Download Cardiovascular Effects after Inhalation of Large Doses of Albuterol

FUNDAMENTAL AND APPLIED TOXICOLOGY 4 0 , 5 2 - 6 2 (1997)
ARTICLE NO F A 9 7 2 3 7 0
Cardiovascular Effects after Inhalation of Large Doses of Albuterol Dry
Powder in Rats, Monkeys, and Dogs: A Species Comparison
Janet M. Petruska,*' J. Gregory Beattie.t Bruce O. Stuart,* Sudhakar Pai,* Karen M. Walters,*
Christopher M. Banks,! George W. Lulham.t and Elmer J. Mirro*
*Drug Safety and Metabolism, Schering-Plough Research Institute, P.O. Box 32, 144 Route 94, Lafayette, New Jersey 07848;
and tClinTrials BioResearch Ltd, Montreal, Quebec, Canada
Received December 16, 1996; accepted September 3, 1997
study do not suggest a cardiotoxicity risk in the human population
after repeated exposures to clinical doses of albuterol currently
Cardiovascular Effects after Inhalation of Large Doses of Albuterol Dry Powder in Rats, Monkeys, and Dogs: A Species Comparison. Petruska, J. M., Beattie, J. G., Stuart, B. O., Pai, S., Walters,
K. M., Banks, C. M., Lulham, G. W., and Mirro, E. J. (1997). Fundam. Appl. Toxicol. 40, 52-62.
used in the treatment of asthma,
c 1997 sockty of Toxicology.
For almost 30 years, albuterol has been used as a prophyAlbuterol is a quickly acting /32-adrenergic agonist bronchodila- lactic and rescue medication for the relief of bronchospasm
tor widely used by asthmatics. Because recent case-control studies associated with reversible obstructive airway disease (Anonhave suggested a relationship between the increase in mortality of ymous, 1971). Albuterol possesses greater than a 500-fold
asthmatics over the past decade and the use of /92-adrenergic ago- activity toward /32-adrenergic receptors in comparison to / 3 r
nists in the control of asthma, concern has developed regarding adrenergic receptors (Jack, 1991). Because /3 -adrenergic re2
the potential cardiotoxlcity of /92-specific adrenergic agonists, inceptors predominate in the smooth muscle of the bronchi
cluding albuterol. The aim of this investigation was to assess the
potential for cardiotoxicity of inhaled albuterol dry powder in rats, and activation of these receptors results in bronchodilation
monkeys, and dogs. All species were exposed to an aerosol of (Barnes, 1995), albuterol is well-suited for its indication.
albuterol 1 h per day, 7 days per week, for at least 2 weeks. Control Albuterol also is rapidly acting, potent, efficacious, and has
groups were exposed to filtered conditioned air and handled in a long history of safe use (Price and Clissold, 1989).
the same manner as the albuterol-exposed animals. Plasma conConcern has developed in the past decade regarding the
centrations of albuterol confirmed systemic exposure. The daily safety of /?2-adrenergic bronchodilators, including albuterol,
inhaled dose received by the animals was calculated based on given their widespread use and the increase in asthma mormeasured respiratory minute volumes, published respiratory tract tality (Sears and Taylor, 1994). Recent case-control studies
deposition data, as well as HPLC-determined particle size distribuhave suggested a link between the clinical use of albuterol
tion data and aerosolized albuterol concentrations. Multiples of
the maximum daily clinical dose (presentation of IS /xg/kg in a and the increase in asthma mortality (Spitzer et al., 1992;
70-kg human) were approximately 0.25- to 2500-fold in the rat, Coughlin et al., 1995). In addition, reports describing inhala9- to 100-fold in the monkey, and 0.5- to 90-fold in the dog. No tion or ingestion of doses of albuterol exceeding the maxifindings attributed to albuterol were observed in the monkey. mum clinical daily dose and the subsequent tachycardia,
Tachycardia and transient hypokalemia occurred in rats at multi- hypokalemia, and potential for eliciting arrhythmias have
ples of 1.5 times or greater of the maximum clinical dose. Absolute caused concern (Leitch et al., 1976; Lewis et al., 1993;
and relative heart weights increased in rats receiving multiples of Wong et al., 1990). The objective of this investigation was
47 times or greater of the maximum human dose. In the absence to evaluate the potential for cardiotoxicity of inhaled large
of histopathologic findings, the increases in rat heart weights were
doses of albuterol in rats, monkeys, and dogs. The animals
considered a physiologic hypertrophic response to tachycardia. In
dogs tachycardia and transient hypokalemia occurred at all doses were exposed to albuterol dry powder using well-charactertested. Slight to mild fibrosis in the papillary muscles of the left ized exposure systems. Measurements of plasma concentraventricle of the heart occurred in dogs at multiples ^ 19 times the tions of albuterol and serum potassium, evaluation of electroclinical dose. The cardiovascular effects observed were consistent cardiographic data, and evaluation of heart tissue for organ
with the known pharmacologic action of 4?2-adrenergic agonists. weight and histopathologic changes were performed.
