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Muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers
Journal of the Autonomic Nervous System, 24 (1988) 147-155 Elsevier
147
JAN 00861
Muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers Kanigula Mubagwa and Jeremy Adler Department of Physiology, University of Zimbabwe, Mount Pleasant, Harare (Zimbabwe) (Received 29 April 1988) (Revised version received and accepted 5 August 1988)
Key words: Chloroquine; Muscarinic antagonist; Cardiac cycle; Beat-to-beat variation of R-R interval
Abstract An investigation was undertaken into possible vagolytic effects of chloroquine. A single oral dose of chloroquine was given to healthy human volunteers and its influence on vagally mediated heart rate changes studied. Chloroquine at a dose of 600 mg significantly increased lying and standing heart rates, reduced the beat-to-beat variation of the R-R interval and reduced the heart rate changes induced by deep breathing, by the Valsalva manoeuvre and by standing. Chloroquine at a dose of 225 mg did not produce significant changes of these parameters. The effects of 600 mg chloroquine resemble those obtained with atropine and are consistent with an antimuscarinic receptor effect.
Introduction Chloroquine is widely used for its antimalarial and anti-inflammatory actions (e.g. in rheumatoid arthritis and in lupus erythematosus). It is also reported to have local anaesthetic [16], anticoagulent [16] and uterotropic [4,15] effects. On the heart chloroquine has an antiarrhythmic action [2] probably due to its local anaesthetic (quinidine-like) properties. Chloroquine has also been shown to cause tachycardia in anaesthetised frogs, an effect involving, at least in part, a central sympathetic stimulation [3]. Recent data suggest that chloroquine may have a muscarinic receptor antagonist action [14,21]. Thus the tachycardia
Correspondence: K. Mubagwa, Department of Pharmacology University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
observed in frogs could be due partly to a vagolytic action of chloroquine. Tachycardia following chloroquine administration has not been reported in man and other atropine-like effects of the drug have not been investigated. We examined the effect of clinical doses of chloroquine on human volunteers, using cardiovascular reflexes which are known to involve vagally mediated changes in heart rate. The results show that chloroquine produces changes similar to those o b t a i n e d with m u s c a r i n i c r e c e p t o r antagonists.
Materials and Methods Subjects Twelve healthy male volunteers (age: 19-51 years) drawn from the University staff were used in the present study. Informed consent and ap-
0165-1838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
148
proval of the Faculty's Ethics Committee were obtained.
Recordings Standard lead electrocardiograms (ECGs) were recorded from the volunteers (electrocardiograph EK-8, Burdick) under various conditions (see below), before and after chloroquine ingestion. The duration of the cardiac cycle was obtained from the interval between QRS complexes (R-R interval). The R peak of the QRS complex of the ECG triggered a pulse which was fed to a computer (Sinclair QL) and was used to measure cardiac cycle duration. The R-R interval values were graphically displayed on the screen of the computer monitor and were stored for later analysis. QRS Peak Detection. Detection was achieved in two steps. First, a simple voltage gate and a heavily DC filtered ECG signal were used to detect the occurrence of a QRS complex. Since accurate discrimination (5:2 ms) is required for some of the data, it was necessary to detect the peak of the R wave. The detection of a QRS complex initiated the generation of a staircase voltage that tracked the R wave. The interval between steps of the staircase depended on the slope of the unfiltered R wave. When the slope decreased and the interval between steps exceeded a preset value, an output pulse was generated. This was made to correspond with the peak of the R wave. The system allows for the detection of the R peak of the E C G in the face of significant DC shifts but without any distortion due to filtering. Details of the gate will be published separately. R-R Interval Measurement. The detection of an R peak generated a "ITL pulse which was fed into a control line of a parallel i n p u t / o u t p u t port (M6821) attached to a computer via an expansion unit (CST Q + 4). A software clock running at 3600 Hz was used to count the intervals between pulses. Experimental protocol On the day of the experiment the subjects had a fight brealffast that avoided caffeine. They were not allowed to consume food, tea or coffee, or to engage in physical exercise for the duration of the experiment, l~aring ~ day tht~y were ~ b j e e t e d to
a repetitive series of tests. Once stable control data were obtained, typically after 3 - 4 tests, the subjects ingested chloroquine phosphate (Datlabs, Bulawayo) dissolved in water. Testing was carried out 10 min later and at hourly intervals for 3 - 4 hours after chioroquine ingestion. All 12 subjects were used for testing with 600 mg chloroquine (base). Seven of them took part in testing with 225 mg chloroquine (base), and another group of 7 underwent testing on an occasion without chioroquine administration (before or at least 15 days after testing with chioroquine).
Tests R-R intervals were measured continuously under each of the following conditions: (1) while lying at rest, (2) while lying and breathing deeply, (3) on standing from a seat, (4) during sustained standing, and (5) during a Valsalva manoeuvre. These tests were run sequentially and each sequence was always preceded by a 5-min recumbent rest. Depending on heart rate each sequence of tests would take 20-30 rain. Lying at rest. 250 R-R intervals were recorded while the subjects were lying on a couch and breathing normally. Deep Breathing. 80 R-R intervals were recorded while the subjects remained lying and were asked to maximally inhale and exhale on command, at a frequency of 6 cycles per minute [11,221. Sit-Stand Test. 50 R-R intervals were recorded while the subjects sat on a 30-era ~ seat. 50 R-R after standing, 50 R-R intervals while again seated and further 300 R-R intervals while in standing position. T o reduce noise in the ECG signal, recording was made from lead i (fight and left arms) and the subjects were asked to move with limp arms. Valsalva Manoeuvre. Recordings were made while the subjects were asked to blow into a leaky tube connected to the column of a sphygmomanometer and to maintain a pressm'e of 48 n m ~ H g for 15 s. The manoeuvre was performed 3 times with at least 60 heart beats between manoeuvres. A leak in the tube connected to the manometer ensured that the mouth alone ceamot be used to generate the pressure. The subjects were asked to
149 avoid a deep intake of air after straining was released.
