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Department of Medicine, University of California, San Diego, California 92093-0618; Bristol-Myers Squibb Co. (D.B.W.-I., H.Z., T.V., G.J.G.), Princeton, New Jersey 08543; and Metabolic Research Unit (J.D.B.) and Departments of Pharmaceutical Chemistry (G.C., T.S.S.) and Molecular and Cellular Pharmacology (G.C., T.S.S.), University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Wolfgang H. Dillmann M.D., Department of Medicine, 9500 Gilman Drive (BSB 5063), La Jolla, California 92093-0618. E-mail: wdillmann{at}ucsd.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TRß. Little is known about effects of ligands that
preferentially interact with the two different TR subtypes. In the
current study the comparison of the effects of the novel synthetic
TRß-selective compound GC-1 with T3 at
equimolar doses in hypothyroid mice revealed that GC-1 had better
triglyceride-lowering and similar cholesterol-lowering effects than
T3. T3, but not GC-1, increased heart rate and
elevated messenger RNA levels coding for the If channel
(HCN2), a cardiac pacemaker that was decreased in hypothyroid mice.
T3 had a larger positive inotropic effect than GC-1.
T3, but not GC-1, normalized heart and body weights and
messenger RNAs of myosin heavy chain and ß and the sarcoplasmic
reticulum adenosine triphosphatase (Serca2). Additional dose-response
studies in hypercholesteremic rats confirmed the preferential effect of
GC-1 on TRß-mediated parameters by showing a much higher potency to
influence cholesterol and TSH than heart rate. The preferred
accumulation of GC-1 in the liver vs. the heart probably
also contributes to its marked lipid-lowering effect vs.
the absent effect on heart rate. These data indicate that GC-1 could
represent a prototype for new drugs for the treatment of high lipid
levels or obesity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TRß.
Recently, the ligand-binding domains of both TR and TRß have been
crystallized and structurally characterized in detail (10, 11). In
addition, it has become apparent in animal models, in which either
TR or TRß is deleted, that distinct and differential cardiac
effects are exerted by these isoforms. For example, in TR1-deleted
animals marked bradycardia occurs (12, 13). In contrast, in mice with
deletion of TRß there is no lowering of heart rate, but the animals
exhibit inner ear deafness (14).
Recently, the novel thyroid hormone analog GC-1 has been synthesized
(15). In this analog the three iodines of T3 are
replaced by methyl and isopropyl groups. A methylene linkage replaces
the biaryl ether linkage between the two phenol groups, and the amino
acid side-chain has been changed to an oxyacetic acid group (Fig. 1
). GC-1 has approximately a 10 times
higher binding affinity to TRß1 compared with TR1 and shows
TRß-selective actions in cells in culture. However, it is unclear
what influence GC-1 has on T3mediated changes in
cardiovascular action and lipid levels in vivo. To explore
its in vivo actions, GC-1 was administered to hypothyroid
mice and hypercholesteremic rats, and its effects were compared with
those of equimolar doses of T3. The results show
that significant and distinct differences in T3
vs. GC-1 action occur.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo hemodynamic measurements. Heart rate was calculated from electrophysiological tracings. Mice were lightly sedated with ketamine (0.033 mg/g BW) and pentobarbital (0.033 mg/g BW) and restrained (17). Four needles were placed sc on each limb close to the trunk, and 6 min after injection of sedatives an electrocardiogram (ECG) was obtained. Heart rate was calculated from the RR interval. For measurement of in vivo hemodynamics, mice were anesthetized with a mixture of ketamine (0.1 mg/g BW) and xylazine (0.007 mg/g BW) and placed under a dissecting microscope (18). ECG electrodes were connected to the limbs, and the body temperature was monitored with an anal temperature probe and kept at 37 C. The animals were intubated and ventilated with room air. A bilateral vagotomy was performed. The right jugular vein and right carotid artery were cannulated with polyethylene catheters, and the carotid line was connected to a pressure gauge. Arterial pressure was measured at this time point. Next, a left-sided thoracotomy was performed. The pericardium was opened, and a high fidelity pressure transducer (1.8 French, Millar Instruments, Houston, TX) was inserted into the left atrium and advanced into the left ventricle. Continuous left ventricular (LV) pressure, differential LV pressure, aortic pressure, and ECG were recorded on a personal computer. After recording the measurements, increasing doses of isoproterenol were infused through the venous line (5, 50, 500, and 1000 ng). The following parameters were analyzed: heart rate (HR), carotid arterial pressure, LV end-systolic pressure (LVESP), and the maximum values of LV pressure derivatives (dP/dtmax, dP/dtmin). Effects of isoproterenol on hemodynamic parameters were analyzed 45–60 sec after drug administration.
