments, administering the first
agent lowers serum ceruloplasmin levels. In some embodiments, the
first agent is administered orally. In other embodiments, the first
agent is administered by injection; in further embodiments, the
injection is chosen from intravascular, intramuscular, or
subcutaneous injection.
[0010] In any of the embodiments described above, the inflammatory
or fibrotic disease can be chosen from pulmonary disease including
pulmonary fibrosis and acute respiratory distress syndrome, liver
disease including liver cirrhosis and hepatitis C, kidney disease
including renal interstitial fibrosis, scleroderma, cystic
fibrosis, pancreatic fibrosis, keloid, secondary fibrosis in the
gastrointestinal tract, hypertrophic burn scars, myocardial
fibrosis, Alzheimer's disease, retinal detachment inflammation
and/or fibrosis resulting after surgery, and graft versus host and
host versus graft rejections.
[0011] The present invention also provides any of the embodiments
described above, where the method further comprises administering
to the patient a therapeutically effective amount of at least a
second agent, where the second agent is chosen from
anti-inflammatory agents, anti-fibrotic agents, and
anti-angiogenesis agents. In some of these embodiments, the second
agent is chosen from a steroid, a NSAIDS (non-steroidal
anti-inflammatory drugs), a chemotherapeutic agent as used in some
auto-immune diseases, and an antibody or antisense agent directed
to specific cytokines or to cytokine receptors or to other
molecules which enhance inflammation and/or fibrosis.
[0012] The present invention also provides a method of prophylactic
or therapeutic intervention in a patient at risk of developing an
inflammatory and/or fibrotic disease, comprising administering to
the patient at risk for developing an inflammatory and/or fibrotic
disease a biologically effective amount of at least a first agent
that binds or complexes copper. In some embodiments, the first
agent is a thiomolybdate; in other embodiments, the thiomolybdate
forms a thiomolybdate-copper-protein complex in a patient. In yet
other embodiments, the thiomolybdate is tetrathiomolybdate. In some
embodiments, the patient is a human; in other embodiments, the
patient is a non-human animal. In some embodiments, the
biologically effective amount of the first agent is between about
20 mg and about 200 mg per patient. In some embodiments,
administering the first agent lowers endogenous copper levels; in
other embodiments, administering the first agent lowers serum
ceruloplasmin levels. In some embodiments, the first agent is
administered orally. In other embodiments, the first agent is
administered by injection; in further embodiments, the injection is
chosen from intravascular, intramuscular, or subcutaneous
injection. In yet other embodiments, the method further comprises
administering to the patient a therapeutically effective amount of
at least a second agent, where the second agent is chosen from
anti-inflammatory agents, anti-fibrotic agents, and
anti-angiogenesis agents. In some of these embodiments, the second
agent is chosen from a steroid, a NSAIDS (non-steroidal
anti-inflammatory drugs), a chemotherapeutic agent as used in some
auto-immune diseases, and an antibody or antisense agent directed
to specific cytokines or to cytokine receptors or to other
molecules which enhance inflammation and/or fibrosis.
[0013] The present invention also provides a composition comprising
a combined therapeutic amount of at least a first agent that binds
or complexes copper and at least a second agent, where the second
agent is chosen from anti-inflammatory agents, anti-fibrotic
agents, and anti-angiogenesis agents. In some embodiments, the
first agent is a thiomolybdate; in some further embodiments, the
thiomolybdate is tetrathiomolybdate. In other embodiments, the
second agent is chosen from a steroid, a NSAIDS (non-steroidal
anti-inflammatory drugs), a chemotherapeutic agent as used in some
auto-immune diseases, and an antibody or antisense agent directed
to specific cytokines or to cytokine receptors or to other
molecules which enhance inflammation and/or fibrosis.
[0014] The present invention also provides a therapeutic kit
comprising, in at least a first suitable container, a
therapeutically effective combined amount of at least a first agent
that binds or complexes copper, and at least a second agent, where
the second agent is chosen from anti-inflammatory agents,
anti-fibrotic agents, and anti-angiogenesis agents. In some
embodiments, the first agent is a thiomolybdate; in some further
embodiments, the thiomolybdate is a tetrathiomolybdate. In other
embodiments, the second agent is chosen from a steroid, a NSAIDS
(non-steroidal anti-inflammatory drugs), a chemotherapeutic agent
as used in some auto-immune diseases, and an antibody or antisense
agent directed to specific cytokines or to cytokine receptors or to
other molecules which enhance inflammation and/or fibrosis. In yet
other embodiments, the kit further comprises appropriate
instructions and labels for use of the agents.
[0015] The agents for use in the present invention, such as copper
binding or complexing thiomolybdates, of which tetrathiomolybdate
is an example, lower endogenous copper levels; although it is not
necessary to understand the mechanism of these agents, and the
invention is not intended to be limited to any particular
mechanism, it is contemplated that in some embodiments, the agents
lower endogenous copper levels by forming a "tripartite
agent-copper-protein complex" that is subsequently cleared from the
body. The copper bound in these "tripartite agent-copper-protein
complex" is not reversibly released from these complexes, and are
thus distinguished from reversible bipartite copper chelation.
[0016] The present invention further provides method and
compositions for treating an inflammatory diseases in a patient,
comprising administering to the patient having an inflammatory
disease a biologically effective amount of at least a first agent
that binds or complexes copper, under conditions such that the
level of at least one inflammatory cytokine in the patient is
reduced. In some of these embodiments, the inflammatory cytokine
comprises TNF.alpha.; in other embodiments, the inflammatory
cytokine comprises I1-1.beta.. The present invention is not limited
however to providing methods and compositions that lower the levels
of the aforementioned cytokines.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the protective effect of tetrathiomolybdate
(TM) therapy against bleomycin induced weight loss, as shown in the
bottom panel. The data is shown as the mean weights for the mice in
each group at several time points during experiment 2, and at the
time of sacrifice at 21 days. The bleomycin group showed severe
weight loss, and TM protected against weight loss in a
dose-dependent manner, with the 0.9 mg dose being fully
protective.
[0018] FIG. 2 shows the relationship of excess hydroxyproline (a
measure of fibrosis) to TM maintenance dose in experiment 2. The
solid squares represent the mean values of 4 mice which received
bleomycin and were treated with varying doses of TM, ranging from 0
to 0.9 mg/mouse/day during the maintenance phase of therapy. The
open circles represent mice which did not receive bleomycin but
received the maintenance doses of TM indicated. Regression of the
hydroxyproline data shown in the solid squares in TM doses from 0.3
to 0.9 provided an F statistic of 14.8, with a p value of
<0.002.
[0019] FIG. 3 shows the hydroxyproline results from experiment 3,
according to the different starting times of TM therapy. The solid
diamonds show the mean hydroxyproline data at sacrifice (day 21) of
four mice in each group, except for +day 7, which had five mice,
all treated with bleomycin, and all treated with TM at varying
starting times as shown in the figure. The solid square represents
the mean value of four mice which received bleomycin only, and the
open circle the mean of four mice who received saline only. The
open triangles represent the means of three to four mice which
received no bleomycin but received TM at varying starting times as
controls for the bleomycin/TM mice. Regression of the four data
points shown by the solid diamonds yielded a significant F
statistic (p<0.05). The data from the bleomycin/TM groups at
days +4 and +7 were pooled and the mean compared to the bleomycin
mean by t test and found to be significantly different
(p=0.05).
[0020] FIG. 4 shows the protective effect of administration of TM
against concanavilin A induced cirrhosis of the liver.
Abbreviations: ConA, indicates concanavilin A treated group;
Saline, indicates saline treated control groups; ALT, represents
alanine amino transferase, SF, represents Sigma-Frankel units
(described further in the Examples).
[0021] FIG. 5 shows mean TNF.alpha. RNA levels in the lungs of the
four groups of mice of Experiment 1. Results are shown as the
threshold cycle (CT) at which an increase of reporter fluorescence
(ARn) can first be detected. Amounts of TNF.alpha. mRNA were
normalized to GAPDH signals and expressed as
2.sup.-.DELTA..DELTA.CT. Statistical evaluation using Scheffe's
correction for multiple comparisons reveal that bleomycin versus
bleomycin/TM (p<0.04), bleomycin versus saline (p<0.02) and
bleomycin versus TM (p<0.02) are all significantly different.
Bleomycin/TM versus saline is not significantly different.
[0022] FIG. 6 shows mean TGF.sub..beta. protein levels in the lungs
of four groups of mice from Experiment 1. Lung TGF.beta. protein
levels were assayed by ELISA, and the results were expressed as
ng/lung.
[0023] FIG. 7 shows mean TGF.sub..beta. protein levels in the lungs
of the four groups of mice of Experiment 2. Lung TGF.beta. protein
levels were measured using the cell line transfected with the PAI-1
promoter-luciferase construct. Results were expressed as relative
light units ("RLU")/lung.
[0024] FIG. 8 shows mean SMA levels in the lungs of the four groups
of mice of Experiment 2. Lung .alpha.-smooth muscle actin protein
levels were measured by ELISA, and the results were expressed as
the absorbance at 405 nm.
[0025] FIG. 9 shows mean hydroxyproline levels in the lungs of the
four groups of mice of Experiment 2. Results are expressed as
.mu.g/lung. Statistical evaluation using Scheffe's correction for
multiple comparisons reveal that bleomycin versus bleomycin/TM
(p<0.04), bleomycin versus saline (p<0.02) and bleomycin
versus TM (p<0.02) are all significantly different. Bleomycin/TM
versus saline is not significantly different.
[0026] FIG. 10 shows mean TGF.sub..beta. protein levels in the
lungs of the five groups of mice of Experiment 3. Lung homogenates
were analyzed for TGF.beta. protein using an ELISA kit, and the
results were expressed as ng/lung. The bleomycin mean was
significantly different from the mean of the bleomycin/TM-5 group
by t test (p=0.04). Regression of the TGF.sub..beta. protein levels
on starting time of TM in the four TM treated groups revealed a
significant F value (p=0.05).
[0027] FIG. 11 shows the results of an experiment wherein Con A
given for four weeks prior to initiation of TM in an animal
model.
[0028] FIG. 12 shows serum ALT data at week 12 in the CT mouse
model of liver damage.
[0029] FIG. 13 shows the hydroxyproline content of 0.05 g of liver.
Column 2 shows the hydroxyproline level in control liver, column 1
shows the effect of CT, and column 3 shows the protection of TM
against fibrosis from CT.
DEFINITIONS
[0030] To facilitate an understanding of the present invention, a
number of abbreviations, terms and phrases as used herein are
defined below.
[0031] The term "inflammatory" is used to refer to pertaining,
characterized by, causing, resulting from, or becoming affected by
inflammation. An inflammation is a fundamental pathologic process
consisting of a dynamic complex of cytologic and chemical reactions
that occur in the affected blood vessels and adjacent tissues in
response to an injury or abnormal stimulation caused by a physical,
chemical, or biologic agent; these reactions include the local
reactions and resulting morphologic changes, the destruction or
removal of the injurious material, and the responses that lead to
repair and healing.
[0032] The term "fibrotic" is used to refer to pertaining to or
characterized by fibrosis. Fibrosis in disease or response to
injury is the dysregulated excessive formation of fibrous tissue as
a reactive process, as opposed to formation of fibrous tissue as a
normal constituent of an organ or tissue, or as a part of normal
repair of tissue.
[0033] The term "disease" refers to an interruption, cessation, or
disorder of body function, systems, or organs. The term "disease"
includes responses to injuries, especially if such responses are
excessive. The term "condition" is used to refer to a disease or a
response to injury. An "inflammatory disease" refers to a disease
caused by or resulting from or resulting in inflammation. A
"fibrotic disease" refers to a disease caused by or resulting from
or resulting in fibrosis. A disease may include a response to
injury, especially where the response is excessive, does not heal
normally, and/or produces symptoms that excessively interfere with
normal activities of an individual (where excessive is
characterized as the degree of interference, or the length of the
interference).
[0034] The term "injury" refers to damage or wound of trauma. A
response to injury may be inflammation and/or fibrosis.
[0035] The term "anti-inflammatory" is used to refer to an effect
or compound which has an effect of preventing, inhibiting,
alleviating or decreasing inflammation or components of an
inflammatory reaction, either completely or partially.
[0036] The term "anti-fibrotic" is used to refer to an effect or
compound which has an effect of preventing, inhibiting, alleviating
or decreasing fibrosis or components of the fibrotic reaction,
either completely or partially.
[0037] The term "biocompatible" refers to compositions comprised of
natural or synthetic materials, in any suitable combination, that
remain substantially biologically unreactive in a subject or
patient. The term "substantially unreactive" means that any
response observed in a subject or patient is a subclinical
response, i.e., a response that does not rise to a level necessary
for therapy.
[0038] The term "biologically active agent" or "therapeutic agent"
refers to an agent that possesses an activity or property capable
of affecting or effecting a biochemical function, such as a
structural (for example, binding ability) or regulatory activity or
a reaction. Biochemical functions include but are not limited to
physiological, genetic, cellular, tissue, and organismal
activities. Moreover, as used herein, the term "agent" refers to
biologically active agents and therapeutic agents, except where
noted otherwise. Biological activities include activities
associated with biological reactions or events in a subject or
patient; preferably such activities can be detected, monitored,
characterized, or measured.
[0039] The term "endogenous copper level" refers to the total
amount of copper in the body of a patient; this amount includes
both tissue and fluid amounts. The amount of copper in the body can
also be divided into the amounts of available and amounts of
unavailable copper. The "copper status" of a patient refers to the
amount of available copper. Copper status is determined in the
blood of a healthy individual, for example, by the concurrent
measurement of plasma copper and ceruloplasmin. Normal plasma
copper is present in two primary pools. Most plasma copper in
normal individuals is part of the ceruloplasmin molecule. This
copper is essentially unavailable for ready exchange with cells.
Another pool of copper is more loosely bound to albumin and small
molecules, such as amino acids. This latter pool of copper is
readily available for cellular uptake. When TM enters the blood it
complexes with the available copper, and renders it, like
ceruloplasmin copper, unavailable for cellular uptake. In TM
treated patients, copper status can be determined by measuring
plasma ceruloplasmin alone. As the level of available copper
decreases, the level of ceruloplasmin also decreases, as the amount
of plasma ceruloplasmin is dependent upon copper availability.
[0040] The term "lowering endogenous copper level" refers to
decreasing the copper level in the body of an animal, typically by
administration of an agent which binds or complexes copper, from
the level existing just before administration of the agent;
copper-binding agents include but are not limited to
thiomolybdates, of which tetrathiomolybdate is an example.
Typically, more than one dose of a copper-binding agent is required
to lower the endogenous copper level.
[0041] The term "therapeutically effective amount" is a functional
term referring to an amount of material needed to make a
qualitative or quantitative change in a clinically measured
parameter for a particular subject. For example, prior to
administration, the subject may exhibit at least one measurable
symptom of disease or response to injury (for example, pulmonary
congestion and/or difficulty breathing; evidence of hepatitis, or
decrease in liver function; evidence or kidney inflammation or
decrease in kidney function; etc), which upon administration of a
therapeutically effective amount the measurable symptom is found to
have changed. A therapeutically relevant effect relieves to some
extent one or more symptoms of a disease or condition or returns to
normal either partially or completely one or more physiological or
biochemical parameters associated with or causative of the
disease.
[0042] In particular, the term refers to an amount of an agent that
binds or complexes copper such as thiomolybdate which amount is
effective to treat an inflammatory and/or fibrotic disease and/or
response to injury upon administration to a patient suffering from
such a disease or response to injury. Treatment includes but is not
limited to preventing the onset or shortening the course or
severity of or reversing the effects of inflammatory and/or
fibrotic disease or response to injury; thus, a therapeutically
effective amount includes a prophylactically effective amount. In
some embodiments, such effects are achieved while exhibiting
negligible or manageable adverse side effects on normal, healthy
tissues of the patient. Thus, the "therapeutically effective
amount" can vary from patient to patient, depending upon a number
of factors, including but not limited to the type of disease, the
extent of the disease, and the size of the patient.
[0043] The term "biologically effective amount" is a functional
term referring to an amount of material needed to make a
qualitative or quantitative change in a biological activity of a
particular subject; such activities include but are not limited to
enzyme activities, production of antigen, and clearance of analyte
from serum.
[0044] In particular, the term refers to an amount of an agent that
binds or complexes copper such as thiomolybdate which amount is
effective to decrease the level of endogenous copper levels upon
administration to a patient.
[0045] The term "therapeutically effective time" refers to the
period of time during which a therapeutically effective amount of a
therapeutic agent or biologically active agent is administered
sufficient to prevent the onset or to shorten the course or
severity of or to reverse the effects of a disease. In particular,
it is the period of time sufficient to both reduce the endogenous
copper level to a target level and/or to maintain the target copper
level to prevent the onset or to shorten the course or severity of
or to reverse the effects of inflammatory and/or fibrotic
disease.
[0046] The term "thiomolybdate" refers to molecules comprising
molybdenum and sulfur, and include but are not limited to species
such as [MoS.sub.4].sup.2- and [MoO.sub.2S2].sup.2-. These
molecules can act as bidentate ligands, and can complex copper.
Examples of thiomolybdates include but are not limited to
tetrathiomolybdate, trithiomolybdate, dithiomolybdate, and
monothiomolybdate. Other examples include complex thiomolybdates,
which include but are not limited to a zinc or an iron between two
thiomolybdate groups, and which contain thiomolybdate capable of
binding or complexing copper. In exemplary complex thiomolybdates,
the molecule may have more than four thio groups related to more
than one molybdenum.
[0047] The term "tetrathiomolybdate" (TM) refers to a compound made
up of molybdenum atom surrounded by four sulfur groups,
[MoS.sub.4].sup.2-.
[0048] The terms "bind," "complex," and "chelate" and any
grammatical equivalents (such as "binds," "binding," etc.) refer to
any type of chemical or molecular interactions of copper with
thiomolybdate which effectively sequester the copper with the
thiomolybdate; when the copper is endogenous to a patient and
thiomolybdate is administered to the patient, the terms refer to
any type of chemical interactions of copper with thiomolybdate
which effectively sequester the copper with the thiomolybdate
rendering the copper unavailable and ultimately removing it from
the patient's body.
[0049] A therapeutically effective amount of a range of values
includes all values in this range. Thus, for example, a
therapeutically effective amount of "between about 20 mg and about
200 mg includes all values in this range, and thus includes amounts
of about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg,
about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg,
about 1100 mg, about 120 mg, about 125 mg, about 130 mg, about 140
mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about
190 mg, or about 195 mg.
[0050] The terms "pharmaceutically acceptable," "physiologically
tolerable," and grammatical variations thereof, as they refer to
compositions, carriers, diluents and reagents, are used
interchangeably and represent that the materials are capable of
administration to or upon a subject or patient, preferably a
mammal, most preferably a human, and that the materials do not
substantially produce, for example, adverse or allergic reactions
when administered to a subject or patient, or can be administered
without the production of undesirable physiological effects such as
nausea, dizziness, gastric upset, toxicity and the like. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0051] The term "aqueous component" refers to the component of a
composition that contains water (or is soluble in water). Where
water is used, it may or may not contain salt(s) and may or may not
be buffered. Thus, a variety of such components are contemplated
including, but not limited to, distilled water, deionized water,
normal saline, and phosphate buffered saline.
[0052] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which a compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable, or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous solution saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions.
[0053] The term "patient" refers to any animal (for example, warm
blooded mammal) comprising humans and non-human animals, where
non-human animals include but are not limited to non-human
primates, rodents, farm animals (for example, cattle, horses, pigs,
goats, and sheep), pets (for example, dogs, cats, ferrets, and
rodents) and the like, that is to be the recipient of a particular
treatment. The terms "patient" and "subject" are used
interchangeably. The term "individual" refers to any animal as
described above who may or may not be a patient. A patient "having"
a disease or condition is a patient "suffering" the disease or
condition, and is "in need" of treatment of the disease or
condition.
[0054] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function. Test compounds comprise both known and potential
therapeutic compounds. A test compound can be determined to be
therapeutic by using the screening methods of the present
invention. A "known therapeutic compound" refers to a therapeutic
compound that has been shown (for example, through animal trials or
prior experience with administration to humans) to be effective in
such treatment or prevention.
[0055] The terms "purify" or "to purify" refer to the removal of
contaminants from a sample. The term "purified" refers to
molecules, such as nucleic or amino acid sequences, that are
removed from their natural environment, isolated or separated. An
"isolated nucleic acid sequence" is therefore a purified nucleic
acid sequence. "Substantially purified" molecules are at least 60%
free, preferably at least 75% free, and more preferably at least
90% free from other components with which they are naturally
associated. As used herein, the term "purified" or "to purify" also
refers to the removal of contaminants from a sample. The removal of
contaminating proteins results in an increase in the percent of
polypeptide of interest in the sample. In another example,
recombinant polypeptides are expressed in plant, bacterial, yeast,
or mammalian host cells and the polypeptides are purified by the
removal of host cell proteins; the percent of recombinant
polypeptides is thereby increased in the sample.
[0056] The term "medical devices" includes any material or device
that is used on, in, or through a patient's body in the course of
medical treatment (for example, for a disease or injury). Medical
devices include, but are not limited to, such items as syringes,
catheters, intravenous administration assemblies including pumps
and monitors, blood sampling equipment, nebulizers, small particle
aerosol generators, inhalers with a propellant, and the like.
