Br. J. Pharmac. (1986), 87, 87-95

Comparison of the parasympatholytic activity ofACC-9358 and disopyramideBarry S. Brown', Richard J. Gorczynski, Robert D. Reynolds2 & Joel E. Shaffer

Department of Pharmaceutical Research, Pharmacology Section, American Critical Care, McGaw Park, IL60085, U.S.A.

1 ACC-9358 (N-[(3,5-di(pyrrolidinylmethyl)-4-hydroxy)benzoyl]aniline) is a newly developedanalogue of changrolin, an antiarrhythmic agent used in the Peoples Republic of China. Sincechangrolin and other antiarrhythmic agents exert parasympatholytic activity which may limit theirclinical usefulness, it was of interest to examine the parasympatholytic effects of ACC-9358. Forcomparative purposes we also studied the parasympatholytic activity of disopyramide.2 In guinea-pig isolated ileal strips, disopyramide, 3-30tgM, and ACC-9358, 100-300 jM, com-

petitively antagonized carbachol-induced contractions with pA2 values of 5.78 and 4.17, respectively.3 In guinea-pig isolated right atria, disopyramide 3-30 jM, competitively antagonized metha-choline-induced slowing of spontaneous beating with a pA2 value of 5.99 whereas ACC-9358,3-300 gM, produced no significant muscarinic blockade in this preparation.4 Disopyramide (1.9-15mgkg-', i.v.), but not ACC-9358 (7.5-1.5mgkg-', i.v.), significantlyincreased rat pupil diameter in vivo.5 Disopyramide and ACC-9358 blocked vagal-induced reductions in heart rate in dogs anaesthetizedwith pentobarbitone. ED50 values were approximately 0.65 and 11.25mg kg-', respectively.6 We conclude that ACC-9358 possesses significantly less parasympatholytic activity than dis-opyramide.

Introduction

Many antiarrhythmic agents alter the activity of theautonomic nervous system. For some agents thisactivity is a component of the therapeutic profile ofthedrug, for example, P-adrenoceptor blockade bypropranolol is an important aspect ofits mechanism ofantiarrhythmic action. However, the autonomiceffects of several other antiarrhythmic drugs areundesirable. Noradrenaline release and adrenergicneuronal blockade by bretylium, a-adrenoceptorblockade by quinidine and the anticholinergic activityofquinidine and disopyramide limit the clinical useful-ness of these agents (Bigger & Hoffman, 1980).Changrolin, a novel class I antiarrhythmic agent

(Xu & Carmeliet, 1981) used in the Peoples RepublicofChina (Xu& Qu, 1978: Li et al., 1979), has also beenreported to possess parasympatholytic activity in vivo(Chen et al., 1979). Stout et al. (1983, 1984, 1985) havestudied the structure-activity relationships of a seriesof synthetic analogues of changrolin in an attempt to

'Author for correspondence2Present address: Medical Department, American CriticalCare, McGaw Park, IL 60085, U.S.A.

identify a compound which has antiarrhythmicactivity similar to changrolin, but with less parasym-patholytic activity than changrolin. From thesestudies one compound, N-[(3,5-di (pyrrolidinyl-methyl)-4-hydroxy)benzoyllaniline (ACC-9358, Fig-ure 1), has been selected for clinical development(Stout et al., 1985).The objectives of this study were to examine the

parasympatholytic activity of ACC-9358 both in vitro

Figure 1 Structure of ACC-9358.

© The Macmillan Press Ltd 1986

88 B.S. BROWN et al.

and in vivo and compare this activity with that ofdisopyramide, the antiarrhythmic agent for whichanticholinergic activity is most clinically restrictive.

Methods

In vitro

Guinea-pig ileum Male Hartley guinea-pigs(300-400 g) were killed by a sharp blow to the head. Amidline abdominal incision was made to expose theileocaecal junction. An approximately 7 cm section ofileum was isolated immediately proximal to theileocaecal junction. The 2-3 cm of ileum nearest thecaecum were discarded. The remaining tissue wasplaced in a physiological saline solution (PSS) andrefrigerated overnight. The composition of PSS was(in mmol -L'): NaCI 120, NaHCO3 25, KCl 4.7,MgSO4 0.57, KH2PO4 1.2, CaCl2 1.96, dextrose 11.1and hexamethonium bromide 0.1. The next day eachileal strip was transferred into fresh oxygenated (95%02, 5% CG2) PSS at room temperature. A 1 cmsegment ofileum was mounted on a muscle holder andplaced in a 30 ml vertical muscle bath containingoxygenated PSS maintained at 37TC. Developed ten-sion was recorded on a Gould 2800S physiologicalrecorder using a Gould-Statham UC-2 or UC-3 forcetransducer. Under a resting tension of 1 g, each tissuewas allowed to stabilize for 30 min.