Data analysis Data analysis was carried out off line using a computer program developed by one of us (J.A.). Lying and Sustained Standing. Mean, standard deviation and frequency distribution histogram of the R-R interval [19] were obtained. In addition we produced frequency distribution histograms of the beat-to-beat variation (i.e. of the changes in R-R interval from one beat to the next) and calculated the mean absolute beat-to-beat R-R interval variation (MABBV), according to the following formula: MABBV=
.= I R R i + I - RRil / N
where X is a summation sign, i is an index used to number R-R intervals, IRRi+I - RRil is the absolute value of the difference between two successive R-R intervals, and N is the total number (usually 250) of R-R intervals. The MABBV is a measure of the spread of the frequency distribution histogram. A similar measure, the standard deviation of the difference between any R-R interval and the next, was used by others [10]. Since the MABBV considers only changes in R-R interval from one beat to the next it will mostly reflect high frequency changes, which are attributed to the activity of the parasympathetic nervous system [1,20]. That the beat-to-beat variation [22] and its mean absolute value (MABBV; J. Adler, unpublished results) provide a measure of vagal tone is supported by the fact that they are reduced following atropinization. Deep Breathing. The average ratio of the longest to the shortest R-R interval during each 10-s respiratory cycle was determined [see 22]. Sit-Stand Test. The ' 3 0 : 1 5 ratio' [7,8] was determined by taking the ratio between the longest R-R interval 22-36 beats after standing and the shortest R-R interval 10-20 beats after standing. The increase in R-R interval (after an initial decrease) on standing is of vagal origin [8]. Falsalva Manoeuvre. The ratio between the longest R-R interval after the manoeuvre and the
shortest R-R interval during and in the 5 s after cessation of straining [6,13] was determined. The period just after the release of the pressure was included in addition to the period of straining because R-R intervals often continued to decline immediately after the pressure is released. The mean of 3 consecutive Valsalva manoeuvres was used in the analysis of chloroquine effects. Statistics. For each subject, the quantitative indices obtained in the tests after chloroquine administration were expressed as percentages of the mean value of the same indices before chloroquine. When no chloroquine was administered, the indices were also normalized by taking the initial measurement as 100%. Statistical significance (at the 95% confidence level) of the difference between data obtained after chloroquine treatment and those obtained in control conditions at similar time intervals was tested using Student's t-test for samples of unequal sizes.
Results
The changes described below were consistently induced by chloroquine in 10 out of 12 subjects. In two subjects no change in some parameters was observed 3 - 4 h after chloroquine administration.
Lying and standing R-R intervals Fig. 1A shows cardiac cycle lengths recorded in a lying subject during quiet breathing and during maximal deep breathing. Under control conditions (i.e. before chloroquine administration), the mean R-R interval of the subject was 946 + 55 ms (mean + SD, n = 250), and 4 h after 600 mg chloroquine administration it declined to 750 + 18 ms. The R-R interval after treatment with chloroquine is significantly different from the control value ( P < 0.001). Thus, chloroquine decreased the mean cardiac cycle length measured in lying position. Fig. 1B shows frequency distribution histograms of the R-R intervals. Following chloroquine administration, both the mean value and the variation of R-R interval had decreased. Similar results were obtained in the same subject for the R-R intervals measured in steady upright position (791
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Fig. 1. Effect of cliloroquine on the cardiac cycle (R-R interval) in a lying subject. A: 250 heart beats were recorded during quiet breathing (left of vertical line) followed by 80 beats during maximal deep breathing (right of vertical line), before and 4 h after chloroquine (600 nag) administration. The means and standard deviations of R-R intervals (indicated by horizontal lines) during quiet beathing are shown. Small horizontal bars indicate the longest and the shortest R-R intervals in each respiratory cycle (6/rain), which are used to produce the respiratory ratio. The average ratio (with number of cycles analysed) is also shown. B: frequeaey distribution histograms of the R-R intervals recorded during the periods of quiet breathing displayed in A (bin size: 5 ms).
± 41 ms before, and 620 + 20 ms 4 h after chloroquine). The average effect of a 600-rag chloroquine dose on lying cardiac cycle in 12 different subjects is illustated in Fig. 2A, which also shows cardiac cycle durations measured in 7 of the subjects on occasions when no chloroquine was administered and the values measured when a 225-mg chloroquine dose was given. When no chloroquine was administered, the cardiac cycle duration remained practically constant throughout the day. The low (225 nag) chloroquine dose tended to produce a decrease of cardiac cycle duration but the R - R interval values were not significantly different from those obtained under control conditions. When
120
the higher (600 mg) chloroquine dose was given, a progressive decrease of the cardiac cycle duration was obtained. The cardiac cycle values measured 2, 3 and 4 h after chloroquine were significantly different from control values ( P < 0.01, P < 0.001, P < 0.05, respectively). The r e s d t s obtained in standing position are summarised in Fig. 2B and are qualitatively similar to those obtained in lying position.
Beat-to.beat changes in R-R interval One possible explanation for the results~ described above is a decrease by chloroquine of vagal influences on the heart. As mentioned in Materials and Methods, the beat-to-beat variation
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Fig. 2. R-R intervals (mean + S.E.M.) measured during quiet breathing: untreated ( o , n = 7); chloroqu!ne 225 rag (EL n = 7); chloroqttine 600 mg (O; n= 12). The R-Rinterval values are normalised as a ~ dinitialor p r ~ v a h i ~ , i' P < 0.05; ** P < 0.01. A: data white in lying potatO. B: data while ~axting.
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Fig. 3. Frequency distribution histograms of the beat-to-beat variation in R-R interval of two standing subjects, before and after chloroquine (600 mg). Bin size: 10 ms on left, 2.5 ms on right.
in R-R interval allows measurement of vagal tone. The R-R interval recordings of Fig. 1A and their frequency distribution (Fig. 1B) already suggest
~ 140 I-
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that variability in cardiac cycle duration was reduced by chloroquine administration. Fig. 3 shows the frequency distributions of the beat-to-beat variation in R-R interval measured in two standing subjects before and after chloroquine administration. The beat-to-beat R-R interval changes appear to be normally distributed both before and after chloroquine administration, but after chloroquine the variability in beat-to-beat change became progressively less pronounced. Fig. 4A shows that the MABBV (which is proportional to the spread of the frequency distribution histogram) tended to increase with time under control conditions while it decreased after 225 mg or 600 mg chloroquine. The values measured 1, 2, 3 and 4 h after 600 mg chloroquine were significantly different from those obtained in control conditions at corresponding times ( P < 0.05 after 1 h, P < 0.001 after 2-4 h). Similar results were obtained for the mean absolute beat-to-beat variation measured in lying position (Fig. 4B). The MABBV values were higher in the lying position (50.1 + 5.8 ms, mean + S.E.M.) than in the standing (19.9 + 4.4 ms). For the data in lying position, there was a significant negative correlation between the magnitude of chloroquine-induced decrease in MABBV and the pretreatment R-R interval value (r = -0.911, P < 0.001, n = 11), and between the decrease in MABBV and the pretreatment MABBV value (r = 0.619, P < 0.05, n = 11).