Northern blot analysis. Messenger RNA levels for
sarcoplasmic reticulum calcium adenosine triphosphatase (Serca2),
myosin heavy chain (MHC), MHCß, and the
hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2)
were determined. RNA was isolated from myocardial tissue by a
guanidinium thiocyanate method (19), and polyadenylated RNA was
obtained using an oligo(deoxythymidine) kit (QIAGEN,
Chatsworth, CA). mRNA was separated on a 1% agarose gel and
transferred to a nylon membrane. A 1.6-kb fragment of the 5'-end of the
rat Serca2 complementary DNA, a 224-bp fragment of the second exon of
mouse HCN2 complementary DNA, and oligonucleotides specific for the rat
MHC and rat MHCß were used to make
32P-labeled probes (Multiprime DNA labeling
systems, Amersham Pharmacia Biotech, Aylesbury, UK).
Radioactivity was assessed on a blue film (Eastman Kodak Co., Rochester, NY), and the resulting image was quantified with
Image 1.61 software.
Measurements of cholesterol and triglycerides in mice. Blood was collected from all mice at the end of the study; the serum was separated by centrifugation and immediately frozen in liquid nitrogen. Triglycerides and cholesterol were measured by enzymatic methods (Roche, Basel, Switzerland).
Statistical analysis. Parameters in control, hypothyroid mice and in hypothyroid mice treated with different doses of GC-1 and T3 were analyzed by ANOVA and Fisher’s post-hoc tests. P < 0.05 was assumed significant. Data are given as the mean ± SEM.
Dose-response studies in hypercholesteremic rats
Male Sprague Dawley rats (Harlan Sprague Dawley, Inc.), weighing 300–400 g, were used for this study. The
rats were fed a diet containing 1.5% cholesterol and 0.5% cholic acid
(Harlan Teklad Rodent Chow, Madison, WI) for 2 weeks before drug
treatment and for the entire course of the study. The rats were given
via oral gavage either vehicle (n = 6; a separate vehicle group
was used for the T3-treated group and the GC-1
treated group, also n = 6) or the following drug treatment groups:
1.54–924 nmol/kg·day T3 (n = 5/group) or
46.2–2920 nmol/kg·day GC-1 (n = 5/group). The doses correspond
to 1.0–601.5 ng/g·day T3 and 15.2–958.9
ng/g·day GC-1. The vehicle or drugs were administered once per day
for 7 days. Vehicle consisted of 5% cremaphor, 5% ethanol, 10%
m-pyrol, and 80% water. The animals were weighed each day before drug
administration. After 7 days, the rats were anesthetized using 40 mg/kg
pentobarbital sodium, ip, and the heart rates were measured by ECG
(Gould Instruments, Valley View, OH). A 3-ml blood sample was taken via
the inferior vena cava for each animal. The blood samples were
centrifuged at 2000 rpm for 20 min, and the plasma was used for
cholesterol, triglyceride, and TSH measurements. Plasma cholesterol
measurement was performed using a Cobas Mira S analyzer
(Roche, Indianapolis, IN). Plasma TSH was measured using a
RIA kit designed for rat TSH (Amersham Pharmacia Biotech,
Arlington Heights, IL).
The doses chosen were based on the lowest dose of T3 (1 µg/kg·day or 1.54 nmol/kg·day). For the purpose of comparison between GC-1 and T3, all doses are denoted as nanomoles per kg/day. Increasing doses of T3 or GC-1 were then given in half-log increments.
