Full text of DOI:10.1038/sj.bjp.0703766

British Journal of Pharmacology (2001) 132, 73 ± 84
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Characterization of a novel mechanism accounting for the adverse
cholinergic e€ects of the anticancer drug irinotecan
1
1
Corrado Blandizzi, Barbara De Paolis, Rocchina Colucci, Gloria Lazzeri, Fabio Baschiera
&
1
1
1
,
* Mario Del Tacca
1
1
Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine,
University of Pisa, Via Roma 55, I-56126 Pisa, Italy
1
This study investigates the mechanisms accounting for the adverse cholinergic e€ects of the
antitumour drug irinotecan. The activity of irinotecan and its active metabolite, 7-ethyl-10-hydroxy-
camptothecin (SN-38), was assayed in models suitable for pharmacological studies on cholinergic
system.
2
Irinotecan moderately inhibited human or electric eel acetylcholinesterase activity, SN-38 had no
e€ect, whereas physostigmine blocked both the enzymes with high potency and ecacy.
Irinotecan and SN-38 did not a€ect spontaneous or electrically-induced contractile activity of
3
human colonic muscle. Acetylcholine and dimethylphenylpiperazinium (DMPP) caused phasic
contractions or relaxations, respectively. Physostigmine enhanced the motor responses elicited by
electrical stimulation.
4
Although irinotecan and SN-38 did not modify the basal contractile activity of guinea-pig ileum
longitudinal muscle strips, irinotecan 100 mM moderately enhanced cholinergic twitch contractions.
Acetylcholine or DMPP caused phasic contractions, whereas physostigmine enhanced the twitch
3
responses. Electrically-induced [ H]-acetylcholine release was reduced by irinotecan (100 mM) or
physostigmine (0.1 mM).
5
Intravenous irinotecan stimulated gastric acid secretion in rats, but no e€ects were obtained with
SN-38, physostigmine or i.c.v. irinotecan. Hypersecretion induced by irinotecan was partly prevented
by ondansetron, and una€ected by capsazepine. In the presence of atropine, vagotomy and systemic
or vagal ablation of capsaicin-sensitive a€erent ®bres, irinotecan did not stimulate gastric secretion.
6
The present results indicate that irinotecan and SN-38 do not act as speci®c acetylcholinesterase
blockers or acetylcholine receptor agonists. It is rather suggested that irinotecan promotes a
parasympathetic discharge to peripheral organs, mediated by capsaicin-sensitive vagal a€erent ®bres,
and that serotonin 5-HT
irinotecan.
British Journal of Pharmacology (2001) 132, 73 ± 84
3
receptors are implicated in the genesis of vago-vagal re¯ex triggered by
Keywords: Irinotecan; adverse e€ects; acetylcholinesterase; acetylcholine release; vagus nerve; capsaicin-sensitive sensory
®
bres; stomach; ileum; colon
Abbreviations: AChE, acetylcholinesterase; DMPP, dimethylphenylpiperazinium; SN-38, 7-ethyl-10-hydroxycamptothecin
Introduction
Irinotecan is a semisynthetic derivative of the plant alkaloid
camptothecin and, like its parent compound, it is endowed
with antitumour activity which depends on the blockade of
DNA-topoisomerase I (Tanizawa et al., 1994). Irinotecan is
not directly responsible for relevant cytotoxic properties, but
acts as a prodrug. Upon i.v. infusion, this camptothecin
analogue is subjected to the enzymatic action of esterases
which, through the cleavage of the di-piperidin carbonyloxy
moiety, generate the major metabolite SN-38 (7-ethyl-10-
hydroxycamptothecin), which mediates most of the anti-
tumour activity exerted by irinotecan (Rothenberg, 1997).
Irinotecan demonstrated antineoplastic activity both in
preclinical and clinical studies, and it is currently undergoing
phase II and phase III clinical trials for various neoplastic
diseases (Rothenberg, 1997). In addition, irinotecan has been
approved as second-line treatment for patients with advanced
colorectal adenocarcinoma and is being investigated as ®rst-
line therapy for the same disease (Rothenberg & Blanke,
1999; Douillard et al., 2000).
Clinical studies have shown that patients treated with
irinotecan frequently experience acute side e€ects, such as
bradycardia, hypotension, hypersalivation, abdominal
cramps, diarrhoea, diaphoresis, visual accommodation dis-
turbances and other signs that are consistent with the picture
of a cholinergic syndrome (Gandia et al., 1993; Rowinsky et
al., 1994). In support of this view, the above symptoms can
be prevented or ameliorated by administration of the
anticholinergic drug atropine (Rothenberg et al., 1993).
At present, three distinct mechanisms are proposed to
explain the cholinergic syndrome associated with irinotecan
treatment. First, data obtained from in vitro experiments on
*
Author for correspondence at: Divisione di Farmacologia
Chemioterapia, Dipartimento di Oncologia, dei Trapianti, e delle
Nuove Tecnologie in Medicina, Universita di Pisa, Via Roma 55, I-
6126 Pisa, Italy; E-mail: m.deltacca@do.med.unipi.it
e
Â
5

74
C. Blandizzi et al
Irinotecan and adverse cholinergic effects
acetylcholinesterase (AChE), isolated from human erythro-
cytes or electric eel, suggest that irinotecan directly inhibits
the activity of this enzyme, thus preventing the breakdown of
acetylcholine at level of cholinergic synapses (Kawato et al.,
section of the whole wall of the proximal or distal colon from
a macroscopically normal region. They were placed immedi-
ately into pre-oxygenated Krebs solution and transported on
ice back to the laboratory. The tissue was laid ¯at in Krebs
solution, the mesentery, mucosa and submucosa were gently
removed, and muscular regions between the taenia coli were
cut into strips of approximately 3 mm width and 30 mm
length along the longitudinal axis (Crema et al., 1968). The
experimental protocol was approved by Pisa University
Hospital Ethics Committee.
1
993; Dodds & Rivory, 1999; Morton et al., 1999). Second,
on the basis of the structural similarity between the di-
piperidin carbonyloxy side chain of irinotecan and dimethyl-
phenylpiperazinium (DMPP),
a well known agonist of
ganglionic nicotinic receptors (Hayashi et al., 1983), it has
been speculated that the mechanism of cholinergic syndrome
might be ascribed to a direct stimulation of nicotinic
autonomic ganglia (Gandia et al., 1993). Third, radioligand
binding experiments suggest that irinotecan may interact with
cholinergic muscarinic receptors, thus causing their direct
activation (Kawato et al., 1993).
Overall, the pathophysiological mechanisms underlying the
cholinergic syndrome induced by irinotecan still remain
obscure. In addition, it seems quite surprising that the same
drug may activate the endogenous cholinergic pathways
acting simultaneously at three distinct levels. Accordingly,
the present study was designed in order to investigate the
e€ects of irinotecan and SN-38 on several in vitro and in vivo
preparations, all regarded as standard experimental models
for pharmacological studies concerning the cholinergic
system.
