Simple, noninvasive method to measure the antibronchoconstrictor activity of drugs in conscious guinea pigs

Simple, Noninvasive Method to Measure the Antibronchoconstrictor Activity of Drugs in Conscious Guinea Pigs

GISELA DANKO AND RICHARD W.

CHAPMAN

A simple,

is described

noninvasive

bronchoconstrictor were

placed

method

activity

in a head-out

and a head

chamber

69% NZ, 21% OJ an increase

of drugs

was used to deliver

in V, that was

aerosol

reduced

histamine-induced

reduction

oral administration

with

measurement guinea

a hypercapnic

(0.05%

(61

t

The

3%)

pigs

of tidal volume

(Vr),

gas mixture

by exposure

(10%

COz,

of CO* caused

to histamine.

The

1 hr after

(p < 0.05) reduced

albuterol

anti-

guinea

for 30 s). Inhalation

of Vr was significantly

the bronchodilators

of the oral

pigs.

for measurement

plethysmograph

and histamine

for

in conscious

(1 and 10 mg/kg)

and ami-

(100 mg/kg),

nophylline

the calcium antagonist nifedipine (30 mg/kg), and the chlorpheniramine (0.5 mg/kg). These results demonstrate the oral

antihistamine

antibronchoconstrictor this technique

activity

of drugs

are that it is simple

in this model.

to operate,

The major

is noninvasive,

advantages

of

and does not require

anesthesia. Key Words:

Ventilatory

Antihistamine;

Guinea

responses

to CO,;

Histamine

aerosol;

Bronchodilators;

pigs

INTRODUCTION One

of the basic therapeutic

lization

of bronchodilator

formed

by measuring

approaches

drugs.

Preclinical

to reversible evaluation

airways of these

disease

is the uti-

drugs is often

per-

their ability to inhibit or reverse bronchoconstriction in guinea meapigs (Daly et al., 1971; Giles et al., 1973, 1977; Brittain et al., 1976). Although

surement

of bronchoconstriction

in guinea

pigs is well

documented,

many of the

techniques are limited since they require either the use of anesthesia, extensive surgical preparation, are technically difficult to perform, or require specific and expensive methodologies (Agrawal, 1981; Amdur and Mead, 1958; Daly et al., 1971; Douglas et al., 1972; Johanson and Pierce, 1971; Pennock et al., 1979). It was the purpose of this study to describe a simple, noninvasive method for measuring bronchoconstriction in conscious guinea pigs and to evaluate the sensitivity of this method for measuring the antibronchoconstrictor activity of drugs. Bronchoconstriction was induced by exposure to histamine aerosol and measured as a reduction

of tidal volume

From the Department Jersey, USA.

during

inhalation

of Allergy and Inflammation,

of a carbon

Schering-Plough

Address reprint requests to: Dr. Richard Chapman, Schering-Plough field, NJ 07003, USA. Received May 28, 1987; accepted October 2, 1987.

dioxide-enriched

Corporation,

Bloomfield,

gas New

Corp., 60 Orange Street, Bloom-

165 Journalof

Pharmacological

Methods

0 1988 Elsevier Science Publishing

19, 165-173 (1988) Co., Inc., 52 Vanderbilt

0160~5402/88/$3.50 Avenue, New York, NY 10017

166

C. Danko and R. W. Chapman mixture

using similar

methodologies

to those

scious

guinea

pigs (Wong

and Alarie,

Alarie,

1985).

To evaluate

the

bronchoconstrictor

albuterol

and the antihistamine

tidal volume

sensitivity

Shaper

of this

during

and aminophylline,

chlorpheniramine

carbon

dioxide

described

for use in con-

et al., 1983, 1984;

method

activity of drugs, we have studied

of the bronchodilators pine,

previously

1982;

for

Shaper

and

the

anti-

measuring

the potential

inhibitory

effects

the calcium

antagonist

nifedi-

on histamine-induced-reduction

of

inhalation.

METHODS Animals Male

Hartley

guinea

weight from 350-450 night, but had water

pigs (Hazleton

Research

g were used in these ad libitum.

Pulmonary

Measurements

The guinea

pigs were

placed

inside

Products,

studies.

PA) ranging

were

available

(Buxco

a commercially

Sharon, CT) head-out, pressure plethysmograph was connected to a 5-L reservoir bottle, which

Denver,

The animals

fasted

in

over-

Electronics,

(Figure 1). The plethysmograph was filled with copper gauze and

covered with foam rubber to keep the interior temperature of the plethysmograph constant. A latex collar was placed over the animal’s head to provide an airtight seal between validated

the guinea pig and the plethysmograph. The integrity of this seal was by injecting 5 ml of air into the plethysmograph, with the animal inside

it, and an airtight pressure. A differential range

?2

system was produced pressure

cm H20)

and measured

transducer

was connected

the pressure

change

when

there was no decay in plethysmograph

(Validyne, to an outlet inside

Northridge, port

CA;

Model

MP-45-1;

in the plethysmograph

the plethysmograph

relative

wall

to atmos-

FIGURE 1. Schematic drawing of the body plethysmograph for the measurement of tidal volume (VT). The head chamber was used to facilitate the inhalation of carbon dioxide (10% COJ enriched gas and exposure to histamine (0.05% for 30 s) aerosol.

