Theobromine inhibits sensory nerve activation and cough

Abstract

Cough is a common and protective reflex, but persistent coughing is debilitating and impairs quality of life. Antitussive treatment using opioids is limited by unacceptable side effects, and there is a great need for more effective remedies. The present study demonstrates that theobromine, a methylxanthine derivative present in cocoa, effectively inhibits citric acid-induced cough in guinea-pigs in vivo. Furthermore, in a randomized, double-blind, placebo-controlled study in man, theobromine suppresses capsaicin-induced cough with no adverse effects. We also demonstrate that theobromine directly inhibits capsaicin-induced sensory nerve depolarization of guinea-pig and human vagus nerve suggestive of an inhibitory effect on afferent nerve activation. These data indicate the actions of theobromine appear to be peripherally mediated. We conclude theobromine is a novel and promising treatment, which may form the basis for a new class of antitussive drugs.

Full text

©2004 FASEB
The FASEB Journal express article 10.1096/fj.04-1990fje. Published online November 17, 2004.
Theobromine inhibits sensory nerve activation and cough
Omar S. Usmani,* Maria G. Belvisi,
†
Hema J. Patel,
†
Natascia Crispino,
†
Mark A. Birrell,
†
Márta Korbonits,
‡
Dezső Korbonits,
§
and Peter J. Barnes*
*
Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London,
London, United Kingdom;
†
Respiratory Pharmacology Group, National Heart and Lung Institute,
Imperial College London, London, United Kingdom;
‡
Department of Endocrinology, St.
Bartholomew’s Hospital, London, United Kingdom;
§
Chinoin Co. Ltd., Budapest, Hungary
Corresponding author: Professor Maria G. Belvisi, Respiratory Pharmacology Group,
Department of Airway Diseases, National Heart and Lung Institute, Dovehouse Street, London
SW3 6LY. E-mail: m.belvisi@imperial.ac.uk
ABSTRACT
Cough is a common and protective reflex, but persistent coughing is debilitating and impairs
quality of life. Antitussive treatment using opioids is limited by unacceptable side effects, and
there is a great need for more effective remedies. The present study demonstrates that
theobromine, a methylxanthine derivative present in cocoa, effectively inhibits citric acid-
induced cough in guinea-pigs in vivo. Furthermore, in a randomized, double-blind, placebo-
controlled study in man, theobromine suppresses capsaicin-induced cough with no adverse
effects. We also demonstrate that theobromine directly inhibits capsaicin-induced sensory nerve
depolarization of guinea-pig and human vagus nerve suggestive of an inhibitory effect on
afferent nerve activation. These data indicate the actions of theobromine appear to be
peripherally mediated. We conclude theobromine is a novel and promising treatment, which may
form the basis for a new class of antitussive drugs.
Key words: vagus • methylxanthines
ough is a protective, primitive reflex, in healthy individuals (1). However, when cough
serves no useful role, it is the most common respiratory complaint for which medical
attention is sought (2). Persistent cough can be debilitating, socially distressing, and
adversely impairs quality of life (3). Cough leads patients to use over-the-counter remedies as
first-line treatments; in the United States alone, sales for these over-the-counter remedies exceed
$2 billion dollars (4). A recent meta-analysis, however, established that evidence regarding the
effectiveness of such remedies was inconclusive (5).
Narcotic agents with a morphine skeleton, such as the opioids codeine and dextromethorphan,
are the most widely used antitussives in cough remedies, but they have unpredictable efficacy
and undesirable central nervous and peripheral side effects that often lead to their discontinuation
(6, 7). The 2nd International Cough Symposium concluded that there is a great need for effective
C
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new cough treatments, as well as a better understanding of the complex genesis and
pathophysiology of cough to guide the development of pharmacological approaches (8).
The cough reflex is initiated by stimulation of two different classes of sensory afferent fiber,
namely the myelinated rapidly adapting receptors (RAR) and nonmyelinated C-fibers with
bronchial or pulmonary endings (9, 10). Inappropriate activation of these nerves can occur in
allergic diseases (e.g., asthma) and chronic obstructive pulmonary disease (COPD) and lead to
many of the symptoms such as coughing. However, the mechanisms involved in the abnormal
functioning of airway nerves have not yet been described. They are thought to involve the release
of inflammatory mediators which sensitize the nerve fibers leading to increased electrical activity
of these fibers and an increase in the release of various neurotransmitters from the nerve endings
(6).