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Fig. 4. Effect of chloroquine on the mean absolute beat-to-beat variation (MABBV). Means and S.E.M. expressed as a percentage of initial or pretreatment value: untreated ( o , n = 7); 225 mg chloroquine (D, n = 7); 600 mg ehloroquine (o, n = 12). * P < 0.05; • * P < 0.01; * * * P < 0.001. A: in standing position. B: while lying.
152
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heart rate (see Materials and Methods), a value of 1.68 is obtained under control conditions in the subject. This ratio decreased to 1.38 4 h after chloroquine (600 mg) administration. Similar resuits were obtained in most subjects and are summarized in Fig. 5. The values obtained 2, 3 and 4 h after 600 mg chloroquine were significantly different from control values ( P < 0.01, P < 0.001, P < 0.01, respectively). Those obtained after 225 mg chloroquine were not different from control values.
4
time (hours)
Standing-induced bradycardia
Fig. 5. Effect of chloroquine on the respiration ratio. Means and S.E.M. of the respiration ratio are expressed as a percent,age of initial or pretreatment values: untreated (o, n = 7): 225 mg chloroquine (12, n = 7); 600 nag chloroquine (O, n = 12). * * P < 0.01; * * * P < 0:001.
Effect of respiration on R-R interoal Fig. 1A also illustrates the effect of chloroquine on the cardiac cycle changes induced by deep breathing. Before chloroquine a d m ~ s t r a t i o n maximal deep b r e a ~ g induced large fluctuations of the cardiac cycle length. Using the ratio between the longest and the shortest cardiac cycle length for eaeh respiratory excursion to quantitatively assess the influence of deep respiration on
In order to further investigate the possibility that chloroquine decreases vagal influences on the heart, the bradycardia produced after standing was analysed. Fig. 6A illustrates the changes in R-R interval produced in one subject following postural change before and after treatment with 600 mg chloroquine. On standing there was a tachycardia (decrease in R - R interval) that was followed by a recovery (increase in R-R interval). The magnitude of this bradycardia was reduced 4 h after chloroquine administration, as seen from the 30:15 ratios which decreased from 1.16 and 1.34 to 1.12 and 1.21 after chloroquine. Similar results were obtained with 600 mg chtoroquine in most subjects and are summarised in Fig. 6B. The
120 1 B
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Fig. 6. Effect of chlofoquine c~ the sUmaln~-~,~,~_,,.~ bradycardhL A: traces before and 4 h after chloroquix~ (600 nag). The subject Standln~ is indtcat~ by vm,tical bars. Small hcxizccaat bars indicate the shortest R-R intervals 10-20 beats after standing and the longest R-R interval 22-36 beats aftor s ~ , The ratim ~ ~ ~ (30 : 15 ratio) are l i w m above the m l c i m p . 8: mean m~l S,F-M of 30 : 15 ratios ~ as a p ~ m u t ~ of tl~ i z ~ z l ~ ~ v~li~: mm,mk~ (o, n-7~: 22.~:.mI~ l o r ~ (~ n--~; ~ O O m ~ ~ ~ , - n - , lt). was inithdly seated, stood on beat 50, sat on beat 100 and stood on beat 150.
* P < 0.05; ** P <0.01.
153 110
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1
2
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Fig. 7. Effect of chloroquine on cardiac cycle changes induced by the Valsalva Manoeuvre. A: R - R intervals during two successive Valsalva manoeuvres, before and 3 h after treatment with chloroquine (600 mg). Vertical bars m a r k start of manoeuvres. Small horizontal bars indicate shortest R - R intervals during manoeuvre and longest intervals after manoeuvre. The ratio between these intervals (Valsalva ratio) for each manoeuvre is shown above the tracing. B: mean and S.E.M. of the Valsalva ratios are expressed as a percentage of the control or pretreatment values: untreated ( o , n = 7); 225 m g chloroquine (D, n = 7); 600 mg chloroquine (e, n = 12).
