|
Wang EQ and Fung HL
Effects of Obesity on the Pharmacodynamics of Nitroglycerin in Conscious Rats
AAPS PharmSci
2002;
4
(4)
article 28
( https://www.aapspharmsci.org/scientificjournals/pharmsci/journal/ps040428.htm
).
Effects of Obesity on the Pharmacodynamics of Nitroglycerin in Conscious Rats
Submitted: May 31, 2002; Accepted: September 9, 2002; Published: October 7, 2002
Ellen Q. Wang
1
and Ho-Leung Fung
2
1
Department of Pharmacokinetics, Dynamics, & Metabolism, Pfizer Global Research & Development, Eastern Point Road, MS4111, Groton, CT 06340
2
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, SUNY, Buffalo, NY 14260-1200
|
Correspondence to:
Ellen Q. Wang
Telephone:
Facsimile:
E-mail: ellen_q_wang@groton.pfizer.com
|
Keywords:
nitrate tolerance
Zucker
noninsulin dependent diabetes mellitus
nitric oxide
obesity
|
Abstract
Literature reports have suggested that hemodynamic response toward organic
nitrates may be reduced in obese patients, but this effect has not been studied. We
compared the mean arterial pressure (MAP) responses toward single doses of
nitroglycerin (NTG, 0.5-50 µg) in conscious Zucker obese (ZOB), Zucker lean
(ZL), and Sprague-Dawley (SD) rats. NTG tolerance development in these animal groups
was separately examined. Rats received 1 and 10 µg/min of NTG or vehicle infusion,
and the maximal MAP response to an hourly 30 µg NTG IVchallenge dose (CD) was
measured. Steady-state NTG plasma concentrations were measured during 10 µg/min
NTG infusion. The E
max
and ED
50
values obtained were 33.9 ± 3.6 and 3.5 ± 1.7 µg for SD rats, 33.2 ± 4.1 and
3.0 ± 1.4 µg for ZL rats, and 34.8 ± 3.9 and 5.3 ± 2.8 µg for ZOB rats,
respectively. No difference was found in the dose-response curves among these 3
groups (P > .05, 2-way ANOVA). Neither the dynamics of NTG tolerance development, nor
the steady-state NTG plasma concentrations, were found to differ among these 3 animal
groups. These results showed that ZOB rats are not more resistant to the hemodynamic
effects of organic nitrates compared with their lean controls. Thus, the acute and
chronic hemodynamic effects induced by NTG are not sensitively affected by the
presence of obesity in a conscious animal model of genetic obesity.
Introduction
Organic nitrates such as nitroglycerin (NTG) are used in the management of
various cardiovascular diseases. Clinical use of these drugs appears to ignore body
weight as a determinant of administered dose, but no study has appeared to provide
the scientific basis for this practice. Obesity is now categorized as an epidemic in
the United States, about 25% of adult women and 20% of adult men are clinically obese
with a body mass index of at least 30.
1,2
Obesity is often associated with increased mortality because of the increased
risk of a number of diseases including noninsulin dependent diabetes mellitus
(NIDDM), sleep apnea, pulmonary dysfunction, and cancer.
2
The presence of obesity also leads to a number of physiological changes in
cardiovascular function, resulting in higher incidence of many cardiovascular
diseases, such as atherosclerosis, coronary artery disease, and hypertension.
1-4
In addition to increased health risks, obesity may affect drug therapy because
of changes in drug pharmacokinetics (PK) and pharmacodynamics (PD). The volumes of
distribution of many highly lipophilic drugs, such as barbiturates and
benzodiazepines, have been shown to be increased in obesity, resulting in much longer
plasma half-life.
5
Dosage adjustments are often made for aminoglycosides in obese patients to
account for the altered PK profiles observed with this class of agents.
6
However, little is known about the effects of this disease state on the PK and
PD of NTG. It is therefore unknown whether obesity, as such, may require adjustments
in the administered dose of NTG to elicit adequate efficacy.