Recording of contractile activity under basal conditions or
electrical transmural stimulation The motor activity of
colonic muscle strips was recorded according to the
procedure previously described (Crema et al., 1968). The
colonic preparations were set up in organ baths of 10 ml
capacity (over¯ow system), containing Krebs solution main-
tained at 378C and bubbled continuously with 95%
O +5%CO . The Krebs solution had the following composi-
2
2
tion (mM): NaCl 113, KCl 4.7, CaCl2 2.5, KH PO4 1.2,
2
4 3
MgSO 1.2, NaHCO 25, and glucose 11.5 (pH 7.4+0.1).
Each colonic preparation was connected vertically to an
isotonic transducer (Basile, Comerio, Italy) under a constant
tension of 1 g, and was allowed to equilibrate for at least
60 min, with intervening washings at intervals of 15 min. The
mechanical activity of the longitudinal muscle was recorded
by a polygraph (Basile, Comerio, Italy). A pair of coaxial
platinum electrodes was positioned at distance of 10 mm
from the longitudinal axis of each preparation in order to
deliver transmural electrical stimulation by means of a BM-
ST6 stimulator (Biomedica Mangoni, Pisa, Italy). Electrical
stimuli were applied as single trains lasting 30 s, consisting of
square wave pulses (1 ms duration, 30 mA intensity) at the
frequency of 10 Hz. At the end of the equilibration period,
each colonic preparation was repeatedly challenged with
electrical stimulations, and the experiments were started only
when constant and reproducible contractile responses were
obtained (usually after two or three electrical stimulations).
In experiments designed to examine the e€ects of irinotecan,
SN-38, acetylcholine or DMPP on basal motor activity, the test
drugs were added to the bathing ¯uid according to a non-
cumulative dosing schedule. Unless otherwise stated, increasing
concentrations of a given drug were applied at intervals of
20 min, with at least two intervening washings, and the contact
time ranged from 30 s to 5 min. The potencies of test drugs, for
which full concentration-response curves could be obtained,
were estimated as EC50. In agonist-antagonist interaction
experiments, the antagonist was added to the bath 20 min
before agonist. In experiments aiming to assay the e€ects of
irinotecan. SN-38, physostigmine or atropine on the electri-
cally-induced motor activity, each test drug was added to the
bath 20 min before the application of electrical stimulation. In
this case the drug-induced variations of contractile activity
were expressed as percentage of control responses elicited by
transmural electrical stimulation alone. Drugs were always
given in volumes 41% of total bath volume (10 ml).
Methods
Acetylcholinesterase assay
The activity of AChE was determined by means of a
colorimetric assay based upon the enzymatic conversion of
acetylthiocholine to thiocholine, which reacts with 5,5'-
dithiobis-2-nitrobenzoic acid to generate the chromogen
compound 5-thio-2-nitrobenzoate (Ellman et al., 1961).
Brie¯y, AChE obtained from human erythrocytes or electric
eel was used, the assay solution was maintained at the
temperature of 378C throughout the assay and consisted of
0
dithiobis-2-nitrobenzoic acid and 3 u ml
.1 M phosphate bu€er (pH 8.0) containing 0.01 M 5,5'-
71
AChE. The
reaction was started by addition of 0.075 M acetylthiocholine
iodide to the assay solution, and the formation of 5-thio-2-
nitrobenzoate was followed for 5 min by means of a
spectrophotometer (Uvikon 810, Kontron Instruments,
Milan, Italy) as changes in absorbance at 412 nm. Irinotecan,
SN-38 or physostigmine were incubated with AChE in the
assay solution for 20 min, and the enzyme reaction was then
initiated by addition of the substrate. The e€ects of test drugs
were expressed as percentage of AChE activity determined in
control experiments, and their potency in inhibiting AChE
was expressed as EC50 (concentration of the drug that
produces 50% of the maximal response for that drug). The
per cent maximum inhibition of the control AChE activity
(Emax) was also evaluated.
Isolated human colon
Isolated guinea-pig ileum
Tissue preparation Specimens of human colon were obtained
from patients undergoing surgery for benign or malignant
conditions of the colon or rectum. Specimens were collected
directly from the surgery room and generally consisted of a
Animal care and tissue preparation Male albino guinea-pigs,
300 ± 350 g body weight, were used throughout the study.
They were fed standard laboratory chow and tap water ad
British Journal of Pharmacology vol 132 (1)

C. Blandizzi et al
Irinotecan and adverse cholinergic effects
75
libitum and were not used for at least 1 week after their
delivery to the laboratory. The animals were housed, four in
a cage, in temperature controlled rooms on a 12-h light cycle
at 22 ± 248C and 50 ± 60% humidity. Their care and handling
were in accordance with the provisions of the European
Community Council Directive 86 ± 609, recognized and
adopted by the Italian Government. At the time of the
experiment, the whole ileum was excised from the small
intestine with the exception of the distal 10 cm, and
longitudinal muscle strips with myenteric plexus attached
were prepared as previously reported (Colucci et al., 1998).
The ileal preparations were then used in order to assess the
e€ects of test drugs on basal motor activity as well as on
electrically-induced twitch contractions or acetylcholine
release. Experiments aiming to assay the e€ects of irinotecan,
SN-38, acetylcholine or DMPP under basal conditions were
performed as reported above. Twenty-four hours before
experiments, some animals were treated with reserpine
(preperfusion), after which 3-min fractions were collected
for 90 min. During the superfusion period, the ileal
preparations were subjected to electrical ®eld stimulation,
71
delivered as square wave pulses (10 V cm ) of 1 ms
2
duration at 1 Hz (180 pulses) in the 3rd (S ) and 20th (S )
1
collection periods. At the end of superfusion, the radio-
activity of fractions was determined by liquid scintillation
counting (Betamatic, Kontron Instruments, Milan, Italy),
and the radioactive content of ileal strips was also measured.
For this purpose, each preparation was incubated in 1 ml of
10% trichloroacetic acid at room temperature for 30 min.
An aliquot of the supernatant (50 ml) was added to 5 ml of
scintillator and the tritium content of tissue was measured
by liquid scintillation spectrometry.
Irinotecan, SN-38 or physostigmine were added to the
superfusing solution in the 12th collection period, namely
1 2
between S and S , and exposure to each drug continued until
the end of experiment. Tritium e‚ux into the superfusate was
calculated as the fraction of tritium present in the ileal strip
at the onset of the respective collection period (fractional
71
(
4 mmol kg s.c.) in order to promote a complete depletion
of serotonin from myenteric serotonergic neurons, as
previously reported by Galligan et al. (1986). Longitudinal
muscle strips prepared from ileum of reserpinized animals
were then used to assay the e€ects of irinotecan on twitch
motor activity.