[0057] As used herein, the term "sample" is used in its broadest
sense. In one sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from an animal,
including a human; a particular biological sample may be a fluid
(for example, blood, plasma and serum), a solid (for example,
stool), or a tissue; other biological samples may be obtained from
other biological sources, such as food, and may be a liquid food
(for example, milk), or a solid food (for example, vegetables).
Environmental samples include environmental material such as
surface matter, soil, water, crystals, and industrial samples.
These examples are not to be construed as limiting the sample types
applicable to the present invention.
DESCRIPTION OF THE INVENTION
[0058] The present invention provides methods to prevent and/or
treat excessive inflammation that is part of the illness in many
diseases, and/or part of the responses to injuries, by
administering therapeutically effect amounts of a copper binding or
complexing agent such as thiomolybdate, of which tetrathiomolybdate
(TM) is an example, to a patient in need thereof.
[0059] The present invention also provides methods to prevent
and/or treat excessive fibrosis that is part of the illness, and/or
part of the response to injury, in many diseases by administering
therapeutically effect amounts of a copper binding or complexing
agent such thiomolybdate, of which tetrathiomolybdate (TM) is an
example, to a patient in need thereof.
[0060] Over the last several years, a common biochemical pathway
has been elucidated for diseases which begin with inflammation, and
which, if the patient survives, often lead to a disabling or
potentially even lethal fibrosis. A partial list of such diseases
or conditions includes but is not limited to liver cirrhosis, renal
interstitial fibrosis (often a final common pathway for many types
of renal damage), systemic sclerosis (frequently complicated by
pulmonary fibrosis), keloid, hypertrophic burn scarring, and
excessive fibrosis in various parts of the intestinal tract in some
patients after disease or injury. The fibrosis may also be fatal,
such as it is in ARDS and hepatitis C. A key player in all of these
diseases is transforming growth factor beta (TGF.sub..beta.). The
presence, activation, or production of TGF.sub..beta. activates
connective tissue growth factor (CTGF), which then stimulates
collagen production as well as other molecules of fibrosis. The
over-activity of this pathway is a common feature of fibrotic
diseases in all organs of the body. The activation of this pathway
thus activates a host of inflammatory and fibrosis-inducing
cytokines. TGF.sub..beta. may also activate cytokines other than
through CTGF. While all of these factors and cytokines are normal
substances, and have important physiological functions, it has been
discovered that their over-production and dysregulation play a
central role in producing the aftermath diseases discussed
above.
[0061] Treatment methods of the present invention lower endogenous
copper levels and abrogate and treat diseases which begin with
inflammation and which, upon patient survival, often lead to
disabling or even lethal fibrosis. In some embodiments, such
therapy involves administering a copper binding or complexing
thiomolybdate, of which tetrathiomolybdate is an example. In the
following description, it is understood that tetrathiomolybdate is
simply an embodiment of the use of a copper binding or complexing
thiomolybdates. While an understanding of the mechanism is not
necessary to practice the present invention and the present
invention is not limited to any particular mechanism, it is
contemplated that reduction of endogenous copper levels alters the
regulation of CTFG, TGF.sub..beta., SPARC and/or heparin. The
contemplated altered regulation of these molecules is based upon
observations that CTGF is a cysteine-rich protein and that
cysteine-rich proteins are often involved in copper binding and
copper dependence, that TGF.sub..beta. can be stimulated or
activated by secreted protein acidic and rich in cysteine (SPARC),
a known copper dependent cytokine, and that TGF.sub..beta. itself
is dependent upon interaction with heparin molecules, many of which
are known to bind copper and require copper for activity.
[0062] Tetrathiomolybdate (TM) is a unique anticopper drug which
forms a stable tripartite complex with protein and copper. In the
gut, if given with food, it prevents the absorption of copper from
food and from endogenous secretions (saliva, gastric juice, etc).
If given away from food, it is partially absorbed and inactivates
copper in the blood by forming a three way complex with serum
albumin. Since the free copper of the blood is in equilibrium with
free copper in organs, it is possible to quickly titrate the body's
free copper. TM is the most potent and rapidly acting anticopper
drug available.
[0063] TM was developed for treating an inherited disease of copper
toxicity, Wilson's disease, as described subsequently. The
development of the treatment of Wilson's disease with TM has
demonstrated that TM is safe and effective, and that it is
particularly useful in the acutely ill, copper-toxic patient. The
preparation of an NDA for the treatment of Wilson's disease with TM
is underway.
[0064] The ability of copper reducing agents to be effective at
abrogating and treating diseases and/or responses to injuries which
begin with inflammation and which may lead to disabling fibrosis
(or inflammatory and/or fibrotic diseases), was initially evaluated
during the development of the present invention by studying the
effects of TM treatment on pulmonary disease and liver disease in
good animal models which exist for both diseases, as described
further below.
[0065] The profibrotic pathway involving TGF.sub..beta. and CTGF is
central to pathological fibrosis in many organs besides the lungs
(W A Border and N A Noble, N. Engl. J. Med., 331(19):1286-1292
[1994]; and D R Brigstock, Endocr. Rev., 20(2):189-206 [1999]), as
is described further below. The effectiveness of TM in treating
pulmonary fibrosis, as described below, shows that TM therapy finds
use in treating these other and similar diseases of excessive
fibrosis and/or inflammation. Other diseases amenable to TM therapy
include but are not limited to renal fibrosis and Alzheimer's
disease, as described further below. It is therefore contemplated
that in other embodiments of the present invention, the
administration of therapeutically effective amounts of TM to
patients susceptible to pathological fibrosis in other organs
prevents and/or treats these diseases.
[0066] Other approaches to treatment of inflammatory and fibrotic
diseases include other copper-lowering drugs, antibodies or
antisense molecules to key cytokines, such as to TGF.sub..beta. or
to CTGF, and other drugs which shut down the system. However,
certain of these approaches may work better than others. Other
copper lowering drugs include penicillamine, trientine, and zinc.
Both penicillamine and trientine are relatively toxic, and
trientine is also relatively slow acting; moreover, zinc is also
slow acting. Other potential drugs include antibodies or antisense
molecules to key cytokines, such as to TGF.sub..beta. or to CTGF.
For example, it is known that antibody to TGF.sub..beta. is
effective in treating the bleomycin mouse model. However,
antibodies and antisense molecules are difficult to deliver and
sustain at therapeutically effective levels in clinical situations.
Other potential drugs shut down the TGF.sub..beta. system. For
example, a drug called perfenidone is effective in the bleomycin
mouse model, and may affect the transcription of TGF.sub..beta..
However, at present, perfenidone does not appear to be in clinical
use, and little is known about it.
[0067] In contrast to these other approaches, the therapeutic use
of TM has been demonstrated to be safe and effective, as described
further below, and studies conducted during the development of the
present invention have demonstrated that therapeutically lowering
endogenous copper with TM will beneficially affect a series of
diseases dependent upon the TGF.sub..beta. pathway. Therefore, the
present invention provides methods of treating inflammatory and/or
fibrotic diseases and/or responses to injuries by therapeutically
lowering endogenous copper levels in a patient in need thereof.
Preferably, such conditions are dependent upon the TGF.sub..beta.
pathway. In some embodiments, the therapy comprises administering
therapeutically effective amounts of a copper binding or complexing
agent for a therapeutically effective time. Exemplary agents
include but are not limited to thiomolybdates, of which tetra-,
tri-, di-, and monothiomolybdates are non-limiting examples.
I. Pulmonary Diseases
[0068] For examining pulmonary disease, the bleomycin mouse model
was used. Pulmonary fibrosis, idiopathic or otherwise, is commonly
progressive and essentially untreatable with a fatal outcome (R K
Coker and G J Laurent, Thorax., 52(3):294-296 [1997]; and K Zhang
and S H Phan, Biol. Signals., 5:232-239 [1996]). It is clear from a
rather wide body of work that the underlying mechanism involves
dysregulation and overproduction of certain cytokines (R K Coker
and G J Laurent, Thorax., 52(3):294-296 [1997]; K Zhang and S H
Phan, Biol. Signals., 5:232-239 [1996]; S H Phan, Thorax.,
50(4):415-421 [1995]; R E Smith et al., J. Leukoc. Biol.,
57(5):782-787 [1995]; and C M Hogaboam et al., Proc. Assoc. Am.
Physicians, 110(4):313-320 [1998]). A central mechanism is
hypothesized to involve continued overproduction of transforming
growth factor beta (TGF.sub..beta.), which in turn increases the
production and/or activity of connective tissue growth factor
(CTGF) (N Khalil and A H Greenberg, Ciba Found. Symp. 157:194-211
[1991]; W A Border and N A Noble, N. Engl. J. Med.,
331(19):1286-1292 [1994]; M Denis, Immunology, 82(4):584-590
[1994]; J A Lasky et al., The American Physiological Society,
L365-L371 [1998]; D R Brigstock, Endocr. Rev., 20(2):189-206
[1999]; J T Allen et al., Cell Mol. Biol., 21:693-700 [1999]; and J
F Pittet et al., J. Clin. Invest., 107:537-1544 [2001]).
[0069] Bleomycin when given to cancer patients produces pulmonary
fibrosis in about 3% of the patients (M Ishizuka et al., J.
Antibiot. (Tokyo), 20:15 [1967]). Based upon these observations, a
mouse model of pulmonary fibrosis has been developed, in which
intratracheal instillation of bleomycin uniformly produces
pulmonary fibrosis (R E Smith, J. Leukoc. Biol., 57(5):782-787
[1995]; N Khalil and A H Greenberg, Ciba Found. Symp., 157:194-211
[1991]; J A Lasky, The American Physiological Society, L365-L371
[1998]; D R Brigstock, Endocr. Rev., 20(2):189-206 [1999]; J T
Allen et al., Am. J. Respir. Cell Mol. Biol., 21:693-700 [1999]; S
H Phan and S L Kunkel, Exp. Lung. Res., 18:29-43 [1992]; and J.
Clin. Invest. 107:537-1544). Thus, the mice develop a severe lung
inflammation followed by fibrosis in 2-3 weeks, at which time they
are sacrificed. Fibrosis is quantified in lung tissue by measuring
hydroxyproline, a key component of the collagen that is deposited
in fibrotic lung. The mouse bleomycin model is believed to be a
good model for human pulmonary fibrosis. The hypothesis that
TGF.sub..beta. is central to pulmonary fibrosis has been validated
by studies showing that inhibition of TGF.sub..beta. by
pharmacological means or by antibodies greatly reduces the
pulmonary fibrosis produced by bleomycin or other methods of lung
injury in the mouse (M Denis, Immunology, 82(4):584-590 [1994]; and
J F Pittet et al., J. Clin. Invest., 107:537-1544 [2001]).
[0070] Using the bleomycin mouse model to examine the effects of TM
treatment in developing the present invention, it was observed that
copper-lowering therapy with TM can dramatically prevent most of
the lung damage and fibrosis from tracheal bleomycin instillation
in mice (as described in Example 1 and as shown in Table 1).
Moreover, there is a strong dose-response relationship between the
amount of TM administered and the degree of pulmonary protection
(as shown in Table 3 and FIG. 2). TM therapy also protects against
bleomycin induced weight loss (as shown in FIG. 1). TM treatment
can be initiated, for example, up to at least seven days after the
bleomycin instillation, and still offer significant protection
against pulmonary damage (as shown in FIG. 3). These results
indicated that TM treatment completely abrogated fibrosis and
markedly attenuated inflammation in an animal model that is
directly relevant to ARDS and pulmonary fibrosis in humans.
[0071] Although it is not necessary to understand the mechanism in
order to practice the invention, and it is not intended that the
invention be limited to any particular mechanism, it is
hypothesized that the mechanism of the protection effected by TM
therapy is due to the inhibition of one or more steps in the
profibrotic pathway which involves activation of TGF.sub..beta.,
which in turn activates CTGF, which then activates the formation of
collagen and other profibrotic molecules, as described above.
[0072] It is also possible that the mechanism of this effect
involves primarily suppression of inflammation, as for example by
inhibiting proinflammatory cytokines. If the inflammatory reaction
to bleomycin is mitigated by TM therapy, the signaling to the
fibrotic pathway might be lessened, resulting in less fibrosis.
However, the observation that TM therapy initiated significantly
after bleomycin instillation (as, for example, at day 7) still has
a significant effect in inhibiting fibrosis (as described in
Example 1 and shown in FIG. 3) suggests that suppression of
inflammation is not the sole effect of TM. That is because TM
therapy initiated on day 7 would not reduce copper levels at the
therapeutic area until about day 11, by which time all or most of
the inflammation and inflammatory stimuli would have subsided. The
positive results observed when the drug treatment is initiated
significantly after bleomycin instillation thus suggests that TM
can act by direct inhibition of the fibrotic pathway. Of course, TM
therapy might result in inhibition of both inflammation and
fibrosis.
[0073] Irrespective of the pathway involved, or of the underlying
molecular mechanism, the fact that TM therapy can so markedly
inhibit fibrosis in this model confirms the use of this approach to
preventing and treating pulmonary fibrosis in human patients. The
experimental results indicate that TM therapy is effective after
injury (as, for example, as is shown in FIG. 3), which supports its
efficacy in clinical use. The use of TM has previously been proven
to be remarkably safe, as demonstrated by its considerable
experimental use in humans for treatment of Wilson's disease (G J
Brewer et al., Arch. Neur., 53:1017-1025 [1996]; and G J Brewer,
PSEBM, 223(1):39-49 [2000]) and for treatment of cancer (G J Brewer
et al., Clin. Cancer., 6:1-10 [2000]; and G J Brewer, Soc. for Exp.
Biol. and Med., 226:665-673 [2001]). The only side effect of
lowering copper levels by TM therapy in cancer has been
over-treatment, which leads to an easily reversible bone marrow
depression. The use of serum ceruloplasmin as a surrogate to
monitor copper status has proven to be effective, reliable, and
easy to use (G J Brewer et al., Clin. Cancer, 6:1-10 [2000]).
[0074] Based upon these results, clinical use of TM therapy for
ARDS and/or pulmonary fibrosis in human patients is contemplated.
It is therefore contemplated that in other embodiments of the
present invention, the administration of therapeutically effective
amounts of TM to patients susceptible to pulmonary fibrosis
prevents and/or treats this disease.
II. Liver Disease
[0075] For examining liver disease, studies of the mouse model of
liver damage (hepatitis) followed by cirrhosis were undertaken. Two
of four appropriate mouse models were involved. In one model,
concanavilin A (ConA) treatment was utilized to produce cirrhosis.
The Con A was injected intravenously once weekly into mice, and
produced a hepatitis, which is manifested by an increasing level of
transaminase enzymes in the blood. TM therapy almost completely
inhibits this increase, indicating suppression of inflammation (as
is described in Example 7 and as is shown in FIG. 4). The results
indicated that TM treatment completely abrogated fibrosis and
markedly attenuated inflammation in a model that is directly
relevant to hepatitis in humans. It is therefore contemplated that
in other embodiments of the present invention, the administration
to patients in need thereof of therapeutically effective amounts of
TM after liver damage due to hepatitis prevents or decreases
subsequent cirrhosis.
III. Kidney Disease
[0076] After kidney injury of almost any type, a diffuse
interstitial fibrosis (believed to be due to over-activity of the
TGF.sub..beta. pathway) produces kidney failure. It is therefore
contemplated that in other embodiments of the present invention,
the administration to patients in need thereof of therapeutically
effective amounts of TM after kidney injury prevents or decreases
fibrosis, thereby preventing or abrogating kidney failure
subsequent to kidney damage.
IV. Alzheimer's Disease
[0077] TGF.sub..beta. has been implicated in Alzheimer's plaque
formation. Moreover, copper has been implicated in the
precipitation of the amyloid into the plaques in the course of the
disease. It is therefore contemplated that in further embodiments
of the present invention, the administration to patients in need
thereof of TM results in lowering copper levels, thus preventing
any further precipitation; this results in arresting the
Alzheimer's disease, and in some cases allows some recovery from
the disease.
[0078] According to R S Turner (Neurologic aspects of Alzheimer's
disease, In: Interdisciplinary handbook of dementia: psychological,
neurologic, and psychiatric perspectives. John Wiley & Sons.
Lichtenberg Pa., Murman DL. and Mellow AM), Alzheimer's disease
(AD) currently affects about 2-3% of individuals at age 65, and the
incidence approximately doubles for every 5 years of age afterward.
The prevalence of AD approaches 50% of those over age 85 (as
reported by D A Evans et al., JAMA, 262:2551-2556 [1989]). AD is
not inevitable with aging, however, and "escapees" warrant further
epidemiologic and genetic study. In 1990, there were an estimated 4
million people in the U.S. with AD. Because of an expanding
population and increasing life expectancy, the number of affected
individuals is projected to increase to 14 million in the U.S. in
2050. Women make up a larger proportion of patients who live and
die with AD due to a higher relative risk and longer life
expectancy than men. In 2001, the annual costs for care of a
patient with AD were approximately $28,000 for formal care and
$11,000-$35,000 for informal care (D P Rice et al., Am. J. Manag.
Care, 7:809-818 [2001]). The high prevalence of AD results in an
enormous economic impact. As the elderly population also increases
in less affluent countries, large numbers of patients with AD will
emerge and face intense competition from the younger populace for
scarce health care resources. The slow progression of disease (with
an average of 7 years, and a range of 2-18 years) engenders many
years of health care costs. As dementia becomes severe and patients
become progressively more dependent on caregivers for basic
activities of daily living, expenditures increase. A major cost for
many patients in the latter stages of AD is assisted living and
nursing home care.
[0079] Genetically, AD is a multifactorial disease, with the
possible involvement of several genetic components (E K Luedecking
et al., Hum. Genet., 106:565-569 [2000]). Three causative genes at
chromosomes 21, 14, and 1 have been identified in the early-onset
form of AD. These three genes, amyloid precursor protein (APP),
presenilin-1 (PS1), and presenilin-2 (PS2), account for most of the
cases of autosomal dominant familial AD (C L Lendon et al., JAMA,
277:825-831 [1997]). Familial AD, however, accounts for <1% of
all AD cases. Additionally, the apolipoprotein E4 allele is a risk
factor for late-onset AD (W J Strittmatter et al., Proc. Natl.
Acad. Sci. USA, 90:1977-1981 [1993]). However, mutations in these
genes do not explain the occurrence of disease in all patients (E K
Luedecking et al., Hum. Genet. 106:565-569 [2000]).
[0080] Biochemically, AD is characterized by the deposition of beta
amyloid protein (A.beta.) within the neocortex, associated with
neuronal demise and oxidative stress (A I Bush, Bio-inorganic
Chemistry, 184-191 [2000]). The deposition of A.beta. is considered
to be closely related to the primary pathogenesis of AD. For
example, familial AD-linked mutations of APP, PS1, and PS2,
increase both cerebral A.beta. burden and A.beta.1-42 production,
underscoring the role that A.beta. metabolism plays in AD
pathogenesis (C S Atwood et al., Met. Ions Biol. Syst., 36:309-364
[1999]). Moreover, the deposition of A.beta. in the neocortex of
transgenic mice overexpressing A.beta. is accompanied by many of
the other neuropathological features of AD, including intraneuronal
tau abnormalities and neuronal loss (M E Calhoun et al., Nature,
395:755-756 [1998]), as well as signs of oxidative damage similar
to those seen in AD-affected brain (M A Smith et al., J.
Neurochem., 70:2212-2215 [1998]). The length of the A.beta. species
is considered to be one important factor in AD pathogenesis as
A.beta.1-42, a minor free soluble species in biological fluids, is
enriched in amyloid deposits. Many studies have now confirmed that
A.beta. is neurotoxic in cell culture. Hence, there is a compelling
argument to consider A.beta. deposition as a therapeutic target in
AD (A I Bush, Metals and neuroscience. Bio-inorganic chemistry,
184-191 [2000]).
[0081] For examining the effects of TM on Alzheimer's disease, the
transgenic mouse model Tg2576 is used. Transgenic (tg) mouse models
have proven to be useful tools in testing hypotheses of AD
pathogenesis as well as testing novel therapeutic strategies
(Turner RS. Commentary). Tg human amyloid precursor protein (hAPP)
mice recapitulate some but not all features of human AD, and may
therefore be best described as developing a partial AD-like
phenotype with aging. However, the distribution of amyloid
pathology in tg hAPP mouse brain is remarkably similar to the human
disease. One of the more widely studied hAPP tg mouse lines--Tg2576
mice developed by Hsiao et al. (K Hsiao, Exp. Gerontol., 33:883-889
[1998]; and K Hsiao et al., Science, 274:99-102 [1996])--expresses
the familial AD gene hAPP swe (Swedish mutation;
APP.sub.K670N/M671L in the APP770 numbering system) in a C57B6/SJL
genetic background. The neuron-specific prion protein promoter
drives expression of the transgene. With aging, Tg2576 mice exhibit
a phenotype that includes learning and memory deficits, an abnormal
pattern of glucose metabolism in brain, and pathologic changes
including amyloid plaque deposition, elevated A.beta.40 and
A.beta.42 levels, neuritic changes, phosphorylated tau epitopes,
.alpha.-synuclein positive dystrophic neurites, gliosis, and
inflammatory responses; however, aging mice develop neither
neurofibrillary tangles nor significant neuronal loss (R S Turner,
Commentary; K Hsiao, Exp. Gerontol., 33:883-889 [1998]; and K Hsiao
et al., Science, 274:99-102 [1996]). Cholinergic abnormalities in
the immediate vicinity of amyloid plaques are apparent in
immunostained brain sections from older HAPP tg (C
Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA,
94:13287-13292 [1997]) and hPresenilin-1 (mutant)/hAPP double tg
mice (T P Wong et al., J. Neurosci., 19:2706-2716 [1999]).