We hypothesize that agents that inhibit sensory nerve activity, that is nerve depolarization, will
also inhibit the cough reflex. Although many compounds demonstrate promising characteristic
antitussive effects in animal models, few have shown any clinical benefit (11). In this paper, we
describe theobromine, a methylxanthine alkaloid derivative predominant in cocoa, as a novel and
promising therapy for the treatment of cough. Theobromine was developed alongside other
methylxanthines for respiratory disorders, but disappeared from clinical use because of its low
potency as a bronchodilator (12). Recent studies, however, demonstrate a unique antitussive
effect, unlike the other methylxanthines, in a series of pharmacological studies in the guinea pig
cough model using a synthetic analog (13, 14).
We, therefore, sought to investigate the action of theobromine in the guinea pig cough model in
vivo, and in isolated human and guinea-pig vagus nerve preparations in vitro. We observed
theobromine inhibited cough in our human trial at concentrations that do not have central side
effects in man and thus is unique in the field of cough therapy. Furthermore, our data show the
antitussive mechanism of action is probably due to direct inhibition of sensory nerve activation.
Our study reveals a promising prospect for a potentially new antitussive that is acutely needed in
the clinical arena for treatment of patients with both acute and chronic cough.
METHODS
In vivo animal experiments were carried out according to the Institutional Guidelines for Care
and Use of Experimental Animals and approved by the animal Ethics Committee of Chinoin
(Chinoin Co. Ltd., Budapest, Hungary). For the human studies, written informed consent was
obtained from all patients, and approved by the Ethics Committee of the Royal Brompton and
Harefield Hospital National Health Service Trust (London, United Kingdom).
Citric acid-induced cough in the guinea pig
Cough was induced in conscious guinea pigs by a previously described method (14). Briefly,
female Dunkin Hartley guinea pigs (250–300 g) were individually placed in transparent
chambers and exposed to 0.78 M citric acid aerosol solution for 3 min. Coughs were counted by
a trained observer and recognized from the characteristic opening of the mouth and posture of
the animal. Animals with six or more coughs were selected and orally dosed with theobromine or
codeine in a suspension of 0.1% methylcellulose vehicle (treatment groups) or with 0.1%
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methylcellulose alone (control group). A second citric acid exposure was evoked one hour after
dosing in dose–response experiments, and at 0.5, 1, 2, 3, 4, and 6 h in time-duration experiments.
The antitussive activity was calculated as the percentage decrease of the number of coughs
between the second and the first challenge. The drug-treated groups were compared with the
control group using the Kruskal-Wallis test followed by the Conover Inman test for pair-wise
comparisons in the dose–response experiments, or by using the Student's t test in the time-
duration experiments (Graph Pad Prism Software, San Diego, CA). Statistical significance was
taken at P < 0.05.
Capsaicin cough challenge in human subjects
Ten healthy nonsmoking subjects (mean age (±
SD) 38 ± 8.1 year, six females) participated in a
randomized double-blind crossover study. The antitussive effect of a single-dose of theobromine
(1000 mg) was compared with codeine phosphate (60 mg) and placebo during three study visits,
each separated by a washout period of a week. The dose of theobromine was determined in a
pilot study. Interestingly, a previous study documented a bronchodilator effect of theobromine
within a similar dose range (at 500–1000 mg) in patients with asthma (12). The main outcome
measure was the capsaicin concentration required to induce five coughs (C5). The capsaicin
inhalation challenge was performed according to a modified version of our previously published
protocol (15). Briefly, subjects inhaled nebulized single-dose doubling concentrations (0.5–500
µM) of capsaicin delivered via a breath-activated dosimeter (P. K. Morgan Ltd., Gillingham,
UK), at one-minute intervals. Capsaicin doses were alternated at random with 0.9% saline doses
to minimize conditioned responses during the study. Coughs were counted for 1 min after the
inhalation by direct observation. The C5 was determined, and the capsaicin dose was repeated
for confirmation. The C5 was obtained on two screening occasions, and subjects were eligible to
enter the study if their C5 was in the middle of the dose response (up to ≤31 µM) and
reproducible to ≤1 doubling difference between both screening visits. Subjects were asked to
refrain from tea, coffee, chocolate, and caffeine-like substances 12 h before each visit. At each
treatment visit, the C5 threshold was determined two hours postdosing, which coincides with the
plasma peak of theobromine (16). Subjects were questioned about adverse effects, including
cardiovascular, central nervous, and gastrointestinal symptoms. Individual capsaicin C5 data was
logarithmically transformed to normalize the data, and the means were used to test the null
hypothesis using two-way ANOVA, with factors for subject and treatment (Graph Pad Prism
Software). Modified t tests to compare subsequent groups of interest were undertaken using the
Bonferroni method to adjust for the P values and multiple comparisons. Statistical significance
was taken at P < 0.05.