71
rate; min ). The increase in tritium out¯ow, evoked by
electrical stimulation, was calculated as percentage of the
tritium content of the tissue at the onset of electrical
stimulation. The e€ects of test drugs on the evoked tritium
out¯ow were expressed as ratio of the percentage release
during S over that obtained during S (S /S ), namely in the
Recording of twitch contractions The twitch motor activity
was recorded as previously described (Colucci et al., 1998).
Brie¯y, ileal longitudinal muscle strips, weighing 60 ±
2
1
2
1
presence and absence of drug respectively (Colucci et al.,
1998).
1
20 mg were set up in organ baths containing oxygenated
Krebs solution, and connected vertically to isometric
transducers (Basile, Comerio, Italy). Recurrent phasic
contractions of the longitudinal muscle (twitch responses)
were evoked by the application of transmural electrical
stimulation consisting of square wave pulses (1 ms duration,
In situ perfusion of rat stomach
Animal care and surgical preparation The experiments were
carried out on male Wistar rats weighing 200 ± 220 g. Their
care and handling were as reported above. Continuous
perfusion of the stomach in situ was carried out as previously
described (Blandizzi et al., 1997). Brie¯y, the animals were
3
0 ± 40 mA intensity) at the frequency of 0.1 Hz. The
preparations were stimulated for 60 min and were washed
four times at 15-min intervals before the beginning of
experiments. Irinotecan, SN-38 or physostigmine were
added cumulatively to the bathing ¯uid in 17log unit
increments. Unless otherwise stated, a period of 3 ± 10 min
was allowed to elapse between subsequent increments of
concentration in order to enable the full e€ect of the drug
to develop. The e€ects of test drugs were expressed as
percentage of the control twitch response.
71
anaesthetized with urethane (13.5 mmol kg
i.p.) and the
trachea was cannulated by a polyethylene catheter to ensure a
patent airway. A polyethylene catheter was introduced into
the oesophagus and advanced as far as 5 mm beyond the
gastroesophageal junction. After a midline laparatomy, a
second polyethylene catheter was introduced into the
duodenum and pushed forward until its tip was about
5 mm beyond the pylorus.
3
Measurement of [ H]-acetylcholine release The measure-
3
ment of [ H]-acetylcholine release was carried out as
Evaluation of gastric acid secretion The stomach lumen was
71
perfused continuously at a rate of 1 ml min
with saline
previously described (Colucci et al., 1998). Longitudinal
muscle strips of guinea-pig ileum, weighing 60 ± 120 mg,
were incubated for 30 min in oxygenated Krebs solution at
solution (154 mM NaCl) at 378C (pH=7.0+0.2), and 15-min
e‚uent fractions were collected. The e‚uent samples were
then used for the quantitative evaluation of acid secretion.
For this purpose, after the surgical preparation of animals,
basal gastric secretion was allowed to stabilize for 30 min and
two consecutive 15-min e‚uent fractions were then collected
in order to assess the basal secretory values. Acid secretion
was monitored at 15-min intervals for additional 180 min.
The acidity in the gastric perfusate was measured with an
autotitrator pH meter (PHM 85, Radiometer, Copenhagen,
Denmark) by automatic potentiometric titration to pH 7.0
3
[
78C (preincubation period), and then loaded with methyl-
H]-choline (3 mCi ml ) for 45 min in 3 ml of Krebs
3
71
solution. Electrical ®eld stimulation of 0.1-ms duration at
1
Hz was applied to ileal preparations for 30 min during the
loading period. At the end of incubation, the ileal strips
were washed ®ve times, transferred to another organ bath
71
(
5-ml capacity) and superfused at a ¯ow rate of 1 ml min
2
with Krebs solution at 378C, aerated with 95% O +5%
+
71
CO . The superfusing solution contained 3 mM hemicholi-
with 0.01 N NaOH and was expressed as mEqH 15 min .
The acid output obtained during the whole 180-min period
2
3
nium-3 in order to inhibit the reuptake of [ H]-choline
3
liberated by the hydrolysis of released [ H]-acetylcholine.
The ®rst 60-min collection of e‚uent was discarded
following the collection of basal e‚uent samples was also
+
71
calculated and expressed as mEqH 180 min .
British Journal of Pharmacology vol 132 (1)

76
C. Blandizzi et al
Irinotecan and adverse cholinergic effects
Experimental procedures In the ®rst set of experiments, the
e€ects of irinotecan, SN-38 or physostigmine on basal gastric
secretion were assayed in rats with intact vagus nerves. All
drugs were administered by i.v. route as a bolus immediately
after the collection of basal e‚uent samples. In experiments
investigating the involvement of muscarinic, serotonin 5-HT3
or vanilloid receptors in the gastric secretory responses
elicited by irinotecan, the animals were pretreated with the
respective antagonists, atropine, ondanstron or capsazepine,
immediately before the collection of the second basal e‚uent
sample.
Under full aseptic conditions, the cervical vagi were exposed
by making a midline neck incision and carefully freeing the
vagal trunk from the carotid artery for a distance of 2 ±
3 mm. Para®lm was placed around the vagus nerve and a
pledget of cotton wool soaked in capsaicin was placed around
the nerve trunk for 30 min. Further drops of capsaicin were
applied every 5 min, the maximum amount of capsaicin
applied being 0.1 ml (3 mmol per rat). The area was then
thoroughly rinsed with olive oil followed by saline and dried
with sterile swabs, and the neck incision was closed. Animals
in which the vagi were treated with 10% Tween 80 in olive oil
served as vehicle controls. Ten days after this treatment the
animals were used for measurement of acid secretion.
In an additional group of experiments, performed on rats
subjected to either systemic or vagal selective capsaiciniza-
tion, the vagus nerves were cut at cervical level, as reported
above. The distal end of the left vagus nerve was then placed
on a bipolar platinum electrode and immersed in paran oil.
After the collection of basal samples, gastric acid secretion
was evoked by continuous electrical stimulation of the left
vagus nerve (180 min). The stimulus parameters were square-
wave pulses 0.5 ms in duration, delivered at 5 Hz with
supramaximal intensity (10 V) by means of a Grass S5
stimulator (Grass Instruments, Quincy, MA, U.S.A.).
The second set of experiments was designed in order to
detect changes in gastric secretion following direct i.c.v.
administration of irinotecan to rats with intact vagus nerves.
