corky ipf
Tetrathiomolybdate therapy protects against
bleomycin-induced pulmonary fibrosis in mice
GEORGE J. BREWER, MATTHEW R. ULLENBRUCH, ROBERT DICK, LEOVIGILDO OLIVAREZ, and
SEM H. PHAN
ANN ARBOR, MICHIGAN
Tetrathiomolybdate (TM), a drug developed for the treatment of Wilson’s disease,
produces an antiangiogenic effect by reducing systemic copper levels. Several
angiogenic cytokines appear to depend on normal levels of copper for activity. In
both animal tumor models and in cancer patients, TM therapy has proved effective
in inhibiting the growth of tumors. We have hypothesized that the activities of fibrotic
and inflammatory cytokines are also subject to modulation by the availability of
copper in a manner similar to angiogenic cytokines. As a first step in evaluating
whether TM plays a therapeutic role in diseases of inflammation and fibrosis, we
studied the effects of TM on a murine model of bleomycin-induced pulmonary
fibrosis. Oral TM therapy resulted in dose-dependent reduction in serum ceruloplasmin,
a surrogate marker of systemic copper levels. Significant decreases in systemic
copper levels were associated with marked reduction in lung fibrosis as
determined on the basis of histopathologic findings and a biochemical measure of
fibrosis. The protection afforded by TM was also reflected in significantly reduced
bleomycin-induced body-weight loss. In the next phase of this work, we will seek to
determine the mechanisms by which TM brings about this therapeutic benefit. (J Lab
Clin Med 2003;141:210-6)
Abbreviations: CTGF _ connective-tissue growth factor; SEM _ standard error of the mean;
SPARC _ secreted protein, acidic and rich in cysteine; TGF _ transforming growth factor; TM _
tetrathiomolybdate
Pulmonary fibrosis, idiopathic or otherwise, is
commonly progressive and essentially untreatable,
with a fatal outcome.1,2 It is clear from a
rather wide body of work that the underlying mechanism
involves dysregulation and overproduction of certain
cytokines.1-5 A central mechanism appears to involve
continued overproduction of TGF-_, which could
in turn increase the production, activity, or both of
CTGF.6-12
Bleomycin, when given to cancer patients, produces
pulmonary fibrosis in about 3%.13 On the basis of these
observations, a mouse model of pulmonary fibrosis has
been established in which intratracheal instillation of
bleomycin uniformly produces pulmonary fibrosis.4,6,9-
12,14 This model is thought to be a model of human
pulmonary fibrosis. The hypothesis that TGF-_ is central
to pulmonary fibrosis has been validated by studies
showing that inhibition of TGF-_ by pharmacologic
means or by antibodies greatly reduces the pulmonary
fibrosis produced by bleomycin or other methods of
lung injury in the mouse.8,12
In previous studies by members of our group, it has
From the Departments of Human Genetics and Pathology, University
of Michigan.
Supported by National Institutes of Health grants HL28737,
HL31963, and HL52285. The University of Michigan has recently
licensed the antiangiogenic uses of tetrathiomolybdate to Attenuon,
LLC. Dr. Brewer and Mr. Dick have equity in this company.
Submitted for publication July 19, 2002; revision submitted October
4, 2002; accepted November 18, 2002.
Reprint requests: George J. Brewer, MD, Department of Human
Genetics, University of Michigan Medical School, 4909 Buhl, Ann
Arbor, MI 48109-0618; e-mail: brewergj@umich.edu.
Copyright © 2003 by Mosby, Inc. All rights reserved.
0022-2143/2003 $30.00 _ 0
doi:10.1067/mlc.2003.20
210
been shown that copper-decreasing therapy with TM, a
copper-specific agent, can prevent, at least for a time,
the growth of many types of advanced and metastatic
cancers through an antiangiogenic mechanism.15-19 The
hypothesis underlying this approach is that one or more
copper-containing or copper-binding angiogenic promoters
(eg, vascular endothelial growth factor, fibroblastic
growth factor, angiogenin, angiotropin, SPARC,
or others) require higher levels of copper to be active
than are required for basic cellular needs for copper.
15,19 We hypothesize that use of the anticopper drug
TM drops the patient into an “antiangiogenic window”
of copper levels in which angiogenesis is inhibited but
copper deficiency is not severe enough to cause side
effects.
As we examined the series of cytokines involved in
the pathogenesis of fibrosis, it appeared to us that one
or more of them had a high probability of sharing the
copper dependency of the angiogenic cytokines discussed
above and that copper-decreasing therapy with
TM might abrogate the development of pulmonary
fibrosis. In this work, we have shown that TM therapy
does indeed inhibit development of pulmonary fibrosis
in the bleomycin mouse model.
