Clarithromycin inhibits myometrial contractions in isolated human myometrium independent of stimulus with use of oscillograph

Clarithromycin Inhibits Myometrial Contractions in Isolated
Human Myometrium Independent of Stimulus
Department of Obstetrics and Gynaecology, and 1Department of Pharmacology, Faculty ofMedicine, Firat University, Elazig, Turkey Summary
Erythromycin has a well-known dual effect on the contractility of the gastrointestinal system and recently has also been
shown to inhibit contractions of the rat myometrium. The aim of the present study was to investigate the effects of
clarithromycin on oxytocin, prostaglandin F2α (PGF2α) and KCl-induced contractions of human myometrium in vitro.
Myometrial strips were obtained from pregnant women undergoing elective Cesarean section and the strips were
suspended in a jacketed organ bath filled with Krebs solution at 37 oC (pH 7.4) and continuously aired with 95 %
oxygen and 5 % carbon dioxide. Isometric contractions were measured using a force displacement transducer.
Oxytocin, PGF2α, KCl and clarithromycin were applied to the tissue bath and the amplitude and frequency of
contractions were evaluated at 20-min intervals. Freidmann analysis of variance, Kruskal Wallis and Wilcoxon Rank
tests were used for statistical analysis of the data. Clarithromycin dose dependently inhibited the amplitude of
contractions independent of the stimulus. Pre-treatment with apamin prevented clarithromycin-induced effects on
amplitude and frequency of contractions. We conclude that the macrolide antibiotic clarithromycin may have a direct
inhibitory effect on contractions of human myometrium.
Key words
Myometrium • Clarithromycin • Oxytocin • Isometric contraction • Human
Introduction
longitudinal smooth muscle of the guinea-pig smallintestine (Minocha et al. 1991) and in bronchial smooth It is well established that the macrolide antibiotic muscle (Tamaoki et al. 1995). However, the exact
erythromycin has a dual effect on the contractility of mechanism of inhibition induced by erythromycin is not smooth muscle. In smooth muscles of the stomach and known. Furthermore, a recent study has reported that duodenum, it has a stimulatory effect that was suggested erythromycin inhibits myometrial contractions of rat to be mediated by motilin receptors (Peeters et al. 1989, independent of stimulant (Granovsky-Grisaru et al. Collard et al. 1999). It also has a direct inhibitory effect 1998). Since structural analogs of erythromycin are not in guinea-pig and human gallbladder, the rat urinary antibiotic but are prokinetic, the modulatory effect of bladder smooth muscle (Nissan et al. 1999), in the erythromycin on smooth muscles contractions is not  2002 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic related to its antimicrobial activity (Omura et al. 1987).
Table 1. Socio-demographic characteristics of sample
Clarithromycin is the new member of the macrolide family of antibiotics with a broad spectrum of activity invitro against clinically important gram-positive and gram- Mean ±S.D.
negative aerobes and anaerobes. The modulatory effect ofclarithromycin on contractility of smooth muscle has been poorly studied compared to erythromycin, with the exception of one study evaluating its effect on The aim of the present study was to investigate whether clarithromycin has any similar effect as itsprogenitor, erythromycin, on myometrial contractility. Toour knowledge, our study shows for the first time the The contractile activities were recorded using a Harvard inhibitory effects of clarithromycin using human isolated Universal Oscillograph (Harvard Apparatus Limited, Kent, England). The myometrial strips were initiallyplaced under 2.0 g tension and a 90-min equilibration period was allowed before the start of each experiment.
Most of the strips developed spontaneous contractions The protocol of this study was approved by the within 30 to 90 min and strips with no spontaneous Firat University Local Ethics Committee for Research on activity in this period were discarded. After development Human Subjects and a written informed consent was of spontaneous contractions 800 mU/l oxytocin, PGF2α obtained from each subject donating a tissue sample.
(1 µM) or KCl (30 mM) was applied to amplify these Patients undergoing elective Caesarean section, gestation period of 37-42 weeks, in the Department of Obstetrics The amplitude of contractions was evaluated at and Gynecology at The Firat University Medical Center 20 min periods before (control) and after application of were included in this study. Patients with medical clarithromycin. The first minute of the control period was problems, including diabetes, hyperthyroidism, taken as the starting point for this comparison. The mean hypertension, pre-eclampsia or connective tissue diseases amplitude of the contractions under control conditions were excluded from this study. None of the subjects were and after application of clarithromycin were determined under any regular medication. Socio-demographic and compared. The frequencies of contractions were also characteristics of sample donating patients are given in evaluated at 20 min intervals before and after application Table 1. Two myometrial strips were used from each of clarithromycin. In some experiments, after amplification of spontaneous contractions with agonists A single full-thickness myometrial strip was (oxytocin, PGF2α or KCl), apamin was applied for 5 min removed from the upper margin of the lower uterine segment incision at the time of the Caesarean section The agents used in the present study included after the infant had been delivered. The strip was than clarithromycin lactobionate (Abbot Laboratories, immediately placed in a Krebs’ solution. Small strips intravenous injection, Wiesbaden, Germany), oxytocin (10 x 2 x 2 mm) of myometrial tissue were cut from (Synpitan iv, 5IU/ml, Deva, Istanbul, Turkey), PGF2α, uterine samples. Each strip was placed in a 20 ml KCl (Sigma) and apamin (Alomone Labs, Israel).