Due to the lack of toxdcologically relevant findings in rats and
monkeys and the wide safety margin in dogs, the findings in this
METHODS
Inhalation and exposure system. Seven-week-old Sprague-Dawley
rats (Crl:CD (SD)BR/viral antibody-free, Charles River, Canada), young
' To whom correspondence should be addressed. Fax: (201) 579-4211.
0272-0590/97 $23.00
Copyright C 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
52
INHALED ALBUTEROL IN RAT, MONKEY, AND DOG
adult cynomolgus monkeys (Charles River, TX) and 6- to 7-month-old
beagle dogs (HRP, MI) were exposed to aerosols of micronized albuterol
dry powder (99.9% purity), 1 h/day, for 7 days/week, for approximately 2
weeks. In all studies, a control group consisting of animals exposed to
filtered conditioned air only was included and was handled in the same
manner as the albuterol-exposed animals, with the exception of exposure
to drug.
For all studies, the aerosol of albuterol dry powder was generated using
a fluid energy air mill (Trost Mill, Gem T Research model, Garlock, Inc.,
Newton, PA) (Cheng et al., 1985). Different concentrations of aerosolized
albuterol were obtained by controlling the rate at which the mill was supplied the albuterol dry powder. For each daily exposure, concentrations of
albuterol were continually estimated during exposures using a real-time
aerosol monitor (RAM-S, MIE Inc., MA) and confirmed by HPLC analyses
of filter samples collected quarter hourly at the animal's breathing point
during the exposure period. In addition, once weekly, particle size analyses
were performed using an Andersen 1ACFM cascade impactor (Andersen
Instruments, Atlanta, GA) situated at the animal's breathing point. The
effective cutoff diameters (ECD50) for each stage were 9.0, 5.8, 4.7, 3.3,
2.1, 1.1,0.7, and 0.4 ^m. The mass median aerodynamic diameter (MMAD)
and geometric standard deviation (GSD) were calculated as well as mean
MMADs and GSDs. The mean particle size distribution data were used for
the calculations of total achieved dose levels of albuterol.
Rats, positioned in restraint cones, were exposed using "flow-through"
nose-only inhalation chambers (ADG Developments, England). Monkeys
were positioned on slings with only their heads exposed in a stainless steel
chamber to the appropriate aerosols. Dogs were positioned on slings and
were exposed using masks fitted to the mouth and nose. For dog exposures,
the aerosol was delivered from a central chamber through tubing to which
the masks were attached. The care and use of all animals were conducted in
accordance with the regulations of the USDA Animal Welfare Act (relevant
sections of 9 CFR, Parts 2 and 3) and the Canadian Council on Animal
Care.
For all studies, animals were acclimated to the exposure systems prior
to initiation of exposures. The airflow was maintained so as to provide
more than adequate oxygen to all animals during each day's exposure. Prior
to initiation of exposures, at intervals during the dosing period and after
completion of dosing, filter samples were obtained to determine the uniformity of aerosol concentrations within each dose group and the uniformity
of particle size distributions within each dose group and/or study. Analyses
confirmed that aerosol concentrations were uniform within each dose group
within a study and that particle size distributions were uniform within each
dose group and among all groups within each study. Animals were placed
onto the exposure systems only after the target aerosol concentrations were
reached, and were rotated to a different position for the following day's
exposure. All aerosol exhausted from the exposure systems was purified
using a 5-/im coarse filter and an absolute filter (99.97% efficient at 0.3
fim) before expelling the air from the building.
HPLC analyses of fitters collected from the exposure system. Filters
were extracted using a solution of dodecyl sulfate, glacial acetic acid, methanol, and distilled water. The albuterol extracted from the filters was separated by reverse phase HPLC using a Bondapak phenyl column (Waters
Co., MA) and a mobile phase consisting of dodecyl sulfate, glacial acetic
acid, methanol, and distilled water. An internal standard was included in
each separation and the peak area ratios of albuterol to the internal standard
were calculated. Albuterol was detected at 280 nm and quantitated using a
standard curve.
Respiratory minute volume measurements.
Respiratory minute volume
(RMV) measurements were obtained prior to initiation of exposure for four
rats/sex/group and on all monkeys and dogs. All RMV measurements were
obtained using the Buxco Electronics LS-20 system (Sharon, CT). The
same rats also had RMV measurements during the exposure period during
Week 1 or 2. Monkeys and dogs had RMV measurements during Week 1
or 2 within 2 h following the exposure period.
Calculation of achieved dose levels.
buterol were calculated according to
53
Total achieved dose levels of al-
total achieved dose
_ RMV (L/min) X albuterol cone. (/*g/L) X 60 min X deposition factor
mean body weight of animal (kg)
For the dog and monkey studies, the individual mean RMV was used in
the equation. For the rat studies, the mean RMV for each sex in each dose
group was used. The overall mean albuterol concentrations for each dose
group were used in the equation. Sixty minutes was the dajly duration of
dosing for all studies. Individual mean body weights over the course of the
dosing period also were utilized.