Determination of T3,
T4, and GC-1 levels in plasma and
tissues
Male Sprague Dawley rats were anesthetized with 30 mg/kg
pentobarbital, ip. Either T3 or GC-1 (10
µmol/kg) was injected directly into the right jugular vein (n =
3/group). Plasma and tissue samples were collected 1 h after
treatment. For various tissues, samples were weighed and homogenized
with 3 vol deionized water. For T3 and
T4 assays, 100-µl samples were treated with 250
µl acetonitrile containing 100 ng/ml internal standard (IS-I). For
GC-1 assays, 25-µl samples were treated with 50 µl acetonitrile
containing 100 ng/ml internal standard (IS-II). After centrifugation to
remove precipitated proteins, the clear supernatant was dried with
nitrogen in silanized glass vials and then reconstituted with 50 µl
acetonitrile-water (50:50). Due to endogenous production of
T3 and T4 hormones,
standard curve and quality control samples for
T3 and T4 measurements were
prepared using acetonitrile-water (50:50) mixture in the same fashion
as for the tissue treatment. A 10-µl aliquot of the reconstituted
solution was analyzed by liquid chromatography/tandem mass
spectrometry.
Sample analysis was performed using a Shimadzu LC system
(Shimadzu Scientific, Kyoto, Japan) with an Inertsil (MetaChem
Technologies, Torrance, CA) ODS-2 HPLC column (2 x 50 mm, 5
µm) interfaced to a Micromass Quattro (Micromass UK Limited,
Manchester, UK) tandem mass spectrometer. The mobile phases consisted
of 0.1% formic acid in water (A) and 0.1% formic acid in methanol
(B). With a flow rate of 0.3 ml/min, a linear gradient was started from
95% A/5% B to 5%A/95% B over 2.5 min and was held at 5% A/95% B
for an additional 1.5 min. The mobile phase was then returned to
initial conditions, and the column was reequilibrated for 2 min. The
total analysis time was 6 min. MS/MS conditions were as followings:
electrospray ionization; ultra high purity nitrogen as
nebulizing gas (100 liters/h) and desolvation gas (900 liters/h);
desolvation temperature, 350 C; and source temperature, 150 C.
(M+H)+ species for T3,
T4, GC-1, and internal standards were selected in
MS1 and collisionally dissociated with argon at a pressure of 1.5
x 10-3 T to form specific product ions that
were subsequently monitored by MS2. Instrument parameters for
each compound are shown in Table 1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); body weight, heart
weight, and heart weight to body weight ratio (HW/BW ratio) were
markedly decreased compared with those in euthyroid age-matched mice.
Treatment of hypothyroid mice with 1 x T3,
4.5 x T3, and 9 x T3
increased body weight, heart weight, and HW/BW ratio. Treatment with
1 x GC-1 and 4.5 x GC-1 did not increase body weight or HW/BW
ratio. At the 9 x GC-1 dose, heart weight was significantly
increased, and there was a trend (albeit insignificant) for an increase
in body weight. No toxic effects were observed in mice treated with
GC-1. The appearance and behavior of GC-1-treated mice were similar to
those of T3-treated mice.
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) was markedly decreased to 335 ±
21 beats/min from a heart rate in control mice of 472 ± 26
beats/min (P < 0.001 control vs. hypothyroid).
Whereas treatment with 1 x T3 lead to an
increase to 502 ± 21 beats/min (P < 0.001
hypothyroid vs. 1 x T3-treated),
mice treated with the equimolar amount of 1.8 ng/g BW GC-1
(1 x GC-1) retained a low heart rate of 358 ± 28 beats/min
(P < 0.01 control vs. 1 x GC-1-treated).
Higher equimolar doses of the compounds let to similar increases in
heart rate; 4.5 x T3 and 4.5 x GC-1
increased heart rates to 536 ± 24 and 527 ± 35 beats/min,
respectively, and treatment with 9 x T3 and
9 x GC-1 resulted in heart rates of 584 ± 28 and 570 ±
31 beats/min, respectively. All other hemodynamic parameters measured,
systolic and diastolic arterial pressure (Pa sys
and Pa dias), end-systolic left ventricular
pressure (LVESP), and maximum speeds of contraction
(dP/dtmax) and relaxation
(dP/dtmin), were also severely decreased in
hypothyroid mice (Table 3). 1 x GC-1 showed a positive inotropic
effect (Figs. 2 and 5) by increasing
dP/dtmax to 4424 ± 510 from 2945 ±
273 mm Hg/sec in hypothyroid mice (P < 0.05
hypothyroid vs. 1 x GC-1-treated), although this was less
than the effect of 1 x T3 that resulted in a
dP/dtmax of 6224 ± 888 mm Hg/sec
(P < 0.05 1 x T3-treated
vs. 1 x GC-1-treated). Treatment with
1 x T3, but not with 1 x GC-1, also
increased Pa sys, Pa dias,
LVESP, and dP/dtmin (P < 0.05
hypothyroid vs. 1 x T3-treated).