values measured 1, 2, 3 and 4 h after treatment with chioroquine were significantly different from control values ( P < 0.05, P < 0.001, P < 0.001, P < 0.05, respectively). Chloroquine also reduced the rate of development of the tachycardia seen just after standing (Fig. 6A), an effect also seen after atropinization [J. Adler, unpublished results]. Effect of Valsalva manoeuvre on R - R interval
Fig. 7A illustrates R-R interval changes induced by the Valsalva manoeuvre in one subject, before and 3 h after treatment with chioroquine. After chloroquine administration, there was a decrease in the magnitude of the post straining bradycardia. In the example shown the mean ratio between the longest cycle length obtained immediately after straining and the shortest cycle length obtained at the end of the straining was 2.65 in control conditions, but it decreased to 2.27 after chloroquine treatment. The results obtained in control conditions and following treatment with 225 mg or 600 mg chloroquine are summarised in Fig. 7B. With the higher chloroquine dose there was a tendency for a decrease in the Valsalva ratio although the differences between values measured
after chloroquine and those measured in control did not reach statistical significance. Discussion
The results show that following 600 mg chloroquine administration, there was (1) an increase of heart rate, (2) a decrease of the mean beat-to-beat R-R interval variation, (3) a decrease of the influence of deep breathing on heart rate, and (4) a decrease of the bradycardia that is obtained following standing or at the end of the Valsalva manoeuvre. There was no change (or there was a change in the opposite direction) in these parameters when no chloroquine was administered and immediately after chloroquine administration. This excludes the possibility that the observed changes are the result of an increase in sympathetic tone due to hypoglycaemia (following food restriction during the experiment) or to stress (following ingestion of chloroquine). The effects of chloroquine are dose-dependent since the above changes were not statistically significant with a 225 mg dose. The effects of chloroquine are consistent with a vagolytic action. It is unlikely that they are due to
154
increased sympathetic tone [3] since a change in sympathetic influence on the heart is not expected to induce a decrease in the beat-to-beat R-R interval variation [20] and to alter the respiratory ratio [12] and the 30 : 15 ratio [8]. The effects are therefore probably due to a decrease in the parasympathetic influence on the heart. Such a decrease could be due to an inhibition by chloroquine of the neural cardio-inhibitory afferent mechanisms, of the vagal center itself, or of the vagal efferents to the heart. The action of chloroquine could also be localized within the heart, i.e. chloroquine could decrease the responsiveness of the cardiac tissue to vagal influences. The last possibility is consistent with recent studies which have demonstrated that chloroquine binds to muscarinic receptors [21, F. Dondo and K. Mubagwa, unpublished results] and inhibits muscarinic agonist-induced negative chronotropic effects in isolated guinea pig hearts [F. Dondo and K. Mubagwa, unpublished results). This suggests that the effects of chloroquine are due, at least in part, to an action at the cardiac cell muscarinic receptors. An inhibition of other muscarinic receptor-mediated effects has been observed in chick oesophagus muscle [14]. The lack of effect in some subjects (2 out of 12), could be due to a low plasma chloroquine concentration following poor absorption. Plasma chloroquine levels were not determined in the present study. Under normal conditions, with the acidity of the gastric cavity (pH 2) most (99%) of the ingested chloroquine (pK a 8.1 and 10.6) is expected to be absorbed. Pharmacokinetic data suggest that, following a single oral dose of 250 or 620 mg, a peak plasma chloroquine concentration of 200/~g/1 (0.4/LM) is reached in 3-6 h [17] or 1-2 h [9]. It remains possible, however, that there was poor or slow absorption in some individuals. After absorption, chloroquine reaches the plasma where a large part of the absorbed amount is bound to proteins. With time the drug is also accumulated in the lysosomes of many tissues [5]. If these last two processes are very marked in some individuals, they could also result in low plasma free chloroquine concentrations. Another possibility to explain the lack of response in some subjects is to assume that they have a very high vagal tone, and that chloroquine
acts by a competitive mechanism. In this case the chloroquine concentration achieved by a single 600-mg dose may not produce significant displacement of the neurotransmitter from its receptor. In favour of this hypothesis is the observation that. in the two subjects who did not respond to chloroquine, the mean beat-to-beat R-R interval variation was highest, suggesting that vagal tone was high in these individuals. This is also supported by the high negative correlation between relative decrease in lying mean absolute beat-to-beat variation and the original value of the mean absolute beat-to-beat variation. The greater percentage fall in MABBV. produced by chloroquine, in the standing compared to the lying position (see Fig. 4), also indicates that high vagal tone is less susceptible to antagonism. The likely peak plasma concentration of chloroquine after a single 600-mg oral dose. about 200 /~g/1 [17], is similar to the concentrations reported in rheumatoid patients receiving 250 mg/day [9] and in volunteers receiving 620 mg/day for 14 days [18]. However. in these two studies plasma samples were taken just prior to the daily dose (i.e. 24 h after the last chloroquine ingestion) and therefore underestimate the peak plasma concentration. This suggests that the peak plasma concentrations occurring in these studies exceed those obtained in the present study. The finding that no effect was obtained with the lower dose of chloroquine indicates that low doses (e.g. those used for low-dose antimalarial prophylaxis) do not produce atropine-like side effects. However, when higher doses are used for prophylactic, therapeutic or criminal reasons, the antimuscarinic effects can be marked and this should be borne in mind by the clinician.
Acknowledgements This work was supported by grants (no. 2.901.1.2980 and 2.901.1.2720) from the University of Zimbabwe Research Board. We would like to thank the volunteers who participated in this study, as well as Miss P. Vdlah, Mr. A. Mtttamba and Mr. T. Saruziwo for their help in the collection of data.
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References 1 Akselrod, S., Gordon, D., Ubel, F.A., Shannon, D.C., Barger, A.C. and Cohen, R.J., Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control, Science, 213 (1981) 220-222. 2 Burrel, Z.L. and Martinez, A.C., Chloroquine and hydroxychloroquine in the treatment of cardiac arrhythmias, N. Engl. J. Med, 258 (1959) 798-800. 3 Chinyanga, H.M., Fletcher, J.J. and Vartanian, G.A., Increase of electrocardiogram voltage and contractile force of the frog's heart induced by chloroquine, West Afr. J. Pharmacol. Drug Res., 3 (1976) 90-101. 4 Chinyanga, H.M., Kurantsin-Mills, J. and Vartanian, G.A., The inhibitory action of chloroquine to guinea pig uterine contractions, West Aft. J. Pharmacol. Drug Res., 1 (1974) 50-57. 5 De Duve, C., De Barsy, T., Poole, B., Trouet, A., Tulkens, P. and Van Hoof, J., Lysosomotropic agents, Biochem. Pharmacol., 23 (1974) 2495-2531. 6 Ewing, D.J., Campbell, I.W., Burt, A.A. and Clarke, B.F., Vascular reflexes in diabetic autonomic neuropathy, Lancet, 2 (1973) 1354-1356. 7 Ewing, D.J., Campbell, I.W. and Clarke, B.F., Heart-rate response as a test for autonomic neuropathy, Brit. Med. J., 4 (1978) 1700. 8 Ewing, D.J., Campbell, I.W., Murray, A., Neilson, J.M.M. and Clarke, B.F., Immediate heart-rate response to standing: simple test for autonomic neuropathy in diabetes, Brit. Med. J., 1 (1978) 145-147. 9 Frisk-Holmberg, M., Bergkvist, Y., Domeij-Nyberg, B., Hellstrom, L. and Jansson, F., Chloroquine serum concentration and side effects: evidence for dose-dependant kinetics, Clin. Pharmacol. Ther., 25 (1979) 345-350. 10 Gundersen, H.J.G. and Neubauer, B., A long-term diabetic autonomic nervous abnormality, Diabetologia, 13 (1977) 137-140. 11 Hirsch, J.A. and Bishop, B., Respiratory sinus arrhythrnia