Measurement of sensory nerve depolarization in isolated vagus nerve preparations
Male Dunkin-Hartley guinea pigs (300–350 g) housed in a temperature-controlled (21°C) room
with food and water freely available, were killed by cervical dislocation, and the vagus nerves
caudal to the nodose ganglion were carefully dissected and placed in oxygenated Krebs–
Henseleit solution (KHS) of the following composition (mM): NaCl - 118; KCl - 5.9; MgSO
4
-
1.2; NaH
2
PO
4
- 1.2; CaCl
2
- 2.5; glucose – 6.6; NaHCO
3
– 25.5
;
and bubbled with 95% O
2
and
5% CO
2
(BDH, Poole, UK). Human trachea, with branches of the cervical vagus still attached,
was obtained from a patient (male 44 year with emphysema) undergoing heart-lung
transplantation. Both human and guinea pig vagus nerve trunk segments (40–50 mm) were
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placed in oxygenated KHS, carefully desheathed, and mounted in a ‘grease-gap’ recording
chamber as described previously (17, 18). Briefly, each nerve was drawn longitudinally through
a narrow Perspex channel and petroleum jelly applied at the center of the channel creating an
area of high resistance, and electrically isolating the extracellular space between the two ends of
the nerve. Nerves were constantly superperfused with KHS at 37°C. Silver (Ag/AgCl) electrodes
(Mere 2 Flexible reference electrodes, World Precision Instruments, Stevenage, UK), made
contact at each nerve trunk end, and recorded DC potential and nerve depolarization via a DAM
50 differential amplifier (WPI) onto a calibrated pen-chart recorder (Lectromed Multi-Trace 2,
Letchworth, UK). Control sensory nerve depolarizations were induced by perfusion of the vagus
nerve with a pre-established submaximal concentration of capsaicin (1 µM in guinea pig) (18)
and (10 µM in human) (18) applied for 4 min, after which the tissue was washed until the
baseline response of the nerve was regained. After two reproducible control responses were
obtained to capsaicin, preparations were then perfused for 20 min with theobromine (0.01–100
µM), codeine (0.01–100 µM), or the relevant vehicle control (0.1% DMSO), following which
capsaicin (1 µM) was reapplied in the continued presence of the test agents. Experiments were
randomized so that different concentrations of different drugs were tested on vagus nerves from
the same animal on the same day. The data were subjected to a paired two-tailed t test since the
response to a stimulant was measured before and after drug intervention within the same nerve.
pD
2
values (–log of the EC
50
defined as the concentration of drug required to elicit 50% of the
maximum inhibition) and statistical significance, taken at P < 0.05, were calculated using
‘GraphPad Instat
TM
’ (Graph Pad Prism Software).
RESULTS
Theobromine as a potential antitussive
Several synthetic antitussives are characterized by the presence of a 1,2,4-oxadiazole ring in their
chemical structure (Fig. 1A
). With the ‘renaissance’ of the methylxanthine, theophylline (19), for
the treatment of asthma in the seventies, a series of novel compounds with an oxadiazolylalkyl
substituent at the N7 atom on the basic xanthine skeleton (Fig. 1B
), were synthesized (Fig. 1C),
and investigated as potential antiasthmatic and antitussive agents.