For this purpose, a chronic cannula, to be used as a guide for
microinjection of drugs, was implanted into the right lateral
ventricle of the brain, during a short anaesthesia with
71
pentobarbitone sodium (120 ± 160 mmol kg i.p.) (Blandizzi
et al., 1995). Brie¯y, a cut was made in the scalp in order to
expose the intersection of the coronal and sagittal sutures. A
hole was then drilled through the skull 1.5 mm lateral and
1
mm posterior to bregma. A stainless steel cannula (24
gauge), equipped with a perspex base, was introduced
through the hole in the skull to a depth of 4 mm, and ®xed
to the skull surface with screws and dental cement. Animals
were allowed to recover from surgery for 6 days. At the time
of experiment, drug solutions (2.5 ml) followed by 2.5 ml of
saline solution were injected into the lateral ventricle of the
brain by means of a 10-ml Hamilton microsyringe connected
to the guide cannula by polyethylene tubing. The solutions
were injected within 20 s. At the end of each experiment, the
correct location of the cannula in the lateral ventricle was
checked by injecting 10 ml of dye (0.1% toluidine). The
visualization of dye on the walls of the lateral ventricle
indicated the exact location of the i.c.v. cannula.
The third series of experiments was performed on rats
whose vagus nerves were carefully separated from the carotid
arteries and cut at the cervical level 30 min before the
collection of basal e‚uent samples began. The e€ects of
irinotecan on gastric secretion were reassessed in animals that
underwent this vagotomy procedure.
Drugs
The following drugs and reagents were used: acetylcholine
chloride, acetylthiocholine iodide, human erythrocyte acet-
ylcholinesterase, electric eel acetylcholinesterase, 5,5'-dithio-
bis-2-nitrobenzoic acid, 1,1-dimethyl-4-phenylpiperazinium
iodide, urethane ethyl carbamate, physostigmine sulphate,
tetrodotoxin, hemicholinium-3 bromide, capsaicin, amino-
phylline, terbutaline, capsazepine, reserpine (Sigma Chemicals
Co., St. Louis, MO, U.S.A.); hexamethonium 2-chloride
3
(RBI, Natick, MA, U.S.A.); methyl-[ H]-choline chloride
(80 Ci mM) (Amersham Laboratories, Des Plaines, IL,
U.S.A.); atropine sulphate (BDH Chemicals, Poole, U.K.);
pentobarbitone sodium (Clin-Midy, Paris, France); ondanse-
tron (Glaxo Wellcome, Verona, Italy); irinotecan, SN-38 [7-
ethyl-10-hydroxycamptothecin] (kindly provided by Rhone-
Poulenc Rorer, Vitry-Alfortville, France). Other reagents
were of analytical grade. Unless otherwise stated, the drugs
were dissolved in distilled water or saline solution. SN-38 was
dissolved in dimethylsulphoxide and then diluted in water.
5,5'-Dithiobis-2-nitrobenzoic acid was dissolved in phosphate
bu€er 0.1 M (pH 7.0). Human erythrocyte acetylcholinester-
ase was dissolved in water by 5% Triton X-100. Electric eel
acetylcholinesterase was provided as aqueous solution
containing ammonium sulphate. All drugs administered by
i.v., i.p. or s.c. route were injected in a volume of 0.25 ml per
rat. Capsaicin to be administered by s.c. route was dissolved
The fourth set of experiments was designed in order to
assess whether irinotecan was able to a€ect gastric acid
secretion in the presence of systemic ablation of capsaicin-
sensitive sensory nerve ®bres (Holzer, 1991). For this
71
purpose, animals were given a total dose of 400 mmol kg
capsaicin s.c., as previously reported (Blandizzi et al., 1997).
Ten days after this treatment the animals were used for
measurement of acid secretion. One day before the experi-
ment, the ecacy of capsaicin treatment was checked by
71
instilling a drop of capsaicin solution (0.3 mmol ml in saline
solution) into one eye of each rat. Capsaicin-treated rats were
expected not to react by wiping their eyes but, whenever an
animal responded with wiping, the a‚icted eye was
immediately and extensively rinsed with water.
71
(37.5 mmol ml ) in a vehicle composed by 10% ethanol,
10% Tween 80 and 80% saline solution (v/v/v). The total
71
dose of capsaicin (400 mmol kg ) was administered under
ether anaesthesia in four injections over two consecutive days
The ®fth series of experiments was performed to investigate
the e€ects of irinotecan on acid secretion in rats subjected to
perivagal application of capsaicin, which produces a selective
functional impairment of vagal capsaicin-sensitive a€erent
7
1
71
(®rst day: 80 mmol kg in the morning and 80 mmol kg in
71
the late afternoon; second day: 80 mmol kg in the morning
71
and 160 mmol kg
in the late afternoon). Capsaicin
®bres (Holzer, 1991). In accordance with the procedure
reported by Raybould & Tache (1989), rats were anaesthe-
tized with pentobarbitone sodium (120 ± 160 mmol kg i.p.).
(30 mmoles) to be used for perivagal application was
sonicated for 10 min in 0.1 ml Tween 80, then 0.9 ml of
olive oil were added and the mixture sonicated for further
Â
71
British Journal of Pharmacology vol 132 (1)

C. Blandizzi et al
Irinotecan and adverse cholinergic effects
77
10 min. In order to counteract the respiratory impairment
induced by capsaicin, the rats received atropine
drug, caused a marked and concentration-dependent inhibi-
tion of AChE function, with an EC50 value of 26.6 nM
(95%CL: 11.5 ± 61.2) and the maximal e€ect (793.4%) at
3 mM (Figure 1A). Similar results were obtained on the
activity of electric eel AChE: SN-38 was without e€ect,
irinotecan caused an inhibition of 715.7% at 100 mM, and
physostigmine blocked the enzymatic activity with an EC50 of
16.3 nM (95%CL: 13.6 ± 19.7) and a maximal inhibition at
71
71
(
0.6 mmol kg
i.p.), terbutaline (0.5 mmol kg
i.p.) and
i.p.) 10 min before the ®rst
71
aminophylline (72 mmol kg
and third s.c. injection as well as the perivagal application of
capsaicin.
Statistical analysis
1
mM (796.1%) (Figure 1B).
The results are given as means of 6 ± 8 experiments+
s.e.mean. The signi®cance of di€erences was evaluated by
Student's t-test or one-way analysis of variance (ANOVA)
followed by post hoc analysis by Student-Newman-Keuls test,
and P values lower than 0.05 were considered signi®cant: `n'
indicates the number of experiments. EC50 values were
interpolated from concentration-response curves. All statis-
tical procedures, curve ®tting and calculations of 95%
con®dence limits (95% CL) for EC50 values were performed
by means of personal computer programs (GraphPad
Isolated human colon
Spontaneous contractile activity During the 60-min equili-
bration period in carbogenated Krebs solution, most human
colonic muscle strips developed varying degrees of sponta-
neous activity, which was variable among di€erent tissues
and often among di€erent preparations from the same colon
specimen. Most of the strips displayed a rapid spontaneous
activity, with an amplitude less than 10% of the contractile
response obtained by electrical stimulation, and generally
stable throughout the experiment or with a trend to reduce.