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in humans: how breathing pattern modulates heart rate, Am. J. PhysioL, 241 (1981) H620-H629. Katona, P.G. and Jih, F., Respiratory sinus arrhythmia: nonmvasive measure of parasympathetic cardiac control, J. AppL PhysioL, 39 (1975) 801-805. Levin, A.B., A simple test of cardiac function based upon the heart rate changes induced by the Valsalva maneuver, Am. J. Cardiol., 18 (1966) 90-99. Lot, T.Y. and Bennet, T., Comparison of the effects of chloroqnine, quinacrine and quinine on autonomic neuroeflector mechanisms, Med BioL, 90 (1982) 307-315. Makinde, J.M. and Ohia, S.E., Response of the non-pregnant and pregnant rat uterus to chloroquine in vitro, Afr. J. Med Sci., 12 (1983) 41-45. Mandel, E.H., A new local anesthetic with anticoagulent properties, Arch. Dermatol., 81 (1960) 260-263. McChesney, E.W., Banks Jr, W.F. and McAufiff, J.P., Laboratory studies of the 4-aminoquinoline antimalarials. II. Plasma levels of chloroqnine and hydroxychloroquine in man after various oral dosage regimens, Antibiot. Chemother., 12 (1962) 583-594. McChesney, E.W., Fasco, M.J. and Banks, Jr, W.F. The metabolism of chloroquine in man during and after repeated oral dosage, J. Pharmacol. Exp. Ther., 158 (1967) 323-331. Murray, A., Ewing, D.J., Campbell, I.W., Nelson, J.M.M. and Clarke, B.F., RR interval variations in young male diabetics, Brit. Heart J., 37 (1975) 882-885. Pomeranz, B., Macaulay, R.J.B., Caudill, M.A., Kutz, I., Adam, D., Gordon, D., Kilborn, K.M., Barger, A.C., Shannon, D.C., Cohen, R.J. and Benson, H., Assessment of autonomic function in humans by heart rate spectral analysis, Am. J. Physiol., 248 (1985) H151-H153. Schmidt, H. and Oettling, G., Chloroqnine is a muscarmic antagonist. Binding and dose-response studies with chick embryo cells, Eur. J. Pharmacol., 133 (1987) 83-88. Wheeler, T. and Watkins, P.J., Cardiac denervation in diabetes, Brit. Meal J., 4 (1973) 584-586.
147
JAN 00861
Muscarinic antagonist action of clinical doses of chloroquine in healthy volunteers Kanigula Mubagwa and Jeremy Adler Department of Physiology, University of Zimbabwe, Mount Pleasant, Harare (Zimbabwe) (Received 29 April 1988) (Revised version received and accepted 5 August 1988)
Key words: Chloroquine; Muscarinic antagonist; Cardiac cycle; Beat-to-beat variation of R-R interval
Abstract An investigation was undertaken into possible vagolytic effects of chloroquine. A single oral dose of chloroquine was given to healthy human volunteers and its influence on vagally mediated heart rate changes studied. Chloroquine at a dose of 600 mg significantly increased lying and standing heart rates, reduced the beat-to-beat variation of the R-R interval and reduced the heart rate changes induced by deep breathing, by the Valsalva manoeuvre and by standing. Chloroquine at a dose of 225 mg did not produce significant changes of these parameters. The effects of 600 mg chloroquine resemble those obtained with atropine and are consistent with an antimuscarinic receptor effect.
Introduction Chloroquine is widely used for its antimalarial and anti-inflammatory actions (e.g. in rheumatoid arthritis and in lupus erythematosus). It is also reported to have local anaesthetic [16], anticoagulent [16] and uterotropic [4,15] effects. On the heart chloroquine has an antiarrhythmic action [2] probably due to its local anaesthetic (quinidine-like) properties. Chloroquine has also been shown to cause tachycardia in anaesthetised frogs, an effect involving, at least in part, a central sympathetic stimulation [3]. Recent data suggest that chloroquine may have a muscarinic receptor antagonist action [14,21]. Thus the tachycardia
Correspondence: K. Mubagwa, Department of Pharmacology University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
observed in frogs could be due partly to a vagolytic action of chloroquine. Tachycardia following chloroquine administration has not been reported in man and other atropine-like effects of the drug have not been investigated. We examined the effect of clinical doses of chloroquine on human volunteers, using cardiovascular reflexes which are known to involve vagally mediated changes in heart rate. The results show that chloroquine produces changes similar to those o b t a i n e d with m u s c a r i n i c r e c e p t o r antagonists.
Materials and Methods Subjects Twelve healthy male volunteers (age: 19-51 years) drawn from the University staff were used in the present study. Informed consent and ap-
0165-1838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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proval of the Faculty's Ethics Committee were obtained.
Recordings Standard lead electrocardiograms (ECGs) were recorded from the volunteers (electrocardiograph EK-8, Burdick) under various conditions (see below), before and after chloroquine ingestion. The duration of the cardiac cycle was obtained from the interval between QRS complexes (R-R interval). The R peak of the QRS complex of the ECG triggered a pulse which was fed to a computer (Sinclair QL) and was used to measure cardiac cycle duration. The R-R interval values were graphically displayed on the screen of the computer monitor and were stored for later analysis. QRS Peak Detection. Detection was achieved in two steps. First, a simple voltage gate and a heavily DC filtered ECG signal were used to detect the occurrence of a QRS complex. Since accurate discrimination (5:2 ms) is required for some of the data, it was necessary to detect the peak of the R wave. The detection of a QRS complex initiated the generation of a staircase voltage that tracked the R wave. The interval between steps of the staircase depended on the slope of the unfiltered R wave. When the slope decreased and the interval between steps exceeded a preset value, an output pulse was generated. This was made to correspond with the peak of the R wave. The system allows for the detection of the R peak of the E C G in the face of significant DC shifts but without any distortion due to filtering. Details of the gate will be published separately. R-R Interval Measurement. The detection of an R peak generated a "ITL pulse which was fed into a control line of a parallel i n p u t / o u t p u t port (M6821) attached to a computer via an expansion unit (CST Q + 4). A software clock running at 3600 Hz was used to count the intervals between pulses. Experimental protocol On the day of the experiment the subjects had a fight brealffast that avoided caffeine. They were not allowed to consume food, tea or coffee, or to engage in physical exercise for the duration of the experiment, l~aring ~ day tht~y were ~ b j e e t e d to
a repetitive series of tests. Once stable control data were obtained, typically after 3 - 4 tests, the subjects ingested chloroquine phosphate (Datlabs, Bulawayo) dissolved in water. Testing was carried out 10 min later and at hourly intervals for 3 - 4 hours after chioroquine ingestion. All 12 subjects were used for testing with 600 mg chloroquine (base). Seven of them took part in testing with 225 mg chloroquine (base), and another group of 7 underwent testing on an occasion without chioroquine administration (before or at least 15 days after testing with chioroquine).