The deposition factor was calculated for each species based upon published respiratory tract deposition data. Total or regional deposition curves
were constructed for each species encompassing the range of ECD50s of
the stages in the Andersen 1ACFM cascade impactor. For the rat, the
regional deposition curves of the Fischer 344 rat following inhalation of
monodisperse aerosols were used (Raabe et al., 1988). The doses calculated
for each region in the rat were summed to give a total achieved dose of
albuterol. A curve of total respiratory tract deposition in cynomolgus and
rhesus monkeys was used to determine the deposition factor for the monkey
exposures (Palm et al., 1956). Because the monkey deposition data were
similar to those found in the human and because no data were available
for particles with an aerodynamic size greater than 5.0 (im for monkeys,
the deposition factor for particles greater than 5.0 /im was assumed to be
100%, as is approximated for the human (Palm et al., 1956). For the dog
studies, a total deposition curve was constructed from particle size diameters
ranging from 0.02 to 2.45 fxm using the deposition data of Kanapilly et al.
(1982), Wolff et al. (1981, 1982), and Cuddihy et al. (1973). Due to the
lack of suitable published canine particle size deposition data for aerodynamic sizes greater than 2.45 fim, human oral inhalation data (Phalen et
al., 1991) was used to extend the deposition curve prepared from the canine
data over the range of aerodynamic particle sizes separated by the Andersen
cascade impactor.
The mean relative quantities of albuterol aerosol collected at each stage
of the cascade impactor during the weekly particle size analyses were used
in the calculation of achieved doses. The mean quantity of aerosol collected
at each stage of the impactor was multiplied by the deposition factor interpolated from the curves of deposition data for each species. In order that the
proportion of particles of aerodynamic size below 0.4 ^m not be excluded
from the calculation of the total deposition factor and because the Andersen
cascade impactor could not separate particles of aerodynamic sizes between
0 and 0.4 fim, the fraction of particles collected below the final stage of
the cascade impactor were added to the fraction of particles collected on
the last stage of the impactor (ECD50 of 0.4 fim). The products of the
quantity of albuterol at each stage (and below for the last stage of the
impactor) and of the deposition fraction at each impactor stage were
summed to give a deposition factor. This deposition factor was used in the
equation to determine the dose for each animal. Mean total achieved dose
levels were calculated for each sex in each dose group.
Cardiovascular studies. Prior to initiation of the daily exposure and at
intervals during and postcxposurc (Exposure Days 1-2, 13-15), electrocardiograms (EKGs) were obtained for 3 - 9 animals/sex/group. Electrocardiographic recordings were made using limb leads I, n, and III for the rats;
limb leads I, II, III, aVR, aVL, and aVF and chest leads V,, V 2 , and V3
for the monkeys; and limb leads I, II, in, aVR, aVL, and aVF and chest
leads rV 2 , V 2 , and V, o for the dogs. All EKGs were obtained while the
animals were positioned in the restraints used for the exposures. The Cambridge Model MC 6400 analog instrument (Cambridge, England) and the
Schiller Model CS 612 digitized instrument (Schiller-Reomed AG, Switzerland) were used for recording the EKGs. Heart rate values were obtained
from the EKGs and the waveforms were qualitatively evaluated.
54
PETRUSKA ET AL.
Determination of plasma albuterol concentrations.
Prior to exposures
and at intervals up to and including 24 h following exposures, heparinized
blood samples were collected from rats (while under ether-induced anesthesia from the abdominal aorta), monkeys (via femoral venipuncture), and
dogs (via jugular venipuncture). The plasma was separated by centrifugation, frozen at approximately -80°C, and later assayed for the presence of
albuterol using a HPLC method with a limit of quantitation of 2.0 ng/ml.
Briefly, the method involved adsorption onto a C-8 solid-phase cartridge,
followed by an extensive washing procedure. Albuterol and the internal
standard were eluted with methanolic ammonium acetate, the eluate was
evaporated, and an aliquot of the reconstituted extract was analyzed by
HPLC with fluorometric detection. The assay was validated for rat, monkey,
and dog plasma. In addition, for each study, plasma collected from control
animals (not exposed in the exposure system to control air or albuterol)
served as assay blanks.
Determination of serum potassium concentrations.
On Days 1 and 14
or 15 for rats and dogs and on Day 13 for monkeys, serum was collected
at the same intervals used for collection of plasma for determination of
albuterol concentrations and analyzed for potassium using a Hitachi-717
analyzer (Mito, Japan).
Histopathology and organ weight assessment of heart tissue. Following completion of the dosing (during Week 3) or a 4-week postdose recovery
period, the animals were fasted overnight and weighed. They were then
anesthetized, exsanguinated, and subjected to a complete necropsy. The
hearts were dissected and weighed; relative heart weights were calculated
by dividing the absolute heart weight by the terminal body weight of the
animal. The hearts were fixed in 10% neutral buffered formalin, embedded
in paraffin wax, sectioned, and stained with hematoxylin and eosin. Deeper
sections of the heart (specifically sections containing the left ventricular
papillary muscle) of monkeys and dogs also were stained.