jacketed tissue bath containing Krebs’ solution at 37 0C Statistical analysis: The results are expressed as means and pH 7.4. The composition of the Krebs’ solution was ± S.D. All statistical analysis was performed using the (in mmol/l): sodium chloride 121, potassium chloride 4.5, statistical program SPSS for Windows (version 6.0.1, sodium bicarbonate 15.5, sodium phosphate 1.2, calcium chloride 2.5, magnesium chloride 1.2, and glucose 11.5.
The Krebs’ solution was constantly gassed with 95 % O2– Wilcoxon Rank test. Figures were made using Origin 5 % C02. A silk thread was used to attach the myometrial version 5.0 (Microcal Software Inc. Northampton, USA).
strips to a fixed hook and an isometric force displacementtransducer (Harvard Apparatus Limited, Kent, England).
Clarithromycin Inhibits Contractions of Isolated Human Myometrium 241
tracing showing the effectof 0.5 mM clarithromycinon oxytocin-induced myo-metrial contractile activityreported in grams oftension generated. Sponta-neous contractions werenoted during period pre-ceding 20-min time point;subsequently oxytocin andclarithromycin was ap-plied at points indicated bythe arrow. dependent. 0.1 mM clarithromycin had no significant A total of 300 myometrial strips from 150 effect on either parameter of agonist-induced contractions patients were used in this study. 284 out of these strips (Tabs. 2, 3, p>0.05). The inhibition of the amplitude and developed spontaneous contractions (in 95 % cases).
increased frequency of contractions were significant after Each dose of clarithromycin was tested on 30, application of 0.2 mM clarithromycin (Tabs. 2, 3, 20, and 10 myometrial strips contracted by oxytocin, The inhibitions of the amplitude of oxytocin- Myometrial strips were contracted by application induced contractions were 5 %, 15 %, and 44 % after of oxytocin (800 mU/l), PGF2α (1 µM) and KCl (30 mM).
applications of 0.2 mM, 05 mM (Fig. 1) and 1 mM The mean peak amplitude and frequency values after clarithromycin, respectively (Tab. 2). The effects of 0.2 each agonist and the dose-dependent effects ofclarithromycin on these parameters are given in Table 2.
Table 2. Dose-dependent effects of clarithromycin on peak amplitude of agonist-induced (oxytocin, PGF2α or KCl)
myometrial contractions.
Oxytocin
P
P
P
Cla: clarithromycin, PGF2α: prostaglandin F2α, KCl: potassium chloride. Data are presented as mean ± SD (each doseof clarithromycin was tested on n= 30 strips for oxytocin-induced, n= 30 strips for PGF2α-induced and n= 10 strips forKCl-induced contractions, lactobionat was tested on n=7 strips for each agonist, apamin+ 1 mM clarithromycin was tested on n=8 strips for each agonist), p<0.05 statistically significant. mM, 0.5 mM, and 1 mM clarithromycin enhanced the The effects of clarithromycin on the PGF2α- frequency of oxytocin-induced contractions by 24 %, induced contractions led to a 7 %, 12 % and 55 % 66 % and 79 %, respectively (Tab. 3).
decrease in amplitude (Tab. 2), while an increase of 29 %, 59 % or 83 % of frequency was found for 0.2, 0.5 investigate the possible role of Ca2+-activated K+ channels on the inhibitory action of clarithromycin on the clarithromycin was applied to myometrium contracted amplitude of agonist-induced contractions using apamin.
with KCl there was an inhibition in amplitude of 18 %, For these experiments, 1 mM dose of clarithromycin were 36 % and 65 % (Tab. 2), and an increase of 12 %, 47 % applied on oxytocin, PGF2α or KCl-induced contractions.
and 59 % in frequency of contractions (Tab. 3).
After pre-treatment with apamin (1 µM) for 5 min, When lactobionat, the vehicle of clarithromycin, application of clarithromycin had no significant effect on was applied in comparable concentrations (1 mM), it had amplitude and frequency of contractions independent of no significant effect on either amplitude or frequency of stimulant (n=8 for each agonist, Tabs. 2, 3).
agonist-induced contractions of myometrium (n= 7 foreach agonist, Tabs. 2, 3).