Tests R-R intervals were measured continuously under each of the following conditions: (1) while lying at rest, (2) while lying and breathing deeply, (3) on standing from a seat, (4) during sustained standing, and (5) during a Valsalva manoeuvre. These tests were run sequentially and each sequence was always preceded by a 5-min recumbent rest. Depending on heart rate each sequence of tests would take 20-30 rain. Lying at rest. 250 R-R intervals were recorded while the subjects were lying on a couch and breathing normally. Deep Breathing. 80 R-R intervals were recorded while the subjects remained lying and were asked to maximally inhale and exhale on command, at a frequency of 6 cycles per minute [11,221. Sit-Stand Test. 50 R-R intervals were recorded while the subjects sat on a 30-era ~ seat. 50 R-R after standing, 50 R-R intervals while again seated and further 300 R-R intervals while in standing position. T o reduce noise in the ECG signal, recording was made from lead i (fight and left arms) and the subjects were asked to move with limp arms. Valsalva Manoeuvre. Recordings were made while the subjects were asked to blow into a leaky tube connected to the column of a sphygmomanometer and to maintain a pressm'e of 48 n m ~ H g for 15 s. The manoeuvre was performed 3 times with at least 60 heart beats between manoeuvres. A leak in the tube connected to the manometer ensured that the mouth alone ceamot be used to generate the pressure. The subjects were asked to
149 avoid a deep intake of air after straining was released.
Data analysis Data analysis was carried out off line using a computer program developed by one of us (J.A.). Lying and Sustained Standing. Mean, standard deviation and frequency distribution histogram of the R-R interval [19] were obtained. In addition we produced frequency distribution histograms of the beat-to-beat variation (i.e. of the changes in R-R interval from one beat to the next) and calculated the mean absolute beat-to-beat R-R interval variation (MABBV), according to the following formula: MABBV=
.= I R R i + I - RRil / N
where X is a summation sign, i is an index used to number R-R intervals, IRRi+I - RRil is the absolute value of the difference between two successive R-R intervals, and N is the total number (usually 250) of R-R intervals. The MABBV is a measure of the spread of the frequency distribution histogram. A similar measure, the standard deviation of the difference between any R-R interval and the next, was used by others [10]. Since the MABBV considers only changes in R-R interval from one beat to the next it will mostly reflect high frequency changes, which are attributed to the activity of the parasympathetic nervous system [1,20]. That the beat-to-beat variation [22] and its mean absolute value (MABBV; J. Adler, unpublished results) provide a measure of vagal tone is supported by the fact that they are reduced following atropinization. Deep Breathing. The average ratio of the longest to the shortest R-R interval during each 10-s respiratory cycle was determined [see 22]. Sit-Stand Test. The ' 3 0 : 1 5 ratio' [7,8] was determined by taking the ratio between the longest R-R interval 22-36 beats after standing and the shortest R-R interval 10-20 beats after standing. The increase in R-R interval (after an initial decrease) on standing is of vagal origin [8]. Falsalva Manoeuvre. The ratio between the longest R-R interval after the manoeuvre and the
shortest R-R interval during and in the 5 s after cessation of straining [6,13] was determined. The period just after the release of the pressure was included in addition to the period of straining because R-R intervals often continued to decline immediately after the pressure is released. The mean of 3 consecutive Valsalva manoeuvres was used in the analysis of chloroquine effects. Statistics. For each subject, the quantitative indices obtained in the tests after chloroquine administration were expressed as percentages of the mean value of the same indices before chloroquine. When no chloroquine was administered, the indices were also normalized by taking the initial measurement as 100%. Statistical significance (at the 95% confidence level) of the difference between data obtained after chloroquine treatment and those obtained in control conditions at similar time intervals was tested using Student's t-test for samples of unequal sizes.
Results
The changes described below were consistently induced by chloroquine in 10 out of 12 subjects. In two subjects no change in some parameters was observed 3 - 4 h after chloroquine administration.
Lying and standing R-R intervals Fig. 1A shows cardiac cycle lengths recorded in a lying subject during quiet breathing and during maximal deep breathing. Under control conditions (i.e. before chloroquine administration), the mean R-R interval of the subject was 946 + 55 ms (mean + SD, n = 250), and 4 h after 600 mg chloroquine administration it declined to 750 + 18 ms. The R-R interval after treatment with chloroquine is significantly different from the control value ( P < 0.001). Thus, chloroquine decreased the mean cardiac cycle length measured in lying position. Fig. 1B shows frequency distribution histograms of the R-R intervals. Following chloroquine administration, both the mean value and the variation of R-R interval had decreased. Similar results were obtained in the same subject for the R-R intervals measured in steady upright position (791
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Fig. 1. Effect of cliloroquine on the cardiac cycle (R-R interval) in a lying subject. A: 250 heart beats were recorded during quiet breathing (left of vertical line) followed by 80 beats during maximal deep breathing (right of vertical line), before and 4 h after chloroquine (600 nag) administration. The means and standard deviations of R-R intervals (indicated by horizontal lines) during quiet beathing are shown. Small horizontal bars indicate the longest and the shortest R-R intervals in each respiratory cycle (6/rain), which are used to produce the respiratory ratio. The average ratio (with number of cycles analysed) is also shown. B: frequeaey distribution histograms of the R-R intervals recorded during the periods of quiet breathing displayed in A (bin size: 5 ms).