METHODS
Mice. Female CBA/J mice, 8 to 10 weeks old, were from
Jackson Laboratories (Bar Harbor, Maine). At the start of the
experiments, the mice weighed 21.4 _ 1.7 g (mean _SD).
Bleomycin treatment. We administered bleomycin on day
0 by means of endotracheal instillation through the oral cavity
after exposing the mouse’s airway by pulling its tongue. Each
mouse received 0.001 units/gm body wt of bleomycin (Bristol-
Myers, Evansville, Ind) in 30 _L sterile saline solution.
Control mice were administered an equal volume of sterile
saline solution.
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.
Three experiments were carried out. Experiment 1 comprised
four 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. (All groups started with seven mice, but
two mice from group 1 died before the end of the experiment
as a result of the illness produced by bleomycin, and one
mouse each from groups 3 and 4 died as a result of technical
problems encountered during TM gavage.) The mice in
groups 3 and 4 were started on TM therapy at a dose of 0.7
mg/day, 7 days before the administration of bleomycin, and
were continued on this dose throughout the study until being
killed at day 21, except for a 3-day period (days 9-11 after
bleomycin) when they were each given a dose of 1.2 mg
TM/day. We temporarily increased the dose to ensure that
copper concentrations would be adequately decreased in this,
our first experiment.
Experiment 2 was a TM dose-response study comprising
five groups, each containing four bleomycin-treated mice.
Group 1 was a control, given bleomycin but no TM. Each
mouse in groups 2 through 5 received an identical load dose
of 1.2 mg TM/day for 3 days before the administration of
bleomycin. Groups 2 through 5 were administered 0.3, 0.5,
0.7, or 0.9 mg of TM/mouse/day, respectively, until the mice
were killed at day 21. Eighteen mice that received no bleomycin
were divided into five groups and given either no TM
(as in group 1, above) or loading doses and varying doses of
TM (as in groups 2 to 5, above) and served as controls for TM
therapy.
In experiment 3, the starting times for TM treatment varied.
Twenty-one mice were divided into five groups of four to five
mice each, all of which received bleomycin. Group 1 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 being
killed at day 21. However, starting times of TM treatment
were varied, beginning 5 days before the administration of
bleomycin in group 2, then beginning 4, 7, and 14 days after
the administratino of bleomycin in groups 3, 4, and 5, respectively.
Eighteen mice that received no bleomycin were divided
into five groups and given either no TM (as in group 1,
above) or doses of TM and starting times comparable to those
in groups 2 through 5, above, and served as controls for TM
therapy.
Copper status. In the presence of TM therapy, copper
status can’t 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, we have found that serum
ceruloplasmin is a good surrogate marker of copper status15
because the liver secretes this copper-containing protein into
the blood at a rate dependent on copper availability. We
monitored copper status by assaying serum ceruloplasmin on
the basis of its oxidase activity20 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.
Hydroxyproline assay. We assessed the extent of fibrosis
by assaying hydroxyproline content of whole-lung homogenates
at the time of sacrifice, as previously described.21 Hydroxyproline
was expressed as micrograms of hydroxyproline
per mouse lung (the lung tissue included both lungs).
Microscopic evaluation of the lungs. For structural evaluation
of fibrosis, we inflated lungs with formalin after killing
the mice. After overnight fixation, the lungs were embedded
in paraffin. We prepared sections for Hematoxylin-and-eosin
staining, as well as for Masson trichrome staining for the
evaluation of collagen deposition.21
Statistical analysis. For comparisons of means, we used
analysis of variance, followed by Scheffe´’s test for multiple
comparisons when appropriate. For the dose-response and
varying starting times for TM studies, we also used regression
analysis to evaluate statistical significance.
J Lab Clin Med
Volume 141, Number 3 Brewer et al 211
RESULTS
Experiment 1. As shown in Table I, at the time of
killing, the mean body weight of bleomycin-treated
animals (group 1) was significantly less than that of
saline solution–treated controls (group 2). TM treatment
protected against some of the bleomycin-induced
weight loss (Table I), as shown by the lack of a significant
difference in weight between the bleomycin/TM
(group 3) and saline solution (group 2) means. TM
alone (group 4) tended to produce some weight loss in
experiment 1 (Table I). The mean hematocrit of bleomycin-
treated animals (group 1 of Table I) was significantly
increased compared with that in the other three
groups.