Table 3. Dose-dependent effects of clarithromycin on frequency of agonist-induced (oxytocin, PGF2α or KCl)
myometrial contractions.
Oxytocin
P
P
P
Cla: clarithromycin, PGF2α: prostaglandin F2α, KCl: potassium chloride. Data are presented as mean ± SD (each doseof clarithromycin was tested on n= 30 strips for oxytocin-induced, n= 30 strips for PGF2α-induced and n= 10 strips forKCl-induced contractions, lactobionat was tested on n=7 strips for each agonist, apamin+1 mM clarithromycin was tested on n=8 strips for each agonist). Frequency reflects number of contractions in 20 minute-periods. p<0.05 Discussion
reports about inhibitory effects of neomycin, gentamycinand clindamycin on myometrial contractions induced by The data presented in this study demonstrate the oxytocin, KCl or aluminium fluoride (Phillipe 1994, ability of clarithromycin to inhibit oxytocin, PGF2α, and KCl-induced contractile activity of uterine tissues from The finding of inhibition at peak amplitude of pregnant women at term. However, clarithromycin also myometrial contractions by clarithromycin is consistent caused an increase in frequency of contractions with a result of the previous study where other macrolide independently of stimulant. The inhibitory effect of erythromycin was used (Granovsky-Grisaru et al. 1991).
clarithromycin was dose dependent, significant inhibition However, contrary to erythromycin, clarithromycin on peak amplitude started after 0.2 mM whereas 0.1 mM increased the frequency of contractions. This may be due had no significant effect either on amplitude or to species differences but there may also be a mechanistic difference involved. Similar to clarithromycin, Previous studies have shown inhibitory effects erythromycin inhibited the amplitude but increased the of antibiotics on myometrial contractions. The finding of frequency of oxytocin-induced contractions in isolated the inhibitory effect of erythromycin on rat myometrial pregnant human myometrium (Celik et al. 2001).
contractions by Granovsky-Grisaru et al. (1998) was the Another possible explanation of the different effects of starting point of the present study. Similarly, there are these two macrolides could be due to the different uterus Clarithromycin Inhibits Contractions of Isolated Human Myometrium 243
parts being studied. We obtained myometrial samples role in modulation of uterine contractility (Anwer et al. from the lower uterine segment whereas Granovsky- Grisaru et al. (1991) used the uterine horns. However, the We have no explanation for the increase in explanation by anatomical difference is not very frequency of contractions after clarithromycin convincing because there were no significant differences application. This could be the result of increased in the contractile rate and force production produced by pacemaker activity that is considered to be related with myometrium from the upper and lower segments when T-type Ca2+ channels (Wray 1993). But the amplitude of biopsies were obtained from women undergoing classical contractions gradually decreased (Fig. 1). Since apamin Caesarean section (Luckas et al. 2000).
prevented the inhibitory effect of clarithromycin, it seems The inhibitory activity of clarithromycin in the likely that this is related to the increase in frequency and uterine tissue may be the result of activation of adenylate inhibition via activation of Ca2+-activated K+ channels.
cyclase, but also from other effects such as the opening of Clarithromycin may be activating the T-type calcium potassium channels and a decrease of intracellular free channels and the resultant increase in free intracellular Ca2+ levels (Wray 1993). Although no direct evidence is Ca2+ may in turn activate the Ca2+-activated K+ channels.
available, it is also possible that clarithromycin may The inhibitory effects of clarithromycin on the inhibit Ca2+ entry through the L-type calcium channels contractility found in this study made us to consider the and thereby cause inhibition of amplitude of agonist- possibility that this could be used in the treatment of induced myometrial contractions (Kaya et al. 1999, preterm labour. There are some limitations regarding this Sanborn 2001). Inhibition of KCl-induced contractions point. Firstly, the dose of clarithromycin used in this by clarithromycin provides evidence for the latter study is higher than the levels achieved after its therapeutic administration (its peak plasma concentration is 2.4 µg/ml after a 500-mg oral dose) (Cheng et al. contractions induced by oxytocin, PGF2α, and KCl 1998). Secondly, the use of clarithromycin in pregnancy suggesting that the effect of clarithromycin is has not been studied thoroughly and available data on independent of the stimulant. High concentrations of KCl clarithromycin are too limited for recommendation its use induce contractions by membrane depolarization and a during pregnancy. It was found in a study performed on a subsequent increase in free intracellular Ca2+ restricted population of patients that clarithromycin concentration resulting from influx via membrane Ca2+ increased spontaneous miscarriage in the first trimester channels (Wray 1993, Trujillo et al. 2000). Oxytocin without any evidence of teratogenic actions (Einarson et causes increase in intracellular free Ca2+ mediated by al. 1998). Further studies need to be performed in order inositol 1,4,5-triphosphate (IP3) formation while to determine the effects of clarithromycin in this respect prostaglandins increase intracellular free Ca2+ via voltage and relation to the gestational age should be addressed.