RESULTS
Achieved Aerosol Concentrations and Dose Levels of
Albuterol, Plasma Albuterol Concentrations, and
Particle Size Distributions
All animals survived the dosing regimen. A summary of
mean albuterol aerosol concentrations, achieved doses of
albuterol, C ^ plasma concentrations following a single 1h exposure and particle size distribution data is presented in
Table 1. Mean achieved albuterol aerosol concentrations
were within 20% of target aerosol concentrations. The coefficient of variation for achieved albuterol aerosol concentrations varied no more than 20% for all studies. Mean MMADs
ranged from 1.0 to 2.1 /zm for all species and mean GSDs
were no greater than 2.1. Within each exposure system for
each species, the MMADs increased slightly as the albuterol
aerosol concentration increased. This is commonly seen in
dry powder exposure systems, as agglomeration may occur
as the aerosol concentration is increased. For each species,
the slight differences in MMADs did not result in a significant difference in total percentage deposition of albuterol
among the dose groups.
Rats were exposed to target albuterol aerosol concentrations ranging from 0.25 to 1000 ^g/L- The mean total doses
the rats received ranged from 5.1 fig/kg daily to approxi-
TABLE 1
Summary of Mean Aerosol Concentrations, Achieved Dose Levels, Cm Plasma Albuterol Concentrations,
and Particle Size Analyses
Species
Rat
Monkey
Dog
Target
albuterol
Achieved
albuterol
aerosol
cone. (/ig/L)
aerosol
cone. (/*g/L)
0.25
1
4
12
36
100
300
1000
4
12
36
0.5
4
16
64
0.233
1.2
4.5
11.6
39.8
106
303
1098
4.2
12.9
36.5
0.53
4.3
16.6
65.8
Day 1 C '
(ng/ml)
Achieved dose level
(/jg/kg/day, mean ± SD)
Males
5.1
12
84
178
987
2,720
9,220
38,120
132
419
1,542
15
82
352
1,375
±
±
±
±
±
+
±
±
+
±
±
±
±
±
±
0.35
0.7
4.7
12.5
62.6
130
460
2530
10.3
50.8
218
10.9
24.3
176
497
Females
5.8
22
73
245
1,098
2,040
9,160
34,440
141
495
1,138
9
66
280
1,349
±
±
±
±
±
±
±
±
±
±
±
±
+
±
+
0.37
1.7
3.8
18.1
60.8
120
570
1960
27.3
85.5
306
2.2
22
88.3
385
Males
Females
BLD'
3.34
11.4
30.7
145
200
594
1962
15.1
32.4
112
BLD
7.17
35.9
123
BLD
4.36
11.2
30.2
173
364
835
1711
13.5
46.2
109
BLD
5.85
35.9
108
MMAD* (/mi) ± GSEr
1.3
1.3
1.4
1.6
1.7
1.7
1.8
2.1
1.3
1.6
1.7
1.0
1.5
1.4
1.9
+
±
±
+
+
±
+
±
+
+
±
±
+
±
+
1.8
1.9
1.9
1.9
1.8
1.8
1.7
1.9
2.1
1.9
1.9
1.9
2.1
2.0
1.8
Note. Achieved albuterol aerosol concentrations were determined daily. Achieved doses of albuterol were calculated for 10 rats/sex/group, 3 or 6
monkeys/sex/group, and 3 or 6 dogs/sex/group. Cnax plasma concentrations following a single 1-h exposure to albuterol dry powder were obtained from
1 to 5 rats/sex/group, 3 or 6 monkeys/sex/group, and 6 or 9 dogs/sex/group. Particle size analyses were performed weekly.
* Coa, is the maximal plasma albuterol concentration. This occurred immediately following exposure.
* MMAD is the mass median aerodynamic diameter.
c
GSD is the geometric standard deviation.
* BLD is below the limit of detection of the plasma albuterol assay.
55
INHALED ALBUTEROL IN RAT, MONKEY, AND DOG
•
50
I -
A
c
o
•
h
c
a>
u
o
0
"g 40-
U
A
•
•
•
30-
|0.
o
D
20-
3
*
A
•
A
A
a
It
=
.1
•
*
0
3
6
9
12
15
18
Achieved Albuterol Aerosol Concentration (//g/L)
FIG. 1. Individual male Cm, plasma albuterol concentrations in rats (•), monkeys (A), and dogs ( • ) following a single 1-h exposure to albuterol
dry powder aerosol. The achieved albuterol aerosol concentrations are shown in Table 1. N = 4 - 5 males/group for rats, N = 3 or 6 males/group for
monkeys, and N = 6 or 9 males/group for dogs.
mately 38 mg/kg/day. The maximum daily clinical dose of
marketed albuterol is approximately 15 /ig/kg in a 70-kg
human, assuming that the total presented dose of 1080 fig
administered via oral inhalation from a metered dose inhaler
(Physician's Desk Reference, 1996) is inhaled and deposited
in the body via the airways and gastrointestinal tract. Therefore, the total achieved dose levels in rats corresponded to
multiples of the maximum human dose ranging from approximately 0.25 to 2500 times.