Following stabilization, cumulative concentrationsof carbachol (2 x 10-8M-5 X 10-6M final concentra-tion) were added to each muscle bath. Each successive-ly higher concentration of carbachol was added whenthe steady state response to the previous concentrationwas reached. Each muscle bath was then flushed withfresh PSS and carbachol administration was repeated.The ileal responses to the second series of carbacholadministrations were used as pretreatment controlvalues since preliminary experiments indicated thatresponses to a second and third carbachol dosingseries were nearly identical, whereas ileal responses tothe first carbachol sequence were often exaggerated.Following the control (second) carbachol dosingseries, each muscle bath was flushed with fresh PSS.Vehicle or a single concentration of ACC-9358 ordisopyramide was then added and 45-60 min later athird cumulative dosing of carbachol was carried out.

Guinea-pig right atrium Male Hartley guinea-pigs(300-500 g) were killed by a sharp blow to the head.The heart was rapidly removed and placed in oxygen-ated (95% 02, 5% CO2) Krebs-Henseleit (K-H)solution. The composition of the K-H solution was(mmoll-'): NaCl 118.4, NaHCO3 25, KC1 4.7,MgSO4 1.2, KH2PO4 1.2, CaCI2 2.5, dextrose 11.7 andNa2EDTA 0.03. The right atrium was isolated, moun-

ted on a muscle holder and placed in a 30 ml verticalmuscle bath containing K-H solution maintained at37°C. Under a diastolic tension of 0.5 g, developedtension was recorded on a Gould 2800S physiologicalrecorder with a Gould-Stratham UC-2 or UC-3 forcedisplacement transducer to monitor spontaneous rate.

Following a 60 min equilibration period, duringwhich the atria were washed several times with freshK-H solution, cumulative concentrations of metha-choline (10-7M-10-4M final concentration) wereadded to each muscle bath. Each successively higherconcentration of methacholine was added when theatrium had reached a steady state response to theprevious concentration. After each atrium reached amaximum response (i.e., no spontaneous rate) metha-choline was washed out and the atrium was allowed toattain its initial beating frequency. At this time, eithervehicle or a single concentration of ACC-9358 ordisopyramide was added and 60 min later a secondcumulative dosing of methacholine was performed.

In vivo

Rat pupil diameter The pupil diameter of femaleSprague Dawley rats (150-220 g) was measured withan American Optical stereo star zoom microscope (at10 x magnification) equipped with a micrometer discin one eyepiece which was calibrated for this study.Each rat was hand-held in the supine position with theexperimenter's hand resting on the stage of themicroscope. The right eye of each animal was illumin-ated with a constant intensity light from a Model 650American Optical illuminator fixed to the base of themicroscope. Pupil diameter was determined beforedrug or saline administration and at predeterminedtimes following intravenous injection of a fixedvolume (0.01 ml g-' body weight) of isotonic saline,ACC-9358 or disopyramide. Each animal receivedeither saline or a single dose of ACC-9358 or dis-opyramide.

Vagally-induced bradycardia Mongrel dogs (8 to16 kg, either sex) were anaesthetized with pentobar-bitone, 30mg kg' i.v., followed by a constant in-fusion, 4mg kg- I h- ', beginning 60 min after induc-tion of anaesthesia. Dogs were ventilated via a cuffedendotracheal tube with a Harvard Apparatus res-pirator at 20 cycles min' and 15cc kg-' strokevolume. A femoral vein was catheterized for adminis-tration of test compound. Electrodes were attached tothe limbs for recording lead II of the ECG. Each vagusnerve was isolated in the cervical region and cutbetween two ties. Platinum hook electrodes wereinserted into the distal portion of the right vagus nervefor electrical stimulation to produce a slowing of heartrate. The voltage which caused maximum slowing ofheart rate (frequency 1 Hz, pulse duration 2.5 ms) was