The positive inotropic response was assessed by the increase in
arterial pressure and the maximal speed of contraction after
administration of isoproterenol. Administration of increasing doses of
isoproterenol iv resulted in positive inotropic effects in all groups.
The hemodynamic effects of 500 ng isoproterenol are presented as the
percent increase from the basal level; arterial pressure increased from
basal levels in control, hypothyroid,
1 x T3-treated, and 1 x GC-1-treated mice
by 25 ± 5%, 23 ± 2%, 25 ± 5%, and 36 ± 9%
respectively, dP/dtmax increased by 16 ±
6%, 25 ± 11%, 14 ± 5%, and 17 ± 9% respectively.
These data show that the sympathetic responses of the hypothyroid,
1 x GC-1, and 1 x T3 groups were not
different from that of the control group.
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mRNA. Treatment of hypothyroid mice with
3.5 ng/g BW T3 (1 x T3)
completely reversed the pattern of MHC isoform expression; control
MHC mRNA levels were restored, and MHCß mRNA was no longer
detected. In contrast, treatment with 1 x GC-1 did not increase
MHC expression, and the decrease in MHCß mRNA was smaller than
that in 1 x T3-treated mice. Treatment with
9 x GC-1 increased MHC mRNA to euthyroid levels, but MHCß mRNA
was still detectable (Figs. 3 and 5
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and 5).
Lipid-lowering effects in mice
Beneficial effects on the lipid profile were equal or greater in
GC-1-treated mice than in T3-treated mice (Figs. 4 and 5);
cholesterol levels were elevated in hypothyroid mice to 165 mg/dl
compared with 74 ± 3 mg/dl in euthyroid mice (P
< 0.0001). Both T3 and GC-1 treatment at all
doses decreased cholesterol to normal levels. Triglycerides were
elevated in hypothyroid mice to 126 ± 15 mg/dl compared with
70 ± 15 mg/dl in euthyroid mice (P < 0.05). GC-1
reestablished normal triglyceride levels at all doses, whereas
T3 treatment failed to decrease triglyceride
levels from the elevated hypothyroid levels.
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. These data are shown as the percent
change for each of these parameters relative to their respective
vehicle-treated group values. Plasma cholesterol was reduced by
T3 and GC-1 in a dose-dependent manner. The
vehicle-treated group values for cholesterol were 187 ± 25 and
180 ± 24 mg/dl for the T3 and GC-1 studies,
respectively. For the purpose of comparison, ED50
values for cholesterol reduction are shown in Table 4. ED50 was
defined as the dose of drug causing a 50% reduction in cholesterol
compared with the vehicle-treated group value, and this was chosen
because it was approximately half of the maximal effect (80–90%
reduction). As shown in this table, the ED50 for
cholesterol lowering was 12-fold lower with T3
than with GC-1. For the T3-treated group,
cholesterol was significantly reduced (compared with vehicle,
P < 0.05) starting at the 15.4 nmol/kg·day dose. For
GC-1, significant cholesterol lowering vs. that in
vehicle-treated animals was seen at the 154 nmol/kg·day dose and
higher doses (P < 0.05).
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). Heart rates were 364 ± 9 and 378 ± 8
beats/min for the vehicle-treated groups for the
T3 and GC-1 studies, respectively. Heart rate was
increased by T3, and significance was noted
starting at the 46.2 nmol/kg·day dose (P < 0.05
vs. vehicle). No significant changes in heart rate were
observed for GC-1 with the doses used in this study. Potencies for
changes in heart rate were expressed as ED15,
which is the dose causing a 15% increase in heart rate relative to the
time-matched vehicle group value. The ED15 was
chosen because a 10–15% increase in heart rate is viewed as being the
lower end of clinically relevant changes in heart rate. In addition,
the potency ratios for ED15 (heart
rate)/ED50 (cholesterol) are shown in Table 4.