[0082] Amyloid plaque deposition in aging hAPP tg mice may be
modulated pharmacologically, immunologically, environmentally, and
genetically. For example, amyloid pathology is accelerated in
hPresenilin-1 (mutant)/hAPP double tg mice (L Holcomb et al., Nat.
Med., 4:97-100 [1998]), and absent in murine ApoE null (-/-)/hAPP
tg mice (K R Bales et al., Nature Genet., 17:263-264 [1997]). In
the latter mice, hApoE4 transgene expression promotes more
fibrillar amyloid deposition than hApoE3 (D M Holtzman et al.,
Proc. Natl. Acad. Sci. USA, 97:2892-2897 [2000]). Human
transforming growth factor .beta.1/hAPP double tg mice develop
increased A.beta. deposition within plaques, with a greater
proportion of meningeal and vascular deposition, reflecting a role
of inflammation in amyloidogenesis (T Wyss-Coray et al., Nature,
389:603-606 [1997]). Amyloid pathology in hAPP tg mice may be
prevented by pharmacologic treatment with the phosphatidylinositol
kinase inhibitor wortmannin that inhibits A.beta. production in
vitro (S J Haugabook et al., FASEB Journal, published online 9 Nov.
2000), by the Cu.sup.++/Zn.sup.++-chelator/antibiotic clioquinol
that blocks amyloid fibril formation in vitro (L Helmuth, Science,
1273-274 [2000] editorial), or by the nonsteroidal
anti-inflammatory drug ibuprofen (G P Lim et al., J. Neurosci.,
20:5709-5714 [2000]). The efficacy of this wide variety of
pharmacologic treatments in preventing amyloid deposition in tg AD
mice reveals multiple alternative and competing therapeutic
strategies. Novel therapeutics targeting the recently-identified
.gamma.-secretase complex and .beta.-secretases that generate
A.beta.40 and A.beta.42 from APP and immune-based strategies are
also under experimental investigation (D J Selkoe, Nature, 399
suppl:A23-31 [1999]). It is contemplated that administration of a
copper binding or complexing agent such as thiomolybdate to Tg2576
mice results in a reduction of A.beta.40 and A.beta.42 levels in
brain homogenates.
V. Cancer and Angiogenic Diseases
[0083] Research indicates that angiogenesis is required for cancer
growth, and since adults have little requirement for angiogenesis,
the present invention contemplates that antiangiogenic therapies
might provide successful cancer treatments.
[0084] Copper has been shown to be a stimulus of angiogenesis in a
rabbit cornea model in which copper sulfate, or a copper containing
molecule, ceruloplasmin, were both angiogenic (A Parke et al., Am.
J. Clin. Path., 137:1121-1142 [1988]; K S Raju et al., J. Natl.
Cancer Inst., 69:1183-1188 [1982]). When rabbits were made
partially copper deficient with penicillamine and a low copper
diet, an angiogenic molecule, PGE1, placed in the cornea, showed
markedly reduced angiogenesis. Brem and colleagues implanted brain
tumors in the brains of copper deficient rabbits and rats, and
showed markedly reduced growth and invasive properties of the
tumors in the copper deficient animals compared to controls (S S
Brem et al., Am. J. Path., 137:1121-1147 [1990]; S S Brem et al.,
Neurosurgery, 26:391-396 [1990]). Some embodiments of the present
invention provide TM compositions that are more potent and much
safer anticopper drugs then penicillamine and trientine and which
are contemplated for use in treating cancer. Indeed, in five
different rodent cancer models, TM has shown dramatic effects on
inhibition of tumor growth, including; the HER2/neu transgenic
mammary model (Q Pan et al., Cancer Res., 62:4854-4859 [2002]); a
head and neck model (C Cox et al., Laryngoscope, 111:696-701
[2001]); a prostate model (K van Golen et al., Neoplasia,
4(5):373-379 [2002]); a lung model (M Khan et al., Neoplasia, 4(2):
1-7 [2002]); and an inflammatory breast model (Q Pan Q et al.). TM
has also shown positive effects in spontaneous canine cancers. A
clinical phase 1/2 study of a variety of metastatic and advanced
cancers has shown positive results, with an average of 11 months
freedom from progression in evaluable patients, and long term
stabilization in three patients (G J Brewer et al., Clin. Cancer
Res., 6:1-10 [2000]). A number of phase 2 studies of specific
cancers are under way.
[0085] While not being limited to any mechanism, the present
invention contemplates that the antiangiogenic mechanism of TM
appears to involve the copper dependence of a large number of
angiogenic promoters (G J Brewer, EBM, 226:665-673 [2001]). In
addition, lowering copper levels to a midrange inhibits nuclear
factor kappa B (NFKb), a type of master switch for cytokine
transcription. These mechanisms may make copper lowering therapy a
more global inhibitor of angiogenesis than other approaches. In
some embodiments, copper is maintained in the midrange by using
ceruloplasmin (Cp) levels as a surrogate marker of copper
status.
[0086] The present invention also contemplates the antiangiogenic
therapeutic effects of TM in diseases of neovascularization besides
cancer (e.g., animal models of retinopathy). In retinopathy of
prematurity, newborn mice exposed to hyperoxia for five days
develop a marked retinopathy after four days exposure to room air,
with a peak of a major angiogenic stimulus, vascular endothelial
growth factor (VEGF), at 24 hours. TM treatment has shown strong
inhibition of the VEGF peak and a dramatic reduction in retinal
neovascularization.
[0087] The invention's success with the antiangiogenic use of TM
through its inhibition of angiogenic cytokines led to investigation
of key cytokines of fibrosis and inflammation, which become
dysregulated in a series of diseases of fibrosis and inflammation,
that may be similarly copper dependent, and treatable with TM.
VI. Primary Biliary Cirrhosis
[0088] The specific cause of primary biliary cirrhosis (PBC)
remains unknown, although it is though to be an autoimmune disorder
(R T Chung and D K Podolsky, Cirrhosis and its complications:
Primary biliary cirrhosis. In: Harrison's Principles of Internal
Medicine 15th edition, E Braunwald et al., (Eds). McGraw-Hill
Companies, Inc, New York, pp. 1757-1758 [2001]). A circulating IgG
antimitochondrial antibody (AMA) is found in more than 90% of
patients, and only rarely in other diseases. PBC is often
associated with other autoimmune disorders such as autoimmune
thyroiditis, type I diabetes mellitus, and other autoimmune
syndromes.
[0089] The disease is divided into four stages. Stage I is a
necrotizing inflammatory process of the portal triads, with
destruction of smaller bile ducts, a heavy infiltrate of
inflammatory cells, mild fibrosis, and part of the time,
cholestasis. In stage II, the inflammatory reaction decreases, the
number of bile ducts is reduced, and small bile ductules
proliferate. In stage III, which results from progression over
months to years, there is a decrease in interlobular ducts, loss of
liver cells, and increase in periportal fibrosis leading to a
fibrotic network. Stage IV is micronodular or macronodular
cirrhosis.
[0090] Clinically the disease may begin with symptoms of itching or
fatigue. Often it is picked up by an elevated serum alkaline
phosphatase on routine screening. Ninety percent of PBC patients
are women. Over a period of months to years the disease may
progress and produce jaundice. Eventually signs of hepatic failure
and of portal hypertension appear. Progression is somewhat
variable, with some patients dying or requiring transplant in 5
years, while others have a more protracted course.
[0091] A presumptive diagnosis may be made on the basis of an
elevated alkaline phosphatase usually in a woman, with or without
jaundice, and a positive AMA test. However, since false positives
do occur, diagnosis should always be confirmed by a liver biopsy
showing typical findings of PBC.
[0092] Treatment with the bile acid ursodeoxycholic acid (ursodiol)
is useful for symptomatic and sometimes biochemical improvement,
but has not been shown to alter the progressive course of the
disease. No other treatment, aside from liver transplantation, has
been shown to be effective.
[0093] Neuman et al. (M Neuman et al., J. Gastro. and Hepat.,
17:196-202 [2002]) report that an increase in serum levels of both
TNF.alpha. and TGF.beta. in PBC (e.g., TNF.alpha. is 324 pg/ml in
PBC versus 77 in normal controls). Second, they conclude that serum
TNF.alpha. and TGF.beta. levels reflect disease severity. Third,
they find that ursodiol therapy significantly decreases serum
TNF.alpha. and TGF.beta. levels in PBC, but not to normal levels
(for example, TNF.alpha. after 2 years of ursodiol therapy was 124
pg/ml, still significantly higher than control values). Since in
animal model work both in the lung and the liver TM is able to
essentially normalize TNF.alpha. and TGF.beta. levels in affected
organs after injury, it is contemplated that TM therapy is
beneficial in PBC.
[0094] The prevalence of PBC in the U.S. is 65.4 cases for women
and 12.1 cases for men (40.2 overall) per 100,000 population. At
402 cases/1 million, and using a U.S. population of 288 million,
this calculates out to about 116,000 case prevalence in the U.S.,
well below the 200,000 figure required to qualify as an orphan
disease.
VII. Agents that Bind or Complex Copper
[0095] A. Thiomolybdates
[0096] The present invention provides methods to prevent and/or
treat inflammation and/or fibrosis by administering a
therapeutically effective amount of at least one copper binding or
complexing agent that includes but is not limited to a
thiomolybdate, to a patient in need thereof. Thiomolybdates are
molecules comprising molybdenum and sulfur, and include but are not
limited to species such as [MoS.sub.4].sup.2- and
[MoO.sub.2S2].sup.2-. These molecules can act as bidentate ligands,
and can complex copper. Examples of thiomolybdates include but are
not limited to tetrathiomolybdate, trithiomolybdate,
dithiomolybdate, and monothiomolybdate. Other examples include
complex thiomolybdates, that include but are not limited to a zinc
or an iron between two thiomolybdate groups, and that contain
thiomolybdate capable of binding or complexing copper. In exemplary
complex thiomolybdates, the molecule may have more than four thio
groups related to more than one molybdenum. In the following
description, it is understood that tetrathiomolybdate is simply an
embodiment of the use of a copper binding or complexing
thiomolybdates. It is also understood that any thiomolybdate may be
utilized as one or several of different salts, such as those
described for TM below.
[0097] Tetrathiomolybdate (TM) is a compound made up of molybdenum
atom surrounded by four sulfur molecules. Various salts of TM are
available; salts of TM include but are not limited inorganic
cations such as ammonium, zinc, and iron ions, and organic cations
such as tetraethyl, tetrapropyl and choline ions. Different salts
have differing properties of solubility in water and ingestible
solvents (such as alcohol), stability upon storage alone or in
formulations, bioavailability to a patient, and toxicity to a
patient. Thus, depending upon the use and formulation, any
particular salt is selected to maximize solubility in water (or a
solvent miscible with water and which can be tolerated by a
patient, such as alcohol), to maximize stability upon storage, as
for example as the compound or as part of a formulation, to
minimize toxicity to a patient, and to maximize bioavailability
after administration to a patient.
[0098] In some embodiments, the salt of TM is an ammonium salt. TM
as the ammonium salt can be purchased from Aldrich Chemical Company
(catalog number W 180-0; Milwaukee, Wis.; available in one kilogram
bulk lots) as a black powder that is moderately water soluble,
yielding a bright red solution; these preparations are also
certified pure for human use. The ammonium salt of TM has one
undesirable property, that of mild air instability. Thus, the bulk
drug should be stored in the absence of oxygen, or the oxygen will
gradually exchange with the sulfur, rendering the drug ineffective
over time. The bulk drug is therefore stored under argon. Stability
assays developed by the inventors indicate that this drug is stable
for several years under argon (G J Brewer et al., Arch. Neurol.,
48(1):42-47 [1991]). Capsules can be filled by hand, and the drug
is stable in capsules for several months at room temperature.
[0099] Alternatively, TM, which is generally synthesized as the
ammonium salt, may be more stable under air as a different salt.
Thus, other salts have been prepared and evaluated for solubility,
stability and anticopper activity. In other embodiments, the salt
tetrapropyl tetrathiomolybdate (TPTM) has met all desired
properties. In other embodiments, the salt choline TM has suitable
desirable properties. In yet other embodiments, the salt tetraethyl
TM has suitable properties. These exemplary salts of TM have
suitable solubility in water, and behave similarly to ammonium salt
of TM in in vitro copper complexing studies.
[0100] Other pharmaceutically acceptable salts include but are not
limited to include salts formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, histidine,
procaine and the like.
[0101] Although it is not necessary to understand the underlying
mechanism to practice the invention, and the invention is not
intended to be so limited, it is believed that TM acts by forming a
tripartite complex with copper and protein (Mills et al., J. Inorg.
Biochem., 14:189 [1981]; Mills et al., J. Inorg. Biochem., 14:163
[1981]; and Bremner et al., J. Inorg. Biochem., 16:109 [1982]). It
is further believed that TM has two mechanisms of action. Given
with meals, it complexes both copper in food and endogenously
secreted copper with itself and food protein, and prevents the
absorption of copper. Patients can be put into an immediate
negative copper balance with TM by administering it with meals.
Given between meals, the TM is absorbed into the bloodstream, and
complexes serum copper with itself and albumin, rapidly rendering
the copper unavailable for cellular uptake. Since free copper in
organs is in equilibrium with free copper in blood, free copper in
the organs will quickly be reduced to very low levels if the blood
copper is bound. This complex is cleared through the kidney and the
liver. No matter what its mechanism of action is, TM is a potent
and rapidly acting anticopper agent. It is also contemplated that
other thiomolybdates complex copper through similar though not
necessarily identical mechanisms. In some hypotheses, some
thiomolybdates directly bind copper. In yet other hypotheses, a
tripartite copper-thiomolybdate-protein complex is degraded in the
body, but the thiomolybdate still results in lowering endogenous
copper levels. For example, trithiomolybdate, dithiomolybdate, and
monothiomolybdate compounds, like tetrathiomolybdate, are believed
to form a tripartite complex with copper and protein that renders
the copper unavailable, and eventually leads to clearance of the
copper-complex.
[0102] The only known toxicity of TM discovered in animal studies
is through its anticopper effects. Animals given TM in sufficient
quantity to produce severe copper deficiency suffer from a variety
of copper deficiency-related problems, including anemia (Mills et
al., J. Inorg. Biochem., 14:163 [1981]; and Bremner et al., J.
Inorg. Biochem., 16:109 [1982]). However, none of these occur if
the animal is copper supplemented (Mills et al., J. Inorg.
Biochem., 14:189 [1981]), or maintained at a moderate copper level.
Tetrathiomolybdate (TM) is a drug that the inventors have developed
as an orphan therapy for Wilson's disease, as described further
below. The drug does an excellent job of gaining quick control over
copper toxicity and preventing the neurological worsening that
occurs 50% of the time during initial treatment with a commonly
used drug for Wilson's disease, penicillamine (Brewer et al., Arch.
Neurol., 48(1):42-47 [1991]; Brewer et al., Arch. Neurol.,
51(6):545-554 [1994]; and Brewer et al., Arch. Neurol.,
53:1017-1025 [1996]). So far, the inventors have treated 79
Wilson's disease patients with TM, generally for an eight-week
period. TM thus fills a very important niche in the initial
treatment of Wilson's disease. The Wilson's disease work has
provided extensive experience with TM therapy in the human, and
provides documentation of TM's extremely low level of toxicity in
humans.
[0103] In the studies of treating human patients with Wilson's
disease studies, one side effect occasionally observed is a
reversible anemia, due to TM's anticopper effects. Given in too
high a dose, TM renders the bone marrow severely or totally copper
deficient. Since copper is required for erythropoiesis, an anemia
develops. That anemia is rapidly reversible by simply stopping TM.
In the Wilson's disease studies, this over-treatment effect of TM
has been diminished by simply reducing the dose to 60 mg per day
from the standard 120 mg per day. A second side effect seen during
treatment with TM of Wilson's disease, but not in treatment of
cancer, is a mild increase in serum transaminase levels (Wilson's
patients already have liver disease). This mild increase is
diminished or removed by reducing the dose of TM. In humans without
Wilson's disease, such as patients with inflammatory and/or
fibrotic diseases, a level of mild copper deficiency at a
pre-anemia state can be established simply by carefully monitoring
ceruloplasmin (Cp) levels during TM therapy. The level of
ceruloplasmin is reduced to and maintained at a targeted level; in
some embodiments, this targeted level is between about 5 and 15
mg/dl.
[0104] TM is eventually metabolized to elemental molybdenum (Mo),
so the potential toxicity of Mo has to be considered. However, it
turns out that Mo is quite innocuous at the levels produced from
breakdown of TM used at the therapeutic regimes described herein.
In one example, up to 50 mg of Mo/day is administered for two
weeks, then no more than about 25 mg/day is administered for
maintenance. High doses of 350 to 1400 mg/day of Mo were previously
used for 4-11 months in patients with Wilson's disease, without
toxicity (Bickel et al., Quart. J. Med., 50:527 [1957]). Thus,
because about 37% of TM by weight is Mo, the dose range of 25-50
mg/day poses no predictable problems, and should be entirely
safe.
[0105] B. Monitoring Copper Levels
[0106] The mechanism of action of TM is to lower systemic or
endogenous copper levels. Copper status is evaluated by measuring
the level of ceruloplasmin, a copper-containing serum protein
secreted by the liver, as the amount of ceruloplasmin is dependent
upon copper availability. Measuring total serum copper is not a
good indicator for evaluating copper status, because the TM complex
with copper accumulates in the blood before it is cleared from the
body, thus elevating serum copper in spite of reduced copper
availability. Thus, the serum ceruloplasmin, which is directly
dependent upon liver copper status, is an accurate indicator of
copper status or availability in preferred embodiments.
VIII. Combination Therapy
[0107] In the present invention, it is initially contemplated that
a method to treat inflammation and/or fibrosis comprises
administration of at least one copper binding or complexing agent,
which include but are not limited to thiomolybdates of which TM is
an example; in this method, treatment is accomplished by
administering a single copper binding or complexing agent. It is
also contemplated that a combination of more than one copper
binding or complexing agent may be administered to a patient; the
different agents are chosen from different thiomolybdates,
different salts of different thiomolybdates, other copper binding
or complexing agents, or any combination thereof. Thus, in some
embodiments, the agents comprise a combination of at least two
different thiomolybdates, such as a tetrathiomolybdate and a
trithiomolybdate; in other embodiments, the agents comprise a
combination of at least one thiomolybdate and at least one other
copper binding or complexing agents. In yet other embodiments, the
agents comprise a combination of at least two different salts of a
single thiomolybdate, such as a tetraethyl- and a
tetrapropyl-tetrathiomolybdate; in yet other embodiments, the
agents comprise a combination of at least two different
thiomolybdates, of which at least one thiomolybdate comprises at
least two different salts; in yet other embodiments, the agents
comprise a combination of at least one thiomolybdate, which
comprises a combination of at least two different salts, and at
least one other copper binding or complexing agent.
[0108] Moreover, it is also contemplated that the methods of the
present invention may be combined with other methods generally
employed in the treatment of the particular disease or disorder
that the patient exhibits. This is particularly true for treatment
of diseases for which decreasing copper levels ameliorates does not
eradicate the disease; in those cases, it may be advantageous to
use additional compounds which eradicate the disease. In other
cases, it may be useful to administer drugs in addition to TM in
order to obtain additive or synergistic effects. For example, in
connection with inflammation, the methods of the present invention
include classical or new approaches in treating and/or preventing
inflammation. Thus, in some embodiments, the present invention
provides a method of treating and/or preventing inflammation
comprising administering at least one copper binding or complexing
agent as described above to a patient in need thereof, and
administering at least one other known or discovered antiflammatory
drug; known antiflammatory drugs include but are not limited to
steroids, NSAIDS (non-steroidal anti-inflammatory drugs), and
chemotherapeutic agents as are used in some auto-immune diseases.
In other examples, in connection with fibrosis, the methods of the
present invention include classical or new approaches in treating
and/or preventing fibrosis. Thus, in some embodiments, the present
invention provides a method of treating and/or preventing fibrosis
comprising administering at least one copper binding or complexing
agent as described above to a patient in need thereof, and
administering at least one other known or discovered anti-fibrotic
drugs; anti-fibrotic drugs include but are not limited to
antibodies or antisense agents directed to specific cytokines or to
their receptors, as well as to other molecules which enhance
fibrosis. In these embodiments, it is contemplated that the
administration of other anti-inflammatory and/or anti-fibrotic
drugs are not known to be detrimental in themselves, and that
administration of other anti-inflammatory and/or anti-fibrotic drug
do not substantially counteract the effectiveness of the endogenous
copper lowering therapy by administering copper binding or
complexing agents. By substantially counteracting the effectiveness
of the endogenous copper lowering therapy, it is meant that the
combined therapy lowers endogenous copper sufficiently to observe
an amelioration of at least one symptom of a disease or condition.