Target aerosol concentrations for monkeys ranged from 4
to 36 fxg/L albuterol. The mean total achieved doses in monkeys ranged from 132 /xg/kg/day to approximately 1.5 mg/
kg/day, corresponding to multiples of the maximum human
dose ranging from approximately 9- to 100-fold.
Dogs were exposed to target albuterol aerosol concentrations ranging from 0.5 to 64 //g/L. The mean total achieved
doses the dogs received ranged from approximately 9 fig/
kg daily to 1.4 mg/kg/day. The achieved dose levels in dogs
corresponded to approximately 0.5 to 90 times the maximum
daily clinical dose.
Albuterol was not detected in the plasma of rats exposed
to a target aerosol concentration of 0.25 //g/L albuterol and
of dogs exposed to a target aerosol concentration of 0.5
fig/L albuterol. For all species, T^ occurred immediately
following exposure to albuterol. Mean and individual C^
values (Fig. 1) increased in a dose-related manner with respect to achieved albuterol aerosol concentration and
achieved dose. Additionally, across all species, C,™, plasma
concentrations were comparable at similar achieved albuterol aerosol concentrations. Plasma samples collected fol-
lowing up to 3 months of consecutive daily exposures to
albuterol aerosol revealed that albuterol did not accumulate
after repeated dosing (data not shown).
Electrocardiography
There were no sex-dependent or day-related differences
in heart rates in any species; therefore heart rates for males
after one or two exposures only are shown (Fig. 2). Due to
the marked variation in and high values of resting monkey
heart rates prior to exposure to albuterol, heart rate differences following exposure to albuterol in monkeys could not
be discerned. In rats during and immediately following exposure, tachycardia was evident at target albuterol aerosol concentrations of s i fig/L after 1 and 15 consecutive days of
exposure. Tachycardia occurred at all doses in dogs after 2
and 13 days of exposure. Heart rates in rats and dogs returned
to preexposure values at 6 h following exposure to albuterol.
There were no albuterol-related EKG waveform findings
in rats and monkeys (Table 2). However, dogs exposed to
target albuterol aerosol concentrations of 0.5, 16, and 64 figl
L exhibited ventricular extrasystole and/or reversal of the
polarity of T waves in chest leads rV2 and V,o.
Serum Potassium
There were no sex-dependent or day-related differences
in serum potassium values within each species; therefore,
only male values from Day 1 are shown (Fig. 3). A minimal, transient hypokalemia occurred in rats exposed to
albuterol aerosol concentrations of 1 fig/L and above after
Beats per Minute
1
s
o
Beats p«r Minute
Beats per Minute
a
c
3
<D
itt
•
I
I
I
57
INHALED ALBUTEROL IN RAT, MONKEY, AND DOG
TABLE 2
Electrocardiographic Findings
Waveform findings
Heart rate findings
Species
Rat
Monkey
Dog
Target albuterol
aerosol cone. Qig/L)
0
0.25
1
4
12
36
100
300
1000
0
4
12
36
0
0.5
4
16
64
Males
T
T
T
T
T
T
Females
Males
Females
T"
T
t
T
r
1/4* Ventricular extrasystole
T
T
1/3 Ventricular extrasystole
1/6 Ventricular extrasystole
T
T
T
T
T
1/9 Positive T wave Vi0
2/9 Ventricular extrasystole
1/6 Negative T wave rV2
1/6
1/6
1/6
2/6
2/6 Negative T wave rV2
1/6 Ventricular extrasystole
Reversal of T waves leads rV2 and V10
Positive T wave lead V,o
Reversal of T waves leads rV2 and V,o
Ventricular extrasystole
Note. N = 4/sex/group for rats, N - 3 or 6/sex/group for monkeys, and N = 6 or 9/sex/group for dogs.
° t, Increased relative to preexposure and concurrent control values.
b
The numerator refers to the number of animals with the finding. The denominator refers to the number of animals examined.
1 and 15 days of dosing. However, serum potassium values for most rats remained within the normal physiologic
range. In dogs, a hypokalemia of a greater magnitude occurred at all dose levels after 1 and 14 days of exposure.
Many values were below the normal physiologic range.
The hypokalemia in dogs occurred immediately following
exposure and at 6 h postexposure. However, at 23 h postexposure, dog serum potassium values were within normal
physiologic range. No changes occurred in monkey serum
potassium concentrations following exposure to albuterol
at any dose level.