The ratio was approximately 10-fold higher for GC-1 compared with
T3, suggesting that GC-1 was significantly more
selective for lowering cholesterol than it is for causing
tachycardia. TSH was reduced by both GC-1 and T3 in a dose-dependent manner, although T3 was approximately 20-fold more potent than GC-1. T3 significantly reduced TSH starting at the 4.62 nmol/kg·day dose, whereas GC-1 did so starting at the 154 nmol/kg·day dose (P < 0.05 compared with their respective vehicle group values). The potency for TSH suppression was expressed as ED30, because the maximal percent suppression was approximately 60% in these animals. As TSH suppression and cholesterol lowering are both presumably TRß-mediated effects, it would be expected that the potency ratios for TSH vs. cholesterol lowering should be similar for T3 and GC-1. GC-1 was slightly more selective for lowering cholesterol than was TSH, although this difference was only 2-fold, which is not pharmacologically significant.
Tissue distribution of GC-1 and T3
Measurements of GC-1 and T3 in plasma and
tissues revealed distinct differences in their organ distributions
(Table 5). The tissue to plasma ratios in
the liver were similar for GC-1 and T3 (2.09
± 0.53 and 5.86 ± 1.93, respectively). In contrast the
tissue/plasma ratio in the heart was 30 times higher for
T3 (3.61 ± 0.69 ng/g) than for GC-1
(0.12 ± 0.002 ng/g). Absolute values for plasma levels were 75
times different (3188 ± 445 ng/ml for T3
and 42 ± 8 ng/ml for GC-1).
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Discussion |
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gene
(TR1) vs. those of the c-erbAß gene (TRß1)
or its splice variant TRß2 leads to distinct thyroid hormone effects
(21, 22). It is therefore of interest to develop new
T3 agonists and antagonists with preferred
binding and action through the various TR isoforms and explore
influences of these analogs on specific thyroid hormone-responsive
physiological functions.
Described in this report are the effects of the
T3 analog GC-1, which exhibits similar binding
affinity and actions in cell culture as T3 for
the TRß1 subtype, but approximately 10 times weaker binding and
actions in cell culture for the TR1 subtype (15). Upon
administration of T3 at a dose that largely
restored a euthyroid status (3.5 ng/g BW, defined as
1 x T3) and equimolar amounts of GC-1 (1.8 ng
GC-1/g BW, defined as 1 x GC-1), distinct differences in the
responses of specific thyroid hormone-responsive parameters were noted.
Thus, 1 x GC-1 lowered cholesterol levels to the euthyroid range, as
did administration of 1 x T3. Triglyceride
levels were lowered significantly with GC-1 compared with
T3, which exerted no significant effect on
triglyceride levels. 1 x GC-1 did not increase heart rate from the
hypothyroid level, but T3 at this dose
significantly increased heart rate and restored it to the normal
euthyroid level. The mRNA coding for the pacemaker channel HCN2, which
was severely decreased in hypothyroid mice, was restored by
1 x T3 to control levels and further increased
with higher T3 doses, whereas 1 x GC-1 did not
affect HCN2, and only higher GC-1 doses restored mRNA HCN2 levels.
These effects of T3 and GC-1 on HCN2 paralleled
their effects on heart rate. Restoration to control values by
1 x T3, but not by 1 x GC-1, was also seen
for mRNAs of MHC, MHCß, and Serca2 and for body weight, heart
weight, and HW/BW ratio. Cardiac contractility was increased by
1 x GC-1 and 1 x T3, although this effect
was larger and included more parameters with
1 x T3.
The potential mechanism underlying this and other differential
responses between GC-1 and T3 could be linked to
differences in TRß isoform distribution in specific organs. Findings
by other investigators have shown that TRß receptors are the
predominant isoform in the liver, accounting for 80% of
T3 receptor binding (23). In addition, the
T3-mediated lowering of cholesterol levels is
most likely due to an elevated clearance rate of cholesterol mediated
by increased expression of hepatic low density lipoprotein receptors
(24) and an increase in specific lipid-lowering liver enzymes (6, 25, 26). The strong effect of GC-1 on lowering cholesterol, which is equal
to that of T3, and the even more enhanced effect
of GC-1 on lowering triglyceride levels, which exceeds the effect of
T3, is therefore compatible with a
TRß-preferred action of GC-1. In contrast to the liver, close to half
of the T3 receptors in the heart are of the
TR1 subtype (23). The target genes of thyroid hormone effects on the
heart rate are not known, although HCN2 and HCN4 are possible
candidates. As mentioned previously, mice with deletion of the TR
(12), but not the TRß (14), receptor have marked bradycardia. The
absence of GC-1 effects on increasing heart rate from the hypothyroid
value is compatible with the previous finding that the
EC50 on TR1 for GC-1 is approximately 10 times
greater than that of T3, and
T3 action on heart rate is mediated through
TR1. In contrast, a T3 dose of 3.5 ng/g BW
returned heart rate from a decreased hypothyroid rate to the normal
euthyroid range. These differential effects of T3
vs. GC-1 on heart rate are compatible with selective TRß
actions of GC-1. However, in this context we cannot exclude
tissue-selective differences in the uptake of the various compounds as
contributors to the differential responses.