When the spontaneous activity was greater than 30% of
contractions evoked by electrical stimulations, the strips were
discarded. Some colonic preparations which displayed slow
phasic contractions with shorter and more frequent contrac-
tions superimposed were also discarded.
TM
Prism , software package version 2.01 for Windows 95,
TM
and InStat , software package version 2.01 for MacIntosh;
both from GraphPad Software Inc., San Diego, CA, U.S.A.).
Results
Acetylcholinesterase activity
The reference drugs used in the present study changed the
spontaneous contractile activity of human colonic muscle in
di€erent ways: (1) exogenously applied acetylcholine (0.01 ±
30 mM) evoked concentration-dependent phasic contractions
(Figure 2A) which were antagonized by atropine 1 mM (not
shown). Maximal responses occurred at the concentration of
10 mM, with an estimated EC50 value of 0.47 mM (95% CL:
0.41 ± 0.54; n=8); (2) DMPP (0.01 ± 100 mM) elicited phasic
relaxations, at concentrations of 10 ± 100 mM (Figure 2B),
which were prevented by hexamethonium 10 mM (not shown).
Irinotecan, at concentrations ranging from 0.01 to 100 mM,
slightly reduced the activity of human erythrocyte AChE,
with an inhibitory e€ect of 721.5% observed at the highest
concentration tested (Figure 1A). Under the same conditions,
no signi®cant changes in the enzymatic activity were detected
when SN-38 (0.01 ± 100 mM) was incubated with human
erythrocyte AChE in the assay solution for 20 min. By
contrast, physostigmine (0.0001 ± 30 mM), used as reference
Figure 1 E€ects of irinotecan (0.01 ± 100 mM), SN-38 (0.01 ± 100 mM) or physostigmine (0.0001 ± 30 mM) on the enzymatic activity of
human erythrocyte (A) or electric eel (B) AChE. Each point represents the mean of 6 ± 8 experiments+s.e.mean (vertical lines).
British Journal of Pharmacology vol 132 (1)

78
C. Blandizzi et al
Irinotecan and adverse cholinergic effects
Figure 2 Isolated human colon. Upper panels: Representative trace recordings showing the e€ects of acetylcholine (ACh, 1 mM)
A), dimethylphenylpiperazinium (DMPP, 30 mM) (B), irinotecan (100 mM) (C) or SN-38 (100 mM) (D) on the spontaneous
contractile activity of longitudinal muscle preparations. Lower panels: Representative trace recordings showing the contractile
(
responses of longitudinal muscle strips to the application of transmural electrical stimulation (TES: 1 ms, 10 Hz, 30 s), either alone
(E) or in the presence of atropine (1 mM) (F), physostigmine (0.1 mM) (G) or irinotecan (100 mM) (H), w=washing.
By contrast, 0.1 to 100 mM irinotecan or SN-38 failed to
modify the spontaneous motor activity of colonic prepara-
tions (Figure 2C,D).
both applied at 0.01 ± 100 mM for 20 min, did not signi®cantly
modify phasic contractile responses elicited by electrical
stimulation of human colonic preparations (Figure 2H).
Contractile activity induced by transmural electrical stimula-
tion Trains lasting 30 s evoked motor responses which
consisted of two components: a fast phasic contraction
observed during the delivery of electrical stimulation,
followed by a further contraction at the end of stimulation
Isolated guinea-pig ileum
Spontaneous contractile activity During the equilibration
period, longitudinal muscle strips of guinea-pig ileum
developed a spontaneous contractile activity, which remained
stable throughout the experiment without interfering with the
motor responses elicited by test drugs. Exogenous acetylcho-
line (0.01 ± 30 mM) as well as DMPP (0.1 ± 100 mM) caused
fast and concentration-dependent phasic contractions (Figure
3A,B), which were antagonized by atropine 1 mM and
hexamethonium 10 mM, respectively (not shown). Acetylcho-
line 10 mM caused maximal responses with an EC50 value of
0.35 mM (95% CL: 0.21 ± 0.59). DMPP induced maximal
contractions at 30 mM, with an EC50 value of 1.94 mM (95%
CL: 1.38 ± 2.74). Under the same conditions, basal motor
activity of longitudinal muscle strips did not change in the
presence of irinotecan or SN-38 (0.01 ± 300 mM for each drug)
(Figure 3C,D).
(
(
aftercontraction) (Figure 2E). In the presence of atropine
1 mM), the initial phasic contraction was abolished, or
reversed into a relaxation, and only the aftercontraction
was evident (Figure 2F). In this respect, it has been
previously shown that, besides acetylcholine, the application
of electrical stimulation to smooth muscle preparations of
human colon promotes also the release of non-adrenergic
non-cholinergic (NANC) neural mediators which account for
the occurrence of relaxations and aftercontractions (Crema et
al., 1968; Maggi et al., 1997).
Under these conditions, physostigmine (0.01 ± 0.1 mM)
greatly enhanced the stimulated motor responses
(
+46.3+5.1% at 0.1 mM; P50.05), without a€ecting the
resting tone (Figure 2G). Concentrations higher than 0.1 mM
could not be tested since they were associated with
increments of basal contractile tone. Irinotecan or SN-38,
Twitch contractions Electrical stimulation of ileal prepara-
tions at low frequency induced recurrent phasic contractions
British Journal of Pharmacology vol 132 (1)

C. Blandizzi et al
Irinotecan and adverse cholinergic effects
79
Figure 3 Isolated guinea-pig ileum. Upper panels: Representative trace recordings showing the e€ects of acetylcholine (ACh, 1 mM)
A), dimethylphenylpiperazinium (DMPP, 10 mM) (B), irinotecan (100 mM) (C) or SN-38 (100 mM) (D) on the spontaneous
(
contractile activity of longitudinal muscle preparations. Lower panels: Representative trace recordings showing the e€ects of
physostigmine (0.01 ± 0.1 mM) (E) or irinotecan (10 ± 100 mM) (F) on the cholinergic twitch contractions evoked by transmural
electrical stimulation of longitudinal muscle preparations (1 ms, 0.1 Hz), w=washing.
of longitudinal muscle, which were abolished by tetrodotoxin
1 mM), or atropine (0.1 mM), but una€ected by hexametho-
superfused ileum strips were stimulated electrically, the
tritium e‚ux increased from 0.0020+0.00012 to
(
71
nium (10 mM), indicating, the involvement of postganglionic
cholinergic nerves (not shown). Physostigmine 0.01 ± 0.1 mM
increased the amplitude of twitch responses (+39.6+5.4%;
P50.05) (Figure 3E). Higher concentrations of physostig-
mine could not be tested, since they caused marked and
persistent increments of the basal tone associated with a
parallel decrease in the amplitude of the superimposed twitch
activity. Under these conditions, irinotecan or SN-38, both
tested at 0.01 ± 100 mM, did not modify the cholinergic twitch
contractions (not shown). However, in a minority of cases
0.0061+0.00028 min
(P50.001). The increase in tritium
out¯ow evoked by electrical stimulation was observed in four
consecutive 3-min fractions: the release reached a peak during
this time, and then declined exponentially to the prestimula-
tion value. Under control conditions, the evoked tritium
out¯ow was 2.86+0.34% for S and 2.69+0.37% for S , not
1
2
signi®cantly di€erent from each other, and the calculated
ratio S /S was 0.94+0.05 (Figure 4A). It has been previously
2
1
shown that, after incubation of ileal preparations with
radiolabelled choline, the application of electrical stimulation
at 1 ms and 1 Hz evokes a signi®cant release of radioactivity,
which is tetrodotoxin-sensitive and consists mostly of
radiolabelled acetylcholine (Vizi et al., 1984).