In the embodiments in which at least one additional
anti-inflammatory and/or anti-fibrotic drug is administered in
combination with the administration of a copper binding or
complexing agent, there is no requirement for the combined results
to be additive of the effects observed when each treatment is
conducted separately, although this is evidently desirable, and
there is no particular requirement for the combined treatment to
exhibit synergistic effects, although this is certainly possible
and advantageous. It is also contemplated that the administration
of the different agents or drugs occurs simultaneously, as for
example administering the combination of agents and drugs at the
same times, and/or at different times during the course of therapy;
any combination of administration is contemplated.
IX. Pharmaceutical Compositions and Kits
[0109] Pharmaceutical compositions of the present invention will
generally comprise an effective amount of an agent for use in the
present invention, such as copper binding or complexing
thiomolybdates, of which tetrathiomolybdate is an example,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium.
[0110] A. Oral Formulations
[0111] In preferred embodiments of the present invention, TM is
administered orally. Oral administration is effected by a number of
means, such as by feeding tubes for administration into the
gastrointestinal track, and preferably the duodenum, or by tablets
or powders or solutions for administration by mouth. A feeding tube
may be preferred for an acute disease, whereas administration by
mouth may be preferred for chronic diseases and/or for maintenance,
once an appropriate level of copper has been attained. Oral
pharmaceutical formulations include but are not limited to tablets
or other solids, time release capsules, liposomal forms and the
like. Other pharmaceutical formulations may also be used, dependent
on the condition to be treated.
[0112] As described in detail herein, it is contemplated that
certain benefits will result from the manipulation of the agents
for use in the present invention, such as copper binding or
complexing thiomolybdates, to provide them with a longer in vivo
half-life. Slow release formulations are generally designed to give
a constant drug level over an extended period. Increasing the
half-life of a drug, such as agents for use in the present
invention, such as copper binding or complexing thiomolybdates, is
intended to result in high plasma levels of TM upon administration,
which levels are maintained for a longer time, but which levels
generally decay depending on the pharmacokinetics of the construct.
Slow release formulations of the instant compositions and
combinations thereof are contemplated for some uses in the present
invention.
[0113] Appropriate solutions of the agents for use in the present
invention, such as copper binding or complexing thiomolybdates, of
which tetrathiomolybdate is an example, pharmaceutical forms
suitable for administration, compositions comprising the agents,
formulations with the agents, and carriers may be similar to those
described below.
[0114] B. Parenteral Formulations
[0115] In addition to the compounds formulated for parenteral
administration, the agents for use in the present invention, such
as copper binding or complexing thiomolybdates, may be formulated
for parenteral administration, e.g., formulated for injection via
the intravenous, intramuscular, sub-cutaneous or other such routes,
including direct instillation into a disease site. The preparation
of an aqueous composition that contains one or more agents for use
in the present invention, such as copper binding or complexing
thiomolybdates, as an active ingredient will be known to those of
skill in the art in light of the present disclosure. Typically,
such compositions can be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for using to prepare
solutions or suspensions upon the addition of a liquid prior to
injection can also be prepared; and the preparations can also be
emulsified.
[0116] Solutions of the active compounds as freebase or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0117] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form should be sterile
and should be fluid to the extent that easy flow through a syringe
exists. It should be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0118] Compositions comprising the agents for use in the present
invention, such as copper binding or complexing thiomolybdates, can
be formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts have been described above.
[0119] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. Proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the contamination with microorganisms can be obtained by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it may be desirable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0120] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the various sterilized active
ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0121] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. Formulations are easily administered in
a variety of dosage forms, such as the type of injectable solutions
described above, but drug release capsules and the like can also be
employed.
[0122] Suitable pharmaceutical compositions in accordance with the
invention will generally include an amount of one or more of the
agents for use in the present invention, such as copper binding or
complexing thiomolybdates, admixed with an acceptable
pharmaceutical diluent or excipient, such as a sterile aqueous
solution, to give a range of final concentrations, depending on the
intended use. The techniques of preparation are generally well
known in the art as exemplified by Remington's Pharmaceutical
Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated
herein by reference. It should be appreciated that endotoxin
contamination should be kept minimally at a safe level, for
example, less that 0.5 ng/mg protein. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0123] The therapeutically effective doses are readily determinable
using an animal model, as shown in the studies detailed herein.
Experimental animals with induced inflammatory and/or fibrotic
diseases are frequently used to optimize appropriate therapeutic
doses prior to translating to a clinical environment. Such models
are known to be very reliable in predicting effective
anti-inflammatory or anti-fibrotic strategies. For example,
bleomycin mice, such as described in Example 1, are appropriate
models of pulmonary fibrosis in humans. One can use such
art-accepted mouse models to determine working ranges of agents for
use in the present invention, such as copper binding or complexing
thiomolybdates, that give beneficial anti-inflammatory and/or
anti-fibrotic effects with minimal toxicity.
[0124] C. Therapeutic Kits
[0125] The present invention also provides therapeutic kits
comprising agents for use in the present invention which bind or
complex copper, such as thiomolybdates, and of which
tetrathiomolybdate is an example, as described herein. Such kits
generally comprise, in suitable container means, a pharmaceutically
acceptable formulation of at least one agent for use in the present
invention, such as copper binding or complexing thiomolybdates, in
accordance with the invention. The kits may also comprise other
pharmaceutically acceptable formulations, such as any one or more
of a range of anti-inflammatory and/or anti-fibrotic drugs.
[0126] The kits may have a single container means comprising an
agent that binds or complexes copper, such as a thiomolybdate, with
or without any additional components, or they may have distinct
container means for each desired agent. In some embodiments, kits
of the present invention comprise an agent for use in the present
invention, such as copper binding or complexing thiomolybdates,
packaged in a kit for use in combination with the co-administration
of a second agent, such as an anti-inflammatory or anti-fibrotic
agent as described above. In such kits, the components may be
pre-complexed, either in a molar equivalent combination, or with
one component in excess of the other; or each of the components of
the kit may be maintained separately within distinct containers
prior to administration to a patient.
[0127] When the components of the kit are provided in one or more
liquid solutions, the liquid solution is generally an aqueous
solution, with a sterile aqueous solution being particularly
preferred. However, the components of the kit may be provided as
dried powder(s). When reagents or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. One of the components of the
kit may be provided in capsules for oral administration.
[0128] The container means of the kit will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which an agent for use in the present
invention, such as copper binding or complexing thiomolybdates, and
any other desired agent, may be placed and, preferably, suitably
aliquoted. Where additional components are included, the kit will
also generally include a second vial or other container into which
these additional components are placed, enabling the administration
of separated designed doses. The kits may also comprise a second
and/or third container means for containing a sterile,
pharmaceutically acceptable buffer or other diluent.
[0129] The kits may also comprise a means by which to administer an
agent for use in the present invention, such as copper binding or
complexing thiomolybdates, to an animal or human patient, e.g., one
or more needles or syringes, or even an eye dropper, pipette, or
other such like apparatus, from which the formulation may be
injected into the animal or applied to a feeding tube or ingested
orally. The kits of the present invention will also typically
include a means for containing the vials, or such like, and other
component, in close confinement for commercial sale, such as, e.g.,
injection or blow-molded plastic containers into which the desired
vials and other apparatus are placed and retained.
[0130] For human use, in preferred embodiments, the kits further
comprise appropriate instructions and labels (e.g., as required by
the FDA) for use of copper binding or complexing agents as
described herein.
X. Inflammatory Disease, Fibrotic Diseases, Injury Response, and
Treatment Thereof
[0131] The compositions and methods provided by this invention are
broadly applicable to the treatment of any inflammatory and/or
fibrotic disease, which includes response to injury. In some
embodiments, the inflammatory and/or fibrotic disease is a result
of the activation or over-activation of transforming growth factor
beta (TGF.sub..beta.). Exemplary fibrotic diseases that may be
treated by a method of the present invention include, but are not
limited to, pulmonary disease including pulmonary fibrosis and
acute respiratory distress syndrome, liver disease including liver
cirrhosis and hepatitis C, kidney disease including renal
interstitial fibrosis, scleroderma, cystic fibrosis, pancreatic
fibrosis, keloid, secondary fibrosis in the gastrointestinal tract,
hypertrophic burn scars, myocardial fibrosis, Alzheimer's disease,
retinal detachment inflammation and/or fibrosis resulting after
surgery, and graft versus host and host versus graft
rejections.
[0132] Currently, most of these diseases do not have an effective
treatment. However, even if another treatment is perceived to exist
in connection with a certain category of patients or for a certain
type of disease, the perceived treatment does not in any way negate
the basic utility of the methods of the present invention in
connection with the treatments of all patients having an
inflammatory and/or fibrotic disease.
[0133] It is contemplated that the methods of the present invention
are widely or entirely applicable to the treatment of all
inflammatory and/or fibrotic diseases, irrespective of the
particular phenotype or localization of the inflammation or
fibroses themselves. However, the particular type of disease may be
relevant to the use of the methods of the present invention in
combination with secondary therapeutic agents, as described
above.
[0134] It is further contemplated that certain types of
inflammatory and/or fibrotic diseases may be more amenable to
treatment with a method of the present invention. Thus, some
diseases may respond to treatment with less effect on inflammation
and/or fibrosis. Although it is not necessary to understand the
underlying mechanism, and the invention is not intended to be
limited to any particular mechanism, it is contemplated that this
might be due to slight differences in the cytokine and other
pathogenic mechanisms from one disease to another. This phenomena
is observed in experimental animals, and may occur in human
treatments. Such considerations are taken into account in
conducting both the pre-clinical studies in experimental animals
and in optimizing the doses for use in treating any particular
patient or groups of patients.
[0135] There are realistic objectives that may be used as a
guideline in connection with pre-clinical testing before proceeding
to clinical treatment. However, this is generally more a matter of
cost-effectiveness than overall usefulness, and is a means for
selecting the most advantageous compounds and doses. In regard to
their basic utility, any composition or combination comprising a
copper binding or complexing agent such as thiomolybdate that
results in any consistent anti-inflammatory and/or anti-fibrotic
effects defines a useful compound. Even in those circumstances
where the anti-inflammatory and/or anti-fibrotic effects are
towards the low end of the range, it may be that the therapy of the
present invention is as or even more effective than other known
therapies in the context of particular anti-inflammatory and/or
anti-fibrotic targets, and especially where other factors (such as
desirable or undesirable side effects, or quality of life) may be
important. Even if it becomes evident to the clinician that
particular anti-inflammatory and/or anti-fibrotic diseases cannot
be effectively treated in the intermediate or long term, it does
not negate the utility of the therapy of the present invention,
particularly where it is about as effective as other known
strategies, or where it is effective after other conventional
therapies have failed. It is not predicted that resistance to
therapy of the present invention can develop.
[0136] In the present invention, an agent that binds or complexes
copper such as a thiomolybdate is administered in a therapeutically
effective amount to a patient suffering from an inflammatory and/or
fibrotic disease. The term "therapeutically effective amount" is a
functional term referring to an amount of material needed to make a
qualitative or quantitative change in a clinically measured
parameter for a particular subject. For example, prior to
administration, the subject may exhibit measurable symptoms of
disease (for example, pulmonary congestion and/or difficulty
breathing; evidence of hepatitis, or decrease in liver function;
evidence or kidney inflammation or decrease in kidney function;
etc), which upon administration of a therapeutically effective
amount the measurable symptom is found to change over time. A
therapeutically relevant effect relieves to some extent one or more
symptoms of a disease or condition or returns to normal either
partially or completely one or more physiological or biochemical
parameters associated with or causative of the disease.
[0137] In particular, the term refers to an amount of an agent that
binds or complexes copper such as thiomolybdate effective to treat
an inflammatory and/or fibrotic disease upon administration to a
patient suffering from such a disease. Treatment includes but is
not limited to preventing the onset or shortening the course or
severity of or reversing the effects of inflammatory and/or
fibrotic disease; thus, a therapeutically effective amount includes
a pro phylactically effective amount. Such effects are achieved
while exhibiting negligible or manageable adverse side effects on
normal, healthy tissues of the patient. Thus, the "therapeutically
effective amount" can vary from patient to patient, depending upon
a number of factors, including but not limited to the type of
disease, the extent of the disease, and the size of the
patient.
[0138] An objective of the therapeutic regimes of the present
invention is to reduce the endogenous copper level to a target
level, and then to maintain that level for a period of time
sufficient to prevent the onset or to shorten the course or
severity of or to reverse the effects of inflammatory and/or
fibrotic disease. The period of time sufficient to both reduce
endogenous copper level and to maintain it to prevent the onset or
to shorten the course or severity of or to reverse the effects of
inflammatory and/or fibrotic disease is referred to as a
"therapeutically effective time". As described earlier, the level
of endogenous copper can be monitored by measuring blood
ceruloplasmin (Cp) levels. In some embodiments, the levels of blood
ceruloplasmin decrease by 10%; in other embodiments, these levels
decrease by about 25%; in yet other embodiments, these levels
decrease by about 50%; in still other embodiments, these levels
decrease by about 90%. Alternatively, ceruloplasmin levels decrease
to between about 5 to 15 mg/dl. The time period in which to reduce
endogenous copper levels will vary, depending upon the disease and
the patient's general health and condition; typically, this time
depends upon the amount of copper-binding agent per dose, and
frequency of dose administration per treatment period. Generally,
for acute diseases, such as ARDS, it is desirable to decrease
endogenous copper levels as rapidly as possible; this is because
patients are at risk of dying quickly, and it is therefore
desirable to initiate quick intervention. Under these
circumstances, it is preferable to use initially a much higher
loading dose of a copper-binding or complexing agent than might be
used for a chronic disease or condition. For both acute and chronic
diseases, initial doses of copper binding or complexing agents
might be higher and administered more frequently in order to fairly
rapidly decrease endogenous copper to the target levels; these
doses are referred to as induction doses. Subsequent doses of
copper binding or complexing agents to maintain endogenous copper
at the target level may be lower, and administered less frequently;
these doses are referred to as maintenance doses.
[0139] In designing appropriate doses of the agents that bind or
complex copper such as thiomolybdate and combinations therewith,
and/or that effectively lower endogenous copper, one may readily
extrapolate from animal studies, as for example as described
further below, in order to arrive at appropriate doses for clinical
administration. To achieve this conversion, one would account for
the mass of the agents administered per unit mass of the
experimental animal, and yet account for the differences in the
body surface area between the experimental animal and the human
patient. All such calculations are well-known and routine to those
of ordinary skill in the art. Accordingly, using the information
provided herein, it is contemplated that useful daily doses of the
agents that bind or complex copper such as thiomolybdate, and/or
that effectively lower endogenous copper, for use in human
administration would be between about 20 mg and about 200 mg per
patient per day. Notwithstanding this stated range, it is
contemplated that, given the parameters and guidance described
above, further variations in the active or optimal ranges are
encompassed within the present invention.
[0140] Induction doses contemplated are generally about 180 mg per
day. Daily maintenance doses contemplated are generally between
about 20 mg and about 180 mg; between about 25 and about 160 mg;
between about 50 and about 150 mg; between about 30 and about 125
mg; between about 40 mg and about 100 mg; between about 35 and
about 80 mg; between about 20 and about 65 mg; between about 30 mg
and about 50 mg; about 40 mg; or in any particular range using any
of the foregoing recited exemplary doses or any value intermediate
between the particular stated ranges. Although daily doses in and
around about 60 mg to about 120 mg, or in and around about 20 to
about 180 mg, are typical, it is contemplated that lower doses may
be more appropriate in combination with other agents, or under
conditions of maintenance, and that high doses can still be
tolerated, particularly given the fact that the agents that bind or
complex copper such as a thiomolybdate and/or that effectively
lower endogenous copper for use in the invention are not themselves
cytotoxic. Even if certain adverse side effects do occur, this
should not necessarily result in toxicity that cannot be
counteracted by normal homeostatic mechanisms, which is believed to
lessen the chances of significant toxicity to healthy tissues.
[0141] The values described above can also be expressed in terms of
mg/kg of body weight. As described above, the biologically or
therapeutically effective amount can vary, depending upon the size
of the animal or human patient. However, taking the average weight
of a human male as about 70 kg, the biologically or therapeutically
effective amount of the agent that binds or complexes copper such
as a thiomolybdate for an average human male would be between about
0.3 mg/kg and about 3 mg/kg.
[0142] Another objective of the therapeutic regimes of the present
invention is generally to produce the maximum anti-inflammatory
and/or anti-fibrotic effects while keeping the dose below the
levels associated with unacceptable toxicity. However, as noted
above, in acute diseases or conditions, it may be necessary to
administer initial high doses to rapidly decrease endogenous copper
levels. In addition to varying the dose itself, the administration
regimen can also be adapted to optimize the treatment strategy. A
currently preferred maintenance treatment strategy is to administer
between about 20 mg and about 200 mg of the agents that bind or
complex copper such as a thiomolybdate or combination thereof, or
which effectively lower endogenous copper, from about 3 to about 6
or more times per day, approximately half of the doses with meals,
and approximately half of the doses between meals. In administering
the particular doses themselves, one would preferably provide a
pharmaceutically acceptable composition to the patient
systemically. Oral administration is generally preferred. An
exemplary induction dosage regime for a patient suffering from a
chronic inflammatory and/or fibrotic disease is about 40 mg three
times daily with meals, and about 60 mg at bedtime. Exemplary
maintenance dosage regimes for a patient suffering from a chronic
inflammatory and/or fibrotic disease is about 20 to 180 mg total
per day, taken approximately proportionately as indicated for the
exemplary induction dosage regime, or taken at fewer times per day,
for example, with breakfast and with dinner.
XI. Efficacy of Tetrathiomolybdate in Lowering Endogenous Copper:
Prior Use to Treat Wilson's Disease
[0143] The efficacy and safety of tetrathiomolybdate to lower
endogenous copper levels in both animal and human patients has been
well documented in its use to treat Wilsons's disease, which is
characterized by an increase in endogenous levels of copper,
generally to toxic levels. A description of Wilson's disease,
previous therapy regimes, the discovery of tetrathiomolybdate, it's
toxicity and efficacy, both alone and in comparison to other
anti-copper agents, and its utility in treating Wilson's disease,
are provided below as part of the description of the methods of the
present invention.
[0144] A. Wilson's Disease and its Existing Treatments
[0145] Wilson's disease is an autosomal recessive disorder of
copper metabolism. In this disorder, the excretion of copper into
the bile appears to be defective, and there is reduced hepatic
incorporation of copper into ceruloplasmin, leading to an
accumulation of toxic levels of copper in plasma and most body
tissues. Wilson's disease usually leads to hepatic and/or
neurologic dysfunction.
[0146] The therapy of Wilson's disease can be divided into two
broad categories (G J Brewer and Yuzbasiyan-Gurkan, Medicine,
71(3):139-164 [1992]; and G J Brewer, Wilson's Disease: A
Clinician's Guide to Recognition, Diagnosis, and Management (Kluwer
Academic Publishers, Boston) [2001]). These two categories are
initial therapy in acutely ill patients, and maintenance therapy.
Initial therapy is that period of time during which a newly
presenting patient is still suffering from acute copper toxicity,
generally the first few weeks to months of therapy. Maintenance
therapy is essentially the rest of the patient's life, or that
period of time after the copper levels have been brought down to a
subtoxic threshold, and the patient is on therapy simply to prevent
the recurrence of copper accumulation and copper toxicity.
[0147] For the maintenance therapy of Wilson's disease, three drugs
were previously used. These include the oldest available drug,
penicillamine (Walshe, Am. J. Med., 21:487 [1956]), a drug called
trien or trientine which was developed for patients who are
intolerant of penicillamine (Walshe, Lancet, 1:643-647 [1982]), and
zinc acetate (G J Brewer and Yuzbasiyan-Gurkan, Medicine,
71(3):139-164 [1992]; Brewer and Yuzbasiyan-Gurkan, in Textbook of
Clinical Neruopharmacology and Therapeutics, 2.sup.nd Edition
(Klawans, Goetz, Tanner, eds; Raven Press, New York; pp. 191-205
[1992]); G J Brewer et al., Annals. Int. Med., 99:314-320 [1983];
Hill et al., Hepatology, 7:522-528 [1987]; Hill et al., Am. J. Med.
Sci., 12:344 [1986]; Brewer et al. (1987) J. Lab. Clin. Med.
109:526-531; Brewer et al. (1987) Proc. Soc. Exper. Biol. Med.
7:446-455; Brewer et al. (1987) Sem. Neurol. 7:209-220;
Yuzbasiyan-Gurkan et al. (1989) J. Lab. Clin. Med. 114:520-526;
Brewer et al. (1989) J. Lab. Clin. Med. 114:633-638; Lee et al.
(1989) J. Lab. Clin. Med. 114:639-645; Brewer et al. (1990) J.
Trace Elem. Exp. Med. 3:227-234; Brewer et al. (1991) J. Lab. Clin.
Med. 118:466-470; Brewer and Yuzbasiyan-Gurkan (1989) Dig. Dis.
7(4): 178-1923; Brewer et al. (1992) JAVMA 201:564-568; G J Brewer
et al., J. Vet. Int. Med., 6:41-43 [1992]; Yuzbasiyan-Gurkan et
al., J. Lab. Clin. Med., 120:380-386 [1992]; Brewer et al., J.
Amer. Coll. Nut., 12(1):26-30 [1993]; G J Brewer et al., Amer. J.