Bronchospasm in Conscious Guinea Pigs 167 pheric pressure. This pressure signal, which was visually displayed on a chart recorder (Model 1610, MFE Corp., Salem, NH), was electrically converted to a signal proportional to volume using a pulmonary function computer (Model 6, Buxco Electronics, Sharon, CT), and the average tidal volume (VT), respiratory rate (f) and minute volume (V) for each minute of breathing were displayed on a printer. A volume calibration was performed before each experiment by injecting known volumes of air into the plethysmograph using a IO-ml syringe. The frequency response of the plethysmograph (Amdur and Mead, 1958) was determined by connecting a small oscillating pump (Model 683, Harvard, South Natick, MA) to an outlet port in the plethysmograph and measuring the injected volume over a range of pump frequencies. The amplitude of the volume signal was linear between 0.5 and 10 ml, and volume was found to be accurately measured for frequencies of up to 5 Hz. Measurement

of Antibroncboconstrictor

Activity

A plexiglass chamber was fitted over the head of the guinea pig to facilitate the inhalation of different gas mixtures and aerosols (Figure I). The animals were exposed to either normocapnic (0% CO*, 21% Op, 79% N2) or hypercapnic (10% COZ, 21% 02, 69% N,) gas mixtures, which were delivered from a compressed gas source (Matheson Gases, Rutherford, NJ) at a constant flow rate of 3 Umin. Aerosols of either isotonic saline or histamine (0.05%/o)were generated from an ultrasonic nebulizer (Model 65, DeVilbiss, Somerset, PA), and delivered to the head chamber at a flow of 0.24 Umin for 30 s. The gas content of the head chamber was evacuated at a constant flow of 3 Umin during inhalation of the gas mixtures alone and at 3.24 Umin during exposure to the aerosols. The guinea pigs were pretreated with oral administration of either drug or vehicle given 1 hr before exposure to histamine. Bronchoconstriction due to histamine was measured as the maximum reduction in tidal volume, which occurred up to 5 min after histamine exposure. The antibronchoconstrictor activity of compounds was determined by comparing the histamine-induced reduction in tidal volume of drugtreated versus placebo-treated animals, and significant differences were determined by analysis of variance and Duncan’s Multiple Range Statistic. Drugs Histamine diHCl (Sigma Chemical Co., St. Louis, MO) was dissolved in isotonic maleate (Schering-Plough Research, saline. Albuterol SO+ chlorpheniramine Bloomfield, NJ), aminophylline (Sigma Chemical Co., St. Louis, MO), and nifedipine (Pfizer Corp., New York, NY) were suspended in methylcellulose and given orally in a volume equivalent of 2 ml/kg. All doses of the drugs are expressed as their free base. RESULTS Effects of Histamine lnh~lation of carbon dioxide caused a prompt and sustained increase in VT, f, and V as illustrated in a representative study in Figure 2. Exposure to histamine

168

C. Danko and R. W. Chapman HISTAMINE

HISTAMINE

I

600r

MINUTES

OF EXPOSURE

TO 10%COe

FIGURE 2. Effect of carbon dioxide (10% CO*) inhalation on tidal volume (VT), respiratory rate (f), and minute volume (V) in a representative study. J denotes the changes induced by exposure to histamine aerosol (0.05% for 30 s).

aerosol (0.05% for 30 s) produced a 61 ? 3% reduction of V,, a 63 i 4% reduction in Vwith no change in f (Table 1). The histamine-induced decrease in VT and V had peak effects after 2-3 min and a duration of at least 5 min (Figure 2). Exposure to a large aerosol concentration of histamine (0.1% for 30 s) caused apnea and death. Exposure to saline aerosol had no effect on the ventilatory responses to CO,.

Bronchospasm in Conscious Guinea Pigs TABLE 1 Change in Tidal Volume, Respiratory Rate and Minute Volume Induced by Aerosolized Saline and Histamine During Carbon Dioxide Inhalation PERCENT CHANGEDUE TO TREATMENT*,~ TREATMEN?

N

VT

f

v

Saline Histamine

5 5

i-2 5 2 -61 2 3d

+2 2 5 0+2

+4 i 5 -63 ? 4d

Abbreviations: N, number; VT, tidal volume; f, respiratory rate; V, minute volume. a Aerosolized saline or histamine (0.05%) given for 30 s. b Percent change = (before treatment) - (peak response after treatment)/(before treatment) x 100. ’ Values are the mean t SE. d p < 0.05 compared to saline.