With two of these compounds selected for preclinical testing, 3,7-dihydro-3-methyl-7-/(5-
methyl-1,2,4-oxadiazol-3yl)methyl/-1H-purine-2,6-dione (Fig. 1D
) (14, 20), and 3,7-dihydro-
1,3-dimethyl-7-/(5-methyl-1,2,4-oxadiazol-3-yl)methyl/-1H-purine-2,6-dione (Fig. 1E) (13),
there was an unexpected correlation between chemical structure and antitussive potency. When
the N1 atom of the xanthine skeleton remained unsubstituted, the antitussive effect of these 1H-
xanthine derivatives increased considerably compared with the corresponding N1-methyl
analogs. The antitussive effect of 3,7-dihydro-3-methyl-7-/(5-methyl-1,2,4-oxadiazol-
3yl)methyl/-1H-purine-2,6-dione was about eightfold stronger than that of 3,7-dihydro-1,3-
dimethyl-7-/(5-methyl-1,2,4-oxadiazol-3-yl)methyl/-1H-purine-2,6-dione (14). This correlation
among the synthetic xanthine derivatives suggests that if the association also applies to the three
natural methylxanthine alkaloids, theobromine (Fig. 1F
), theophylline (Fig. 1G) and caffeine
(Fig. 1H
), then theobromine, which in contrast to theophylline and caffeine is unsubstituted at
N1, should exhibit a therapeutically significant antitussive effect.
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Theobromine inhibits citric acid-induced cough in the guinea pig model
We investigated the antitussive effect of theobromine on citric acid-induced cough in guinea
pigs, using codeine hydrochloride as a positive control. Cough provocation in conscious guinea
pigs is widely used to test the efficacy of new cough treatments (21, 22). Theobromine and
codeine showed a dose-dependent antitussive effect on citric acid-induced cough in guinea pigs
compared with the vehicle-treated control groups (Fig. 2
). The antitussive effect of theobromine
and codeine was similar to each other, but significantly better than vehicle treatment using doses
between 4–64 mg/kg and 8–64 mg/kg, respectively. Vehicle treatment itself caused some
decrease in the numbers of citric acid-induced cough, which were similar within the two
treatment groups (P=0.92) (Fig. 2
). Theobromine at a dose of 32 mg/kg produced a long-lasting
antitussive effect that was statistically significant for up to 4 h after treatment (Fig. 3
). There was
no tachyphylaxis evident in the tussive response elicited by citric acid as can be seen following
repeat exposure to the tussive agent, following vehicle administration, in the time-course
experiment (Fig. 3
).
Theobromine inhibits capsaicin-induced cough in man
We next examined whether theobromine could inhibit induced cough in human subjects.
Capsaicin is a well-established, reproducible, sensory C-fiber stimulant in vitro, widely used as a
tussive stimulant in clinical research to determine the cough threshold (23). In 10 healthy
volunteers, theobromine significantly increased the capsaicin concentration required to induce
five coughs (C5, cough threshold) when compared with placebo (P<0.01) (Fig. 4
). The log C5
(±
SD) was 1.43 ± 0.65, 1.59 ± 0.74, and 1.86 ± 0.58 for placebo, codeine, and theobromine,
respectively. There was no significant difference between codeine and placebo (P=0.25). No
adverse effects, particularly cardiovascular or central nervous, were observed.
Theobromine inhibits capsaicin-induced sensory nerve depolarization of guinea pig vagus
and human vagus nerves in vitro
To ascertain whether the mechanism of action was peripheral or central, the effect of
theobromine (Fig. 5A
) and codeine (Fig. 5B) on capsaicin-induced guinea pig vagus nerve
depolarization was investigated. Perfusion of guinea pig vagus nerve preparations with
theobromine (0.01–100 µM) inhibited capsaicin-induced nerve depolarization in a concentration-
dependent manner (pD
2
=5.2). Maximum inhibition of 94.9 ± 3.8% was observed at the highest
concentration used (Fig. 5A
). Similarly, nerve preparations treated with codeine (0.01–100 µM)
showed markedly reduced sensory nerve depolarization induced by capsaicin in a concentration-
dependent manner (pD
2
=5.6), and complete inhibition of induced nerve depolarization was
achieved at 100 µM (Fig. 5B
).
We also studied the effects of theobromine on depolarization of a human vagus nerve preparation
by capsaicin. Human vagus preparations were used to confirm the observations seen with guinea
pig vagus nerves to provide the appropriate validation of the target in man and confirm clinical
relevance. Theobromine (100 µM) inhibited capsaicin (10 µM)-induced nerve depolarization of
the human vagus by 66.7% (Fig. 6
).