PARASYMPATHOLYTIC ACTIVITY OF ACC-9358 AND DISOPYRAMIDE

determined for each dog and was increased by 20V toprovide supramaximal stimulation voltage. Thefrequency of supramaximal stimulation required todecrease heart rate by approximately 60 beats min-'after 10 s of stimulation was determined for eachanimal.The pretreatment control response to vagal stimula-

tion was taken as the average of three responsesobtained at I min intervals. ACC-9358 or dis-opyramide were administered as bolus injections of0.5 mg kg- ', i.v., each min; isotonic saline was admin-istered at an equal volume and rate (1 ml min '). Fiftyseconds after each bolus injection, vagal stimulationwas performed and the heart rate response wasrecorded on a Beckman R-611 strip chart recorder.

Drugs

Carbamylcholine chloride (carbachol) and metha-choline chloride were purchased from Sigma ChemicalCo. (St. Louis, MO). Disopyramide phosphate(racemic mixture) was a gift from the G.D. Searle Co.(Skokie, IL). Atropine sulphate (injectable) was man-ufactured by Burroughs Wellcome Co. (ResearchTriangle Park, NC). ACC-9358 was synthesized as thefree base in the Medicinal Chemistry Department ofAmerican Critical Care (McGaw Park, IL).

Data analysis

Unless otherwise indicated, all data are expressed asthe mean ± s.e.mean.

In guinea-pig ileum and right atrium studies, agon-ist (carbachol or methacholine) EC50 values weredetermined by least squares analysis for each tissue inthe presence and absence of test compound (ACC-9358 or disopyramide) or vehicle. Schild analysis(Arunlakshana & Schild, 1959) was performed on theresulting concentration-ratios to determine whetherACC-9358 or disopyramide were competitiveantagonists in these models and, if so, their potencies(pA2 values). A two-tailed Student's t test for unpaireddata was used to compare drug-induced with vehicle-induced mean concentration ratios. A P value lessthan 0.05 indicated a significant difference.

In rat pupil diameter studies, changes from pretreat-ment control values were determined at 5, 10, 15, 20,25 and 30 min after drug or saline administration. Aone-way repeated measures analysis of variance(ANOVA) model (Winer, 1971) was used to test thehypothesis that mean values were unchanged overtime. Calculations were performed using the SASGLM procedure (SAS Institute, 1982). If there wasevidence of a statistically significant time effect, Dun-nett's two-sided test (Winer, 1971) was used to deter-

Table 1 Concentration-ratios, slope ratios and Schild analysis for vehicles, disopyramide and ACC-9358 in thecarbachol-stimulated guinea-pig ileum

Concentrationn ratio (X ± s.e.)

5 1.36 ± 0.135 1.29±0.164 1.35±0.20

5 2.83 ± 0.44d6 8.54± 1.65d5 18.59 ± 3.84d

6 1.22±0.136 1.15 ± 0.075 2.02 ± 0.27d5 2.41 ± 0.22d6 4.22 ± 0.42d

Slope ratio(X ± s.e.)

Schild analysisSlope (± 95% CI) pA2 (± 95% CI)

0.99 ± 0.180.93 ± 0.100.98 ± 0.05

0.80 ± 0.170.90 ± 0.130.81 ± 0.07

0.89 ± 0.060.94 ± 0.090.96 ± 0.180.98 ± 0.141.40 ± 0.16

0.99 ± 0.29

0.40 ± 0.25e0.59 ± O.l9

0.74 ± 0.51g

5.78 ± 0.25

4.17 ± 0.34w

aVehicle control for all disopyramide concentrations.bVehicle control for 3, 10, 30 and 100 pM ACC-9358.Wehicle control for 300 M ACC-9358.dSignificantly different from appropriate vehicle control value or unity (P <0.05).eCalculated using all 5 concentrations.fCalculated using the highest 3 concentrations.9Calculated using the highest 2 concentrations.

TreatmentVehiclesH2O0Low HClbHigh HCIC

Disopyramide3pM10fiM30 11MACC-93583 fM10IM30 pMIOO1M300 pM

89

90 B.S. BROWN et al.

mine which mean values obtained at various time-points after drug administration were significantlydifferent from the pretreatment control mean. Themean of the maximum pupil diameter following salineor drug treatment was compared to the pretreatmentmean using Student's t test for paired data. Thesignificance level for ANOVA, Dunnett's andStudent's tests was set at P < 0.05.