Med. Sci., 305(4):199-202 [1993]; In Essential and Toxic Trace
Elements in Human Health and Disease: An Update (Prasad, ed; Allan
R. Liss, New York; PCBR 380:129-145), G J Brewer et al., J. Lab.
Clin. Med., 123:849-858 [1993]; G J Brewer, Nutrition and the MD,
19(12) [1993]; Hoogenraad et al., Lancet, 2:1262-1263 [1978];
Hoogenraad et al., Eur. Neurol., 18:205-211 [1979]; Hoogenraad et
al., J. Neurol. Sci., 77:137-146 [1987]). In the past, it was
generally believed that zinc provided an effective maintenance
therapy with a very low level of toxicity.
[0148] About two thirds of patients who present with Wilson's
disease present with symptoms referable to the brain (G J Brewer et
al., JAMA, 201:564-568 [1992]; Scheinberg and Sternlieb, In: Major
Problems in Internal Medicine, Vol. XXM (W.B. Saunders Company,
Philadelphia) [1984]; and Danks, In: Metabolic Basis of Inherited
Diseased, Vol. 1, Sixth Ed. (Scriver, Beaudet. Sly, Valle, eds;
McGraw Hill, N.Y.; pp. 1411-1431 [1989]; and G J Brewer, Wilson's
Disease: A Clinician's Guide to Recognition, Diagnosis, and
Management (Kluwer Academic Publishers, Boston [2001]). These can
be neurologic symptoms or symptoms of psychiatric nature in the
beginning, with neurologic symptoms later. Therapy for these
patients was not nearly as straightforward as it was for
maintenance phase patients. It was found that approximately 50% of
these patients who were treated with penicillamine became worse
rather than better (G J Brewer et al., Arch. Neurol., 44:490-494
[1987]). Half of these patients who worsen, or about 25% of the
original sample, never recovered to their pre-penicillamine
baseline. In other words, penicillamine induced additional
irreversible damage.
[0149] The mechanisms underlying this worsening are not known with
certainty, although it is likely that the mobilization of hepatic
copper by the drug further elevates brain copper. The inventors
have shown that this mechanism can occur in a rat model. Regardless
of the mechanism, neurologically presenting patients very often
ended up much worse after being treated initially with
penicillamine. In fact, even presymptomatic patients could develop
neurologic disease after being initiated on penicillamine (Glass et
al., Arch. Neurol., 47:595-596 [1990]; and G J Brewer et al., Arch.
Neurol., 51:304-305 [1994]). It was not known whether trientine
exhibits the phenomenon of neurological worsening when used as
initial therapy, because it has not been used very much in this
kind of situation. It would not be surprising if trientine
exhibited this problem to some degree, because its mechanism of
action is believed to be similar to that of penicillamine; however,
it is anticipated that the problematic effects of trientine would
be less serious, as its effects on copper seem to be somewhat
gentler.
[0150] Zinc is not an ideal agent for the initial treatment for
this type of patient. Zinc has a relatively slow onset of action,
and produces only a modest negative copper balance. Thus, during
the several months required for zinc to bring copper down to a
subtoxic threshold, patients may be at risk for further copper
toxicity and worsening of their disease.
[0151] B. Tetrathiomolybdate
[0152] The discovery of TM began with observations of cattle and
sheep which developed copper deficiency when grazing on pasturages
with high molybdenum (Mo) content (Ferguson et al., J. Agr. Sci.,
33:44 [1943]; Dick and Bull, Aust. Vet. J., 21:70 [1945]; Miller
and Engel, Fed. Proc., 19:666 [1960]). It was established that
administration of supplementary Mo impaired copper metabolism in
ruminants (Macilese Ammerman et al., J. Nutr., 99:177 [1969]);
however, Mo had little effect on non-ruminant animals such as rats
(Mills et al., J. Nut., 65:129 [1958]; Cox et al., J. Nutr., 70:63
[1960]). The answer to the different effects of Mo came from
observations which suggested that the administered Mo was converted
to thiomolybdates in the rumen as a result of the high sulfide
metabolism there, and that thiomolybdates were the active
anti-copper agents (Dick et al., J. Agri. Sci., 85:567 [1975]).
This theory was confirmed when thiomolybdate compounds were given
to rats and produced anti-copper effects (Mills et al., J. Inorg.
Biochem., 14:189 [1981]; Mills et al., J. Inorg. Biochem., 14:163
[1981]; and Bremner et al., J. Inorg. Biochem., 16:109 [1982]). The
tetra-substituted compound, tetrathiomolybdate or TM, appeared to
be the most potent of the thiomolybdates initially tested.
[0153] The anti-copper effects TM are believed to be based upon two
modes of action of TM (Mills et al., J. Inorg. Biochem., 14:189
[1981]; and Mills et al., J. Inorg. Biochem., 14:163 [1981];
Bremner et al., J. Inorg. Biochem., 16:109 [1982]; and Gooneratne
et al., Br. J. Nutr., 46:469 [1981]). One mechanism operates in the
gastrointestinal or GI tract, and the other in the blood. In the GI
tract, TM forms complexes with copper and food proteins (or other
proteins) that are not absorbed. This absorption block involves not
only food copper, but also the rather considerable amount of
endogenously secreted copper in saliva, gastric juice and other GI
tract secretions (Allen and Solomons, In: Absorption And
Malabsorption Of Mineral Nutrients, Solomons and Rosenberg (Eds.)
Alan R. Liss, Inc., New York, 12:206 [1984]). Although both TM and
zinc are apparently effective in the GI tract, TM offers several
advantages over zinc. One advantage is that TM is a more effective
blocker of copper absorption than zinc, because zinc acts only in
those areas of the small intestine where metallothionein can be
induced (Yuzbasiyan-Gurkan et al., J. Lab. Clin. Med., 120:380-386
[1992]), where in contrast, TM is effective the entire length of
the GI tract. Another advantage of TM over zinc is that TM acts
immediately; therefore, it does not have a lag period required for
the induction of metallothionein.
[0154] The second mode of action of TM is in the blood. TM given at
times away from meals is relatively well absorbed into the blood.
There it forms complexes with copper and albumin, rendering the
complexed copper unavailable for cellular uptake (Gooneratne et
al., Br. J. Nutr., 46:469[1981]). The normal plasma copper is in
two primary pools. Most of the plasma copper in normal persons is
part of the ceruloplasmin molecule. This copper is essentially
unavailable for ready exchange with cells and is considered
non-toxic. The other pool of copper is more loosely bound to
albumin and small molecules, such as amino acids. This pool of
copper is greatly expanded during acute copper toxicity in Wilson's
disease, and is readily available for cellular uptake and is,
therefore, potentially toxic (Scheinberg and Sternlieb (1984) In:
Major Problems In Internal Medicine, Vol. XXIII, Saunders Company,
Philadelphia). When TM enters the blood, it complexes with this
latter copper and renders it, like the ceruloplasmin copper,
unavailable for cellular uptake and for further toxicity.
[0155] Very good evidence exists that TM-complexed copper is
unavailable for cellular uptake. The most direct evidence is that
in sheep levels of copper in the plasma which would normally be
high enough to produce hemolytic anemia do not do so in the
presence of TM (Gooneratne et al., Br. J. Nutr., 46:469 [1.981]).
It was shown that the TM bound copper does not permeate the
erythrocyte. This is direct evidence that TM-complexed copper does
not permeate cells.
[0156] C. Tetrathiomolybdate Toxicity and Efficacy
[0157] Considerable work on the potential toxicity of TM has been
carried out in rats (Mills et al., J. Inorg. Biochem., 14:189
[1981]; and Bremner et al., J. Inorg. Biochem., 16:109 [1982]).
Approximately 6 mg of TM per kilogram of diet shows substantial
effects on copper levels in rats, including a reduction of plasma
ceruloplasmin and a reduction in liver and kidney copper. At
approximately 12 mg of TM, all of these changes were increased and,
in addition, liver Mo was increased. Mild anemia was present, and
skeletal lesions were present in one of six animals. At
approximately 18 mg of TM, the anemia was severe. Melanogenesis of
hair was impaired, diarrhea was present, growth rate was markedly
impaired, and all animals had skeletal lesions characterized by
dysplasia in the epiphyseal cartilage cells of long bones,
resorption of trabecular bone, and structural changes in
ligaments.
[0158] It was later shown that all of the toxic effects of TM, up
to 36 mg of TM per kilogram of diet, could be prevented by oral
supplementation with copper, or with intraperitoneal injection of
copper (Mills et al., J. Inorg. Biochem., 14:163 [1981]). Thus, it
appears that all the toxic lesions induced by TM are due to copper
deficiency induced by the TM. In support of this hypothesis, almost
all of the above lesions are induced by dietary copper deficiency,
the two exceptions being the skeletal lesions and the enterocyte
mitochondrial damage which leads to diarrhea. The reason that these
last two lesions are seen with TM administration, but may not be
seen in dietary copper deficiency, could be related to the severity
and the rapidity of the copper deficiency induced by TM. With
dietary copper deficiency, there is always some contaminating
copper available, and rapidly dividing cells such as the enterocyte
and epiphyseal cells may obtain enough copper to prevent the
lesions. The prevention of these two lesions as well as all of the
other TM induced lesions by copper supplementation indicates that
the lesions are probably due to copper deficiency.
[0159] Other publications reported the results of examining gut
pathology in rats receiving approximately 18 mg of TM per kilogram
of diet (Fell et al., J. Com. Pathol., 89:496 [1979]). These rats
also received approximately 3 mg of copper per kilogram of diet. In
these rats, observed gut pathology involving cell apoptosis, edema,
and necrosis was attributed to hypocuprosis, although this was not
proven. It is probable that a higher copper supplement was required
for protection, in view of the observations that all such problems
were prevented by adequate copper supplementation (Mills et al. J.
Inorg. Biochem., 14:163 [1981]).
[0160] Wilson's disease patients have a huge store of excess
copper, so none of the TM toxicities due to copper deficiency are a
risk in these patients. Even in the case of the skeletal and
enterocyte lesions, since copper administration is protected, the
Wilson's disease patient with excessive stores of copper should
also be protected.
[0161] The effect of TM on copper loaded sheep has also been
studied (Gooneratne et al., Br. J. Nutr., 46:457 [1981]). It is
well known that sheep are quite susceptible to copper toxicity,
usually developing hepatic failure and hemolytic anemia. The
studies involved loading sheep dietarily with copper to the point
of initiation of hepatic damage, then giving TM intravenously in
doses of 50 or 100 mg 2.times. weekly for up to 11 weeks. Five of
the 26 sheep died during the study. All deaths were attributed to
copper toxicosis based on autopsy results. Three of the five deaths
occurred in control animals who received copper but not TM. One
death occurred after an animal had received only one dose of TM,
and another in an animal who had received only 4 doses of TM. It is
clear that these two animals died from copper toxicity prior to the
ability of TM to rescue them. If animals survived the initial onset
of copper toxicosis, they were protected from further copper
toxicity by TM, even though in some cases copper administration was
continued. These animals tolerated up to 22 injections of TM
without clinical problems.
[0162] Support for the beneficial effect of administering TM by
either intravenous injection (Humphries et al., Vet. Record,
119:596-598 [1986]) or by subcutaneous injection (Humphries et al.,
Vet. Record, 123:51-53 [1988]) in protecting sheep against severe
hepatic copper toxicity has also been shown. TM not only reduced
the amount of hepatic copper, but the actual liver damage. TM was
also used prophylactically to prevent copper toxicity in commercial
sheep flocks. Over 400 animals have been treated with TM with no
adverse side effects (Humphries et al., Vet. Record, 123:51-53
[1988]).
[0163] Preliminary work also indicated that TM may be dramatically
effective against copper toxicity in the LEC rat model (Suzuki et
al., TOXIC, 83:149 [1993]). The genetic defect in these rats has
been recently shown to be due to a defect in the Wilson's disease
gene (Wu et al., Nat. Genet., 7:541 [1994]). These rats develop
severe liver disease and usually die. TM has been very effective in
treating these animals in the late stages of their liver
disease.
[0164] Molybdenum metabolism in sheep has been studied after the
intravenous injection of .sup.99Mo labeled TM (Mason et al., J.
Inorg. Biochem., 19:153 [1983]). There was a rapid disappearance
from plasma during the initial 15 minutes, and then a slow
disappearance with a half-time of about 40 h. The TM was
transformed step wise to molybdate, and over 90% was excreted in
urine compared to 5% in feces. The same group published
subsequently on .sup.99Mo and .sup.35S metabolism after intravenous
injection of double labeled TM in sheep (Hynes et al., Brit. J.
Nutr., 52:149 [1984]). Most of the .sup.99Mo and .sup.35S were
associated initially with albumin. Displaced or unbound TM was
rapidly hydrolyzed to molybdate and sulfate. There was no evidence
of an irreversible interaction of either .sup.35S or .sup.99Mo with
copper and plasma despite the appearance of a TCA insoluble copper
fraction.
[0165] It is clear that in the presence of high levels of copper,
TM administration results in the accumulation of copper complexed
with TM in both the liver and kidneys (Jones et al., Res. Vet.
Sci., 37:273 [1984]; and Bremner and Young, Br. J. Nutr., 39:325
[1978]). However, there is no evidence of a storage disease
associated with this complex. Current theory holds that the complex
is disassociated and that the TM is metabolized to oxymolybdates
and excreted (Mason et al., J. Inorg. Biochem., 19:153 [1983]). The
copper then enters other pathways in the liver. In the presence of
high levels of metallothionein, the copper would most likely be
taken up by metallothionein. In the kidneys, the evidence is that
the copper is simply excreted.
[0166] Two cases of reversible bone marrow depression have been
reported in patients receiving TM for maintenance therapy (Harper
and Walshe, Br. J. Hematol., 64:851-8 [1986]). The inventors have
observed reversible anemia in seven patients. These patients had a
strong response to therapy, and likely ended up with localized,
bone marrow copper deficiency. Since copper is required for heme
synthesis, this appears to be a manifestation of over-treatment, at
least as far as the bone marrow is concerned. Since TM is such an
effective anticopper agent, it would not be unexpected for
over-treatment to occur during maintenance therapy with TM, as was
previously observed (Harper and Walshe, Br. J. Hematol., 64:851-853
[1986]).
[0167] D. Molybdenum (Mo) Toxicity
[0168] About 37% of TM is Mo. The normal intake of Mo is about 350
.mu.g/day (Seelig, Am. J. Clin. Nutr., 25:1022 [1972]), or the
equivalent amount of Mo that would be in about 1.0 mg of TM.
Molybdenum seems to be quite well tolerated by the human.
Relatively high doses of 5-20 mg/kg/day of Mo (equivalent to the Mo
in 1-4 g of TM) were used for 4-11 months in patients with Wilson's
disease in a 1957 study, without known toxicity (Bickel et al,
Quart. J. Med., 50:527 [1957]). However, it was not effective,
because as pointed out earlier, TM is the active metabolite, and
that is formed efficiently from Mo only in ruminants.
[0169] E. Additional Anti-Copper Drugs
[0170] 1. Penicillamine
[0171] Penicillamine is the drug that has been used the most, and
is the best known. However, it should be the last choice for
initial treatment of patients suffering from neurological symptoms
because of the very high risk of worsening their neurologically
symptoms (G J Brewer et al., Arch. Neurol., 44:490-494 [1987]; and
G J Brewer et al., Arch. Neurol., 51:304-305 [1994]). Another
problem with penicillamine is that about a quarter to a third of
patients develop an initial hypersensitivity syndrome which
requires significant interventions, such as temporarily stopping
the drug and restarting it at a lower dose, usually with concurrent
corticosteroid administration. This is a somewhat frightening
experience for patients who are already ill, and prevents the
attending physician in the inventors' study from being blinded.
Finally, there is a long list of other side effects that can occur
with penicillamine during the first few weeks of therapy. These
include bone marrow depression, proteinuria, and auto-immune
disorders.
[0172] 2. Zinc
[0173] Zinc was used for the comprehensive treatment of Wilson's
disease including initial treatment (Hoogenraad et al., Lancet,
2:1262-1263 [1978]; Hoogenraad et al., Eur. Neurol., 18:205-211
[1979]; and Hoogenraad et al., J. Neurol. Sci., 77:137-146 [1987]).
However, zinc was not ideal for initial therapy (by itself) because
it is rather slow acting. Thus, it takes approximately two weeks to
achieve intestinal metallothionein induction and a negative copper
balance in Wilson's patients (Yuzbasiyan-Gurkan et al., J. Lab.
Clin. Med., 120:380-386 [1992]). At the two week point, zinc
immediately reverses the +0.54 mg daily (positive) copper balance
these patients average, but the negative copper balance induced is
rather modest, averaging -0.35 mg daily (negative) copper balance
(G J Brewer et al., J. Trace Elem. Exp. Med., 3:227-234 [1990]; G J
Brewer et al., Amer. J. Med. Sci., 305:(4)199-202 [1993]). Due to
this low rate of copper removal, it takes as long as six months of
zinc therapy to bring urine copper and nonceruloplasmin plasma
copper (the potentially toxic copper measured in the blood), down
to subtoxic levels.
[0174] TM is a more effective blocker of copper absorption than
zinc, since zinc acts only in those areas of the small intestine
where metallothionein can be induced. In contrast, TM works all up
and down the gastrointestinal track. The other advantage of TM over
zinc in this setting is that TM acts immediately. It does not have
a lag period required for the induction of metallothionein.
[0175] 3. Trientine
[0176] Trientine acts by chelation and urinary excretion of copper
(Walshe, Lancet, 1:643-647 [1982]). A therapeutic dose (1,000-2,000
mg/day) usually produces only about half as much cupruresis as a
similar dose of penicillamine. Nonetheless, trientine is capable of
an initial production of a several mg negative copper balance, much
greater than zinc. Typically, this 4-5 mg cupruresis decreases
during the first few weeks of therapy to a more modest, but still
substantial, 2-3 mg. Ingestion of copper is about 1 mg/day, with
obligatory, non-urine losses of about 0.5 mg. Thus a cupruresis of
2-3 mg produces a negative copper balance of 1.5 to 2.5 mg/day.
[0177] Trientine is officially approved for use in patients
intolerant of penicillamine therapy. Because of this, and because
it was introduced much later than penicillamine, it has not been
used and reported on very extensively. It has not had a formal
toxicity study. It appears to have substantially less risk of side
effects then penicillamine. An initial hypersensitivity problem has
not been reported. It does cause proteinuria, after several weeks
of use in about 20% of patients. It can also occasionally produce
bone marrow depression and autoimmune abnormalities, although the
latter is usually after prolonged use.
[0178] So far, trientine has not been reported to cause initial
worsening in neurological patients, but its sole use in this type
of patient is probably very limited. Anecdotally, the inventors
have received patients in transfer who worsened on penicillamine,
were switched briefly to trientine, and when they became worse (or
failed to improve) were transferred to the inventors for TM
therapy. In patients with this history, it is impossible to know if
trientine played any role in worsening. Theoretically, it could,
because as with penicillamine, trientine mobilizes copper,
producing a higher blood level to achieve urinary excretion. But
whether this increased level of blood copper translates into
increased brain levels, and increased neurotoxicity, is
unknown.
XII. Results of Tetrathiomolybdate Therapy for Wilson's Disease
[0179] Over a period of several years, the inventors carried out an
open label study of the use of TM for initial treatment of
neurologically presenting Wilson's disease patients. The inventors
also developed both a spectrophotometric and bioassay for the
activity of the drug, to evaluate its stability and to assure its
potency when administered (G J Brewer et al., Arch. Neurol.,
48(1):42-47 [1991]; and G J Brewer et al., Arch. Neurol.,
51(6):545-554 [1994]). As noted above, TM is unstable in air, and
slowly loses potency when exposed to air. This is apparently due to
the exchange of oxygen molecules with the sulfur molecules,
rendering TM inactive.
[0180] The results in the first patient studied can be used to
illustrate several points. For the first seven days, the patient
received TM only with meals (tid with meals). This produced the
immediate negative copper balance one would expect from the first
mechanism of action (blockade of copper absorption when given with
meals). After the first seven days, TM was given between meals as
well (tid with meals, and tid between meals). This led to the
immediate rise in plasma copper expected from absorption of TM into
the blood, and formation of a complex of copper, TM, and albumin.
The copper complexed with TM and albumin is unavailable for
cellular uptake, and this copper is therefore non-toxic. There is a
1:1 stoichiometric relationship between molybdenum and copper in
this complex. Knowing the molybdenum level in the blood, and the
ceruloplasmin level (ceruloplasmin also contains copper that is
non-toxic), one can calculate how much of the plasma copper is not
bound to one or the other. This so-called "free copper"
(non-ceruloplasmin plasma copper) is the potentially toxic copper.
When reduced to zero, the plasma copper-molybdenum "gap" is closed.
This took 16 days in the first patient (9 days after adding the
between meal doses). Since in the brain (and in other organs), free
copper is in equilibrium with the blood, decreasing the blood free
copper to a low level begins the process of lowering the brain
level of free (toxic) copper.
[0181] The inventors have treated initially 56 Wilson's disease
patients with TM, all of whom presented with neurological or
psychiatric disease, in an open label study. These patients were
all diagnosed by standard criteria. These patients had a
diagnostically elevated hepatic or urine copper, usually both. Some
of them were treated briefly with other agents prior to this trial.
Two patients had psychiatric but not neurological symptoms.