Recently, the gene coding for the cardiac pacemaker channel
If, which appears to play an essential role in
setting the heart rate, has been cloned (27, 28). This
hyperpolarization-activated inward rectifier channel conducts the
monovalent cations potassium and sodium at a 4:1 ratio. This channel
has been termed hyperpolarization-activated cyclic nucleotide-regulated
(HCN) channel. Four isoforms of it occur in the brain (HCN1–HCN4) (27, 28). Two of these, HCN2 and HCN4, are expressed in the heart, with a
marked predominance of HCN2 (Gloss, B., and W. H. Dillmann,
unpublished observations). As the expression of this gene might be
involved in thyroid hormone effects on heart rate, we quantitated the
response of HCN2 to T3 and GC-1 and found a
response in line with that of heart rate. HCN2 mRNA does not
significantly increase upon treatment with 1 x GC-1, in contrast to
treatment with 1 x T3, which results in
elevated HCN2 mRNA levels compared with euthyroid levels. It appears
therefore quite likely that the discrepant heart rate and HCN2
responses to T3 and GC-1 may indicate a
cause-effect relationship between the heart rate and the level of
expression of the HCN2 channel. Thus, HCN2 may be a major target for
thyroid hormone regulation of the heart rate. In line with the effects
on heart rate are other markedly smaller effects of GC-1 vs.
T3 on other cardiac parameters. This includes the
finding that 1 x GC-1 has no significant effect on heart weight and
mRNAs coding for proteins related to cardiac contraction, such as
MHC, MHCß, and Serca2. It is of interest to note that the first
derivative of the rise in systolic pressure,
dP/dtmax, significantly increased by treatment
with 1 x GC-1, although this effect was smaller than that with
1 x T3 treatment. Some specific cardiac
functions, such as dP/dtmax, show a partial
response to a TRß-preferred agent.
Whereas the GC-1-induced lowering of cholesterol shows a similar
response to T3 at doses of 1.8, 8.1, and 16.2
ng/g BW, other responses differ. Thus, GC-1-induced lowering of
triglyceride levels is more efficient at all doses compared with
T3. In contrast, other parameters, such as body
weight or HW/BW ratio, showed no change from the hypothyroid level at
any GC-1 dose, in contrast to T3, which resulted
in marked and progressive increases in these parameters. The mechanisms
underlying these discrepant T3 vs.
GC-1 responses are currently unclear. The GC-1 effect on heart weight,
which only occurs at high GC-1 doses, is in line with a significantly
diminished GC-1 effect on parameters such as heart rate and reversal of
MHC RNA levels and MHCß mRNA levels compared with
T3. In addition, the
T3-induced increase in body weight was already
maximal at doses of 3.5 ng/g BW and did not further increase at higher
T3 doses. It is unlikely that the decreased
response of heart weight or MHC mRNA levels of GC-1-treated mice is
due simply to a much weaker thyromimetic action than
T3, as this would not explain the equivalent
effects of GC-1 and T3 on cholesterol levels and
the greater effects of GC-1 than T3 on
triglyceride levels. Results from mice with deletion of TR
vs. TRß clearly indicate that for some effects
T3 can exert a similar influence, being bound to
either TR1 or TRß1, and the different TR isoforms can exert
functional cross-coverage for each other. In contrast, other effects
can only be fully mediated by T3 bound to either
the TR or the TRß. Another mechanism that has to be considered for
differential effects of GC-1 is the difference in tissue distribution;
tissue/plasma ratios of T3 are similar for heart
and liver, whereas the tissue/plasma ratio of GC-1 for the liver is 17
times higher than that for the heart. This difference in tissue
distribution ratio could contribute to a higher stimulation of hepatic
effects such as lipid lowering by GC-1 without increasing the heart
rate. This observation implies that organ-specific distribution
contributes to the effect of this new thyroid hormone analog. Much
lower absolute levels of GC-1 than of T3 in
plasma and tissues also indicate that GC-1 may be more rapidly
metabolized than T3. Details of
T3 vs. GC-1 pharmacokinetics need to
be clarified by an in-depth study; however, it may be that the
tissue-selective thyromimetic effects of GC-1 arise from a combination
of selective tissue uptake and selective activation of TR
127.