In vagally-induced bradycardia studies, heart rateresponses to vagal stimulation were determined eachminute following commencement of drug or salineadministration. Mean responses over time (orcumulative dose of ACC-9358 or disopyramide) werecompared to pretreatment control responses using theANOVA and Dunnett's procedures as describedabove.

Results

Guinea-pig ileum

In the concentration range of0.02 to 2.0 jIM, carbacholcaused a concentration-dependent increase in tensiondevelopment in the guinea-pig ileum. In all ex-

periments (n = 58) the ED50 of carbachol alone was3.5 ± 0.2 x 10 7M.The relative antimuscarinic activity of dis-

opyramide and ACC-9358 in the guinea-pig ileum wascompared by examining the effects of these agents(and the appropriate vehicles) on the carbachol con-centration-response curve. None of the three vehiclesused in this study caused a significant shift in thecarbachol concentration-response curve (Table 1).Disopyramide, however, caused a concentration-de-pendent rightward parallel shift in the carbacholconcentration-response curve. Schild analysis of thiseffect indicated that disopyramide was a competitiveantagonist of carbachol in this tissue with a pA2 valueof 5.78 ± 0.25 (Table 1). At 3 and 1OiM, ACC-9358had no effect on the carbachol concentration-responsecurve whereas at 30,100 and 300ILM, ACC-9358caused a rightward parallel shift in the curve. Schildanalysis of these effects indicated that ACC-9358 maybe a competitive antagonist ofcarbachol in the guinea-pig ileum, but only at the two highest concentrationstested (Table 1). There was a large degree ofvariabilityin the data at 100 and 300 pIM ACC-9358 and the slopeof the Schild plot using concentration-ratios deter-mined at these concentrations of ACC-9358 was not

120

100

80

60

40

20

7 6 5 4 3

7 6 5

-log [Carbachol] M

4 3

b

d120 -

100 -

80 -

60 -

40 -

20 -

0 r

-O'P

6 5 4

a{d

7 6 5 4 3

-log [Carbacholl M

Figure 2 Concentration-response curves for carbachol in guinea-pig isolated ileum before (0) and after exposure tovehicle (0, a), disopyramide 10 FM (0, b) or ACC-9358 10 JM (0, c) or 300 gM (0, d). Each point represents the meanresponse to each concentration of carbachol (n = 5-6 for each group).

a120 -

100 '

80'60 -

40-

20 -

ncCo0

ua0Co

-5

0)0

EEx

E0

C

120

100

80

60

40

20

PARASYMPATHOLYTIC ACTIVITY OF ACC-9358 AND DISOPYRAMIDE

Table 2 Concentration ratios, slope ratios and Schild analysis for vehicles, disopyramide and ACC-9358 in themethacholine-treated guinea-pig right atrium

Concentrationn ratio (X ± s.e.)

Slope ratio Schild analysis(X ± s.e.) Slope (± 95% CI) pA2 (± 95% CI)

13 1.16±0.26 1.02±0.05

5 5.48 ± 1.13b3 9.23 ± 2.55b7 32.06 ± 6.52b

4 0.95 ± 0.034 1.65 ± 0.656 2.37±0.614 2.24±0.447 2.50±0.82

0.%+± 0.100.88 ± 0.031.02 ± 0.16

0.82± 0.110.97 ± 0.061.11 ± 0.240.95 ± 0.021.10 ± 0.26

1.02 ± 0.27

ND

aVehicle used for all disopyramide and ACC-9358 concentrations.bSignificantly different from vehicle control value or unity (P< 0.05).ND = Not determined.

different from unity. Comparison of calculated pA2values indicated that ACC-9358 was 41 fold lesspotent than disopyramide.

Carbachol concentration-response curves in thepresence or absence of vehicle (water), 1OM dis-opyramide or 10 or 300 M ACC-9358 are shown inFigure 2. This figure clearly illustrates the markeddifference in antimuscarinic activity betweenequimolar (1O0M) concentrations of disopyramideand ACC-9358 and that, even at a 30 times higherconcentration, ACC-9358 shifted the carbachol con-

centration-response curve to a smaller extent than10JIM disopyramide.None of the vehicles or any concentration of

disopyramide or ACC-9358 significantly alteredbaseline tension or the maximum response to carba-chol.