[0182] With three exceptions in the earliest part of the study, all
patients received a dose of 20 mg tid with meals, or qid with three
meals and a snack. Thus, the only difference between a patient
receiving 120 mg and 140 mg total dose is that the former was
receiving 20 mg tid, or 60 mg, with meals, and the latter was
receiving 20 mg qid, or 80 mg with meals plus a snack. The rest of
the total daily dose was divided up into three equal doses and
given between meals.
[0183] The total daily dose was varied considerably among the
patients, from a high of 410 mg to a low of 120 mg. In the end, the
inventors could discern no dose-related correlation with copper
variables, nor with functional variables measured either during the
study or at the one and two year time point.
[0184] Zinc administration was also used in these patients. The
starting time of zinc administration was varied widely and did not
correlate with copper variables, outcome variables or toxicity.
Early zinc therapy should theoretically help preserve liver
function. In these patients, liver function returned to normal by
year 1, but since these tests don't measure the extent of tissue
preservation, it seems likely zinc was somewhat beneficial.
[0185] Measuring trichloracetic acid (TCA) soluble copper of the
plasma is somewhat useful in assessing the impact of TM therapy on
copper metabolism in Wilson's disease. Generally, a high proportion
of plasma copper in these patients is TCA soluble (it averaged 56%
in patients which is 27 p.g/dl). All of the non-ceruloplasmin
plasma copper is TCA soluble, and a somewhat variable portion of
the ceruloplasmin copper is also TCA soluble. Because the
ceruloplasmin levels are usually rather low in Wilson's disease,
most of the plasma copper is TCA soluble. The copper in the
TM/albumin/copper complex in the blood is TCA insoluble. Thus, as
therapy proceeds, the fraction of the plasma copper that is TCA
soluble becomes smaller. During the late stages of TM therapy, the
TCA soluble fraction of plasma copper of the patients averaged 15
pg/dl, a significant reduction from the starting value of 27. The
TCA soluble fraction cannot be used as an absolute endpoint, for
example attempting to reduce it to zero, because a small and
somewhat variable soluble fraction is usually present due to plasma
ceruloplasmin. However, the significant mean reduction from 27 to
15 p.g/dl illustrates the beneficial effect that TM therapy has on
the status of the potentially toxic plasma copper in these
patients. Further evidence of the desirable impact of TM therapy on
copper metabolism is shown by reduction of mean urine copper values
during the latter part of TM therapy, compared to baseline
values.
[0186] TM has a quick and favorable impact on copper metabolism,
reducing the levels of potentially toxic copper of the blood and as
contemplated the rest of the body as well. The primary clinical
objective in treatment of Wilson's disease is to gain control over
copper toxicity while not allowing worsening of the disease or
symptoms. In other words, the prime objective is to protect all
neurological function that is present at the time therapy is
started. This was evaluated weekly by quantitative neurological and
speech exams. Methodology and the neurology rating scale system
have been published (Young et al., Neurol., 36:244-249 [1986]).
During the weeks of TM administration, during which copper
metabolism is being controlled, neurological function, as evaluated
by quantitative neurological exam is protected. Only two patients
(4% of the sample) showed a change of more than 5 units, the
criterion for significant worsening.
[0187] During the years following induction doses which act to
initially lower endogenous copper levers, while the patients are on
maintenance therapy, the brain damage previously induced by copper
is at least partially repaired. This is exemplified by the partial
recovery in neurological scores seen at yearly time-points in
follow-up. It is clear that with the initial TM approach, long-term
recovery is excellent, with most patients showing substantial
neurological recovery. These excellent results are to be contrasted
with results observed with penicillamine therapy. As pointed out
earlier, about 50% of patients initially deteriorate on
penicillamine, and that half of these, or 25% of the original
sample, never recover to their pre-penicillamine baseline.
[0188] The results of TM therapy on speech during the initial 8
weeks of TM therapy were evaluated by quantitative speech exams
performed as described (Brewer et al., Arch. Neurol., 53:1017-1025
[1996]). During the weeks of TM administration, during which copper
metabolism is controlled, neurological function as measured by
quantitative speech exams is also controlled. No patient shows
significant (more than 2.0 units) reduction in scores. During the
following years, while the patients are on maintenance therapy, the
brain damage previously induced by copper is partially repaired.
This is exemplified by the partial recovery in speech scores over
years of follow-up. Long-term recovery is excellent. No patient
shows significantly (more than 2.0 units) less long-term function
than at the time of initiation of therapy, and most show marked
improvement.
[0189] Two undesirable effects from TM therapy were observed in
these patients. One is a reversible anemia/bone marrow depression,
which was exhibited by seven patients. The fall in hemoglobin in
all of these patients was significant, averaging 3.4%. Three of the
patients showed a reduction in platelet count and four of the
patients showed a reduction in white blood cell count that may have
been significant. TM administration was stopped in all seven cases.
Except for two of the patients, stopping TM therapy occurred late
in the 56-day course of TM administration.
[0190] At the time of the anemia, these patients all had zero
non-ceruloplasmin plasma copper and an extremely low TCA soluble
copper. The latter averaged 2.7 in these patients, and the average
value for this variable in the entire group of patients was 27 at
the beginning and 15 at the height of therapy. The cause of the
anemia/bone marrow depression was concluded to be bone marrow
depletion of copper. Since copper is required for heme synthesis
and other steps in cell proliferation, it could be expected that
anemia and bone marrow effect would be the first signs of copper
depletion. This result from copper depletion is a well-known
phenomenon.
[0191] Thus, this undesirable response to TM is not a side effect
but is, rather, due to overtreatment. It is perhaps surprising that
it is possible to produce even localized bone marrow copper
depletion within such a short period of time in Wilson's disease, a
disease in which the body is overloaded with copper. This response
to TM is unique. None of the other anticopper drugs are able to
produce this effect in early therapy. Thus, this speaks to the
potency of TM and the rapidity with which it can control copper
levels. Its also likely that the bone marrow is especially
dependent on plasma copper, and that it is the first pool that it
is reduced to very low levels. At a dose of 180 mg/day or over,
overtreatment occurred in 6 of 37 patients. At a dose of 150 or
lower, only 1 of 13 patients exhibited overtreatment, and that
occurred very late (53 days in the 56 day program).
[0192] The second undesirable effect of TM therapy in these
patients is an elevation of transaminase values in four of the
patients. The serum AST and ALT values were elevated. TM therapy
was discontinued in one patient because of these elevations. During
the period of elevated serum AST and ALT values, the urine copper
increases, contrary to the general trend in other patients, where
it is decreasing. These data support the concept that a hepatitis
is occurring, with release of copper from damaged hepatocytes. It
is not clear why this hepatitis is occurring. However, untreated
Wilson's disease patients have an episodic hepatitis as part of
their history. Since there is little in the way of observation of
untreated patients after diagnosis, no good information exists on
how often episodes of transaminase elevations occur as part of the
natural history of the disease.
[0193] Alternatively, the TM in some cases may be mobilizing
hepatic copper at a faster rate than it can be disposed of, in
which case these patients would be classified as showing a side
effect of treatment. However, the observation in copper-poisoned
sheep, in which the acute hepatitis, liver necrosis, and hemolytic
anemia are rapidly corrected with high doses of TM, argue against
this explanation. All four of these patients were treated with 150
mg TM/day or higher. None of the patients treated with 150 mg or
lower exhibited this response. No other negative effects of TM have
been observed.
EXAMPLES
[0194] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0195] In the experimental disclosures which follow, the following
abbreviations apply: N (normal); M (molar); mM (millimolar); .mu.M
(micromolar); mol (moles); mmol (millimoles); .mu.mol (micromoles);
nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams);
.mu.g (micrograms); ng (nanograms); 1 or L (liters); ml
(milliliters); .mu.l (microliters); cm (centimeters); mm
(millimeters); .mu.m (micrometers); nm (nanometers); .degree. C.
(degrees Centigrade); TM (tetrathiomolybdate); TGF.sub..beta.
(transforming growth factor beta); CTGF (connective tissue growth
factor); Cp (ceruloplasmin); H&E (hematoxylin and eosin); ANOVA
(analysis of variance); SPARC (secreted protein, acidic and rich in
cysteine); IU (international units); ConA (concanavlin A); ALT
(alanine amino transferase); SF (Sigma-Frankel units)
Example 1
Treatment of Pulmonary Fibrosis in the Bleomycin Mouse Model
[0196] Several experiments were carried out in the bleomycin mouse
model of pulmonary fibrosis (Experiment 1: Prophylaxis of TM
Experiment; Experiment 2: Dose Response; and Experiment 3: Effect
of TM as prophylaxis and treatment).
[0197] In the bleomycin mouse model, which is known to be dependent
upon the TGF.sub..beta. pathway, the intratracheal administration
of bleomycin leads to the development of severe lung inflammation
followed by fibrosis in 2-3 weeks, at which time the mice are
sacrificed. Fibrosis is quantified in lung tissue by measuring
hydroxyproline, a key component of the collagen that is deposited
in fibrotic lung.
[0198] The bleomycin control animals showed high levels of
hydroxyproline, and severe histological inflammatory and fibrotic
changes involving whole lobes, while the TM treated bleomycin mice
showed no increase in hydroxyproline, and only small patches of
inflammatory foci. These results are highly significant
statistically.
A. Methods
[0199] Mice. Female CBA/J mice at 8-10 weeks of age were from the
Jackson Laboratories (Bar Harbor, Me.). These mice weighed an
average of 21.4 g at the start of experiments, with a standard
deviation of 1.7 g.
[0200] Bleomycin treatment. This was undertaken on day 0 by
endotracheal instillation through the oral cavity after exposure of
the airway by pulling the tongue. Each mouse received 0.001 units
of bleomycin (Bristol-Myers, Evansville, Ind.)/gm body weight in 30
.mu.l of sterile saline, while control mice received an equal
volume of sterile saline only.
[0201] TM treatment experiments. TM was given in 0.25 ml of water
by intragastric gavage once daily, in the doses and times indicated
in the various studies as described below.
[0202] Three experiments were carried out. In experiment 1, the
effect of TM administered before the administration of bleomycin
was examined; thus, the efficacy of TM as a prophylactic was
evaluated. In experiment 2, the effect of TM at different doses
after bleomycin administration was examined. In experiment 3, the
effect of starting the administration of TM at various times before
and after the administration of bleomycin was examined.
[0203] Copper status. In the presence of TM therapy, copper status
is difficult to assess by measuring serum copper directly, since a
slowly turning over tripartite complex of TM, copper, and albumin
accumulates, causing the serum copper to be elevated even though
availability of copper is decreasing. However, the inventors have
previously determined that serum ceruloplasmin (Cp) is a good
surrogate marker of copper status (G J Brewer et al., Clin. Cancer,
6: 1-10 [2000]), because the liver secretes this copper containing
protein into the blood at a rate dependent upon copper
availability. Copper status was followed by assaying serum
ceruloplasmin (Cp), by measuring its oxidase activity (K H
Schosinsky et al., Clin. Chem., 20(12)1556-1563 [1974]). Blood was
obtained from the tail vein of the mice. To avoid excessive
bleeding, only one mouse from each group was bled at any time point
during the study, and mice were rotated so that different mice were
bled at the different time points. A Cp assay in all mice was done
at time of sacrifice.
[0204] Hydroxyproline assay. The extent of fibrosis was assessed by
assaying hydroxyproline content of whole lung homogenates at the
time of sacrifice as previously described (M Gharaee-Kermani et
al., J. Leukoc. Biol., 64:657-666 [1998]). Results are expressed as
.mu.g hydroxyproline/lung (the lung tissue included both
lungs).
[0205] Microscopic evaluation of the lungs. For morphological
evaluation of fibrosis, lungs were inflated with formalin at the
time of sacrifice, and after overnight fixation were embedded in
paraffin and sections prepared for H&E staining as well as for
Masson-trichrome staining for evaluation of collagen deposition as
previously described (M Gharaee-Kermani et al., J. Leukoc. Biol.,
64:657-666 [1998]).
[0206] Statistics. For comparisons of means, ANOVA was used
followed by Scheffe's test for multiple comparisons when
appropriate. For the dose-response study, regression analysis was
used to evaluate statistical significance. For the third study,
varying time of TM initiation, the standard error was calculated
for each group and a 95% confidence interval for the mean of each
group was determined.
B. Results
Experiment 1
[0207] In this experiment, the effect of TM administered before the
administration of bleomycin was examined; thus, the efficacy of TM
as a prophylactic was evaluated.
[0208] There were four experimental groups of five to seven mice
each. Group 1 received bleomycin, group 2 was a saline control,
group 3 received bleomycin and TM therapy, and group 4 was a TM
therapy control. The TM was given in a dose of 0.7 mg/mouse/day
beginning 7 days prior to bleomycin treatment. During days 9 to 11
after bleomycin, the TM-treated mice received 1.2 mg of TM daily,
to ensure adequate lowering of copper levels, and then received 0.7
mg daily for the duration of the study. The mice were sacrificed 21
days after bleomycin treatment.
[0209] The results from Experiment 1 are shown in Table 1. At the
time of sacrifice, the mean body weight of bleomycin treated
animals (group 1) was significantly less than that of saline
controls (group 2). TM treatment protected against some of the
bleomycin induced weight loss (Table 1), as shown by the lack of a
significant difference in weight between the bleomycin/TM (group 3)
and the saline (group 2) means. TM alone (group 4) tended to
produce some weight loss in experiment 1 (Table 1).
[0210] The mean hematocrit of bleomycin treated animals (group 1 of
Table 1) was significantly increased compared to the other three
groups, probably due to hemoconcentration from not drinking
adequate water near the end of the 21 days.
[0211] The mean ceruloplasmin level of bleomycin/TM mice (group 3
of Table 1) was about 55% that of bleomycin animals (group 1), and
the two were significantly different. TM alone (group 4) resulted
in a mean ceruloplasmin about 80% of saline controls, but this
difference didn't reach statistical significance.
[0212] The hydroxyproline results of experiment 1 are also shown in
Table 1. Therapy with TM almost completely abrogated fibrosis as
measured by this assay. Bleomycin treatment (group 1) produced a
highly significant increase in hydroxyproline compared to saline
controls (group 2), but there was no significant difference between
the TM-treated bleomycin (group 3) and the saline group 2, and the
means were very close to one another. There was a highly
significant difference in the mean values between bleomycin treated
(group 1) and bleomycin/TM treated (group 3) animals. In this
experiment, TM alone seemed to have some effect on increasing
hydroxyproline levels, an effect which wasn't borne out in
experiments 2 and 3.
TABLE-US-00001 TABLE 1 Data from Experiment 1 at the Time of
Sacrifice Treatment 1. Bleomycin 2. Saline 3. Bleomycin/TM 4. TM N
5 7 6 6 Weight (g) 18.6 .+-. 1.4 24.0 .+-. 0.04 21.2 .+-. 0.8 20.4
.+-. 1.0 Hematocrit 55.0 .+-. 2.7 44.8 .+-. 0.2 39.7 .+-. 2.6 44.1
.+-. 1.0 Ceruloplasmin (I.U) 25.3 .+-. 2.8 22.7 .+-. 1.1 13.9 .+-.
3.3 18.1 .+-. 3.4 Hydroxyproline (.mu.g/lung) 252 .+-. 16 156 .+-.
9 162 .+-. 12 193 .+-. 5
TABLE-US-00002 Statistical Analysis Weight Hematocrit Ceruloplasmin
Hydroxyproline P p* p p* p p* p p* 1. Bleomycin versus2. Saline
0.001 0.004 0.001 0.005 0.183 0.90 0.001 0.001 1. Bleomycin versus3
0.06 0.32 0.001 0.001 0.01 0.06 0.001 0.001 Bleomycin/TM 3.
Bleomycin/TM versus2. Saline 0.16 0.62 0.001 0.004 0.06 0.38 0.674
0.980 4. TM versus2. Saline 0.002 0.07 0.24 0.99 0.1 0.70 0.018
0.119 *p value with Scheffe's correction for multiple
comparisons.
[0213] The lung histopathology results from experiment 1 bear out
the hydroxyproline results. Lung sections from bleomycin-treated
and bleomycin plus TM treated mice were examined by staining with
H&E, and photographing at 40.times., 400.times., or 1000.times.
magnification. The results indicate that while scattered patches of
fibrosis and inflammatory cells could still be found in the
TM-treated bleomycin animals, these were substantially smaller with
lesser degrees of cellular infiltration compared to the animals
treated with bleomycin alone. Sections stained with
Masson-trichrome (which is a stain for collagen) revealed much less
collagen deposition in the TM treated group relative to mice
receiving bleomycin only.
Experiment 2
[0214] In this experiment, the effect of TM at different doses
after bleomycin administration was examined. Experimental groups of
four bleomycin-treated and two to four control (non-bleomycin
treated) mice were given varying doses of TM. Initially, All
TM-treated mice received identical loading doses of 1.2
mg/mouse/day for 3 days (minus 5 to minus 3 days) prior to
bleomycin administration. From that point on, groups of mice were
given 0.3, 0.5, 0.7, or 0.9 mg of TM/mouse/day until sacrifice at
day 21 after bleomycin treatment. A group of four bleomycin treated
and a group of four non-bleomycin treated mice received no TM.
[0215] A significant protection against the weight loss caused by
bleomycin was provided by TM treatment (as shown in FIG. 1).
Protection against weight loss from bleomycin by TM is generally
related to the dose of TM. The 0.9 mg TM dose is fully protective,
with a weight curve similar to saline controls, whereas 0.3 mg TM
is only slightly protective. Doses of 0.5 mg and 0.7 mg TM are
intermediately protective.
[0216] Table 2 shows the effect of varying TM dose on ceruloplasmin
levels (data at 8 and 14 days are from single mice; data from 21
days are at the time of sacrifice, and represent the mean and
standard error of four mice in each group). At the end of the
experiment, all four TM treatment groups show relatively low
ceruloplasmin levels. However, at intermediate time points, the 0.9
mg dose shows low levels, the 0.3 mg dose relatively normal levels,
and the 0.5 mg and 0.7 mg doses intermediate levels.
[0217] Finally, the protection against bleomycin-induced fibrosis
as measured by hydroxyproline accumulation is dose dependent, as
shown in Table 3 and FIG. 2. Regression of the hydroxyproline mean
values against TM doses from 0.3 mg to 0.9 mg TM give a highly
significant F statistic (<0.002). Comparison of the individual
0.7 mg and 0.9 mg TM dose means against the bleomycin mean by t
test showed both to be significantly different, whereas the 0.5 mg
dose was not. In this experiment there was no effect of TM
treatment alone on hydroxyproline levels (open circles of FIG. 2).
The histopathological evaluation generally reflects the biochemical
analysis, with diminution of fibrotic lesion size with increasing
dose of TM.
TABLE-US-00003 TABLE 2 Ceruloplasmin Values (International Units)
of TM-Treated Animals During Experiment 2 Days TM Maintenance 8*
14* 21* Dose (mg/day/mouse) Ceruloplasmin Values 0.0 20.8 30.2 22.8
.+-. 2.8 0.3 20.3 23.6 5.4 .+-. 2.7 0.5 14.4 10.6 4.5 .+-. 2.7 0.7
12.6 15.5 4.7 .+-. 2.2 0.9 0.6 3.7 1.8 .+-. 0.7
TABLE-US-00004 TABLE 3 Lung Hydroxyproline Results of Experiment 2
0 TM 0.3 TM 0.5 TM 0.7 TM 0.9 TM Bleomycin Group N 4 4 4 4 4
Hydroxyproline 217 .+-. 8 232 .+-. 4 197 .+-. 15 172 .+-. 22 157
.+-. 14 (Mean .+-. SE) Control Group (Saline only) N 4 2 3 3 2
Hydroxyproline 126 .+-. 14 132 .+-. 3 130 .+-. 5 152 .+-. 10 130
.+-. 8 (Mean .+-. SE)
TABLE-US-00005 Statistical Analysis (t Tests) (Regression of
hydroxy-proline values on TM Dose from 0.3 to 0.9) F Statistic =
14.8; P < 0.002 Comparison t value p value p value * Bleo/0 TM
versusBleo/0.3 TM 0.71 0.485 0.970 Bleo/0 TM versusBleo/0.5 TM 1.00
0.333 0.905 Bleo/0 TM versusBleo/0.7 TM 2.26 0.039 0.323 Bleo/0 TM
versusBleo/0.9 TM 3.01 0.009 0.110 Saline/0 TM versusBleo/0.9 TM
1.72 0.098 * p value with Scheffe's correction for multiple
comparisons.
Experiment 3
[0218] In this experiment, the effect of starting the
administration of TM at various times before and after the
administration of bleomycin was examined.
[0219] All experimental groups of mice treated with TM received a
four day loading dose of 1.2 mg/mouse/day, and then 0.9
mg/mouse/day until sacrifice at day 21 after bleomycin treatment.
However, the starting time of TM treatment was varied beginning
with 5 days prior to bleomycin in the first experimental group,
then 4, 7 and 14 days after bleomycin in experimental groups two,
three, and four, respectively. All experimental groups contained
five mice at the beginning. A control group of five mice received
neither bleomycin nor TM.
[0220] A significant suppression of hydroxyproline production was
observed if the TM was given before bleomycin, or started at day 4
or 7 after bleomycin. Regression of mean hydroxyproline levels on
the day therapy was started gave an F statistic of 21, p<0.05.