The Zucker obese rat (ZOB) is an animal model of genetic obesity and has been
used extensively to study the possible effects of obesity on the pharmacokinetics and
pharmacodynamics of a number of drugs.
7-11
In addition, ZOB has been used as an animal model for NIDDM,
12-16
since these animals exhibit many symptoms that are observed in NIDDM patients,
including insulin resistance, hyperglycemia, and hyperlipidemia. Some reports had
surmised (but not demonstrated) that diabetic patients may be more resistant to
organic nitrates possibly because of increased oxidative stress,
17,18
a mechanism that has been suggested as one of the underlying causes of nitrate
tolerance.
19
Thus, a study examining the acute and chronic pharmacological effects of NTG
in ZOB may not only reveal the effects of obesity on the PK/PD of NTG but may also
provide useful suggestions regarding the possible effects of experimental
NIDDM.
In the present study we examine the role of obesity in affecting NTG PK/PD,
comparing our observed results in ZOB to those obtained from its lean controls
(Zucker lean, ZL rats), as well as those from normal Sprague-Dawley (SD) rats. We
included the last group of animals for comparison because several previous studies in
our laboratory have established basic information on the PK/PD of NTG in this animal
strain.
20,21
Materials and Methods
Animals
All surgical procedures were performed according to protocols approved by
the Institutional Animal Care and Use Committee, University at Buffalo. Male SD
rats weighing 300 to350 g were obtained from Harlan (Indianapolis, IN), male ZL
rats (340-440 g) and ZOB rats (490-590 g) were obtained from Vassar College
(Vassar, NY). Two days prior to the experiment, animals were cannulated at the
left femoral artery for blood pressure measurements or blood sampling, and at the
left femoral vein for bolus drug administration and at the right jugular vein for
drug infusion.
22
Hemodynamic measurements
All hemodynamic studies were carried out in conscious, unrestrained
animals. Food and water were allowed ad libitum. Animals were divided into 2
study groups: (A) NTG dose response group (n = 5-6 in each group) and (B) NTG
tolerance group (n = 3-5 in each group). On the day of the experiment, systolic
and diastolic blood pressures were recorded continuously via the left femoral
artery cannula using a Statham pressure transducer (Ohmeda, Murray Hill, NJ) and
a Gould RS3400 recorder (Gould, Cleveland, OH). Basal systolic and diastolic
blood pressures were recorded for at least 15 to 30 minutes for each animal prior
to the start of the experiment. In group A, intravenous (IV) bolus doses of NTG
(0.5, 1.0, 2.0, 4.0, 6.0, 10, 15, 20, 30, 40, and 50 µg) were given in random
order via the left femoral vein cannula at 30-minute intervals. The injection
volume for all NTG bolus doses was kept at 50 µL, using 5 % dextrose (D5W) as a
diluent. The hemodynamic response of each NTG bolus dose was measured as percent
maximal change in mean arterial pressure (MAP) vs baseline MAP just prior to the
specific dose. In group B, animals received an initial 30 µg of NTG IV bolus
challenge dose (CD), and baseline maximal MAP response to this dose was recorded.
Fifteen minutes later, rats were infused continuously with 1 of the following 3
regimens using an electronically controlled infusion pump (Harvard Instruments,
South Natick, MA): 1 µg/min of NTG, 10 µg/min of NTG, or D5W control.
The infusion time was 8 hours for the 1 µg/min NTG and D5W infusion groups,
and 1 hour for the 10 µg/min NTG infusion group, as previous data showed
that NTG tolerance was readily observed within 1 hour at this higher dose.
