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Archives of Medical Research 38 (2007) 392e397
ORIGINAL ARTICLE
Gastric Emptying Effect by 4-Aminopyridine in Patientswith Chronic Spinal Cord Injury
Israel Grijalva,a Gabriel Guızar-Sahagun,a,c Delia Rodrıguez-Pacheco,b Carlos Francisco-Arguelles,b
Gilberto Castaneda-Hernandez,d and Jose Antonio Palma-Aguirreb
aMedical Research Unit for Neurological Diseases, bDepartment of Pharmacology, Hospital de Especialidades,
Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, MexicocProyecto Camina, A.C., Mexico City, Mexico
dExternal Pharmacology Section, Centro de Investigaciones y Estudios Avanzados, Instituto Politecnico Nacional, Mexico City, Mexico
Received for publication June 24, 2006; accepted November 29, 2006 (ARCMED-D-06-00256).
Background. 4-Aminopyridine (4-AP) given to patients with chronic spinal cord injury(SCI) has shown beneficial effects in some somatic and autonomic functions, althoughpatients often develop dyspeptic symptoms. 4-AP is a potassium-channel blocker capableof altering gastro-pyloric functions as demonstrated experimentally. Our objective was toexamine the influence of 4-AP treatment on gastric emptying in patients with chronic SCI.
Methods. Gastric emptying was measured by the acetaminophen absorption test in 18 pa-tients (9 with cervical and 9 with thoracic injury), and 9 healthy volunteers. Patientsreceived increasing oral doses, 5 mg day�1 of oral 4-AP (5e30 mg day�1), for 12 weeks.Patients were studied before and at the end of the last week of 4-AP treatment, whereashealthy volunteers (without 4-AP treatment) were studied only once. Whole blood sam-ples of 2.5 mL were drawn at 0 (before 1 g of oral acetaminophen) and at 15, 30, 45, 60,75, 90, 105 and 120 min postdose. Acetaminophen concentration in plasma was deter-mined by high-pressure liquid chromatography.
Results. Treatment with 4-AP significantly delayed stomach emptying in patients withchronic SCI, considering the significant decreasing of acetaminophen absorption (t pairedtest, p !0.05). This effect did not correlate either to the level or ASIA score of the injury(linear regression correlation analysis, r2 5 0.003 and 0.015, respectively). No significantdifferences were observed by comparing data of patients before 4-AP treatment withhealthy volunteers.
Conclusions. 4-AP intake in patients with chronic spinal cord injury significantly slowedgastric emptying regardless of level and ASIA score of the injury. � 2007 IMSS. Pub-lished by Elsevier Inc.
Key Words: Acetaminophen absorption test, Autonomic nerve conduction, Paraplegia, Potassiumchannel blocking.
Introduction
Previous reports support the fact that 4-aminopyridine(4-AP) is a pharmacological agent capable of restoringsome neural conduction (1,2) and improving motor andsensory functions in selected patients with chronic spinalcord injury (SCI) (3e6).
Address reprint requests to: Israel Grijalva, UIME Neurologicas, Cal-
zada de Tlalpan 4430, Col. Toriello Guerra. Mexico, D.F. 14050, Mexico;
E-mail: igrijalvao@yahoo.com
0188-4409/07 $esee front matter. Copyright � 2007 IMSS. Published by Edoi: 10.1016/j.arcmed.2006.11.007
SCI affects not only voluntary motor and sensory func-tions below the cord level; it also alters autonomic func-tions, resulting in multiple systemic and metabolicdisturbances (7e10). Abnormalities in gastrointestinal mo-tility, particularly delay of gastric emptying, are commoncomplications after acute and chronic stages of SCI andare closely related to autonomic dysfunction (11e15).
4-AP given to patients with chronic SCI has shown ben-eficial effects on some autonomic functions, particularly onlungs (16) and heart (17,18). However, possible effects of
lsevier Inc.
393Gastric Emptying Effect by 4-AP in SCI Patients
4-AP on gastrointestinal function in such patients have notpreviously been explored.
Biochemical and electrophysiological studies haveshown that a diversity of potassium channels are presentin gastric muscle as well as in myenteric neurons. Potas-sium currents through these channels are important regula-tors of gastric tone and motility (19e22).
4-AP is the classic nonselective pharmacological blockerof voltage-gated potassium channels (23,24). Preliminaryexperimental studies have demonstrated that 4-AP modifiessome physiological gastrointestinal processes (25e29),which can result in slowing gastrointestinal transit (30).