Since the number of animals in each group was relatively small, the
groups +day 4 and +day 7 were combined, and the mean compared to
the bleomycin control group, which yielded a significant t test
(p=0.05). As in experiment 2, there was no effect of TM alone on
hydroxyproline levels (open triangles of FIG. 3). These results
show that the sooner TM was started, the more effective it was in
protecting against hydroxyproline accumulation.
[0221] The levels of cytokines, which are believed to be involved
in pulmonary fibrosis, are also measured.
[0222] In summary, TM treatment completely abrogated fibrosis and
markedly attenuated inflammation in a model that is directly
relevant to ARDS and pulmonary fibrosis patients.
Example 2
Inhibition of TGF.beta., and TNF.alpha. by Tetrathiomolybdate in
the Bleomycin Model of Pulmonary Fibrosis
[0223] This example examines the mechanisms by which TM inhibits
fibrosis in the bleomycin mouse model. This example, and the
experiments described herein, focus on evaluating the possible
inhibition by TM of the action of TGF.beta., and TNF.alpha., which
have been shown to be important in the pathogenesis of fibrosis in
the bleomycin model
A. Methods
[0224] Mice. Female CBA/J mice at 8-10 weeks old, were from the
Jackson Laboratories (Bar Harbor, Me.). At the start of the
experiments, the mice weighed between 20-25 g.
[0225] Bleomycin treatment. Briefly, bleomycin was administrated on
day 0 by means of endotracheal instillation through the oral cavity
after exposing the mouse's airway by pulling the tongue. Each mouse
received 0.001 units/gm body wt of bleomycin (Bristol-Myers,
Evansville, Ind.) in 30 .mu.l sterile saline solution. Control mice
were administrated an equal volume of sterile saline solution.
[0226] TM treatment experiments. TM was given in 0.25 ml of water
once daily by means of intragastric gavage in the doses and times
indicated in the various studies.
[0227] Three experiments were carried out. Experiment 1 comprised
four groups of three mice each. Group 1 received bleomycin only,
group 2 received bleomycin and TM therapy, group 3 received saline
in the trachea rather than bleomycin, and group 4 received TM
therapy only. The mice in groups 2 and 4 each received 1.2 mg of TM
per day for four days prior to bleomycin, and then were given 0.9
mg TM per day until sacrifice at seven days after bleomycin.
[0228] Experiment 2 comprised four groups of five mice each. The
four groups were assigned as in experiment 1 to bleomycin,
bleomycin and TM, saline rather than bleomycin, and TM only. Groups
2 and 4 were started on TM five days after bleomycin treatment at a
dose of 1.2 mg per day for four days, and then 0.9 mg per day until
sacrifice at day 21.
[0229] Experiment 3 involved variable starting times of TM and
comprised five groups of mice. All of the mice received bleomycin.
Group 1 (4 mice) received no TM. Mice in groups 2 through 5
received a 4 day loading dose of 1.2 mg TM/day, then 0.9 mg/day
until sacrifice at day 21. However, starting times of TM treatment
were varied, beginning 5 days before bleomycin in Group 2 (TM-5, 4
mice), then beginning coincident with bleomycin in group 3 (TM+0, 2
mice), beginning 4 days after bleomycin in group 4 (TM+4, 4 mice),
and beginning 7 days after bleomycin in group 5 (TM+7, 5 mice).
[0230] Copper status. In the presence of TM therapy, copper status
cannot be assessed by direct measurement of serum copper because of
the accumulation of a tripartite complex of TM, copper, and albumin
that turns over slowly, causing the serum copper to be increased
even though availability of copper is decreasing. However, it was
found that serum ceruloplasmin is a good surrogate marker of copper
status because the liver secretes this copper-containing protein
into the blood at a rate dependent on copper availability. Copper
status was monitored by assaying serum ceruloplasmin on the basis
of its oxidase activity in blood from the tail vein. To avoid
excessive bleeding, one mouse from each group was bled at each time
point; mice were rotated so that different mice were bled. A
ceruloplasmin assay was conducted in each mouse when it was
killed.
[0231] Hydroxyproline assay. The extent of fibrosis was assessed by
assaying hydroxyproline content of whole-lung homogenates at the
time of sacrifice as described in Gharaee-Kermani, et al., J.
Leukoc. Biol., 64:657-666 (1998). Hydroxyproline was expressed as
micrograms of hydroxyproline per mouse lung (the lung tissue
included both lungs).
[0232] Cytokine assays. Expression of TNF.alpha. and TGF.beta. was
determined in lung tissues from the various experiments. TNF.alpha.
mRNA was measured in total RNA isolated from lung tissue
homogenates. Primer Express 2.0 software (Applied Biosystems,
Foster City, Calif.) was used to design Taqman primers and MGB
probes (6-FAM conjugated) for TNF.alpha., which were then purchased
from Applied Biosystems (PE/ABI, Foster City, Calif.). Primers and
probes for GAPDH were purchased from PE/ABI. GAPDH mRNA was used as
internal control to normalize the amount of input RNA. One-step
real time RT-PCR was undertaken with Taqman One Step RT-PCR Master
Mix (PE/ABI) using a GeneAmp 5700 Sequence Detection System
(PE/ABI). Results were expressed as the threshold cycle (CT) at
which an increase of reporter fluorescence (.DELTA.Rn) can first be
detected. The levels of TNF.alpha. mRNA were normalized to the
internal control GAPDH signals and expressed as
2.sup.-.DELTA..DELTA.CT.
[0233] Lung TGF.beta. levels were measured using either ELISA with
a kit from R&D Systems (Minneapolis, Minn.), or using a cell
line stably transfected with a plasminogen activator inhibitor-1
(PAI-1) promoter-luciferase construct (M Abe et al., Analytical
Biochemistry, 216:276-284 [1994]). Briefly for the latter assay,
mink lung epithelial cells transfected with the PAI-1 promoter
construct were incubated with the indicated activated (by
pre-acidification) test samples diluted (1:2 dilution) in fresh
media. After a 24 hr incubation, the cells were lysed with reporter
lysis buffer (Promega, Madison, Wis.). Luciferase activity was
measured by the luciferase assay system (Promega) and read using a
Reporter microplate luminometer (Turner Designs, Sunnyvale,
Calif.). Human TGF.beta.1 (R&D Systems) was used as a
standard.
[0234] .alpha.-Smooth muscle actin assay. De novo appearance of
myofibroblasts is a hallmark of active fibrosis, and these cells
are known as the primary source of interstitial collagen in
pulmonary fibrosis (See e.g., K Zhang et al., Am. J. Pathol,
145:114-125 [1994]). Since .alpha.-smooth muscle actin is a marker
of myofibroblast differentiation, the level of this protein in lung
tissue homogenates was measured by ELISA as before (use same
reference above for TGF.beta. assay by ELISA).
[0235] Statistical analysis. For comparisons of means, Fisher's t
test was used and analysis of variance, followed by Scheffe's test
for multiple comparisons when appropriate. For varying starting
times for TM (Experiment 3), regression analysis was also used to
evaluate statistical significance.
B. Results
Experiment 1
[0236] In this experiment, TM was started in TM treated animals
four days prior to bleomycin and continued until the animals were
sacrificed at day 7, the point at which TNF.alpha. levels and the
inflammatory response is at its peak.
[0237] At the time of sacrifice, mean Cp levels in TM treated
groups were approximately half of the means in the non-TM treated
groups, and using Scheffe's corrections for multiple comparisons
were significantly lower in the TM treated groups (p<0.001).
This indicates that the copper status in TM treated animals was
lowered appropriately. The mean hematocrit levels in the four
groups at the time of sacrifice were generally similar, with the
value in the bleomycin group slightly higher than the others,
generally attributable to the bleomycin group drinking less as they
begin to get ill. In keeping with this, the mean weight of the
bleomycin group was 2-3 g less than the means of the other three
groups, and was significantly less in all three cases, using
Scheffe's correction (p=0.001 to 0.04). The mean weight in the
bleomycin/TM group was almost exactly the same as the saline
control group demonstrating that TM protected against this aspect
of the bleomycin-induced illness.
[0238] TNF.alpha. mRNA levels in the lungs from the four groups of
animals of Experiment 1 are shown in FIG. 5. The mean lung
TNF.alpha. mRNA levels were markedly and significantly elevated in
bleomycin treated animals versus controls. TM therapy in bleomycin
treated animals almost completely, and significantly, inhibited
this increase in levels. TNF.alpha. protein levels were not
detectable in any of the samples by ELISA using commercially
available kits.
[0239] TGF.beta. protein levels in the lungs of the four groups of
animals of Experiment 1 are shown in FIG. 6. The mean lung
TGF.sub..beta. level was elevated in bleomycin treated animals
compared to controls. TM therapy in bleomycin treated animals
inhibited this increase in response to bleomycin, and the mean
levels in bleomycin versus bleomycin/TM were very close to
statistical significance (p=0.06).
[0240] Lysyl oxidase in the lungs of the four groups of animals was
also measured. There were no significant differences in the means
of the four groups. Specifically, activity was not inhibited in the
TM treated animals compared to non-TM treated animals.
Experiment 2
[0241] In this experiment TM was started in TM treated animals five
days after bleomycin and continued until day 21, when all animals
were sacrificed. The objective was to allow the inflammatory
reaction to peak, which occurs at about day seven, prior to copper
levels dropping into the target range, which is about four days
after TM is started, or day nine of this experiment. Then, at the
time of sacrifice, TGF.beta. was to be measured to test whether it
is inhibited by TM therapy when the inflammatory reaction is
allowed to occur.
[0242] At the time of sacrifice, mean Cp levels in the bleomycin/TM
group was 38% of the mean of the bleomycin group and the two means
were significantly different using Scheffe's correction
(p<0.0001). Similarly, the mean Cp of the TM control group was
50% of the mean of the saline control group, and the two means were
significantly different using Scheffe's correction (p<0.0001).
Thus, the copper status of the TM treated animal was appropriately
lowered by TM therapy. The mean hematocrit of the bleomycin group
was 51% versus44.3 in the bleomycin/TM, 44.2 in the saline, and
43.8 in the TM groups. The mean of the bleomycin group was
significantly different than the means of the other three groups
using Scheffe's correction (p=0.0001 to 0.0002). The higher
hematocrit in the bleomycin group is generally attributable to the
bleomycin group drinking less as they become ill, and TM completely
protected against this effect of bleomycin. In keeping with this,
the mean weight of the bleomycin group at the time of sacrifice was
19.1 g, significantly less than the mean of the saline control
which was 24.5 (p<0.0002). However, in contrast to earlier
experiments where TM completely protected against this weight loss
(e.g. Experiment 1 and studies in reference 20), in this experiment
TM only slightly protected against weight loss, the mean in the
bleomycin/TM group being 19.6, significantly lower than either the
saline or the TM control groups (p=0.0003 to 0.006).
[0243] TGF.beta. protein levels in the lungs from the four groups
of animals of Experiment 2 are shown in FIG. 7. The mean
TGF.sub..beta. levels were almost three times as high in bleomycin
treated animals compared to controls. TM therapy in
bleomycin-treated animals completely inhibited this increase in
TGF.sub..beta. levels, but because of relatively high variances and
small sample sizes, the results were not statistically
significant.
[0244] SMA (.alpha.-smooth muscle actin) protein levels in the
lungs from the four groups of animals of Experiment 2 are shown in
FIG. 8. The mean SMA levels were significantly increased in
bleomycin treated animals compared to saline-treated controls. TM
therapy in bleomycin-treated animals inhibited most of the increase
in SMA levels brought about by bleomycin, and was close to
statistical significance (p=0.09).
[0245] Hydroxyproline levels in the lungs from the four groups of
animals of Experiment 2 are shown in FIG. 9. The mean
hydroxyproline levels were significantly elevated in
bleomycin-treated animals compared to controls. TM therapy in
bleomycin-treated animals completely and significantly inhibited
the increase in hydroxyproline levels brought about by
bleomycin.
Experiment 3
[0246] In this experiment the starting time of TM therapy was
varied. The mean TGF.beta. protein levels in the lungs of the
animals from this experiment are shown in FIG. 10. TM started prior
to bleomycin (Bleo/TM-5) significantly inhibited TGF.beta. levels
when the means were compared (Bleo/TM-5 versus Bleo, p=0.04). This
effect lessened as the starting time of TM therapy was made later
and later, producing a significant regression for Bleo/TM-5,
Bleo/TM+0, Bleo/TM+4, and Bleo/TM+7 data points (p=0.05).
Example 3
Treatment of Chronic Pulmonary Fibrosis Clinical Trial (Treatment
Protocol)
[0247] A protocol is designed to treat patients with chronic
pulmonary fibrosis. The initial protocol is a phase I/II trial of
TM treatment in patients with usual interstitial pneumonia
refractory to previous therapy.
[0248] Idiopathic interstitial pneumonias (IIP) are part of a group
a diffuse parenchymal diseases including usual interstitial
pneumonia (UIP/IPF), respiratory bronchiolitis interstitial lung
disease, cryptogenic organizing pneumonia, alveolar macrophage
pneumonia, acute interstitial pneumonia, lymphocytic interstitial
pneumonia and nonspecific interstitial pneumonia. Usual
interstitial pneumonia (also referred to as idiopathic pulmonary
fibrosis, IPF) is the most common type of IIP and is associated
with the worst prognosis. The median survival for patients with
(UIP) is 2-4 years from the time of diagnosis. UIP typically
affects people 40 and 70 years of age with over two-thirds being
over the age of 60 at the time of diagnosis. There does not appear
to be a specific predilection for a particular ethnicity or race
although IPF may be more common in males.
[0249] Recent studies in the bleomycin mouse model have shown an
antifibrotic and antiinflammatory effect of TM therapy (as
described in Example 1). As described above, TM is an anticopper
drug developed for the treatment of Wilson's disease. TM has also
been shown to produce an antiangiogenic effect in non-Wilson's
disease patients with cancer, and in animal tumor models, by virtue
of lowering systemic copper levels. Angiogenesis, the ability to
grow new blood vessels, is believed to be one component required
for the progression of the fibrotic response that is typical of
IPF. Thus, the rationale for a trial of TM in this disease is based
upon its successful use in the bleomycin mouse model, and its
antifibrotic and antiinflammatory properties, as well as its
antiangiogenic properties.
[0250] Up to twenty patients with UIP that have demonstrated
disease progression with standard therapy are enrolled in the study
during the first year. The patients initially receive TM as an
induction dose to induce a reduction of ceruloplasmin (Cp) levels;
after induction of ceruloplasmin (Cp) to the target range of 5-15
mg/dl of serum, the patients then receive maintenance TM doses to
maintain that Cp target. Continued treatment of the patients occurs
until one of several events occurs: serious further progression of
the pulmonary disease; recovery; or disease stabilization for an
extended period of time.
A. Protocol for Patient Selection
[0251] Only patients with UIP/IPF not associated with any known
precipitating cause are eligible for this study. Patients with
suspected unsual interstitial pneumonia (UIP) are eligible for
initial entry into the study. The criteria for diagnosis are
illustrated in FIG. 1 and Tables 4-6. In general, the diagnosis
will be considered in the setting of one of the following: 1) an
appropriate clinical picture plus a typical histologic picture on
surgical lung biopsy as described in Table 4; or 2) an appropriate
clinical and radiographic picture with bronchoscopic exclusion of
an alternative process as described in Table 5.
[0252] In addition to fulfilling either criteria 1 or 2 above,
patients should meet the inclusion/exclusion requirements listed in
Table 6. Table 7 provides Criteria for disease progression after at
least six months of standard therapy considered in some embodiments
of the present invention.
TABLE-US-00006 TABLE 4 Histologic features of UIP Pertinent
positive features: Remodeling of lung architecture with dense
fibrosis and frequent `honeycomb` fibrosis Fibroblastic foci
typically at the edges of dense scars Patchy involvement and
temporal heterogeneity Distribution which is frequently subpleural,
paraseptal and/or bronchovascular Pertinent negative features:
Absence of active lesions typical of other interstitial diseases
Absence of marked interstitial, chronic inflammation Inconspicuous
or absent granulomas Absence of substantial inorganic dust deposits
(except carbon black pigment) Absence of marked tissue
eosinophilia
TABLE-US-00007 TABLE 5 Adapted ATS criteria for the diagnosis of
UIP in the absence of a surgical lung biopsy Major criteria:
Clinical: Exclusion of other known cause of ILD including collagen
vascular illness, environmental or drug exposure Roentgenographic:
Diffuse reticulonodular pattern on chest x-ray without adenopathy
HRCT features including: Bibasilar interstitial and intralobular
reticular opacities Irregular interlobular septal thickening with
or without traction bronchiectasis Subpleural honeycombing in the
lower lobes Limited ground-glass opacity and no pleural
abnormalities Absence of micronodules, peribronchial nodules,
consolidation, isolated non-honeycomb cysts(5, 6) Physiologic:
Reduced total lung capacity and/or diffusion capacity INCREASED
P(A-A)O.sub.2 AT REST OR WITH EXERCISE Morphological:
Transbronchial biopsy or bronchoalveolar lavage excluding alternate
diagnosis Minor criteria: Age >50 years Insidious onset of
unexplained dyspnea Duration of illness for .gtoreq.3 months
Bibasilar, inspiratory rales
The presence of all major criteria and three of the four minor
criteria increases the likelihood of an accurate diagnosis of
UIP.
TABLE-US-00008 [0253] TABLE 6 Additional inclusion and exclusion
criteria for UIP patients Inclusion criteria Clinical criteria:
Disease progression after at least six months of standard therapy
(Table. 4) Taking <15 mg prednisone for at least 30 days prior
to screening Age 35-80, inclusive Able to understand a written
informed consent and comply with the study protocol Exclusion
criteria Clinical criteria: Significant environmental exposure
Diagnosis of collagen vascular disease Evidence of active infection
Clinically significant cardiac disease Myocardial infarction,
coronary artery bypass or angioplasty within 6 months Unstable
angina pectoris Congestive heart failure requiring hospitalization
within 6 months Uncontrolled arrhythmia Poorly controlled or severe
diabetes mellitus Pregnancy or lactation Women of childbearing
potential not using a medically approved means of contraception
(i.e. oral contraceptives, intrauterine devices, diaphragm,
Norplant .RTM.) Prior treatment with cytotoxic drugs within 6 weeks
Investigational therapy within 6 months of screening Therapy with
zileuton within 6 months of screening Physiologic criteria:
FEV.sub.1/FVC <0.60 Laboratory criteria: Total bilirubin
>1.5.times. upper limit normal AST or ALT >3.times. upper
limit normal Alkaline phosphatase >3.times. upper limit normal
White blood cell count <2,500/mm.sup.3 Hematocrit <30%
Platelets <100,000/mm.sup.3 PROTHROMBIN TIME INR >1.5
TABLE-US-00009 TABLE 7 Criteria for disease progression after at
least six months of standard therapy Disease Progression (any
Standard Therapy: one of the following): prednisone: .gtoreq.1800
mg administered Worsening dyspnea at rest within a six-month period
or with exertion azathioprine: six month course of .gtoreq.10%
decrease in percent therapy predicted FVC cyclophosphamide: six
month course .gtoreq.20% decrease in percent of therapy predicted
DLCO Worsening CXR or HRCT
B. Baseline Studies
[0254] All patients undergo a screening history and physical
examination. The physician discusses therapeutic options with the
patient including standard therapy, experimental protocols, and the
potential for consideration of lung transplantation. During this
visit the inclusion/exclusion criteria are reviewed.
[0255] Flexible fiberoptic bronchoscopy (FFB) is performed in those
patients with suspected UIP using standard technique.
Bronchoalveolar lavage (BAL) is performed using standard technique
(described below) and transbronchial biopsy (TBBx) is performed
using fluoroscopic guidance. Tissue is obtained from a
radiologically affected region of lung parenchyma. Patients are not
asked to undergo a second bronchoscopy if their first bronchoscopy
was performed at an outside institution. The purpose of this
bronchoscopy is to rule out diseases other than UIP (Table 5) as
the tissue samples obtained by bronchoscopy are usually too small
to demonstrate the histopathologic features of UIP (Table 4). BAL
involves the instillation of small volumes of saline solution
through the bronchoscope after it is wedged into a segmental
bronchus. The fluid is immediately aspirated back through the
bronchoscope by suction and collected in a vacuum trap. The
effluent contains cells that are extracted by centrifugation. TBBx
involves the passage of a flexible biopsy forceps through the
bronchoscope and into the pulmonary parenchyma. The location of the
biopsy forceps is noted under fluoroscopy and the biopsy forceps
are positioned appropriately in involved areas as determined by
chest x-ray and HRCT. Five to eight TBBxs are normally obtained
during the course of a diagnostic evaluation for these diseases.
Each biopsy specimen is approximately three millimeters in
diameter. Only those patients who, in the judgment of the pulmonary
physician performing the bronchoscopy, are in stable medical
condition following the procurement of the necessary diagnostic
biopsies undergo additional transbronchial biopsies.
[0256] Surgical lung biopsy specimens are obtained from patients
undergoing surgical lung biopsy as part of the diagnostic work up
for IIP. Lung biopsies are obtained from sites which demonstrate
the following radiographic characteristics: 1) normal appearance,
2) ground glass opacity, and 3) fibrotic disease. When technically
feasible, biopsies are obtained from all lobes in the biopsied
lung. Patients are not required to undergo a surgical lung biopsy
if they can meet the diagnostic criteria (outlined above) for
UIP/IPF without a surgical lung biopsy. For patients that have
undergone a surgical lung biopsy at another institution, their
slides are reviewed to confirm the diagnosis of UIP prior to entry
into this trial.