± 41 ms before, and 620 + 20 ms 4 h after chloroquine). The average effect of a 600-rag chloroquine dose on lying cardiac cycle in 12 different subjects is illustated in Fig. 2A, which also shows cardiac cycle durations measured in 7 of the subjects on occasions when no chloroquine was administered and the values measured when a 225-mg chloroquine dose was given. When no chloroquine was administered, the cardiac cycle duration remained practically constant throughout the day. The low (225 nag) chloroquine dose tended to produce a decrease of cardiac cycle duration but the R - R interval values were not significantly different from those obtained under control conditions. When
120
the higher (600 mg) chloroquine dose was given, a progressive decrease of the cardiac cycle duration was obtained. The cardiac cycle values measured 2, 3 and 4 h after chloroquine were significantly different from control values ( P < 0.01, P < 0.001, P < 0.05, respectively). The r e s d t s obtained in standing position are summarised in Fig. 2B and are qualitatively similar to those obtained in lying position.
Beat-to.beat changes in R-R interval One possible explanation for the results~ described above is a decrease by chloroquine of vagal influences on the heart. As mentioned in Materials and Methods, the beat-to-beat variation
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Fig. 2. R-R intervals (mean + S.E.M.) measured during quiet breathing: untreated ( o , n = 7); chloroqu!ne 225 rag (EL n = 7); chloroqttine 600 mg (O; n= 12). The R-Rinterval values are normalised as a ~ dinitialor p r ~ v a h i ~ , i' P < 0.05; ** P < 0.01. A: data white in lying potatO. B: data while ~axting.
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Fig. 3. Frequency distribution histograms of the beat-to-beat variation in R-R interval of two standing subjects, before and after chloroquine (600 mg). Bin size: 10 ms on left, 2.5 ms on right.
in R-R interval allows measurement of vagal tone. The R-R interval recordings of Fig. 1A and their frequency distribution (Fig. 1B) already suggest
~ 140 I-
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that variability in cardiac cycle duration was reduced by chloroquine administration. Fig. 3 shows the frequency distributions of the beat-to-beat variation in R-R interval measured in two standing subjects before and after chloroquine administration. The beat-to-beat R-R interval changes appear to be normally distributed both before and after chloroquine administration, but after chloroquine the variability in beat-to-beat change became progressively less pronounced. Fig. 4A shows that the MABBV (which is proportional to the spread of the frequency distribution histogram) tended to increase with time under control conditions while it decreased after 225 mg or 600 mg chloroquine. The values measured 1, 2, 3 and 4 h after 600 mg chloroquine were significantly different from those obtained in control conditions at corresponding times ( P < 0.05 after 1 h, P < 0.001 after 2-4 h). Similar results were obtained for the mean absolute beat-to-beat variation measured in lying position (Fig. 4B). The MABBV values were higher in the lying position (50.1 + 5.8 ms, mean + S.E.M.) than in the standing (19.9 + 4.4 ms). For the data in lying position, there was a significant negative correlation between the magnitude of chloroquine-induced decrease in MABBV and the pretreatment R-R interval value (r = -0.911, P < 0.001, n = 11), and between the decrease in MABBV and the pretreatment MABBV value (r = 0.619, P < 0.05, n = 11).
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Fig. 4. Effect of chloroquine on the mean absolute beat-to-beat variation (MABBV). Means and S.E.M. expressed as a percentage of initial or pretreatment value: untreated ( o , n = 7); 225 mg chloroquine (D, n = 7); 600 mg ehloroquine (o, n = 12). * P < 0.05; • * P < 0.01; * * * P < 0.001. A: in standing position. B: while lying.
152
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heart rate (see Materials and Methods), a value of 1.68 is obtained under control conditions in the subject. This ratio decreased to 1.38 4 h after chloroquine (600 mg) administration. Similar resuits were obtained in most subjects and are summarized in Fig. 5. The values obtained 2, 3 and 4 h after 600 mg chloroquine were significantly different from control values ( P < 0.01, P < 0.001, P < 0.01, respectively). Those obtained after 225 mg chloroquine were not different from control values.
4
time (hours)
Standing-induced bradycardia
Fig. 5. Effect of chloroquine on the respiration ratio. Means and S.E.M. of the respiration ratio are expressed as a percent,age of initial or pretreatment values: untreated (o, n = 7): 225 mg chloroquine (12, n = 7); 600 nag chloroquine (O, n = 12). * * P < 0.01; * * * P < 0:001.
Effect of respiration on R-R interoal Fig. 1A also illustrates the effect of chloroquine on the cardiac cycle changes induced by deep breathing. Before chloroquine a d m ~ s t r a t i o n maximal deep b r e a ~ g induced large fluctuations of the cardiac cycle length. Using the ratio between the longest and the shortest cardiac cycle length for eaeh respiratory excursion to quantitatively assess the influence of deep respiration on
In order to further investigate the possibility that chloroquine decreases vagal influences on the heart, the bradycardia produced after standing was analysed. Fig. 6A illustrates the changes in R-R interval produced in one subject following postural change before and after treatment with 600 mg chloroquine. On standing there was a tachycardia (decrease in R - R interval) that was followed by a recovery (increase in R-R interval). The magnitude of this bradycardia was reduced 4 h after chloroquine administration, as seen from the 30:15 ratios which decreased from 1.16 and 1.34 to 1.12 and 1.21 after chloroquine. Similar results were obtained with 600 mg chtoroquine in most subjects and are summarised in Fig. 6B. The
120 1 B
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Fig. 6. Effect of chlofoquine c~ the sUmaln~-~,~,~_,,.~ bradycardhL A: traces before and 4 h after chloroquix~ (600 nag). The subject Standln~ is indtcat~ by vm,tical bars. Small hcxizccaat bars indicate the shortest R-R intervals 10-20 beats after standing and the longest R-R interval 22-36 beats aftor s ~ , The ratim ~ ~ ~ (30 : 15 ratio) are l i w m above the m l c i m p . 8: mean m~l S,F-M of 30 : 15 ratios ~ as a p ~ m u t ~ of tl~ i z ~ z l ~ ~ v~li~: mm,mk~ (o, n-7~: 22.~:.mI~ l o r ~ (~ n--~; ~ O O m ~ ~ ~ , - n - , lt). was inithdly seated, stood on beat 50, sat on beat 100 and stood on beat 150.
* P < 0.05; ** P <0.01.