Guinea-pig right atrium

In the concentration range of 0.1-100JIM, metha-choline produced a concentration-dependent slowingof spontaneously beating guinea-pig right atria. In allexperiments (n = 53) the ED50 of methacholine alonewas 7.6 ± 1.6 x 10-6M.

Analysis of the effect of several concentrations ofdisopyramide or ACC-9358 indicated that 3, 5 and10 tIM disopyramide caused rightward parallel shifts inthe methacholine concentration-response curves witha pA2 value of 5.99±0.27, whereas at 3-100MM,ACC-9358 had no significant effect on the atrialresponse to methacholine (Table 2). These resultsindicate that disopyramide, but not ACC-9358, is a

competitive muscarinic antagonist in the metha-choline-treated guinea-pig right atrium.

Figure 3 illustrates the effects of the vehicle (0.05 NHCl), 3 and 30MM disopyramide and 100pM ACC-9358 on the methacholine concentration-responsecurve in guinea-pig right atria. The vehicle had no

effect on either the control spontaneous atrial rate or

on the bradycardia induced by methacholine. Alth-ough both disopyramide and ACC-9358 slightly re-duced control spontaneous atrial rate, only dis-opyramide caused a rightward shift in the metha-choline concentration-response curve (Figure 3).

Rat pupil diameter

Saline, ACC-9358 (7.5, 15.0mg kg ') or disopyramide(1.9, 3.8, 7.5, 15.Omgkg-') were administered as an

intravenous bolus and their effects on rat pupildiameter were determined for 30 min after administra-tion. Mean pretreatment diameters ranged from0.46 ± 0.02 to 0.52 ± 0.01 mm in the seven experimen-tal groups with no significant differences betweengroups.The maximum response to each treatment, illus-

trated in Figure 4, was reached at 30, 25, 20, 20, 10, 5and 5 min following administration of saline, 7.5 and15.0 mg kg-' ACC-9358, 1.9, 3.8, 7.5 and15.0 mg kg-' disopyramide, respectively. At all dosestested, disopyramide significantly increased pupildiameter compared to pretreatment control values.Conversely, neither saline nor ACC-9358 significantlyaffected pupil diameter at any time period within30 min after administration.

TreatmentVehicleaHCl 0.05 N

Disopyramide3gM10JAM30 gMACC-93583gM10gM30 IM100AM300gM

5.99 ± 0.27

ND

91

92 B.S. BROWN et al.

a Vagally-induced bradycardia

Under the conditions of this study, stimulation of thevagus nerve in a pentobarbitone-anaesthetized dogreduced spontaneous heart rate by approximately 60beats min- '. The effect of repeated bolus intravenousinjections (each minute) of isotonic saline, ACC-9358(0.5 mg kg-' min ') or disopyramide (0.5mg kg-'min ') on the heart rate response to vagal stimulationwas determined for a maximum of 30 min of saline or

.. .. v . .,a.drug administration. Mean pretreatment heart rates7 6 5 were 170±8, 168± 14 and 162±9 beatsmin-' in

saline-, disopyramide- and ACC-9358-treated dogs,b respectively, with no significant differences between

the three experimental groups. Over the time period ofthese experiments both ACC-9358 and disopyramidelowered spontaneous heart rate to no less than 140beats min '. At this heart rate a 60 beat min-' de-crease in rate could readily be achieved and thereforethe direct bradycardiac effects of these agents were notconsidered likely to account for any of the observeddiminution of heart rate responses to vagal stimula-tion.

v . ..v ... .. The effect of vagal stimulation on spontaneous7 6 5 4 3 heart rate before and at various times following

continuous administration of saline, ACC-9358 orc disopyramide is illustrated in Figure 5. At no time did

isotonic saline alter the bradycardiac response to vagalstimulation. Both ACC-9358 and disopyramide,however, significantly attenuated the effects of vagalstimulation. The parasympatholytic effect of diso-pyramide occurred at an earlier time (and consequent-tly a lower cumulative dose) than did that of ACC-9358. In this regard, disopyramide and ACC-9358inhibited the vagally-induced bradycardiac effect by50% at a cumulative dose of approximately 0.65 and

IIII..fe w,11.25mgkg-', respectively, thereby indicating that7 6 5 4 3 the parasympatholytic activity of disopyramide is

-log [Methacholinel M approximately 17 fold greater than that of ACC-9358in this in vivo preparation.