23
The infusion volume was kept at a constant rate of 10 µL/min for all
3 groups of animals. To determine the development of NTG hemodynamic tolerance, a
30-µg NTG IV bolus CD was given hourly, and the hemodynamic response
produced by the hourly CD was compared with that produced before NTG infusion. A
decrease in the maximal MAP response of the bolus CD indicates the development of
nitrate tolerance. MAP was calculated as (diastolic pressure + 1/3 [systolic
pressure — diastolic pressure]). The time-averaged maximal MAP response was
calculated as the sum of the MAP response induced by NTG bolus CD divided by the
number of NTG bolus CD. NTG pharmacokinetics
Steady-state NTG plasma concentrations were determined in all 3 animal
groups (n = 3-4 each). NTG was infused via the right jugular vein at 10
µg/min for 2 hours using a programmable Harvard infusion pump (pump 22,
Harvard Instruments). Aliquots of 0.4 mL blood were taken from the left femoral
artery catheter at 60 and 120 minutes. Plasma was immediately isolated following
each blood sample collection and stabilized in 1N silver nitrate and stored at
-80°C until analysis. NTG concentration in plasma was measured using gas
chromatography with electron capture detection as described previously.
21
Statistical analysis
Data are presented as mean ± SD. The E
max
and ED
50
values from NTG dose-response curves were calculated by fitting the curves
with an E
max
model using WinNonlin (Version 2.1, Pharsight, Palo Alto, CA). Statistical
analysis was performed, where appropriate, using either Student t-test, 1-way
ANOVA, followed by the Student-Newman-Keuls post-hoc test, or 2-way ANOVA.
Differences with P < .05 were considered statistically significant.
Results
The body weights of the ZOB, ZL, and SD rats were 536 ± 36, 393 ± 34, and
346 ± 4.5 g, respectively (ANOVA, P < .001 vs ZL or SD rats). Effects of obesity on NTG dose—MAP response curve after acute
dosing
Figure 1
shows the in vivo NTG dose—MAP response curves in SD, ZL, and ZOB
rats. Bolus injections of NTG caused a dose-dependent increase in maximal MAP
responses in all 3 groups of animals. It was observed that the effects of NTG
bolus on MAP were short-lived for all doses examined. Blood pressure returned to
baseline values within 30 seconds after NTG bolus dosing. The duration of
NTG-induced MAP effect was not dose-dependent (data not shown). The effect of NTG
on maximal-MAP dose response was well described by an E
max
model.
Figure 1
shows that there were no apparent differences in NTG dose-response curves
among the 3 animal groups studied, even when NTG doses were not adjusted for body
weight (P > .05 vs ZL or SD rats, 2-way ANOVA). The E
max
and ED
50
values obtained from the 3 animal groups are listed in
Table 1
. ZOB animals exhibited E
max
and ED
50
values similar to the ZL controls and the SD rats (P > .05,
ANOVA).
Effects of obesity on NTG tolerance development
Figure 2
shows the effects of D5W vehicle infusion on maximal MAP response to bolus
CD of NTG administered at hourly intervals. Consistent with previous reported
studies,
23
a 30-µg NTG IV bolus CD produced an immediate maximal decrease in MAP of
about 23% to 29%. When D5W was infused over 8 hours, repeated bolus CD of 30
µg NTG produced a consistent maximal drop in MAP of about 24% to 27%, and
none of the values observed during the control infusion period was different from
that observed prior to D5W infusion (P > .05, ANOVA). There were no apparent
differences in the maximal MAP response of the hourly NTG IV bolus CD in the ZOB
animals when compared with the ZL and SD rats (P > .05, ANOVA). Consistent
with these results, the time-averaged maximal MAP response (% change from
baseline) of the hourly CD over the entire 8-hour infusion period was 27.1% ±
0.8%, 26.7% ± 4.7%, 26.4% ± 4.1% for SD rats, ZL and ZOB rats,
respectively (
Figure 2B
). ANOVA revealed that this parameter was not significantly different among
the 3 animal groups.
Figure 3
shows the effects of 1 µg/min NTG continuous infusion on the maximal
MAP response of the hourly NTG IV bolus CD. At this low dose, there was no
apparent change over the entire 8-hour infusion period. Each of the hourly
30-µg NTG IV bolus CD produced similar maximal MAP response (P > .05,
ANOVA). ANOVA revealed that the maximal MAP effect of the 30-µg NTG IV bolus
CD from the NTG infused group was not different from the corresponding D5W
controls for all 3 groups of animals (P > .05). The time-averaged values for
maximal MAP response of the hourly CD over the 8-hour infusion period was 25.4% ±
3.5%, 21.9% ± 2.4%, and 19.1% ± 1.8% for SD rats, ZL, and ZOB rats, respectively.