153 110
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s 1
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1
2
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3
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Fig. 7. Effect of chloroquine on cardiac cycle changes induced by the Valsalva Manoeuvre. A: R - R intervals during two successive Valsalva manoeuvres, before and 3 h after treatment with chloroquine (600 mg). Vertical bars m a r k start of manoeuvres. Small horizontal bars indicate shortest R - R intervals during manoeuvre and longest intervals after manoeuvre. The ratio between these intervals (Valsalva ratio) for each manoeuvre is shown above the tracing. B: mean and S.E.M. of the Valsalva ratios are expressed as a percentage of the control or pretreatment values: untreated ( o , n = 7); 225 m g chloroquine (D, n = 7); 600 mg chloroquine (e, n = 12).
values measured 1, 2, 3 and 4 h after treatment with chioroquine were significantly different from control values ( P < 0.05, P < 0.001, P < 0.001, P < 0.05, respectively). Chloroquine also reduced the rate of development of the tachycardia seen just after standing (Fig. 6A), an effect also seen after atropinization [J. Adler, unpublished results]. Effect of Valsalva manoeuvre on R - R interval
Fig. 7A illustrates R-R interval changes induced by the Valsalva manoeuvre in one subject, before and 3 h after treatment with chioroquine. After chloroquine administration, there was a decrease in the magnitude of the post straining bradycardia. In the example shown the mean ratio between the longest cycle length obtained immediately after straining and the shortest cycle length obtained at the end of the straining was 2.65 in control conditions, but it decreased to 2.27 after chloroquine treatment. The results obtained in control conditions and following treatment with 225 mg or 600 mg chloroquine are summarised in Fig. 7B. With the higher chloroquine dose there was a tendency for a decrease in the Valsalva ratio although the differences between values measured
after chloroquine and those measured in control did not reach statistical significance. Discussion
The results show that following 600 mg chloroquine administration, there was (1) an increase of heart rate, (2) a decrease of the mean beat-to-beat R-R interval variation, (3) a decrease of the influence of deep breathing on heart rate, and (4) a decrease of the bradycardia that is obtained following standing or at the end of the Valsalva manoeuvre. There was no change (or there was a change in the opposite direction) in these parameters when no chloroquine was administered and immediately after chloroquine administration. This excludes the possibility that the observed changes are the result of an increase in sympathetic tone due to hypoglycaemia (following food restriction during the experiment) or to stress (following ingestion of chloroquine). The effects of chloroquine are dose-dependent since the above changes were not statistically significant with a 225 mg dose. The effects of chloroquine are consistent with a vagolytic action. It is unlikely that they are due to
154
increased sympathetic tone [3] since a change in sympathetic influence on the heart is not expected to induce a decrease in the beat-to-beat R-R interval variation [20] and to alter the respiratory ratio [12] and the 30 : 15 ratio [8]. The effects are therefore probably due to a decrease in the parasympathetic influence on the heart. Such a decrease could be due to an inhibition by chloroquine of the neural cardio-inhibitory afferent mechanisms, of the vagal center itself, or of the vagal efferents to the heart. The action of chloroquine could also be localized within the heart, i.e. chloroquine could decrease the responsiveness of the cardiac tissue to vagal influences. The last possibility is consistent with recent studies which have demonstrated that chloroquine binds to muscarinic receptors [21, F. Dondo and K. Mubagwa, unpublished results] and inhibits muscarinic agonist-induced negative chronotropic effects in isolated guinea pig hearts [F. Dondo and K. Mubagwa, unpublished results). This suggests that the effects of chloroquine are due, at least in part, to an action at the cardiac cell muscarinic receptors. An inhibition of other muscarinic receptor-mediated effects has been observed in chick oesophagus muscle [14]. The lack of effect in some subjects (2 out of 12), could be due to a low plasma chloroquine concentration following poor absorption. Plasma chloroquine levels were not determined in the present study. Under normal conditions, with the acidity of the gastric cavity (pH 2) most (99%) of the ingested chloroquine (pK a 8.1 and 10.6) is expected to be absorbed. Pharmacokinetic data suggest that, following a single oral dose of 250 or 620 mg, a peak plasma chloroquine concentration of 200/~g/1 (0.4/LM) is reached in 3-6 h [17] or 1-2 h [9]. It remains possible, however, that there was poor or slow absorption in some individuals. After absorption, chloroquine reaches the plasma where a large part of the absorbed amount is bound to proteins. With time the drug is also accumulated in the lysosomes of many tissues [5]. If these last two processes are very marked in some individuals, they could also result in low plasma free chloroquine concentrations. Another possibility to explain the lack of response in some subjects is to assume that they have a very high vagal tone, and that chloroquine
acts by a competitive mechanism. In this case the chloroquine concentration achieved by a single 600-mg dose may not produce significant displacement of the neurotransmitter from its receptor. In favour of this hypothesis is the observation that. in the two subjects who did not respond to chloroquine, the mean beat-to-beat R-R interval variation was highest, suggesting that vagal tone was high in these individuals. This is also supported by the high negative correlation between relative decrease in lying mean absolute beat-to-beat variation and the original value of the mean absolute beat-to-beat variation. The greater percentage fall in MABBV. produced by chloroquine, in the standing compared to the lying position (see Fig. 4), also indicates that high vagal tone is less susceptible to antagonism. The likely peak plasma concentration of chloroquine after a single 600-mg oral dose. about 200 /~g/1 [17], is similar to the concentrations reported in rheumatoid patients receiving 250 mg/day [9] and in volunteers receiving 620 mg/day for 14 days [18]. However. in these two studies plasma samples were taken just prior to the daily dose (i.e. 24 h after the last chloroquine ingestion) and therefore underestimate the peak plasma concentration. This suggests that the peak plasma concentrations occurring in these studies exceed those obtained in the present study. The finding that no effect was obtained with the lower dose of chloroquine indicates that low doses (e.g. those used for low-dose antimalarial prophylaxis) do not produce atropine-like side effects. However, when higher doses are used for prophylactic, therapeutic or criminal reasons, the antimuscarinic effects can be marked and this should be borne in mind by the clinician.
Acknowledgements This work was supported by grants (no. 2.901.1.2980 and 2.901.1.2720) from the University of Zimbabwe Research Board. We would like to thank the volunteers who participated in this study, as well as Miss P. Vdlah, Mr. A. Mtttamba and Mr. T. Saruziwo for their help in the collection of data.
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