The comparative effects of GC-1 and T3 on rats
were determined to show the relationship between heart rate
vs. cholesterol and TSH vs. cholesterol. This was
performed in rats because of the greater reliability of heart rate and
TSH measurements in this species. As TSH and cholesterol lowering are
both thought to be primarily due to TRß activation (12, 14), the
potency ratios for these two plasma parameters should be similar for
T3 and GC-1. The ED30
(TSH)/ED50 (cholesterol) potency ratios were
similar for GC-1 and T3, which is consistent with
them being mediated by the same receptor subtype. GC-1 was
significantly more selective for cholesterol lowering compared with its
tachycardic potency. The selectivity was more than 15-fold, and this
may be conservative, as we could not observe significant tachycardia
even at high doses. These data are consistent with the TRß binding
selectivity for this compound (15). Tachycardia is thought to be
primarily due to TR stimulation, and our results are consistent with
this hypothesis; however, selective tissue uptake may also play a role,
as mentioned previously.
Although GC-1 is relatively selective for TRß, it has a slightly weaker binding affinity (2-fold less potent) compared with T3. The differences in potency for cholesterol and TSH suppression were on the order of 10- to 20-fold less potent for GC-1. These differences in potency of TRß-mediated effects of GC-1 and T3 were not observed in mice. Cholesterol levels were similarly lowered by equivalent T3 and GC-1 doses, and GC-1 was even more effective in lowering triglycerides. The rats were euthyroid and had only elevated cholesterol levels, so an influence on triglyceride lowering could not be assessed. The effects of GC-1 and T3 on cholesterol levels in mice were maximal at the 1x doses; thus, a difference of potency for this parameter might have become apparent at lower GC-1 and T3 doses. T3 and GC-1 were administered orally to rats to determine whether the in vitro selectivity could be observed when administered in the manner in which patients will be treated. The selectivity should not be affected by differences in oral absorption, although the apparent potency could certainly be affected. The results clearly showed that therapeutic cholesterol-lowering activity was observed for GC-1 without concomitant tachycardia, unlike T3; therefore the in vitro studies correctly predicted the selectivity observed in vivo.
Previous reports (7, 8) of thyroid hormone analogs have indicated that some thyroid hormone analogs can lead to lipid lowering without significant cardiac effects. A differential effect on the heart was observed for the compound 3,5-diiodothyropropionic acid, which increased cardiac contractility without increasing heart rate and MHC isoform expression (29). The actual mechanism of action of this compound is also not clear, as 3,5-diiodothyropropionic acid differs in several aspects from the chemical structure of thyroid hormones, and its binding to thyroid receptors was not studied.
There has been extensive interest in using thyroid hormones for treating a variety of indications, including weight loss, lowering plasma lipid levels, and treatment of hypothyroid states in the elderly. Unfortunately, the use of thyroid hormones for these indications has been limited, predominantly by the fact that the compounds have adverse effects on the heart, particularly on heart rate and rhythm. The findings in the current study and in some previous reports (7, 8) indicate that lowering of cholesterol without a significant increase in heart rate is possible. Our report shows that a compound such as GC-1 has TRß-selective actions, with over 14 times greater potency on lowering cholesterol and TSH levels than on heart rate, with similar or greater effects than T3 on triglyceride levels and diminished effects relative to T3 on weight gain and heart rate. These findings provide further support for the idea that compounds with such selectivity can be generated. Whereas the mechanism for these in vivo actions is not fully understood and may involve a combination of selective tissue uptake and selective TR subtype activation, the results suggest that compounds with properties similar to GC-1 may be useful therapeutic agents for the treatment of a variety of thyroid hormone-related metabolic disorders.
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Received September 14, 1999.
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