(
24%) the amplitude of twitch responses increased moder-
ately following the application of irinotecan 10 mM
+18.4+2.3%), and returned toward the basal level when
(
the drug concentration was raised to 100 mM (Figure 3F).
Similar results were obtained when irinotecan was assayed on
twitch contractions evoked by electrical stimulation of ileal
preparations obtained from animals pretreated with reserpine
Neither physostigmine (0.1 mM) nor irinotecan (0.1 ±
100 mM) or SN-38 (0.1 ± 100 mM) signi®cantly modi®ed the
resting over¯ow of tritium (not shown). When tested on
tritium out¯ow evoked by electrical stimulation, physostig-
3
(
not shown).
mine 0.1 mM signi®cantly inhibited the release of [ H]-
acetylcholine (747.7+5.6%) (Figure 4B). Under the same
conditions, irinotecan (10 ± 100 mM) reduced the electrically-
induced tritium out¯ow (731.9+4.2% at 100 mM) (Figure
4C), and SN-38 (1 ± 100 mM) was without e€ect (S2/
3
[
H]-Acetylcholine release In control experiments, after a
0-min initial preperfusion period, the spontaneous tritium
6
71
over¯ow approached a rate of 0.0021+0.00014 min and
remained unchanged throughout the experiment. When the
1
S =0.90+0.07 at 100 mM) (Figure 4D).
British Journal of Pharmacology vol 132 (1)

80
C. Blandizzi et al
Irinotecan and adverse cholinergic effects
3
Figure 4 Isolated guinea-pig ileum. Tritium e‚ux from longitudinal muscle strips preincubated with methyl-[ H]-choline. Abscissa:
time of superfusate collection. Ordinate: e‚ux of tritium at the onset of the respective collection period. Electrical ®eld stimulation
during S and S consisted of 180 pulses (1 ms, 1 Hz). (A) Control experiments. (B, C, D) E€ects of physostigmine (0.1 mM),
1 2
irinotecan (100 mM) and SN-38 (100 mM), respectively. The drugs were present in the superfusion medium during the time indicated
by the horizontal bar. Each point represents the mean of 6 ± 8 experiments+s.e.mean (vertical lines).
Acid secretion from in situ perfused rat stomach
fully prevented gastric hypersecretory e€ect of irinotecan
71
(
subjected to selective ablation of vagal capsaicin-sensitive
10 mmol kg
i.v.) (Figure 6C). Analogously, in rats
In control animals with intact vagus nerves, basal acid
secretion, assessed after 30-min stabilization, accounted for
71
a€erent ®bres, irinotecan (10 mmol kg i.v.) failed to modify
basal secretory output (Figure 6D). In both types of
preparations, the application of electrical stimuli to the distal
trunk of the left vagus nerve, in animals subjected to bilateral
vagotomy, was still able to promote signi®cant and atropine-
sensitive increments of acid secretion (Figure 6C,D).
+
71
3
constant until the end of the experiments (180 min). In
.92+0.47 mEqH 15 min , and this value remained nearly
addition, when control animals underwent bilateral cervical
71
+
vagotomy, basal secretion was 3.4+0.73 mEqH 15 min
This value was similar to that obtained in control rats with
.
intact vagus nerves, and remained at
throughout the experiment.
In animals with intact vagus nerves, irinotecan (5, 10 and
a steady level
Discussion
71
20 mmol kg i.v.) caused a dose-dependent increase in acid
secretion, with the maximal e€ect at the dose of
In a previous investigation, Kawato et al. (1993) suggested
that irinotecan might directly activate muscarinic receptors,
based on the ability of this drug to compete with the binding
of radiolabelled quinuclidinyl benzylate to cell membranes
isolated from rat brain. In the present study, exogenously
applied acetylcholine caused atropine-sensitive phasic con-
tractions of both ileal and colonic muscle strips, with EC50
values in the nanomolar range. However, these stimulant
actions could not be reproduced in the presence of irinotecan
or SN-38, even when they were applied at high concentra-
tions, suggesting that these drugs are devoid of direct
muscarinic agonistic properties.
71
1
secretory rate were observed when irinotecan was injected by
0 mmol kg (Figure 5). No signi®cant changes in the gastric
71
i.c.v. route at 0.01 or 0.1 mmol kg or when animals were
7
1
treated with SN-38 (20 mmol kg
3 mmol kg
i.v.) or physostigmine
i.v.) (Figure 6A). In addition, the excitatory
71
(
e€ect of irinotecan no longer occurred when injected to
animals subjected to bilateral vagotomy or pretreated with
71
atropine (3 mmol kg i.v.), whereas it was partly prevented
7
1
by ondansetron (15 mmol kg
i.v.) and una€ected by
71
capsazepine (50 mmol kg i.v.) (Figure 6B).
In animals with intact vagus nerve, the functional ablation
of capsaicin-sensitive sensory neurons by systemic pretreat-
ment with capsaicin did not modify basal acid secretion, but
Similar conclusions to those drawn for muscarinic
receptors can be applied to the present experiments in which
British Journal of Pharmacology vol 132 (1)

C. Blandizzi et al
Irinotecan and adverse cholinergic effects
81
the activity of AChE with potency values ranging from 0.19
to 0.41 mM (Kawato et al., 1993; Rivory et al., 1996; Morton
et al., 1999). In addition, it has been recently proposed that
irinotecan interacts directly with both human erythrocyte and
electric eel AChE, leading to a non-competitive inhibition of
these enzymes (Dodds & Rivory, 1999).
In the present study, in vitro assays performed on AChE
isolated from human erythocytes or electric eel revealed that
irinotecan acted as a very weak inhibitor of AChE activity,
since the activities of these enzymes decreased by about 15 ±
2
0% only when irinotecan was used at 100 mM. The
remarkable discrepancies between our results and those
obtained in previous studies can be hardly explained.