Figure 3 Concentration-response curves for metha-choline in guinea-pig isolated right atria before (0) andafter exposure to vehicle (0, a), disopyramide 3 JuM (0)and 301uM (-, b), or ACC-9358 100M (0, d). Eachpoint represents the mean response to each concentrationof methacholine (n = 13 for vehicle, n = 5-7 for dis-opyramide, n = 7 for ACC-9358).

To establish further the validity of this procedurefor determining anticholinergic activity we alsoevaluated the effects of atropine in this preparation.Intravenous bolus injections of 12.5, 25.0, 50, or

100ligkg-' atropine significantly increased pupildiameter within 5-0 min of administration to 1.1,2.2, 3.2, 4.2 mm, respectively, from pretreatmentvalues of 0.48 ± 0.03 to 0.51 ± 0.02 mm.

Discussion

It is well known that pharmacological agents can exertparasympatholytic activity via several mechanisms.These include: (1) an antimuscarinic action by com-

petitive or non-competitive blockade of muscarinicreceptors located in the postsynaptic membrane of theeffector organ, or (2) a vagolytic action throughsuppression of impulse conduction along efferentvagal nerves, inhibition of acetylcholine synthesis or

storage, inhibition of release from pre- or postgan-glionic nerve terminals, or by blockade of nicotinic(and, to a small extent, muscarinic) receptors inparasympathetic ganglia. In the present study weexamined the effects of ACC-9358 and disopyramide

200

150

100

50

0

I

X 20040-.0.0150a)

li 100._

4-0

0 50000

1 0

100

c0C.C,)

200

150

100

50

0

PARASYMPATHOLYTIC ACTIVITY OF ACC-9358 AND DISOPYRAMIDE

3.0

2.5

E 2.0E

0)E' 1.5

X 1.0 *c: * -~~~~~~

0.50 ~ U __

7.5 15 1.9 3.8 7.5 15Dose (mg kg- i.v.)

Figure 4 Effect of saline (hatched column), disopyramide (solid column) or ACC-9358 (cross-hatched column) on ratpupil diameter. Each bar represents the mean of the maximum pupil diameter observed within 30 min of saline or drugadministration; s.e.mean shown by vertical lines. (n = number shown in pretreatment column for each group.)*Indicates P< 0.05 compared to pretreatment value (open column) using Student's t test for paired data.

T 100

, 800)

.0

CR60

c

40

0)

20CD

0 o

FigureACC-pentolthe mstimulvertica*P<(nett's

under conditions which specifically allowed for theevaluation of antimuscarinic activity (in vitro ex-periments) and under conditions in which an antimus-carinic or vagolytic action could account for anobserved parasympatholytic effect (in vivo ex-periments).

------ -----iWith the possible exception of very high concentra-tions in the guinea-pig ileum, the results of our in vitro

* *1# ; studies indicated that ACC-9358 was not a com-

*1# - petitive antagonist of exogenously-administered mus-

*#*H carinic agonists in either the ileum or right atrium.nS * * * # Conversely, disopyramide clearly was a competitive

*3i_ # # muscarinic antagonist in both the ileum and rightatrium with a pA2 value of 5.78 and 5.99, respectively,in these tissues. A pA2 value of 5.78 for racemicdisopyramide in intact ileal strips compares well with

,, 1, 5, 0, , 0, values previously reported for the R and S isomers ofo 5 10 15 20 25 30 disopyramide in isolated longitudinal muscle strip

Time (min) preparations from guinea-pig ileum (Giacomini et al.,1980). These investigators reported a pA2 of 5.74 and

5 Effect of saline (0), disopyramide (0) or 6.25 for the R and S isomers, respectively. Further-9358 (0) on vagally-induced bradycardia in the more, a pA2 of 5.99 for racemic disopyramide inbarbitone-anaesthetized dog. Each point represents * '

bean ecreaeinheartrate oducd b v al uinea-pig isolated right atria also agrees well with theiean decrease in heart rate produced by vagal iniiincntn )o .5 0' aaoostlation (n = 4-5 for each grop) g~~maihwb nhibition constant (Ks) of 8.5 x 10- M (analogous tol lines, a pA2 of 6.07) for racemic disopyramide derived from0.05 compared to pretreatment value using Dun- tritiated quinuclidinyl benzilate ([3H]-QNB) dis-two-sided test. placement studies with crude homogenates of guinea-

93

94 B.S. BROWN et al.

pig right atria (Mirro et al., 1980). Thus, underconditions in which disopyramide exerted antimus-carinic activity virtually identical to that previouslyreported, ACC-9358 exerted little, if any, antimus-carinic activity.