Heart Weights and Histopathology
No changes in absolute and/or relative heart weights were
observed in monkeys and dogs exposed to albuterol for approximately 2 weeks. In rats, absolute and/or relative heart
weights were slightly higher than control in males and females following approximately 2 weeks of exposure. Increases in absolute heart weights were approximately 4 -
17% in male rats (Fig. 4) and approximately 5-17% in
females. The increases in relative heart weights in rats were
approximately 1-11% in males and approximately 1-9%
in females.
Inhalation of albuterol aerosols at targeted concentrations
up to and including 1000 and 36 /xg/L in rats and monkeys,
respectively, for approximately 2 weeks, resulted in no histopathologic heart lesions related to exposure to this /?2-adrenergic agonist. Dogs exposed to target aerosol concentrations
of 16 and 64 ybgTL albuterol for 2 weeks exhibited a slight
to mild, focal or multifocal cardiac fibrosis with or without
a slight mineralization of the left ventricular papillary muscle
(Table 3). Following a 4-week postexposure period, one of
three males in the 64 /xg/L albuterol group exhibited a slight
focal fibrosis with slight focal mineralization of the left ventricular papillary muscle.
DISCUSSION
Inhalation of albuterol at multiples up to and including
approximately lOO-fold of the maximum daily clinical dose
FIG. 2. Heart rate versus time in male rats (A), monkeys (B), and dogs (C) during and following a 1-h exposure to filtered conditioned air or albuterol
dry powder aerosol. Data are plotted as the mean + or - the standard error. Rat and monkey data were collected immediately prior to, during, and
following a single 1-h exposure to filtered conditioned air or albuterol dry powder. Dog data were collected before, during, and following the second 1h exposure to filtered conditioned air or albuterol dry powder. N = 4 males/group for rats, N = 3 or 6 males/group for monkeys, and N = 6 or 9 males/
group for dogs.
58
PETRUSKA ET AL.
TABLE 3
Albuterol-Related Heart Tissue Histopathologic Findings in Dogs
Males
Target albuterol
aerosol cone. (/ig/L)
0.5
4
16
64
Females
Week 3
Recovery"
Week 3
Recovery
None
None
None
2/3 Slight focaJ fibrosis with
slight focal mineralization
1/3 Mild multifocal fibrosis with
slight focal mineralization
NP*
None
None
None
None
None
1/3' Slight multifocal fibrosis
1/3 Slight multifocal fibrosis
NP
None
None
1/3 Slight focal fibrosis with
slight focal mineralization
Note. N = 3 dogs/sex/group.
" Three dogs/sex/group in the 4, 16, and 64 ^g/L groups were retained for a 4-week postdose period.
* NP refers to "not performed."
" The numerator refers to the number of animals with the finding. The denominator refers to the number of animals examined.
actuated from a metered dose inhaler resulted in no remarkable findings in cynomolgus monkeys. Tachycardia and a
transient hypokalemia were observed in rats at multiples of
1.5 times or greater than the maximum daily clinical dose.
The tachycardia was no longer apparent at 6 h following
dosing, and the transient hypokalemia was not clinically
significant, as most serum potassium values were within the
normal physiologic range for this species. Rats dosed with
albuterol aerosol at 66 times or greater than the maximum
daily clinical dose had slightly increased absolute and/or
relative heart weights in comparison to filtered conditioned
air control rats. This increase in heart weight also occurred
in rats dosed with albuterol aerosol at 47 times the maximum
daily clinical dose (data not shown). Due to the absence of
albuterol-related histopathologic findings in the heart tissue
of rats receiving =s2500 times the maximum daily dose, the
slight heart weight increases were considered to be due to
a physiologic adaptation related to the tachycardia. Results
of this study indicate that administration of exceedingly large
doses of albuterol via nose-only inhalation to rats is not
cardiotoxic.
Subcutaneous administration of albuterol for 3 days at
doses of 0.5-50 mg/kg to Sprague-Dawley rats resulted in
a slight myocardial necrosis in the apex and papillary muscle,
with concomitant increases at 50 mg/kg in absolute and relative heart weights in comparison to saline controls (Magnusson and Hansson, 1973). The slight myocardial necrosis was
characterized by edema, hemorrhage, and mononuclear cell
infiltration in the necrotic areas. Oral administration of albuterol for 1 month at doses of 17, 50, and 150 mg/kg resulted
in statistically significantly higher absolute and relative heart
weights in rats (increased 13 to 27%) in comparison to saline
controls (Yamada et ai, 1977). Following a 1-month recovery period, heart weights of rats in all dose groups were
comparable to saline controls. Orally administered albuterol
resulted in edema, a slight to moderate focal myocardial
necrosis in the apex and subendocardial layer of the left
ventricle, and hypertrophy of muscle fibers of the left ventricle. At the end of the 1-month recovery period, the incidence
and severity of heart lesions in all groups were markedly
decreased. The cardiotoxicity observed after subcutaneous
and oral dosing in rats occurred at doses greatly exceeding
the maximum daily exposure of inhaled or orally administered albuterol (Physician's Desk Reference, 1996).