However, in the present experiments, physostigmine, a well
known AChE blocker (Taylor, 1996), inhibited the activity of
both human erythrocyte and electric eel AChE with EC50
values similar to those reported by other authors (Yu et al.,
1
988; Rampa et al., 1998), indicating that our assays were
carried out under standard and suitable experimental
conditions. In addition, the following points must be taken
into account: (1) no signi®cant inhibition of AChE activity
was observed either in isolated erythrocytes or in whole blood
samples obtained from cancer patients exposed to i.v.
infusion of irinotecan (Gandia et al., 1993; Blandizzi et al.,
2
000; (2) AChE is widely expressed in mammalian tissues,
and symptoms associated with its pharmacological blockade
usually result not only from the indirect activation of
peripheral muscarinic receptors, but also from an involve-
ment of cholinergic receptors at autonomic ganglia, skeletal
muscle and central nervous system (Taylor, 1996); never-
theless, the acute cholinergic syndrome evoked by irinotecan
is consistent with a picture of systemic parasympathetic
activation (Gandia et al., 1993; Rowinsky et al., 1994), a
feature which can be hardly reconciled with a drug-induced
widespread inhibition of AChE activity.
In the present study, additional evidence to support the
view that irinotecan exerts a negligible inhibitory activity on
AChE came from the experiments performed on longitudinal
muscle preparations of guinea-pig ileum or human colon. The
electrically-induced contractile activity of ileal strips was
weakly stimulated by irinotecan in a minority of experiments
when this drug was applied at high concentrations, whereas
SN-38 was always ine€ective. By contrast, physostigmine,
used at concentrations which do not cause a direct muscarinic
receptor activation (Yamada et al., 1982), signi®cantly
enhanced the cholinergic motor responses of both ileal and
colonic muscles to electrical stimulation. These ®ndings are in
full agreement with previous studies, where the potentiating
actions of physostigmine on human or guinea-pig intestinal
preparations were related to the accumulation of endogenous
acetylcholine following AChE blockade (Cox & Stobart,
1972; Frigo et al., 1973; Yamada et al., 1982). Moreover,
since the electrical stimulation of human colon is also
associated with the activation of intramural NANC pathways
(Crema et al., 1968; Maggi et al., 1997), and in the present
study irinotecan did not modify the pattern of electrically-
induced motor responses from colonic preparations, it can be
suggested that irinotecan does not interfere with functions
mediated by myenteric NANC transmitters.
Figure 5 Anaesthetized rats with in situ perfusion of gastric lumen.
i.v.) on gastric acid
secretion. Each point represents the mean value obtained from 6 ± 8
animals+s.e.mean (vertical lines). The arrow indicates the time of
irinotecan administration. *P50.05: signi®cant di€erence from
control values.
71
E€ects of irinotecan (5, 10 and 20 mmol kg
the e€ects of irinotecan and SN-38 were compared with
motor responses elicited by DMPP. The opposite e€ects
exerted by DMPP on guinea-pig ileum or human colon
preparations in the present series are in agreement with other
studies showing that nicotinic receptor agonists may induce
hexamethomium-sensitive contractile or relaxant responses,
depending on the intestinal region and species investigated
(
Bucknell & Whitney, 1964; Hayashi et al., 1983). Since, in
the present study, irinotecan and SN-38 did not cause
relaxations of human colonic preparations, neither had they
mimicked the fast contractions of longitudinal muscle evoked
by application of DMPP to guinea-pig ileal strips, a possible
implication of neuronal nicotinic receptors in the genesis of
cholinergic symptoms associated with irinotecan treatment
appears to be unlikely. The present results do not support the
hypothesis of Gandia et al. (1993), who speculated that
irinotecan might act as agonist on ganglion nicotinic
receptors, due to the structural similarity of its di-piperidin
carbonyloxy group with DMPP.
An attractive mechanism, proposed to explain the
cholinergic syndrome induced by irinotecan, is based on the
assumption that this camptothecin analogue may block the
activity of AChE, thus preventing the enzymatic breakdown
of endogenous acetylcholine and promoting the tissue
accumulation of this neuromediator over physiological
concentrations (Kawato et al., 1993; Rivory et al., 1996;
Morton et al., 1999). Previous investigations supported this
view showing that, when tested in vitro, irinotecan inhibited
A useful model for testing the e€ects of drugs on
cholinergic neurotransmission consists of the measurement
of acetylcholine release from longitudinal muscle strips
British Journal of Pharmacology vol 132 (1)

82
C. Blandizzi et al
Irinotecan and adverse cholinergic effects
7
1
Figure 6 Anaesthetized rats with in situ perfusion of gastric lumen. (A) E€ects of irinotecan (10 mmol kg
i.v. or 0.01 ±
i.v.) on gastric acid secretion. (B) E€ects of
71
71
71
0
.1 mmol kg
irinotecan (10 mmol kg
ondansetron (15 mmol kg
i.c.v.), SN-38 (20 mmol kg
i.v.) or physostigmine (3 mmol kg
71
71
i.v.) either alone or in the presence of bilateral cervical vagotomy, atropine (3 mmol kg
i.v.),
i.v.) on gastric acid secretion. (C) E€ects of irinotecan
71
71
i.v.) or capsazepine (50 mmol kg
71
71
(10 mmol kg
i.v.) and electrical vagal stimulation, either alone or in the presence of atropine (3 mmol kg
i.v.), on gastric
acid secretion in rats subjected to systemic ablation of capsaicin-sensitive sensory nerve ®bres. (D) E€ects of irinotecan
71
71
(
10 mmol kg i.v.) and electrical vagal stimulation, either alone or in the presence of atropine (3 mmol kg i.v.), on gastric acid
secretion in rats subjected to perivagal application of capsaicin. Each column represents the mean value obtained from 6 ± 8
a
animals+s.e.mean (vertical lines). *P50.05: signi®cant di€erence from control values: P50.05: signi®cant di€erence from
irinotecan alone.