In rat pupil diameter studies, doses ofACC-9358 upto 15 mg kg-' had no mydriatic effects. Conversely,disopyramide in doses as low as 1.9 mg kg-' produceda significant mydriatic effect. These results indicatethat, in the rat, ACC-9358 had neither antimuscarinicnor vagolytic activity. However, since pupil diameteris modulated by both parasympathetic and sympath-etic activity, it is theoretically possible that ACC-9358exerted either (1) x-adrenergic and muscarinic-cholin-ergic antagonism, or (2) suppression of both parasym-pathetic and sympathetic nerve activity, thereby resul-ting in no net change in pupil diameter. The formerpossibility is unlikely since available evidence indicatesthat ACC-9358 is devoid of a-antagonist properties(unpublished observation) and the present studiesindicate little, if any, muscarinic antagonist properties.The possibility that ACC-9358 suppressed both

parasympathetic and sympathetic neural activity can-not be ruled out at present. Accordingly, since ACC-9358 lacked appreciable antimuscarinic activity, itsinhibition of vagally-induced bradycardia in the dog isprobably due to a direct vagolytic action. Supportingthis direct depression of vagus nerve activity arepreliminary experiments showing that ACC-9358 re-duced the hindlimb vasoconstrictor response to

stimulation of the lumbar sympathetic chain in theanaesthetized dog (unpublished observation). Thus, insufficiently high doses, ACC-9358 can be shown tosuppress both parasympathetically- and sympath-etically-mediated responses. It should be pointed out,however, that the doses required to exert vagolytic(EDo= 11.25mg kg-') or sympatholytic (ED50 =25.0 mg kg- ') activity are well above those which exertantiarrhythmic activity (2-3mgkg-') in the pen-tobarbitone-anaesthetized dog (Reynolds et al., 1984).Although the precise mechanism of the vagolytic andsympatholytic activity of ACC-9358 is unknown, itmay be due to a local anaesthetic action since ACC-9358 has been shown to be a potent sodium channelblocker in canine isolated Purkinje fibres (Wine &Brown, 1985).Depending on the tissue studied, ACC-9358 posses-

sed approximately 1/40 the antimuscarinic activity ofdisopyramide and exhibited vagolytic activity in vivoonly at very high doses. In light of the fact that ACC-9358 is approximately equipotent to disopyramide inthe ouabain-induced ventricular tachycardia andcrush-stimulation-induced atrial flutter experimentalarrhythia models (Reynolds et al., 1984), these resultssuggest that the incidence of anticholinergic sideeffects ofACC-9358 should be significantly lower thanthose encountered with disopyramide.The authors wish to acknowledge Samuel V. Calzadilla andMark J. Diemer for their excellent technical assistance andLori Spaulding for preparing this manuscript.

References

ARUNLAKSHANA, 0. & SCHILD, H.O. (1959). Some quan-titative uses of drug antagonists. Br. J. Pharmac.Chemother., 14, 48-58.

BIGGER, T.J. & HOFFMAN, B.F. (1980). Antiarrhythmicdrugs. In The Pharmacological Basis of Therapeutics, 6thEdition. ed. Gilman, A.G., Goodman, L.S. & Gilman, A.pp. 761-792. New York: Macmillan Publishing Co.

CHEN, W., DONG, Y. & DING, G. (1979). Cardiovasculareffect of a new antiarrhythmic drug, changrolin. Actapharmac. Sin., 14, 710-714.

GIACOMINI, K.M., COX, B.M. & BLASCHKE, T.F. (1980).Comparative anticholinergic potencies of R- and S-dis-opyramide in longitudinal muscle strips from guinea pigileum. Life Sci., 27, 1191-1197.

LI, L.Q., QU, Z.X., WANG, Z.M., ZENG, Y.L., DING, G.S., HU,G.J. & YANG, X.Y. (1979). Studies on a new antiarrhyth-mic drug, changrolin, 4-[3',5'-bis (N-pyrrolidinyl-methyl)-4'-hydroxyanilinol-quinazoline. Sci. Sin., 22,1220-1228.

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(Received August 16, 1985.Accepted October 11, 1985.)