Effects of Drugs Oral

administration

of aminophylline

(10-100

mg/kg)

and nifedipine

(IO and 30

mg/kg) caused a dose-related increase in f during inhalation of the normocapnic gas mixture (Table 2). Aminophylline also caused an increase in VT and V. Albuterol (0.3-10

mg/kg)

during

inhalation

and chlorpheniramine

(0.2 and 0.5 mg/kg)

of the normocapnic

did not change

ventilation

gas mixture.

Aminophylline (100 mg/kg) significantly (p < 0.05) attenuated to increase in f due to inhalation of CO, but had no effect on the VT and V responses to CO*. Albuterol (0.3-10

mg/kg),

nifedipine

(IO and 30 mg/kg)

and chlorpheniramine

(0.2 and 0.5 mg/

kg) had no effect on the ventilatory responses to COz (Table 2). The reduction of VT induced by histamine was significantly (p < 0.05) attenuated by oral treatment with the bronchodilators albuterol (1 and IO mg/kg) and aminophylline (100 mg/kg), the calcium antagonist nifedipine (30 mg/kg) and the antihistamine chlorpheniramine (0.5 mg/kg) (Table 3). There was no significant effects of drug treatment

on prehistamine

tidal volume.

Discussion Carbon dioxide inhalation in tidal volume, respiratory

is a potent respiratory stimulant and causes an increase rate, and minute volume. At least a IO-min exposure to

carbon dioxide is required to achieve for ventilation to stabilize at an elevated

steady-state conditions (Jennett, 1981) and plateau. In these studies, we did not exceed

15 min of COz exposure in order to avoid potential changes in plasma bicarbonate and body temperature that are the result of prolonged exposure to CO* (Schaefer et al., 1975). When histamine aerosol was superimposed was a marked reduction of both tidal volume

on the carbon dioxide stimulus, there and minute volume and no consistent

change in respiratory rate. Since histamine would be expected to cause bronchoconstriction and pulmonary edema and increase mucus production (Mills and Widdicombe,

1970;

Persson

et al., 1978),

the

reduction

of tidal

volume

and

minute

169

5 5 S 5 5 5 5 5 5 5 5 5 5 5 0 0.3 1 10 0 10 30 100 0 10 30 0 0.2 0.5

(m&kg)

ORAL DOSE

0.24 0.16 0.14 0.15 0.20 0.19 0.09 0.08d 0.15 0.24 0.09 1.30 t 0.20 1.18 F 0.21 1.19 t 0.25

1.39 ; 1.56 r 1.12 4 1.44 f 1.30 i 1.66 I 1.73 t 1.93 i 1.22 + 1.39 r 1.60 i

Vr unl) 116 It 12 139 t- IO 125 s 7 110 t 9 105 ir 4 144 jl 12d 155 t IOd 176 _i 12d 108 i_ 11 144212 148 i- 13d 105 t: 4 116 t_ 6 105 rt 6

min)

f (breaths/

Abbreviations: N, volume; VT, tidal volume; f, respiratoryrate;V, minute volume. Values are the mean rt SE a Drugs given 1 hour before exposure to COZ. ’ Ventilation during inhalation of 0% CO?, 21% OZ, 79% N2. ’ Ventilation during inhalation of 10% CO2, 21% 02, 69% Nz. d P < 0.05 compared to zero drug.

Chlorpheniramine

Nifedipine

Aminophylline

Albuteroi

N

BASALVENTILATION’

157 212 143 163 138 248 267 341 138 208 239 138 142 131

5 35 _c 16 i 27 A 28 _i 24 i 45" * 22d + 32d t_ 31 2 42 _' 30 -+ 24 t_ 34 _' 34

li (ml/ min) 3.05 i 0.21 2.11 + 0.15 2.12 t 0.32 2.83 i 0.16 2.74 + 0.38 2.05 2 0.35 2.17 5 0.19 2.04 _" 0.28 2.98 i_ 0.33 1.90 r 0.25 2.46 i 0.26 2.74 i 0.38 3.03 i_ 0.25 2.76 t 0.52

30 14 29 37 26 17 6 - 16 45 30 11 26 29 30

tf:7 i- 8 t 13 ?I IO t: 8 + IO * 13 + Ild i_ IO rt 12 -t 13 i 8 t 4 t 9

459 347 409 468 387 346 356 285 501 395 409 387 469 388

f 20 t 28 + 94 + 49 f 64 2 58 + 45 % 60 -t 52 + 30 2 56 + 64 I+_33 _t 60

INCREASE DUE TO CARBON’ ______ .._...___ f (breaths/ \i (ml/ min) min) VT (ml)

and on Ventilatory Responses to Carbon Dioxide

-.__

Effect of Drugs on Basal Ventilation

DRUGS”