The purpose of the present study was to determine theinfluence of 4-AP on gastric emptying in patients withchronic SCI. Taking into account basic experimental re-ports regarding the effects of 4-AP on gastrointestinal func-tion, as well as the association between 4-AP intake andgastric symptoms reported in some clinical studies (6),we hypothesized that this agent given to our patients couldslow gastric emptying. As an alternative hypothesis,we proposed that 4-AP could improve gastric emptying asa result of improved autonomic nerve conduction. To testour hypotheses, we chose a validated method to assessgastric emptying, namely, the acetaminophen absorptiontest (31,32).
Materials and Methods
To assess the influence of 4-AP on gastric emptying time inpatients with chronic SCI, a comparative open labeled clin-ical trial was performed. Patients were recruited from theMedical Research Unit for Neurological Diseases of theSpecialties Hospital, Centro Medico Nacional Siglo XXI,Instituto Mexicano del Seguro Social, Mexico City. Thestudy was carried out in accordance with the recommenda-tions of the Declaration of Helsinki and approved by thehospital Medical Ethics and Research Committee. Allhealthy volunteers and patients gave written informed con-sent for participation.
Gastric emptying was evaluated using the acetamino-phen absorption test (31,32). The absorption of a 1 g fastingdose of oral solution of acetaminophen was studied in 9healthy volunteers and 18 SCI patients. Healthy volunteersserved as controls for normal gastric function and were nottreated with 4-AP; they were subjected to the gastric emp-tying study only once, whereas acetaminophen absorptionstudies were performed twice in SCI patients: the first be-fore 4-AP treatment and the second 12 weeks after 4-APtreatment. SCI patients received oral doses of 4-AP startingat 5 mg day�1, increasing 5 mg per day every week untila dose of 30 mg day�1 was reached in a 6-week period. Pa-tients then received 30 mg day�1 (10 mg each 8 h [6:00,14:00 and 22:00 h]) for 6 weeks. The 4-AP was given asan immediate-release formulation using gelatin capsules
containing 4-AP 5 mg and microcrystalline cellulose asthe excipient. Capsules were prepared at our facilities.The day of the second acetaminophen absorption test (start-ing at 8:00 AM), patients omitted intake of the correspond-ing dose of 4-AP to avoid any interaction with the singledose of acetaminophen given for the study.
Subjects
Table 1 shows spinal cord injury/completeness levels of pa-tients subjected to the study. Age range of healthy male/fe-male volunteers was 18e40 years. Patients of both genderswere from 23 to 48 years old with both complete and in-complete injury and injury levels from C4 to T8; timeelapsed after injury was from 1.5e15 years (mean �SD:7.1 � 3.2 years).
Inclusion criteria were as follows: tetraplegia or paraple-gia for O1.5 years before the study began, aged 18e60years, medically stable and able to breathe independently,stable neurological deficits for O90 days before the study,absence of epileptic history and electroencephalogramwithout epileptic activity. Women had to be postmeno-pausal, surgically sterile, or using an acceptable methodof birth control. Exclusion criteria were as follows: pressureulcers, skin infections, or phlebitis; history of cardiovascu-lar disease; systolic blood pressure O150 or !70 mmHg,diastolic blood pressure O110 or !50 mmHg, or heart rateO110 or !50 beats/min; impaired hepatic function (totalhepatic enzyme or bilirubin levels greater than two timesthe upper limit of normal) or impaired renal function (cre-atinine level greater than two times the upper limit of
Table 1. Demographic and injury characteristics of study patients
(listed according to injury level)
Patient Age Gender
Post-injury
time
(years)
Type of
injury
Injury
level AIS
1 29 Male 11 BA C4 B
2 33 Female 10 MVA C4 B
3 35 Male 7 Diving C4 C
4 40 Male 10 MVA C4 A
5 48 Male 10 MVA C4 B
6 23 Male 1.5 Diving C5 B
7 25 Male 6 Fall C5 B
8 43 Male 15 MVA C5 D
9 29 Male 3 Diving C6 C
10 42 Male 7 Fall T1 D
11 26 Male 4 Gun shot T2 C
12 44 Male 6 Gun shot T2 B
13 35 Male 9 Gun shot T5 B
14 30 Female 11 MVA T5 A
15 44 Female 6 Gun shot T5 C
16 42 Male 6 MVA T6 B
17 23 Male 2.5 Gun shot T7 C
18 25 Female 7 Fall T8 B
AIS, ASIA impairment scale; BA, bicycle-riding accident; MVA, motor
vehicle accident.