Tachycardia and transient hypokalemia also were observed in dogs at all aerosol concentrations of albuterol
tested. The tachycardiac effects of albuterol in dogs have
been observed previously after intravenous administration
of 0.5, 1, and 2 /xg/kg in adult anesthetized mongrel dogs
(Nayler and Mclnnes, 1972), after a single intravenous dose
of 100 //g/kg in anesthetized dogs (Maxwell et ai, 1969)
and following oral or intraduodenal administration of 25
/xg/kg, intratracheal administration of 400 ^g per dog, and
intravenous administration of 0.25, 1, and 4 /ig/kg in anesthetized mongrel dogs (Minatoya, 1978).
The cardiac effects of albuterol are believed to be due to
a reflex tachycardia resulting from albuterol-induced peripheral and coronary vasodilation and from a reduction in peripheral resistance. In anesthetized dogs receiving a single
intravenous dose of 100 /xg/kg albuterol, tachycardia occurred concomitant with an increase in cardiac output and
reductions in femoral arterial pressure and total peripheral
resistance (Maxwell et ai, 1969). In addition, coronary blood
flow was increased and coronary vascular resistance was
decreased in comparison to controls. Intravenous infusion
of 0.86-7.2 /ig/kg albuterol in anesthetized greyhounds also
resulted in increased cardiac output and reductions in femoral arterial diastolic pressure (Loh et al., 1971). Intravenous
dosing of 2 /xg/kg albuterol to anesthetized adult mongrel
dogs resulted in tachycardia, reduced mean arterial pressure,
59
INHALED ALBUTEROL IN RAT, MONKEY, AND DOG
1
I
a.
E
a
1
2
3
23
24
Hours Post-exposure
Opflfl-
»-0.25pg/L
pre-exposure
- * - 1 pg/L
-»-4pg/L
0 hr
Time Post-exposure
6 hr
-»-12pg/L
-•- 36pgrt.
23 hr
|O0 A>g/t BO.5 pglL 114 //g/L • 16 </g/L
FIG. 3. Serum potassium concentrations versus time in male rats (A) and male dogs (B) preceding and/or following a single 1-h exposure to filtered
conditioned air or albuterol dry powder. Data are plotted as the mean + or — the standard error. N = 4 - 9 males/group for rats and N = 5-9 males/
group for dogs.
and increased coronary blood flow (Nayler and Mclnnes,
1972). In the present study, slight reductions in indirect systolic blood pressures measured over the tail occurred on
occasion in addition to tachycardia (data not shown).
Dogs which received 2=0.5 times the human daily dose
of albuterol also exhibited T wave changes in chest leads
rV2 and V| 0 . The reversal of polarity of T waves observed
is considered a secondary effect of the hypokalemia (Detweiler, 1981) and occurred simultaneously with reductions
in serum potassium. Hypokalemia is believed to occur due
to the stimulation of skeletal muscle Na-K-ATPase, re-
sulting in an influx of potassium and resultant reduction
in extracellular potassium (Bradberry and Vale, 1995). The
changes in T waves in chest leads rV2 and V, o arc relevant
in a dog model, as the T waves in leads rV2 and V10 can
reverse polarity due to drug-induced effects, whereas the T
waves in the other leads are labile and may reverse polarity
under normal conditions (Detweiler, 1981). The tachycardia,
hypokalemia, and waveform changes observed were transient, and were not evident within 6-23 h following inhalation of the large doses used in this study.
Tachycardia and hypokalemia are believed to be due to
m
PETRUSKA ET AL.
0.4
0
1
4
12
36 100 300 1000
Target Albuterol Aerosol Concentrations (/sg/L)
IBAbsolute (g) • Relative (g%) I
FIG. 4. Absolute and relative heart weights in male rats following
approximately 2 weeks of exposure to filtered conditioned air or albuterol
dry powder. Data are plotted as the mean + the standard error. N = 20
males/group for the air control group (combined data from two separate
studies) and N = 10 males/group for the albuterol-exposed rats.
an extension of the pharmacologic action of /?2-adrenergic
agents and have been observed in humans following inhalation (Rolf-Smith et al., 1984; Lipworth etal, 1988; Lip worth
and McDevitt, 1989; Wong et al., 1990), intravenous administration (Leitch et al., 1976; Neville et al., 1917; Morice et
al., 1986), and oral ingestion of doses of albuterol exceeding
the maximum daily clinical dose (Lewis et al., 1993; Wiley
et al., 1994). These studies in humans included asthmatic
patients as well as healthy volunteers. The doses used in the
inhalation studies in man were generally greater than the
maximum daily dose and/or were given in greater frequency
than recommended (Physician's Desk Reference, 1996). The
intravenous route of administration is used in asthmatics
only when inhalation of a /?2-agonist fails to provide adequate bronchodilation (Nelson, 1995) and the doses used
in the above referenced studies were far greater than that
administered on a daily basis. The ingestion studies investigated overdoses, which occur rarely in individual patients.
With all routes of administration, the tachycardia and hypokalemia were reversible. The heart rates and serum potassium values returned to normal physiologic values within
no more than a few hours following withdrawal of albuterol.