TABLE 2

Bronchospasm in Conscious Guinea Pigs TABLE 3 Effect of Drugs on Histamine-Induced Volume During Carbon Dioxide Inhalation

Reduction of Tidal

TIDAL VOLUME DECREASEDUE TO ORAL DOSE DRUC~

Albuterol

Aminophylline

Nifedipine

Chlorpheniramine

N

(mgikg)

5 5 5 5 5 5 5 5 5 5 5 5 5 5

0 0.3 ? 10 0 10 30 100 0 10 30 0 0.2 0.5

PREHISTAMINE

HISTAMINES

(ml)

(%)

4.44 3.68 3.25 4.29 4.04 3.71 3.90 3.97 4.20 3.49 4.06 4.04 4.27 3.95

2

2 + ” ” i k ” + k k 2 k ”

0.28 0.19 0.27 0.20 0.58 0.29 0.26 0.25 0.21 0.24 0.32 0.58 0.14 0.65

66 k 6812 24 2 11 2 68 2 79 k 50 k 37 t 74 + 80 2 25 k 68 2 48 2 15 k

6 10’ 2= 2 5 12 13’ 4 6 7= 2 6 4c

Values are the mean 2 SE. N, number. a Drugs given 1 hr before exposure to histamine. b Percent decrease due to histamine aerosol (0.05% for 30 s). c p i 0.05 compared to zero drug.

volume responses to CO2 are most likely due to airway obstruction. Consistent with this hypothesis are studies in guinea pigs that demonstrate that the dose of histamine required to inhibit the ventilatory response to CO, is equivalent to the dose required to increase pulmonary resistance (Shaper et al., 1984). Since histamine does not reduce ventilatory drive (Pack et al., 19821, the reduction of ventilatory responses to CO2 cannot be due to diminished inspirato~ effort. The concentration (0.05%) and duration (30 s) of exposure to histamine was empirically determined in our study to yield a 60430% reduction in tidal volume, which in effect reduced tidal volume to pre-CO2 levels. We did not exceed a 6040% reduction in Vr since the method of measuring tidal volume with a whole body plethysmograph is subject to artifact during intense bronchoconstriction (Epstein et al., 1980; Shaper et al., 1984). Moreover, we found that exposure to histamine of greater than 30 s or increasing the concentration to more than 0.05% caused periodic apnea and death. On the other hand, we found that a duration of histamine exposure of less than 30 s or by reducing the concentration below 0.05% gave variable reductions in tidal volume. The concentration and duration of exposure to histamine required to produce a reproducible fall in tidal volume will, of course, depend upon the method of aerosolization and the exposure conditions (Shaper et al., 1983, 1984; Shaper and Alarie, 1985). Oral pretreatment with the bronchodilators albuterol and aminophylline effec-

171

172

C. Danko and R. W. Chapman tively blocked the histamine-induced reduction in tidal volume. This occurred in the absence of a significant effect of these pharmacologic agents upon the tidal volume response to CO? inhalation, although aminophylline did exacerbate the known

respiratory

the prehistamine

stimulant

actions

treated animals, so it is unlikely inhalation of a reduced amount The

doses

responses

of this drug (Mueller

values for Vr and f were equivalent

of aminophylline

to histamine

that the actions of histamine. and albuterol

are similar

et al., 1981).

in placebo

of aminophylline

that were

required

to the oral bronchodilator

Regardless,

and aminophyllinewere

due to the

to block

doses of these

the

V-r

com-

pounds in guinea pigs (Ciles et al., 1973; Brittain et al., 1976; Giles et al., 1977). Furthermore, the dose of chlorpheniramine that we required to block histamine’s bronchoconstrictor effect is similar to its oral antihistamine dose in guinea pigs (Tozzi et al., 1974). In other words, the technique described herein is sensitive for measuring the oral bronchodilator and antihistamine activity of drugs in guinea pigs. Oral pretreatment with the calcium channel blocker tamine-induced bronchospasm. This finding confirms activity

of nifedipine

in guinea

is the first to demonstrate

nifedipine blocked the histhe antibronchoconstrictor

pigs (Fanta et al., 1982;

its oral activity in conscious

Chapman

guinea

et al., 19841, but

pigs. A relatively

large

oral dose of nifedipine was required to block histamine-induced bronchospasm, but this probably reflects the weak bronchodilator activity of this compound (Triggle, 1983). In summary, we have described a method for measuring the oral bronchodilator and antihjstamine activity of compounds in conscious guinea pigs. The major advantages of this method are that it is simple to operate, is noninvasive, and does not require

anesthesia.

The authors would like to thank Drs. Kreutner and Egan for their valuable comments and Ms. Dawn Vitelli for the preparation of this manuscript.

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