394 Grijalva et al. / Archives of Medical Research 38 (2007) 392e397
normal) !6 months before the study; known allergy to pyr-idine-containing drugs; neurological, degenerative, or psy-chiatric disorders that would impair the patient’s ability tocomplete the protocol; any illness or abnormality thatwould jeopardize patient safety or interfere with the con-duct of the study; history of substance abuse; and inabilityto discontinue excluded concomitant drug therapy. Patientsalso were excluded if they were receiving treatment with anantispasticity compound and could not maintain a stabledaily dosage, were pregnant or lactating, had received anyother investigational drug !30 days before the study, orhad received any drug known to cause significant majororgan toxicity !3 months before the study.
Plasma Collection and Analysis
After 10 h fasting conditions, a heparinized, indwelling ve-nous cannula was placed in a dorsal vein of the forearm topermit sampling of blood for analysis of plasma concentra-tions of acetaminophen. Whole blood samples of 2.5 mLwere withdrawn at 0 (before acetaminophen dose) and at15, 30, 45, 60, 75, 90, 105 and 120 min postdose.
Each blood sample was drawn into a test tube containingsodium heparin and immediately tilted gently 6 to 10 timesto mix the blood and heparin (in order to prevent coagula-tion). Within 10 to 20 min, the sample was centrifuged at1200 rpm for 10 min. The plasma was then transferred intoa plastic vial, labeled, packed in dry ice, and stored at�70�C until analysis.
Acetaminophen concentration in plasma was determinedby a high-pressure liquid chromatography assay with UVdetection according to the methodology by Garcıa-Lopezet al. (33).
Individual plasma acetaminophen concentration vs.time curves were constructed, and conventional pharmaco-kinetic parameters such as maximum plasma concentration(Cmax) as well as the time to reach this maximum (tmax)were directly determined from these plots. The area underthe concentration-time curve (AUC) was estimated by thetrapezoid rule. A paired t-test was used for comparison ofthe results in SCI subjects between pre- and post-4AP-treatment. Comparison between control and SCI basal
measures were also performed by Students t-test in orderto detect possible alterations attributable to the SCI. Usinglinear regression analysis, we correlated AUC (differencesbetween pre- and post-treatment with 4-AP) to the level ofinjury and to the ASIA (American Spinal Injury Associa-tion) impairment scale. The pharmacokinetic and linearregression analysis were performed with the SPSS version10 and the GraphPad Prism version 3.02 software,respectively. Statistical significance was considered to bep !0.05.
Results
Results are summarized in Table 2. Gastric emptying ap-peared to be normal in patients with chronic SCI before4-AP treatment, taking into account that comparisons be-tween control and SCI basal measures were not significant.4-AP treatment significantly delayed gastric emptying,compared with results of the same patients before 4-APtreatment, as demonstrated by decreased acetaminophenabsorption after 4-AP treatment, with both Cmax and AUCvalues ( p !0.05). tmax remained unchanged. 4-AP treat-ment delayed gastric emptying by |20 and 31% in cervicaland thoracic patients, respectively. AUC changes after 4-APtreatment were independent of both the level of injury andthe ASIA impairment scale (r2 5 0.003 and 0.015, respec-tively, Figure 1).
Discussion
Our aim was to assess the effect of 12-week treatment with4-AP on gastric emptying of patients with chronic SCI. Weused the acetaminophen absorption technique, which offersa relatively simple and inexpensive technique that yields anindirect quantitative measurement of gastric emptying asverified by the scintigraphic emptying of liquid and solidmeals (31,32). Our main results deal with reduction in gas-tric emptying induced by 4-AP in patients with SCI at bothcervical and thoracic levels.
We observed that 4-AP treatment significantly decreasedacetaminophen Cmax and AUC values, while tmax valuesremained without changes. This pattern of changes suggests
Table 2. Pharmacokinetic parameters of a single dose of 1 g of oral acetaminophen solution before and after 12 weeks of 4-AP treatment
in patients with chronic spinal cord injury
Pre-treatment Post-treatment (4-AP)
Group n Cmax (mg/mL) tmax (min) AUC(0e2 h) mg/mL/min n Cmax (mg/mL) tmax (min) AUC(0e2 h) mg/mL/min
Patients 18 13.26 � 1.01 45.83 � 5.88 900.3 � 71.55 18 *10.55 � 0.93 45.81 � 3.92 *661.6 � 56.11
Control 9 13.30 � 0.70 42.00 � 5.10 895.0 � 123.8
Healthy volunteers without 4-AP were used as controls.
Cmax 5 maximum plasma concentration; tmax 5 time to reach maximum plasma concentration; AUC 5 area under the plasma concentration-time curve. Data
represent mean � standard error of the mean.
*Significant differences against pre-treatment ( p !0.05).