Dogs dosed with 19 times or greater the maximum clinical
daily dose had a slight to mild, focal or multifocal, cardiac
fibrosis with or without a slight mineralization of the left
ventricular papillary muscle. This finding also was observed
in one dog following a 4-week postdose period. This lesion
is commonly seen following the administration of other vasodilating compounds in the dog (Detweiler, 1989; Dogterom et al., 1992). The combination of waveform changes
present (reversal of polarity of T waves in leads rV2 and V,o
and ventricular extrasystoles) is consistent with the cardiac
lesions observed. Dogs are believed to be uniquely sensitive
to vasodilating agents (Detweiler, 1989), including /32-adrenergic agonists, in comparison to the human population
(Dogterom et al., 1992). Therefore, the heart lesions ob-
served in the dog are believed not to be relevant to the
human population after repeated clinical dosing.
In addition, the safety margin of 19-fold in the dog is
probably closer to 95- to 190-fold when one takes into account that in man only approximately 10-20% of the total
administered dose from a metered dose inhaler is actually
deposited into the lungs and is bioavailable (Melchor et al.,
1993; Newman et al., 1984). When comparing plasma concentrations among the three species tested, cardiotoxicity
was noted in the dog only at mean concentrations of approximately 36 ng/ml and above. These plasma concentrations are
approximately 12-fold greater than that reported for humans
inhaling a dose of 1200 fig from a metered dose inhaler over
a 6-min period (Clark et al., 1996; Clark and Lipworth,
1996). In addition, patients who ingested overdoses of albuterol and had plasma concentrations ranging from 18 to 449
ng/ml survived without any serious cardiac arrhythmias
(Lewis et al., 1993). The magnitude of exposure seen in the
dog in this study is unlikely to occur in the human population
except in the case of an exceedingly large overdose.
There are case-control studies which suggest that repeated exposure to clinical doses of albuterol may be responsible for the increase in asthma mortality (Spitzer et al,
1992; Coughlin et al., 1995). It is important to note that
case-control studies cannot establish a cause and effect relationship (Taubes, 1995). In the Saskatchewan case control
study, there was an increased risk from near death or death
due to fatal asthma with the use of a 1.4 canisters of /?2agonist per month (Suissa et al., 1994). This corresponds
to approximately 10 puffs per day, close to the maximum
recommended daily dose of 12 puffs of 90 /xg each for
albuterol (Physician's Desk Reference, 1996). It was then
hypothesized that the severity of asthma could be linked to
the increased risk for death or near death from asthma, as
more severe asthmatics or those patients with poorly controlled asthma may overuse /32-agonist inhalers (Ernst et
al., 1993). However, not all appropriate indices of assessing
asthma severity (National Asthma Education Program, 1991)
were investigated in the later analysis (Ernst et al., 1993);
therefore, one can surmise that the severity of asthma still
may be a confounding variable in the analysis of the Saskatchewan study data. Another case-control study suggests
that use of /32-agonists, including albuterol, is linked to an
increased risk of developing idiopathic dilated cardiomyopathy (Coughlin et al., 1995). This study employed few subjects and did not take into account the dose and frequency
of /J2-agonist use. In addition, the authors stated that some
of the subjects studied may not have had a history of true
asthma, but rather an episode of cardiogenic bronchospasm
and wheezing.
Autopsies of patients who have died from asthma fail to
show cardiotoxic effects and instead reveal the hallmarks of
death due to asphyxiation (Barger et al., 1988; Johnson et
INHALED ALBUTEROL IN RAT, MONKEY, AND DOG
al., 1984; Molfino et al., 1991; Sly, 1988). Furthermore,
appropriate treatment, including the administration of albuterol, of patients with severe, potentially fatal asthma routinely resulted in improvement of symptoms and prevention
of morbidity and/or mortality (Nelson, 1995). In a recent
study of adequately oxygenated acute severe asthmatics administered doses up to and including 1600 fig of albuterol by
inhalation using an aerosol holding chamber, cardiovascular
mortality and clinically significant arrhythmias were not observed (Newhouse et al., 1996). These human data support
that albuterol is not a cardiotoxic agent.
In summary, albuterol continues to be used safely as a
bronchodilating agent for asthmatics. Due to the lack of
toxicologically relevant findings in rats and monkeys and to
the large safety margin in dogs, albuterol is not believed to
be a cardiotoxic agent as used routinely for the treatment of
asthma.
ACKNOWLEDGMENTS
Dr. D. K. Detweiler qualitatively evaluated all electrocardiograms, Dr.
Winston Evering performed the histopathologic examinations, and Dr. Andras Fabry served as the peer review pathologist. The authors gratefully
acknowledge the technical assistance of Stephen Distilio, Giovana Ciconte,
Steven Russel, Thomas Adcock, Eric Wachman, Yannick Lamarre, Fabien
Dion, Denis Lagace, Use Pigeon, Cecile Begin, and Jacques Royer.
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