preincubated with radiolabelled choline and subjected to
electrical stimulation (Vizi et al., 1984). Previous studies have
clearly established that AChE blockers reduce the electrically-
autonomic responses. Therefore, an in vivo model was used in
order to test this hypothesis, as the anaesthetized rat
represents a useful preparation to assay the in¯uence of
centrally or peripherally applied cholinergic stimuli on gastric
secretory functions (Maggi & Meli, 1986; Blandizzi et al.,
1997). Data obtained from these experiments provided
consistent evidence that irinotecan, but not SN-38, acts at
peripheral sites to promote a re¯ex activation of vagal
cholinergic e€erent out¯ow and that capsaicin-sensitive ®bres
are involved in the a€erent branch of this re¯ex response. In
particular, the following ®ndings support this conclusion: (1)
when administered parenterally, at doses comparable with
those used in clinical practice (Rowinsky et al., 1994; O'Leary
& Muggia, 1998), irinotecan caused a gastric hypersecretory
e€ect which could not be reproduced by its direct injection
into the central nervous system; (2) the stimulant action of
irinotecan was no longer observed in rats pretreated with
atropine or subjected to bilateral vagotomy; (3) in animals
where the functional ablation of capsaicin-sensitive a€erent
®bres was induced by systemic pretreatment with capsaicin,
irinotecan did not modify the acid output, whereas, under the
same conditions, electrical stimulation of the vagus nerve
elicited signi®cant atropine-sensitive secretory responses,
3
induced [ H]-acetylcholine output from ileal preparations,
since the reduced enzymatic breakdown of endogenous
acetylcholine enhances its feedback inhibitory control on its
own release through the activation of muscarinic autorecep-
tors (Somogyi & Vizi, 1988). Accordingly, the results of our
experiments, showing that physostigmine signi®cantly de-
3
creased the evoked [ H]-acetylcholine release, whereas
irinotecan exerted only moderate inhibitory e€ects when
tested at concentrations of 100 mM, further strengthen the
view that irinotecan does not act as a speci®c inhibitor of
AChE. Moreover, it is unlikely that such weak anti-
cholinesterase activity may be relevant in clinical settings,
since i.v. infusion of irinotecan to cancer patients usually
results in drug peak plasma concentrations of 1.8 ± 4.5 mM
(
Rowinsky et al., 1994; Falcone et al., 1999).
Since mechanisms related to cholinergic receptor activation
or AChE blockade do not support the cholinergic syndrome
induced by irinotecan, we hypothesized that this antitumour
drug might stimulate a cholinergic parasympathetic dicharge
either by acting at central nervous sites or triggering re¯ex
British Journal of Pharmacology vol 132 (1)

C. Blandizzi et al
Irinotecan and adverse cholinergic effects
83
indicating that capsaicin did not a€ect the functional integrity
of vagal e€erent ®bres.
There is evidence in the literature to support the view that
abdominal viscera, including the stomach, are supplied with
capsaicin-sensitive a€erent nerve-®bres (Sengupta & Gebhart,
enhancing action exerted by irinotecan on twitch contractions
could be reproduced also in ileal preparations obtained from
reserpinized animals, argue against the possibility that
irinotecan promotes the release of serotonin from myenteric
serotonergic neurons. On the other hand, previous studies
have shown a close relationship between the cytotoxic action
of various antineoplastic drugs and the peripheral serotonergic
system (Andrews et al., 1990, Cubeddu, 1996). In particular, it
has been proposed that enterochroman cells, located in the
mucosa of the upper gut, may act as chemoreceptors which are
able to release serotonin once challenged by cytotoxic drugs.
1994; Szallasi & Blumberg, 1999). These sensory neurons
carry out several regulatory functions by means of bioactive
peptides, whose release can be promoted by activation of
vanilloid receptors or application of various chemicophysical
stimuli (Holzer, 1991; Szallasi & Blumberg, 1999). Moreover,
capsaicin-sensitive nerves constitute the a€erent branch of
autonomic re¯exes and express speci®c receptors on their
peripheral endings, through which various mediators, includ-
ing cholecystokinin and serotonin, can modulate the activity
of central nervous pathways implicated in the control of
peripheral organs (Bozkurt et al., 1999; Szallasi & Blumberg,
3
The released serotonin then activates 5-HT receptors on vagal
a€erent ®bres, which terminate in close proximity of
enterochroman cells, and hence triggers the emetic re¯ex
(Andrews et al., 1990; Cubeddu, 1996).
Clinical studies have shown that treatment with irinotecan
may induce nausea and vomiting, which respond favourably
1
999). In line with these ®ndings, three major mechanisms
can be postulated to account for irinotecan-induced activa-
tion of abdominal a€erents: (1) direct activation of a€erent
nerves; (2) sensitization of the a€erents to endogenous
mediators; (3) release of endogenous mediators which then
3
to serotonin 5-HT receptor antagonists (Rowinsky et al.,
1994), indicating that this camptothecin analogue has the
potential for activating serotonin-mediated emetogenic re-
¯exes. Accordingly, taking into account the results obtained
with ondansetron in the present in vivo experiments, it is
conceivable that irinotecan acts on peripheral chemorecep-
tors, including enterochroman cells, and promotes the
activation of a vago-vagal re¯ex which causes a centrally
driven cholinergic out¯ow. However, since other cytotoxic
drugs known to induce nausea and vomiting, such as
cisplatin, cyclophosphamide, epirubicin and 5-¯uorouracil,
did not a€ect, or rather inhibited, gastric acid secretion in
previous studies (Fabrin et al., 1991, Kakinuma & Ohwada,
1997), it is also likely that, besides serotonin, other
endogenous mediators and pathways can be involved in the
vagal activation promoted by irinotecan.
In conclusion, the present results indicate that the acute
cholinergic syndrome observed in cancer patients during
treatment with irinotecan is not likely to depend on the
blockade of AChE activity by irinotecan or SN-38, and that
both these drugs are devoid of direct cholinomimetic
properties. According to our ®ndings, it is rather proposed
that irinotecan is able to promote a sustained vagal e€erent
discharge to peripheral e€ector organs, mediated by the
activation of capsaicin-sensitive sensory a€erent ®bres. It is
stimulate peripheral a€erent ®bres. Although
a direct
activation or sensitization of capsaicin-sensitive a€erent
nerves by irinotecan cannot be ruled out on the basis of
the present results, these mechanisms are not supported by
previous studies dealing with other cytotoxic drugs. In
particular, cisplatin failed to directly stimulate vagal a€erent
®bres or to sensitize them to the depolarizing action exerted
by serotonin (Woods & Andrews, 1995).
In the present study, a series of experimental data indicates
that the gastric stimulant action of irinotecan was abolished
in animals subjected to perivagal capsaicin application, it was
partly prevented by blockade of 5-HT3 serotonin receptors
with ondansetron and was not modi®ed by capsazepine, a
selective antagonist of vanilloid receptors (Szallasi
&
Blumberg, 1999). Therefore, on the basis of these results, it
can be suggested that the capsaicin-sensitive a€erent ®bres,
which mediate the cholinergic responses triggered by
irinotecan, belong to the vagus nerves, and that endogenous
serotonergic pathways could be involved, at least in part, in
the activation of this re¯ex.
At the level of gastrointestinal tract, serotonin can be
released from enterochroman cells or intramural serotoner-
gic neurons (Gershon et al., 1994). In the present study, the
results of in vitro experiments, showing that the weak
also suggested that serotonin 5-HT receptors are implicated,
3
at least in part, in the genesis of the vago-vagal re¯ex
triggered by irinotecan.
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(Received May 9, 2000
Revised September 1, 2000
Accepted October 12, 2000)
British Journal of Pharmacology vol 132 (1)