The mean ceruloplasmin level of bleomycin/TM
mice (group 3 of Table I) was about 55% that of
bleomycin animals (group 1), and the two were significantly
different. TM alone (group 4) resulted in a mean
ceruloplasmin concentration about 80% that of saline
controls, but this difference was not statistically significant.
The hydroxyproline results of experiment 1 are also
shown in Table I. Therapy with TM almost completely
abrogated fibrosis as measured by this assay. Bleomycin
treatment (group 1) produced a highly significant
increase in hydroxyproline compared with that seen in
saline solution–treated controls (group 2), but we noted
no significant difference between the TM-treated bleomycin
mice (group 3) and the saline-solution group 2,
and the means were very close. The mean values between
bleomycin-treated (group 1) and bleomycin/TMtreated
(group 3) animals were highly significant. In
this experiment, TM alone seemed to have some effect
on increasing hydroxyproline levels, an effect that
wasn’t borne out in experiments 2 and 3.
The results of lung histopathologic study from experiment
1 bear out the hydroxyproline results (Fig 1).
Although scattered patches of fibrosis and inflammatory
cells were found in the TM-treated bleomycin
animals, these were substantially smaller, with lesser
degrees of cellular infiltration compared with those in
the animals treated with bleomycin alone. Masson’s
trichrome–stained sections revealed much less collagen
deposition in the TM-treated group than in the mice
given bleomycin only (data not shown).
Experiment 2. Experiment 2 was a dose-response
study. The overall data on weight, hematocrit, and
hydroxyproline levels at the time of sacrifice are shown
in Table II. Scheffe´’s’ correction for multiple comparisons
showed that the relevant means were not significantly
different from one another, except as noted in
Table II.
We monitored weight throughout experiment 2 (Fig
2), and although the means at the time of death are not
significantly different, it is clear from the data shown in
Fig 2 that TM in doses of 0.5 mg/day on up protected
against bleomycin-induced weight loss, similar to the
results in experiment 1.
Additional analysis of the hydroxyproline data of
experiment 2 is shown in Fig 3. Regression of the
hydroxyproline values against TM doses of 0.3 to 0.9
mg/day yields a highly significant F statistic. In this
experiment, hydroxyproline levels were not affected by
TM treatment alone (Fig 3), in contrast to experiment 1.
The ceruloplasmin data of experiment 2 were separately
analyzed (Table III). All of the means of the
bleomycin TM groups are significantly different than
those of the bleomycin control at the time of killing, but
they do not show much dose-response effect. Dose
response of TM on ceruloplasmin may be better appre-
Table I. Data from experiment 1 at time mice were killed
Treatment parameters Bleomycin Saline solution Bleomycin/TM 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 (IU) 25.3 _ 2.8 22.7 _ 1.1 13.9 _ 3.3 18.1 _ 3.4
Hydroxyproline (_g/lung) 252 _ 16 156 _ 9 162 _ 12 193 _ 5
Statistical analysis (P values) comparisons Weight Hematocrit Ceruloplasmin Hydroxyproline
Bleomycin vs saline solution .001 .004* .001 .005* .183 .90* .001 .001*
Bleomycin vs bleomycin/TM .06 .32* .001 .001* .01 .06* .001 .001*
Bleomycin/TM vs saline solution .16 .62* .001 .004* .06 .38* .674 .980*
TM vs saline solution .002 .07* .24 .99* .1 .70* .018 .119*
*P value with Scheffe´ ’s correction for multiple comparisons.
J Lab Clin Med
212 Brewer et al March 2003
ciated after examination of the individual mouse data
during the course of the experiment, in which the 0.9
group shows low levels, the 0.3 group relatively normal
levels, and the 0.5 and 0.7 groups intermediate levels.
Experiment 3. In experiment 3, the starting time of
TM was varied. As shown in Fig 4, the sooner TM was
started, the more effective it was in protecting against
hydroxyproline accumulation. Regression of hydroxyproline
levels against the day therapy was started
(days _5 to _14) gave an F statistic of 21 (P _ .05).
Because the number of animals in each group was
relatively small, we combined the data on the postbleomycin
TM groups (days _4, _7, and _14), then
compared the mean with that of the bleomycin control,
which yielded a significant t-test result (P _ .05). As in
experiment 2, we detected no effect of TM alone on
hydroxyproline levels (Fig 4). Overall hydroxyproline
values were lower than those in experiments 1 and 2
because of the use of younger animals, weighing 10%
to 20% less.