395Gastric Emptying Effect by 4-AP in SCI Patients
that 4-AP effect on acetaminophen absorption was due toa delayed gastric emptying. A tmax change could be ex-pected if two different acetaminophen formulations werecompared. If one formulation releases the drug faster thanthe other, the amount of drug arriving to the duodenum atshort times will be higher than with the other slow-releasingformulation. Thus, differences in tmax will be observed.
However, in our study, the same acetaminophen formu-lation, a solution, was used under two different conditions.Thus, there were no differences due to drug release, as thedrug is already dissolved. Hence, if gastric emptying is de-layed, and this is the only factor that plays a significant rolein drug absorption, a slower absorption is expected in thepresence of 4-AP. That is, a lower amount of drug will ar-rive to the duodenum at every sampling time. Therefore, theabsorption rate will not be slower at shorter times and fasterat longer times. It will be slower at all times. Hence, therewill be a lower plasma concentration at all sampling timeswithin the 2-h sampling period, but no shift in tmax.
Even though 4-AP has been tested in several studies ofSCI and multiple sclerosis, there are no previous reports re-garding its effect on gastrointestinal functions, althoughfrequent gastric irritation and other symptoms associated
Figure 1. Linear regression correlation analysis of the area under the
concentration-time curve (AUC) with both injury level and to ASIA
impairment scale (AIS). Points represent differences between baseline
(pre-treatment) and final (after 4-AP treatment) values of each studied
patient. There was no correlation between either the level of injury
(r2 5 0.0007) or the AIS impairment scale (r2 5 0.015).
with gastric dysfunction have been reported for patientsreceiving 4-AP (6).
The lack of correlation between AUC changes with boththe level of injury and the ASIA impairment scale suggeststhat direct influence of 4-AP on physiological gastric func-tion, regardless of neurological integrity, is the main causeof the adverse effect we observed. If so, such an effectcould be present in patients treated with 4-AP for otherdiseases, or even in healthy subjects.
The effect of 4-AP on gastric emptying of healthy con-trols was not assessed in this study, taking into account eth-ical issues, particularly the possibility of producing painfuladverse effects. However, experimental studies clearlyshow that 4-AP given orally inhibited gastrointestinal pro-pulsion of a charcoal suspension in healthy conscious mice(30).
To better understand our results, it is important to reviewsome data obtained from basic research. The physiologicalmechanisms that regulate gastric tone and motility are verycomplex. Adequate gastric emptying requires diverse neu-rohumoral events, which act in a coordinated mannerresulting in contraction of antral smooth muscles and relax-ation of the pylorus. Cholinergic and adrenergic, as well asnon-adrenergic, non-cholinergic neurotransmission playimportant roles in regulating contraction and relaxation ofthe gastrointestinal tract.
We propose that 4-AP at doses and times used here willdiminish potassium current properties in gastric muscle andmyenteric neurons, contribute to altering gastric emptying,and may lead to development of dyspeptic symptoms. 4-APis a known potassium-channel blocker (34). Taking intoaccount such a property, it has been used as a pharmacolog-ical tool in multiple studies on gastric function. 4-APinduces electrical and contractile activities on gastricmuscles of the cat (25), blocks voltage-sensitive transientpotassium current in stomatogastric neurons of the lobster,interferes with the pyloric motor rhythm (26), diminishesb3-adrenoceptor-mediated relaxations in guinea pig gastro-intestinal muscle (27), blocks A-type currents (due to Kv4channels) in murine antral muscle, alters the negativeresting membrane potentials (28), and stimulates gastricsecretion in rat (29).
4-AP also increases the release of several neurotransmit-ters including acetylcholine (35), noradrenaline (36), dopa-mine (37,38), and serotonin (39), all closely related togastrointestinal motility (40).
On the other hand, NO neurotransmission influencesgastric emptying and transpyloric flow by altering the pylo-ric tone (41,42). The effects induced by NO in differenttissues depend on direct and/or indirect interactions withpotassium channels. The blockage of potassium channelsproduced by 4-AP inhibits the neuromuscular effects in-duced by NO (43).
In conclusion, the present study extends the knowledgeregarding the effects of 4-AP on gastrointestinal motility
396 Grijalva et al. / Archives of Medical Research 38 (2007) 392e397
in patients with SCI and suggests that the significantlydelayed gastric emptying we described could be relatedto adverse gastrointestinal symptoms reported for somepatients treated with 4-AP.
AcknowledgmentsThe study was supported by the Instituto Mexicano del SeguroSocial and the Centro de Investigaciones y Estudios Avanzadosdel Instituto Politecnico Nacional, Mexico City, Mexico.
The authors thank Susan Holmes for helpful review of themanuscript.
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