ANOVA revealed that the time-averaged maximal MAP responses (percentage change
from baseline) in the ZOB rat were significantly different from the SD rats (P
< .05), but not from the ZL rats (P > .05).
Figure 4
shows the effect of 10 µg/min NTG continuous infusion on the maximal
MAP responses of the hourly NTG IV bolus CD. Consistent with data obtained from
earlier studies,
23
the maximal MAP response of the first hourly NTG IV bolus CD was
significantly attenuated. The decreases in maximal MAP response at 1 hour
compared with baseline MAP response were 60.5%, 58.7%, and 57.0% for SD, ZL, and
ZOB rats respectively (P < .001 vs the corresponding baseline response for
each group of animals, Student t test). However, there was no apparent difference
in the hemodynamic behavior among the 3 animal groups (P > .05, ANOVA). The
time-averaged maximal MAP response of the challenge NTG dose after the 1-hour
infusion period was 18.7% ± 0.8%, 17.9% ± 2.4%, and 18.9% ± 4.9% for SD, ZL, and
ZOB rats, respectively (P > .05, ANOVA).
Effect of obesity on steady-state NTG plasma concentrations after 10
µg/min infusion
Figure 5
shows NTG plasma concentrations at 1 and 2 hours after the start of 10
µg/min infusion. Consistent with the reported half-life of approximately
5 minutes in rats,
20
steady-state NTG concentration was reached at 1 hour of NTG infusion. Our
results indicate that NTG plasma concentrations at 1 and 2 hours were similar in
all 3 animal groups. The time-averaged steady-state plasma concentrations were
84.9 ng/mL, 85.7 ng/mL, and 102.9 ng/mL for SD, ZL, and ZOB rats, respectively
(P > .05, ANOVA).
Discussion
In the present study, we showed that acute in vivo NTG hemodynamic
responses were not significantly altered in the ZOB rats vs ZL and SD controls
(
Figure 1
), whether the MAP response was corrected for body weight or not. In
addition, our results indicated that the time-course and extent of NTG tolerance
development in the ZOB rats were not different from the ZL and SD controls at the
2 infusion doses of NTG examined. Neither dose was corrected for the body weight
difference between ZOB rats and the ZL and SD controls. Consistent with these
data, no difference in the steady-state NTG plasma concentration was observed
among these animal groups. These results suggest that NTG PK and PD are not
highly sensitive toward obesity in a conscious animal model of genetic
obesity.
Obesity is often closely associated with NIDDM, and the body mass index has
been shown to be a good predictor of the risk of diabetes.
2,4
Endothelial dysfunction and altered nitric oxide (NO) activity have been
observed in obesity
24-28
and obesity-associated NIDDM
17,18,
29-32
for endothelium-dependent vasodilators. Moreover, impaired vasodilator
responses to endothelium-independent vasodilators such as NTG and sodium
nitroprusside have been reported for obesity-associated NIDDM.
17,
32,33
The results from the in vivo NTG dose-response curve (
Figure 1
) showed that acute NTG pharmacological effect was not significantly
changed in obesity even when doses were not adjusted for kg body weight,
suggesting that obesity and possibly NIDDM do not lead to any observable
resistance to the hemodynamic effects of NTG. These observations are in agreement
with several reports that only endothelium-dependent vasodilator responses are
altered in obesity.
27,
34,35
Consistent with our data, literature reports also indicated that
endothelium-independent vasodilator responses are not impaired in either animal
models of NIDDM, using ZOB rats,
31.36
or diabetic humans.
37,38
However, our results from the acute NTG hemodynamic study (
Figure 1
) are in contrast to an earlier study by Laight et al,
33
which reported impaired NTG-induced MAP effects in the ZOB rats vs ZL
controls. This apparent discrepancy may be due to the differences in the animal
models employed. Our study was performed in conscious, unrestrained animals,
whereas Laight et al
33
performed their study in anesthetized animals, in which cardiovascular
regulatory controls are diminished. In addition, the magnitude of change shown
in the latter study was quite modest (about 20% decrease in E
max
, with no apparent change in ED
50
). Thus, the findings of Laight et al
33
can also be interpreted as demonstrating only a marginal effect of
obesity on NTG-induced vascular response.