[0257] A baseline dyspnea index (BDI) is collected using the
techniques described by Mahler et al at the time of initial
evaluation. The transitional dyspnea index (TDI) is administered at
times of follow-up.
[0258] A general measure of the patient's perceived health and
daily activities is assessed using Short form 36 question (SF-36)
instrument. Multiple dimensions are assessed, including physical
function, role limitation caused by physical impairment, bodily
pain, general health, vitality, social function, role limitation
caused by emotional impairment, and mental health. The use of this
instrument has been validated in patients with interstitial lung
disease.
[0259] The St. George's Respiratory Questionnaire (SGRQ) disease
specific instrument was designed to assess the impact of
respiratory disease on overall health, daily life and perceived
well-being of the patient. It has been validated in patients with
interstitial lung disease. Three components are measured including
respiratory symptoms, impairment of mobility or physical activity,
and the psychosocial impact of disease.
[0260] Pulmonary function testing is performed according to the
guidelines of the American Thoracic Society. The forced vital
capacity (FVC) and the forced expiratory volume in one second are
measured with a recording spirometer and pneumotachograph. The
maximal values from three maneuvers are reported. Thoracic gas
volume (V.sub.TG) is measured with a body plethysmograph. Diffusing
capacity for carbon monoxide is determined by the single breath
method.
[0261] A baseline high resolution computed tomography (HRCT scan is
performed within six months of beginning the study. A
semiquantitative score is generated by two blinded radiologists who
interpret the HRCT using the scoring system enumerated in Table 8.
The score is reported with an alveolar component which describes
the extent of `ground glass opacity` (ranging from 0 to 5) and an
interstitial component which describes reticular densities (ranging
from 0 to 5). The sum is the total score (ranging from 0 to
10).
TABLE-US-00010 TABLE 8 Components of the HRCT Score Alveolar Score
0 no alveolar disease 1 ground glass opacity involving <5% of
the lobe (minimal, but not normal) 2 ground glass opacity involving
up to 25% of the lobe 3 ground glass opacity involving 25-49% of
the lobe 4 ground glass opacity involving 50-75% of the lobe 5
ground glass opacity involving >75% of the lobe Interstitial
Score 0 no interstitial disease 1 thick interlobular septal
thickening; no discrete honeycombing 2 honeycombing (+/-septal
thickening) involving up to 25% of the lobe 3 honeycombing
(+/-septal thickening) involving 25-49% of the lobe 4 honeycombing
(+/-septal thickening) involving 50-75% of the lobe 5 honeycombing
(+/-septal thickening) involving >75% of the lobe *In addition
to the scores above, each lobe will be scored for the
presence/absence of bronchietasis. The number of lymph nodes >1
cm and <1 cm will be recorded.
C. Administration of Study Drug
[0262] The current cancer therapy induction dose for TM is 40 mg
3.times. daily with meals and 60 mg at bedtime separated from food
(180 mg/day). At this dose, it takes 15-25 days to induce cancer
patients into the Cp target range. This same induction dose is used
for IPF patients. All patients remain on their induction doses
until they reach target Cp levels, then are switched to maintenance
therapy.
[0263] 1. Maintenance
[0264] As soon as patients reach target Cp levels, their TM dose is
be dropped to that dose estimated to be required for maintaining Cp
in the target range. Generally, this dose is 20 mg X 2 with meals
and 20 mg HS, but the dose for any particular patient is customized
up or down if necessary. In the absence of unacceptable toxicity,
patients are kept on maintenance TM until it is obvious that the
disease is seriously progressing, or while improving until recovery
is complete, or if the disease persists in a chronic but stable
form (see study endpoints below).
[0265] 2. Monitoring TM Dose
[0266] There are two important types of measurements in terms of
monitoring and adjusting TM dose. The first is measurement of serum
ceruloplasmin (Cp). The Cp level is a surrogate measure of body
copper status and has worked well in all kinds of applications of
TM therapy, including treating patients with cancer. During
induction, Cp is measured weekly. Once a stable maintenance dose of
TM is underway, the frequency of Cp measurements are decreased to
once every two weeks, then once every four weeks if
appropriate.
[0267] The other type of measurement necessary to monitor TM
therapy is blood counts. The first indication of overtreatment is a
mild anemia and/or leukopenia, which is easily correctable by
lowering the dose, or if more severe, by temporarily stopping the
drug. This is rarely seen at Cp levels over 10, but is more
frequently seen between Cp levels of 5 and 10, and is much more
common at Cp levels below 5. Thus, the target range is Cp levels
between 5 and 15 (to give copper-lowering therapy an optimal chance
to suppress fibrosis and inflammation). Weekly blood counts are
done during induction and the early part of maintenance to help
guide the proper therapeutic dose. During the latter part of
maintenance, blood counts are done at the same frequency as Cp
assays. If there is a mild, replicable, but less than 20% drop of
hemoglobin, WBC, or platelets, the TM dose is decreased based on
clinical judgment. If there is a 20% or more drop in hemoglobin,
WBC, or platelets, the drug is temporarily stopped until count
recovery, and then resumed at 75% (or less) of the previous dose.
If there is a recurrence, the drug holiday is repeated and resumed
at 75% of that previous dose. Dosage adjustments are continued
until a dose is reached at which blood counts are not affected.
[0268] 3. Studies Performed at Visit wo (Three Months)
[0269] At this visit, a history and physical are performed.
Patients are asked to complete the transitional dyspnea index, the
SF-36, and the St. George's respiratory questionnaire. Pulmonary
function studies, including a spirometry and diffusing capacity for
carbon monoxide, are performed. Follow-up pulmonary function
testing is standard clinical practice in patients with UIP.
[0270] 4. Studies Performed at Visit Three (Six Months)
[0271] At this visit, a history and physical are performed.
Patients are asked to complete the transitional dyspnea index, the
SF-36, and the St. George's respiratory questionnaire. Pulmonary
function studies including a spirometry, diffusing capacity for
carbon monoxide, and a six minute hall walk, are performed.
Follow-up pulmonary function testing is standard clinical practice
in patients with UIP.
[0272] 5. Studies Performed at Visit Four (Nine Months)
[0273] At this visit, a history and physical are performed.
Patients are asked to complete the transitional dyspnea index, the
SF-36, and the St. George's respiratory questionnaire. Pulmonary
function studies, including a spirometry and diffusing capacity for
carbon monoxide, are performed. Follow-up pulmonary function
testing is standard clinical practice in patients with UIP.
[0274] 6. Studies Performed at Visit Five (Twelve Months)
[0275] At this visit, a history and physical are performed.
Patients are asked to complete the transitional dyspnea index, the
SF-36, and the St. George's respiratory questionnaire. Pulmonary
function studies, including a spirometry and diffusing capacity for
carbon monoxide, are performed. Follow-up pulmonary function
testing is standard clinical practice in patients with UIP. A
follow up HRCT is also performed to evaluate for changes in the
amount of ground glass and/or fibrosis.
[0276] 7. Study Endpoints
[0277] The primary endpoint of the study is a change in pulmonary
function, where a change in pulmonary function is defined as
either: 1) improvement: defined as a 10% or greater increase in FVC
or a 20% or greater increase in DLCO; or 2) worsening: defined as a
10% or greater decrease in FVC or a 20% or greater decrease in
DLCO; or 3) stable: defined as the lack of a 10% change in FVC or a
20% change in DLCO.
[0278] Pulmonary function is monitored at three month intervals.
For patients with improvement, TM treatment is continued until
pulmonary function stabilizes (lack of improvement in FVC or DLCO
of 5% over a 3 month period). Once these improved patients
stabilize, TM is discontinued for a 3 month period to determine
that stopping the drug does not jeopardize recovery. If these
improved patients decline, then TM treatment is restarted. Patients
remaining stable are treated for 12 months, and then the drug is
stopped and pulmonary function is reassessed at three months. If
these stable patients remain stable off drug, the drug is
permanently discontinued. If these stable patients decline, then TM
treatment is reinstituted. Patients worsening on two consecutive
visits with at least 3 months of having Cp in a therapeutic range
are taken off TM.
[0279] Secondary endpoints of the study include: 1) overall
mortality; 2) changes in quality of life (as assessed by the St.
George's respiratory questionnaire and the SF-36); 3) change in
dyspnea as assessed by the baseline and transitional dyspnea
indexes; 4) change in six-minute walk distance; 5) change in oxygen
requirements at rest and with exercise as determined via a
six-minute walk test; and 6) change in the amount of ground glass
and/or fibrosis as measured by HRCT. Criteria for stopping
administration of the study drug include but are not limited to: 1)
patients mortality or reaching a primary end-point as outlined
above; and 2) any unexpected toxicity potentially attributable to
the study drug (i.e., TM).
Example 4
[0280] Treatment of Liver Cirrhosis: Animal Model
[0281] A study was carried out in a mouse model of concanavilin A
(Con A) production of liver cirrhosis. There were four experimental
groups of several mice each. Two groups received ConA, of which one
group also received TM therapy (4 mice) and while the other group
received saline therapy (6 mice), and two other groups did not
receive ConA, of which one group also received TM therapy (3 mice),
while the other group received saline therapy (6 mice). The Con A
was injected intravenously once weekly into mice (0.3
mg/mouse/week), and produced a hepatitis, which is manifested by an
increasing level of alanine transaminase (ALT) enzymes in the
blood. The TM was given once daily by oral lavage in a dose of 0.7
mg/mouse/day beginning 7 days prior to ConA treatment. During days
9 to 11 after Con A, the TM-treated mice received 1.2 mg of TM
daily, to ensure adequate lowering of copper levels, and then
received 0.7 mg daily for the duration of the study. A blood sample
was obtained 28 days after ConA treatment, and the mice were
sacrificed on the same day. Serum alanine transaminase (ALT) was
measured in Sigma-Frankel (SF) units, where one unit is the
formation of 0.000482 umoles of glutamate/minute at pH 7.5 and
25.degree. C.
[0282] TM therapy almost completely inhibits the ConA induced
increase of serum alanine transaminase (ALT) enzymes (as shown in
FIG. 4). The ALT results indicated that TM treatment completely
abrogated inflammation in a model that is directly relevant to
hepatitis. Histological examination showed inflammation and early
bridging fibrosis in the controls, but not in the TM treated
animals.
Example 5
Treatment of Liver Cirrhosis: Animal Model
[0283] This example describes additional TM treatment experiments
in the model Concanavalin (Con A) mouse model of liver damage. As
described previously, in the Con A mouse model, intravenously
administered Con A produces an inflammatory reaction and cell
damage in the animal's liver that is marked by the release hepatic
transaminase enzymes, such as amino leucine amino transferase
(ALT), into the blood. Several different experiments are reported
in this example
Experiment 1
[0284] Four serial injections of Con A at weekly intervals were
given to the four groups of mice shown in the Table 9. Twenty-four
hours after each of the first three injections, one mouse from each
group was bled, and serum ALT measured. In this experiment, TM was
started in the TM treated mice before the first Con A injection.
After the fourth injection of Con A, all the mice were bled for ALT
and Cp measurements and the experiment terminated. The Cp levels in
TM treated mice were 25-50% of non-TM treated animals. The ALT
results (Table 9) show that after each injection, there is a marked
increase 24 hours later in serum ALT due to Con A, and that this
effect is almost completely blocked by TM therapy. (Statistical
evaluation (t test with Scheffe's correction); means marked "1" are
significantly different (p=0.0001); means marked "2" are
significantly different (p=0.001); means marked "3" are not
significantly different (p=0.4); ALT levels are expressed in
Sigma-Frankel units; each Sigma-Frankel unit equal 0.48 of an
International Unit).
TABLE-US-00011 TABLE 9 Serum ALT Results in Mice 24 Hours After
Each of Four Weekly Serial Injections of Con A Injection Number
4.sup.th Animal Type 1st 2nd 3rd n Mean S.D. Saline Control 35 44
57 6 41.sup.1, 3 5.2 TM only control 85 39 49 3 38 6.2 Con A only
179 265 361 6 168.sup.1, 2 47.9 Con A + TM 52 50 63 4 74.sup.2, 3
10.6
Experiment 2
[0285] In a second experiment, Con A was given in weekly injections
for four weeks, prior to any treatment with TM. The objective was
to see if TM treatment would be useful after considerable organ
damage had been allowed to occur. Then, one group of Con A treated
animals were initiated on TM therapy. The results are shown in FIG.
11. With TM treatment, the ALT values returned to normal, in spite
of continued Con A injections.
Experiment 3
[0286] In a third experiment, two Con A injections one week apart
were given to 10 mice. Five of the mice had begun TM treatment 5
days prior to the first injection. All the mice were sacrificed 2
hrs after the last Con A injection. Plasma levels of TNF.alpha.
were significantly lower (p=0.03) in the TM treated animals, and
plasma levels of IL-1.beta. (interleukin-1.beta., another
inflammatory cytokine) were close to being significantly lower
(p=0.15) in the TM treated group.
Example 6
TM in the Carbon Tetrachloride (CT) Animal Model of Liver
Damage
[0287] The carbon tetrachloride (CT) mouse model produces much more
liver fibrosis (cirrhosis) than the Con A model. In this model
carbon tetrachloride is injected intraperitoneally twice weekly,
and after 12 weeks, a well-established cirrhosis should be present.
This can be evaluated histologically, and can be quantitated by
measuring liver hydroxyproline, a major amino acid in collagen. The
TM was started in TM treated animals at the beginning of week 5 and
the animals were sacrificed at week 12.
[0288] The serum ALT data from this experiment at the time of
sacrifice at 12 weeks are shown in FIG. 12. There were 4 animals in
the CT group, 4 in the CT/TM group, 2 in the olive oil/TM group,
and 1 in the TM group. Since there were so few animals in the last
two groups, and since their data were generally similar, they were
combined. The mean of the CT/TM group was significantly lower
(p<0.0008) than that of the CT group showing significant
protection against liver injury by TM. However, the CT/TM group
mean was significantly higher than the control mean (p<0.004)
showing that the protection was only partial.
[0289] The liver hydroxyproline data from this experiment after
week 12 are shown in FIG. 13. TM clearly blocks all fibrosis, as
evaluated by hydroxyproline. The CT/TM mean was significantly
(p=0.001) lower than the CT mean, and there was no difference
between the CT/TM and control means. This is corroborated by the
histological data. The CT control has a well established cirrhosis
whereas the CT/TM treated liver is essentially normal.
Example 7
Treatment of Renal Fibrosis: Animal Model
[0290] A study is carried out in an animal model of renal fibrosis.
After kidney injury of almost any type, a diffuse interstitial
fibrosis (believed to be due to over-activity of the TGF.sub..beta.
pathway) produces kidney failure.
[0291] Rats are made diabetic by administering streptozotocin;
renal fibrosis is typically well developed by 60 days after
initiation of streptozotocin administration. TM therapy is
initiated at varying times relative to streptozotocin
administration in several groups of rats, and the results compared
to control groups not receiving TM. Typically, at least three mice
are present in each experimental group. In one group, TM therapy is
initiated prior to streptozotocin administration, to determine
whether TM therapy mitigates the pancreatic damage, as it mitigates
hepatitis/cirrhosis in the concanavilin A mouse model described
above. In other groups, TM is started after streptozotocin
administration, at various times, to determine if it can mitigate
the renal fibrosis that develops after streptozotocin
administration.
[0292] The presence and extent of renal fibrosis is determined. The
results show that TM therapy mitigates pancreatic damage if
administered before diabetes is induced, and that TM therapy after
diabetes is induced mitigates the renal fibrosis that develops as a
result of the diabetes.
Example 8
Treatment of Alzheimer's Disease: Animal Model
[0293] A study is carried out in an mouse model of Alzheimer's
disease. The mouse model is the Tg2576 mouse model, as described
above in the Description of the Invention and in further detail in
Gau et al., 2002 (3 Gau et al., Amer. J. Pathology, 160(2):731-738
[2002]; J Q Trojanowski, Amer. J. Pathology, 160(2):409-411
[2002]). These mice begin developing amyloid plaques at about 9
months of age and plaques are well developed by 15 months. The
transgenic mice also develop progressive age-dependent cognitive
and behavioral abnormalities described in the background.
A. Overview of Study Design
[0294] Forty female Tg2576 mice, aged 3 months, are purchased from
Tagonic Inc, and grown until 9 months of age, at which time they
are divided into two groups of 20. One group begins treatment with
TM at 9 months of age, and are treated until sacrifice at age 15
months. TM treatment consists of daily gavage of 0.7 mg TM/mouse in
0.25 ml of sterile water. This dose has been shown to reduce the
serum Cp levels (surrogate marker of copper status) into the target
range of 25-50% of baseline levels. Once weekly, one mouse from the
treatment group and one from the control group are bled from the
tail vein, to determine Cp levels, and blood counts. Mice will be
rotated so that different mice are bled each week. Cp is determined
by an oxidase method. If the Cp levels begin to move out of the
target range, TM dose is adjusted accordingly. Control mice receive
a daily gavage of 0.25 ml of sterile water only. The weight of the
mice is determined weekly. It is contemplated, based upon past
experience, that the weight in mice in the Cp target range will
match the weight of control mice.
B. Studies at Autopsy
[0295] After 6 months of treatment with water or TM, all mice in
both groups are sacrificed painlessly, and the brains removed and
divided sagitally. The left half is utilized for physiological
assays, and is homogenized for measurements of A.beta.40 and
A.beta.42 by ELISA (see below). The right half is utilized for
anatomical assays, and is fixed in 4% paraformaldehyde in phosphate
buffered saline (PBS), pH 7.4, at 4.degree. C. and processed for
paraffin-embedding. Fixed hemi-brains are sectioned coronally at
5-8 .mu.m thickness, and sections processed for hematoxylin and
eosin, Bielschowky silver, and Congo Red staining. Analysis of
amyloid plaque load in mouse brains is determined by
light-microscopic semi-quantitative analysis of brain sections.
Subtle effects of treatment are measured by a more rigorous
quantitative analysis of amyloid plaques in brain sections to
detect a significant difference.
[0296] Brain homogenates of left hemi-brains are used for detection
of human A.beta.40 and A.beta.42 by ELISA, as previously described
(J Gau J et al., Amer. J. Pathology, 160 2:731-738 [2001]).
Homogenates are centrifuged at 7840.times.g for 5 minutes at
4.degree. C. to remove insoluble material. A sandwich ELISA is
performed on the supernatants using BAN50 as the capture antibody
and either horseradish peroxidase-coupled BA-27 or BC-05 as the
detection antibody for A.beta.40 or A.beta.42, respectively. BAN-50
is a monoclonal antibody specific for A.beta.1-10. All samples are
measured in triplicate. Standard curves for the ELISA are
constructed using pure human A.beta.40 and A.beta.42 (Bachem). In
addition to measurements of A.beta., a commercially available ELISA
is used to detect TGF.sub..beta. levels in brain homogenates of the
two groups.
C. Statistical Analysis
[0297] The results show that a mean reduction in A.beta.40 and
A.beta.42 levels of up to about 50% in brain homogenates of treated
compared to untreated Tg2576 mice is observed at about 15 months of
age. Assuming equal variation in treated and untreated animals, 20
animals per group are adequate to readily detect a mean reduction
of about 50%. The results also show that semi-quantitative analysis
of amyloid load in brain sections correlate with reduction of
A.beta. levels measured by ELISA. Subtle effects of TM treatment on
amyloid plaque deposits in brain are measured by a more rigorous
quantitative study; this study utilizes equipment available at the
University of Michigan imaging core facility. The results also show
that a significant reduction in levels of TGF.sub..beta. as
measured by ELISA in TM-treated mouse brain homogenates is
observed.
Example 9
Treatment of Alzheimer's Disease Clinical Trial (Treatment
Protocol)
[0298] A protocol is designed to treat patients with Alzheimer's
disease. Briefly, 40 patients with mild to moderate dementia (MMSE
10-23) due to probable AD are recruited, where patients have been
enrolled and followed for some time in other programs, so that the
clinical diagnosis and baseline studies, including
neuropsychometric tests, are well established. Patients are
randomized to receive placebo or TM, but the pilot study is
single-blinded. The results show that TM treatment arrests or
retards decline over 12 months compared to the expected cognitive
and functional decline in the placebo group. Before and during the
clinical trial, currently approved (FDA) cholinesterase inhibitors
are withheld, but other medications are typically continued.
Inclusion and exclusion criteria routinely used in clinical trials
of patients with AD are also utilized in this trial.
[0299] An induction dose of 20 mg TM 3.times. daily will be
administered with meals and 60 mg HS(HS means at bedtime) will be
administered away from food. When Cp reaches the target range of
10-15 mg/dl (usually in 2-3 weeks), a typical maintenance dose is
used of 20 mg 2.times. daily with meals and 40 mg HS. Cp and blood
counts are followed weekly for a time, adjusting the TM dose as
necessary to maintain target. After a stable Cp and TM dose are
established, Cp and blood counts are followed every 2-4 weeks. The
following battery of neuropyschometric tests are carried out at the
beginning and end of the treatment 12 months later: MMSE, CDR, and
ADAS-Cog. It is contemplated that few drop-outs will occur, since
TM is well-tolerated. For statistical purposes, drop-outs will be
subject to intention-to-treat analysis.
[0300] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the relevant art
are intended to be within the scope of the following claims.
* * * * *