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DISCUSSION
We have demonstrated for the first time that theobromine, a methylxanthine derivative present in
cocoa, effectively inhibited citric acid-induced cough in conscious guinea pigs in vivo, and using
a randomized double-blind study in healthy human volunteers, we showed that theobromine
inhibited capsaicin-induced cough. We also established by using isolated guinea pig vagus in
vitro that the antitussive mechanism of action probably involves direct inhibition by theobromine
of capsaicin-induced sensory nerve activation. Furthermore, we demonstrated the inhibitory
action of theobromine on capsaicin-induced nerve depolarization using an isolated human vagus
nerve preparation, providing in vitro proof of concept for this action in man. However, further
confirmation of this observation is needed as it was only possible to study one human vagus
sample in the course of these studies.
Even in their recognized clinical role as bronchodilators, the underlying mechanism of action by
methylxanthines is unclear, but is most likely related to their phosphodiesterase inhibitory
activity, and also antagonism of bronchoconstrictor adenosine A
1
receptors (20, 24).
Prostaglandins PGI
2
and PGE
2
cause cyclic AMP accumulation in peripheral nerves (25) and
although cAMP-elevating drugs have recently been shown to activate sensory C-fibers in the
isolated rat vagus in vitro (26) little is known regarding the neuromodulatory role of cyclic
nucleotides and adenosine on human sensory nerve function. Interestingly, methylxanthines have
recently been shown to activate human intermediate-conductance, Ca
2+
-activated K
+
channels
(hIK) (27). We have previously proposed that activation of large conductance Ca
2+
-activated
potassium channels might be a common mechanism of G-protein coupled receptor activation
leading to inhibition of afferent and efferent vagal activity (28), but a role for the hIK channel in
the modulation of sensory nerve activity has not yet been demonstrated.
To determine the mechanism of action of theobromine, we determined the effect of theobromine
on the isolated guinea pig vagus nerve preparation, which is a validated in vitro model that
allows evaluation of compounds on sensory nerve depolarization and appears predictive of data
obtained in single sensory fiber recording experiments (18, 28). We previously demonstrated that
both guinea pig and human vagus isolated nerve preparations respond similarly to a variety of
tussive sensory nerve stimulants, including capsaicin, suggesting that human vagal afferent fibers
react to and show similar responses to afferent stimulants acting on guinea pig nerves (M.
Belvisi, unpublished data). Our data demonstrate that guinea pig and human vagus nerves
respond similarly to theobromine inhibition of capsaicin-induced depolarization. We have
previously demonstrated the utility of the isolated vagus preparation as a preclinical in vitro
screen, which can be used to predict the efficacy of test compounds before evaluation in the in
vivo guinea pig cough model. Furthermore, the data obtained in the guinea pig cough model was
similar to that obtained in the clinical studies in agreement with published studies describing
similarities in the responsiveness of the cough reflex between the two species (22). Taken
together, the findings with theobromine in the guinea pig and human cough models in vivo, as
well as in the guinea pig and human nerve preparations in vitro, are consistent with the
hypothesis that the antitussive action was suggestive of a direct inhibition of sensory nerve
activation rather than by a centrally mediated mechanism. Although the evidence presented
suggests that theobromine may inhibit sensory nerve function via a peripheral mechanism, no
evidence has been presented suggesting the absence of a centrally mediated effect and as such,
this remains to be confirmed. However, although the isolated vagus preparation presents us with
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the ideal opportunity to conduct a comprehensive pharmacological assessment, data using this
preparation should be interpreted with some caution as the pharmacological agents are applied to
the axon of the isolated vagus nerve in vitro. Thus, the depolarization signal recorded
extracellularly represents a summation of the changes in membrane potential of all the axons via
activation of receptors expressed in the neuronal membrane of the axon. Conduction velocities of
the fibers are not recorded and action potentials per se are not measured, so identification of the
activation/inhibition of specific populations of sensory nerve fiber is not possible. Furthermore,
the receptor expression and signal transduction mechanisms in the axon may not necessarily
represent the behavior of those elements in the peripheral endings.
In our human study, no adverse effects were observed of the cardiovascular or central nervous
system, and these data support the use of theobromine as an effective antitussive agent in man,
with a safe therapeutic index. We compared theobromine to codeine phosphate, often used as a
benchmark against which new cough treatments are compared (29), and although systemic
opiates suppress capsaicin-induced cough (30), they are associated with many unacceptable
adverse effects when used in effective antitussive doses (7). To achieve maximum sensitivity in
the capsaicin cough challenge methodology, we controlled the capsaicin variability in our study
population by selecting individuals with no more than one difference in the doubling
concentration of capsaicin (31). Subjects in the middle of the cough dose–response were
selected, and those with attenuated or hyperresponsive effects were excluded to obtain
reproducible results. Our population was biased toward women, supported by previous studies
observing greater capsaicin sensitivity in healthy women (32).