DISCUSSION
It is clear from the data presented here that TM
therapy has a major protective effect against the lung
damage and illness produced by intratracheal bleomycin
instillation in mice. Bleomycin treatment causes
much weight loss, but this is protected against by an
adequate dose of TM (Table I, Fig 2). The bleomycininduced
illness produces hemoconcentration by day 21,
probably a result of the animals’ not drinking adequate
water; this is also protected against by TM (Tables I
and II). Bleomycin also causes pulmonary fibrosis,
Fig 1. Effects of TM on histopathologic findings. Lung sections from bleomycin-treated mice (left) and mice
treated with bleomycin plus TM (right) mice were stained with hematoxylin and eosin, then photographed at a
magnification of 40_ (upper), 400_ (middle), or 1000_ (lower).
J Lab Clin Med
Volume 141, Number 3 Brewer et al 213
measured by levels of hydroxyproline, and TM therapy
strongly protects against hydroxyproline accumulation
(Table I, Fig 3). The therapeutic and protective effects
of TM are dose-dependent, as shown in Figs 2 and 3.
TM can be started after the inflammatory damage is
well under way14,21 and still provide benefit, as shown
in experiment 3 (Fig 4). As late as days 4, 7, and 14
after bleomycin administration, TM initiation is still
beneficial.
The main mechanism of action of TM is to decrease
systemic copper levels. That TM worked in this study
to decrease copper availability, as planned, is shown by
the reduced ceruloplasmin levels in the experiments
(Tables I and III). Possible mechanisms by which decreased
copper levels could be therapeutically effective
against the bleomycin damage—in increasing order of
likelihood, in our judgment — are:
(1) Effects of TM on decreasing direct copper interactions
with bleomycin. This seems unlikely; in experiment
3, TM was effective when started several days
after bleomycin-induced injury.
(2) Possible antioxidant effects of TM, countering the
oxidant-damage effects of bleomycin. This seems unlikely
for the same reason given above.
(3) An effect of copper depletion on a copper-dependent
enzyme, such as lysyl oxidase. We measured lysyl
oxidase in the lungs in one experiment and found no
effect of TM (unpublished data). We believe the copper
deficiency isn’t sufficiently severe to affect copperdependent
cellular enzymes, but study of this possible
mechanism should continue.
(4) Effect of TM on proangiogenic cytokines. We
know that many proangiogenic cytokines are copperdependent,
as discussed in the Introduction, and we
Fig 2. Mean weights for mice in each group at several time points
during experiment 2 and at the time they were killed (21 days). The
bleomycin group showed severe weight loss, but TM protected
against weight loss in a dose-dependent manner, with the 0.9-mg dose
being fully protective.
Fig 3. Relationship of excess hydroxyproline (a measure of fibrosis)
to TM maintenance dose in experiment 2. Closed squares, mean
values of four mice given bleomycin and treated with varying doses
of TM, ranging from 0 to 0.9 mg/day during the maintenance phase
of therapy. Open triangles, mice that were not did given bleomycin
but received the maintenance doses of TM indicated. Regression of
the hydroxyproline data (closed squares) on TM doses ranging from
0.3 to 0.9 mg/day provided an F statistic of 14.8 (P _ .002).
Table II. Data from experiment 2 at the time mice were killed
Treatment
Bleomycin
(n _ 5)
Bleomycin/TM 0.3
(n _ 4)
Bleomycin/TM 0.5
(n _ 4)
Bleomycin/TM 0.7
(n _ 5)
Bleomycin/TM 0.9
(n _ 5)
Weight (g) 17.6 _ 0.68 19.5 _ 1.76 21.5 _ 0.87 20.8 _ 1.02 22.4 _ 0.87
Hematocrit (%) 55.0 _ 0.71 46.3 _ 0.25 47.3 _ 0.63 44.3 _ 2.75 42.5 _ 1.32
Hydroxyproline
(_g/lung)
217 _ 7.8 232 _ 3.7 197 _ 15.1 172 _ 22.0 157 _ 14.5
Data approved as mean _ SEM.
Weight: None of the means was significantly different than other means, with Scheffe´ ’s correction for multiple comparisons.
Hematocrit: The means of the bleomycin TM 0.7 mg, bleomycin TM 0.9 mg, and all the saline-solution and saline-solution TM groups were
significantly different than the mean of the bleomycin group with Scheffe´ ’s correction for multiple comparisons.
Hydroxyproline: None of the means of the bleomycin TM groups was significantly different than the bleomycin mean, with Scheffe´ ’s correction
for multiple comparisons. The means of the saline-solution control and the saline-solution TM 0.5 were significantly different than that of
bleomycin.