Figure 2
shows that with vehicle D5W infusion, NTG-induced maximal MAP response was
similar in the ZOB rats, ZL controls, and SD rats throughout the 8-hour infusion
period. These results suggest that in the presence of D5W vehicle infusion, the
30-µg NTG IV bolus, when given at 1-hour intervals, did not induce any
apparent hemodynamic tolerance. At an infusion rate of 1 µg/min NTG (
Figure 3
), vascular tolerance development was not observed for all 3 groups of
animals (P > .05 vs corresponding D5W control group, and P > .05 vs
preinfusion baseline response). These results were in general agreement with our
previous data
23
showing that, in SD rats, administration of a 1 µg/min regimen for 10
hours produced little hemodynamic tolerance. However, when the time-averaged
maximal MAP response was used as an index of NTG hemodynamics, ANOVA revealed
that ZOB rats were significantly different from the SD rats but not from the
control ZL rats. These results suggest that there may be slight differences in
NTG hemodynamics among the different strains of rats. However, these results
also indicated that genetic obesity has no apparent effect on NTG hemodynamics,
as no differences were observed between the ZL and ZOB rats.
When the NTG infusion dose was increased from 1 µg/min to 10
µg/min, hemodynamic tolerance to the maximal MAP lowering effect of the
hourly 30-µg NTG IV bolus was observed within 1 hour of NTG infusion (
Figure 4
). The development of NTG tolerance at this high infusion dose was both
rapid and pronounced. However, the extent of NTG tolerance development in the 3
groups of animals was not different. Laight et al had reported a more severe NTG
tolerance in the ZOB rats compared with the ZL control; however, the difference
reported did not reach statistical significance.
33
The ZOB rats have been shown to manifest pre-existing oxidative stress in
both the myocardium
39
and the plasma.
40
This general state of elevated oxidative stress may lead to in vivo
inactivation of NO. The reactive oxygen species (ROS), particularly superoxide
anion, can inactivate NO via the formation of the highly reactive radical,
peroxynitrite,
19
and deplete intracellular thiols, which are important mediators in NO
metabolism.
41
Results from our study would imply that the increased oxidant stress
present in ZOB does not affect the animal's vascular sensitivity to NTG. One
possible explanation for this observation is that in vivo oxidative stress, even
when it occurs generally in ZOB rats, does not affect the vasculature.
Plasma 8-epi-prostaglandin F
2 , used in the study by Laight et al
33
as an index of oxidant stress, may not be reflective of the oxidant state
in the vasculature. Indeed this explanation was supported by the observation in
the same study that although tiron (a superoxide scavenger) effectively restored
NTG-induced vasodilation in ZOB rats, it was not able to restore plasma
8-epi-prostaglandin F
2 levels to that of ZL rats. It is currently unknown if higher levels of ROS
are indeed present in the vasculature of the ZOB rat since no studies have been
performed to address this issue.
Consistent with data obtained from the in vivo pharmacodynamic studies,
steady-state NTG plasma concentrations were not changed in the ZOB rats. The mean
steady-state plasma concentrations observed in our study were in general
agreement with previous reports in normal SD rats and congestive heart failure
rats.
21
Our results also suggest that obesity and possibly NIDDM did not have
a discernable effect on NTG plasma pharmacokinetics.
In conclusion, results of the present study do not support the view that
NTG-induced hemodynamic effects may be significantly affected by obesity and
experimental NIDDM. The PK/PD of NTG are not sensitively dependent on the body
weight of conscious rats.
Acknowledgements
We thank Ms Sun Mi Fung and Mr David M. Soda for their excellent technical
assistance. This work was supported in part by NIH grant HL22273 and funds from the
University at Buffalo Foundation.
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