In conclusion, the data described demonstrate a significant antitussive effect of theobromine in
healthy subjects when compared with placebo. Further studies are needed to see whether these
effects can be extrapolated to patients with chronic persistent cough, who, interestingly, have
previously been demonstrated to exhibit increased sensitivity to the tussive effect of the inhaled
irritant, capsaicin (33, 34). These data suggest that theobromine could form the basis for
development of novel and safe antitussive agents for the treatment of a very common and
troublesome symptom.
ACKNOWLEGEMENTS
We are grateful for the help of Endre G. Mikus, Péter Arányi, and István Jelinek (Chinoin Co.
Ltd., Budapest, Hungary).
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Received June 25, 2004; accepted October 12, 2004.
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Fig. 1
Figure 1.
Chemical structures of the xanthine derivatives. Synthetic antitussives have a characteristic 1,2,4-oxadiazole
ring (A), and incorporating this into the N7 atom of the basic xanthine skeleton (B), a series of novel compounds were
synthesized (C–E). The antitussive activity of the N1 unsubstituted compound, 3,7-dihydro-3-methyl-7-/(5-methyl-1,2,4-
oxadiazol-3-yl)methyl/-1H-purine-2,6-dione (D), was about eightfold stronger than that of the corresponding N-1-methyl
analog, 3,7-dihydro-1,3-dimethyl-7-/(5-methyl-1,2,4-oxadiazol-3-yl)methyl/-1H-purine-2,6-dione (E)(14)
. The naturally
occurring methylxanthines are shown; theobromine, 3,7-dihydro-3,7-dimethyl-1H-purine-2,6-dione (F), theophylline, 3,7-
dihydro-1,3-dimethyl-1H-purine-2,6-dione (G), and caffeine, 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (H).
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Fig. 2
Figure 2.
Dose–response antitussive effect of theobromine () and codeine (∆) on citric acid-induced cough in
conscious guinea pigs. Theobromine (n=8) and codeine (n=6) significantly inhibited citric acid-induced cough;
theobromine at 4, 8, 16, 32, and 64 mg/kg, and codeine at 8, 16, 32, and 64 mg/kg when individually compared with the
vehicle- (V) treated control groups. The antitussive effect with vehicle was 28.5% and 28% for theobromine and codeine,
respectively. Logarithmic dose values shown represent mean ± SEM, where *** denotes P < 0.001.
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Fig. 3
Figure 3.
Time dependency of the antitussive effect of theobromine () on citric acid-induced cough in conscious
guinea pigs. Theobromine at a dose of 32 mg/kg (n=8) significantly inhibited citric acid-induced cough after 1, 2, 3, and 4
h compared with vehicle control (). Values shown represent mean ± SEM, where *** denotes P < 0.001.
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Fig. 4
Figure 4.
The effect of theobromine, codeine, and placebo on the capsaicin concentration required to induce five
coughs (C5) in 10 healthy volunteers. Values shown represent mean ± SEM, where ** denotes P < 0.01.
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Fig. 5
Figure 5.
Inhibition of nerve depolarization by theobromine (A) and codeine (B). The inhibitory action of theobromine
and codeine on capsaicin-induced nerve depolarization in isolated guinea pig vagus nerve preparations occurs in a
concentration-dependent manner. Nerve depolarization responses were expressed as mV depolarization before (control
response) and after drug additions and then expressed as a percentage change. V represents vehicle. *P < 0.05, **P < 0.01,
***P < 0.001 denote statistical significance compared with control responses in the same tissue before drug treatment.
Values shown represent mean ± SEM percentage change of n = 4 determinations in depolarizations compared with control
response. The pD
2
values (–log
10
of the EC
50
defined as the concentration of drug required to elicit 50% of the maximal
response) are shown.
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Fig. 6
Figure 6.
Theobromine mediated inhibition on capsaicin-induced nerve depolarization of isolated human vagus
nerve. Tracing shows the inhibitory effect of theobromine on human vagus nerve depolarization induced by capsaicin.
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