J Lab Clin Med
214 Brewer et al March 2003
have unpublished evidence that some of these are inhibited
by TM therapy. Angiogenesis may play some
role in the mouse model of bleomycin-induced lung
injury, although it seems unlikely that it plays a central
role. However, antiangiogenic effects of TM may contribute
to TM’s therapeutic effect.
(5) Inhibition of inflammation through inhibition of
proinflammatory cytokines. We know from unpublished
work in a mouse model of liver inflammation
that TM inhibits inflammation. The finding in experiment
3 that TM administration started relatively late
(days 4, 7, and 14) after bleomycin instillation still has
a significant effect in inhibiting fibrosis (Fig 4) argues
against suppression of inflammation as the sole effect
of TM. TM started on day 4 wouldn’t reduce copper
levels to the therapeutic area until about day 7, by
which time all or most of the inflammatory stimuli are
peaking. TM started at days 7 and 14 would not get
copper levels into the therapeutic range until well after
inflammation and inflammatory stimuli would have
subsided.
(6) Inhibition of fibrosis through inhibition of the
profibrotic cytokine pathway. As discussed in the Introduction,
the possibility of copper dependence of this
pathway was the reason we initiated this study. Several
candidate cytokines may be inhibited in this pathway. It
is known that SPARC is one stimulus for TGF-_ expression,
22,23 and SPARC is known to be copper-dependent.
24 SPARC-null mice demonstrate marked mitigation
of bleomycin-induced fibrosis.23 TGF-_ itself
interacts with heparan molecules, some of which are
known to be copper-dependent.25 Finally, CTGF is rich
in cysteine,10 suggesting that it binds copper and is
dependent on copper for activity. We think it most
likely that a combination of antiangiogenic, antiinflammatory,
and antifibrotic effects are involved. These
possible mechanisms are under investigation.
Irrespective of the pathway, or the molecular mechanism,
involved, the fact that TM therapy can so markedly
inhibit fibrosis in this model raises the exciting
possibility of using this approach to preventing and
treating pulmonary fibrosis in patients. The data of
experiment 3, shown in Fig 4, indicate that TM therapy
is effective after injury, which supports its potential
efficacy in the clinical situation. The possibility of early
clinical trials is enhanced by the fact that this drug has
already seen considerable experimental use in human
subjects with Wilson’s disease26,27 and cancer15,19 and
has proved remarkably safe. The only side effect of
copper-decreasing therapy with TM in cancer has been
overtreatment, which leads to easily reversible bonemarrow
depression. The use of serum ceruloplasmin as
a surrogate to monitor copper status in the clinical
situation has proved effective, reliable, and easy to
use.15
It has not escaped our attention that the profibrotic
pathway involving TGF-_ and CTGF is central to
pathologic fibrosis in many organs besides the
lungs.7,10 A partial list includes 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 track in some patients
after disease or injury. If TM treatment is effective in
treating pulmonary fibrosis, there are obviously a host
Fig 4. Hydroxyproline data from experiment 3. TM-treated mice
were given a loading dose of 1.2 mg for each of 4 days, then 0.9
mg/day. Closed diamonds, mean hydroxyproline data at the time
mice were killed (day 21) from four mice in each group, except for
day _7, which had five mice, all treated with bleomyci, and all
treated with TM at varying starting times. Closed squares, mean
values from four mice given bleomycin only; open circles, mean of
four mice given saline solution only. Open triangles, means of three
or four mice given no bleomycin but administered TM at varying
starting times as controls for the bleomycin/TM mice. Regression of
the four data points shown by the yielded a significant F statistic (P _
.05). The data from the bleomycin/TM groups given TM post bleomycin
instillation (_4, _7, and _14 days) were pooled and the mean
compared with the bleomycin mean by the use of Student’s t test and
found to be significantly different (P _ .05).
Table III. Ceruloplasmin values of bleomycin and
TM-treated animals during experiment 2
TM maintenance dose
(mg/day)
Ceruloplasmin values
Day 8* Day 14* Day 21†
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
*Data at 8 and 14 days are from single mice.
†Data from 21 days are at the time mice were killed and represent
the mean _ SEM of four to five mice in each group. At the time of
death, the mean value of each bleomycin/TM-treated group was
significantly different from the mean of the bleomycin group (no
TM treatment) with Scheffe´ ’s correction for multiple comparisons.
J Lab Clin Med
Volume 141, Number 3 Brewer et al 215
of other diseases of excessive fibrosis and/or inflammation
in which TM therapy should be evaluated.
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