or receptor-operated Ca2+ channels (Wray 1993). The There is no report in the literature about the effects of only evidence relating the mechanism underlying the clarithromycin on the contractility of smooth muscles.
inhibitory effect of clarithromycin was that the specific In conclusion, we have shown for the first time Ca2+-activated K+ channel blocker apamin prevented the the inhibitory effects of clarithromycin on agonist- inhibitory effect of clarithromycin on oxytocin-induced induced contractions of the isolated myometrium from contractions suggesting involvement of Ca2+-activated K+ channels. These channels are reported to play important References
ANWER K, OBERTI C, PEREZ GJ, PEREZ-REYES N, MCDOUGALL JK, MONGA M, SANBORN BM, STEFANI E, TORO L: Calcium-activated K+ channels as modulators of human myometrial contractile activity. Am J
Physiol 265: C976-C985, 1993.
CELIK H, AYAR A, SAPMAZ E: Effects of erythromycin on stretch-induced contractile activity of isolated myometrium from pregnant women. Acta Obstet Gynecol Scand 80: 697-701, 2001.
CHENG KL, NAFZIGER AN, PELOQUIN CA, AMSDEN GW: Effect of grapefruit juice on clarithromycin pharmacokinetics. Antimicrob Agents Chemother 42: 927-929, 1998.
CLESHAM GJ: Beta adrenergic agonists and pulmonary oedema in preterm labour. Br Med J 308: 260-262, 1994.
COLLARD JM, ROMAGNOLI R, OTTE JB, KESTENS PJ: Erythromycin enhances early postoperative contractility of the denervated whole stomach as an esophageal substitute. Ann Surg 229: 337-343, 1999.
COPPER RL, GOLDENBERG RL, CREASY RK, DUBARD MB, DAVIS RO, ENTMAN SS, IAMS JD, CLIVER SP: A multicenter study of preterm birth weight and gestational age-specific neonatal mortality. Am J Obstet
Gynecol 168: 78-84, 1993.
CUNNINGHAM FG, MACDONALD PC, GANT NF, LEVENO KJ, GILSTRAP RC, HANKINS GDV, CLARK SL: Williams Obstetrics. 20th Ed, p: 306 Appleton Lange, Philadelphia, 1997.
EINARSON A, PHILLIPS E, MAWJI F, D'ALIMONTE D, SCHICK B, ADDIS A, MASTROIACOVA P, MAZZONE T, MATSUI D, KOREN A: Prospective controlled multicentre study of clarithromycin in
pregnancy. J Perinatol 15: 523-525, 1998.
GIBBS RS, ROMERO R, HILLIER SL, ESCHENBACH DA, SWEET RL: A review of premature birth and subclinical infection. Am J Obstet Gynecol 166: 1515-1528, 1992.
GOODWIN TM, MILLAR L, NORTH L, ABRAMS LS, WEGLEIN RC, HOLLAND ML: The pharmacokinetics of the oxytocin antagonist atosiban in pregnant women with preterm uterine contractions. Am J Obstet Gynecol
173: 913-917, 1995.
GOODWIN TM, PAUL R, SILVER H, SPELLACY W, PARSONS M, CHEZ R, HAYASHI R, VALENZUELA G, CREASY GW, MERRIMAN R: The effect of the oxytocin antagonist atosiban on preterm uterine activity in
the human. Am J Obstet Gynecol 170: 474-478, 1994.
GRANOVSKY-GRISARU S, ILAN D, GRISARU D, LAVIE O, ABOULAFIA I, DIAMANT YZ, HANANI M: Effects of erythromycin on contractility of isolated myometrium from pregnant rats. Am J Obstet Gynecol 178:
171-174, 1998.
KADANALI S, DEMIR N, APAYDIN S: In vitro relaxant effects of gentamycin and clindamycin on human myometrium at term: A preliminary study. New J Med 13: 75-78, 1996.
KATZUNG BG: Basic and Clinical Pharmacology, 7th ed, Appleton & Lange, 1998.
KAYA T, CETIN A, CETIN M, SARIOGLU Y: Effects of endothelin-1 and calcium channel blockers on contractions in human myometrium. A study on myometrial strips from normal and diabetic pregnant women. J Reprod
Med 44: 115-21, 1999.
LETTIERI L, VINTZILEOS A, RODIS JF, ALBINI SM, SALAFIA CMJ: Does "idiopathic" preterm labor resulting in preterm birth exist? Am J Obstet Gynecol 166: 1480-1485, 1993.
LUCKAS MJ, WRAY S: A comparison of the contractile properties of human myometrium obtained from the upper and lower uterine segments. Br J Obstet Gynaecol 107: 1309-1311, 2000.
MCGREGOR JA: Adjunctive erythromycine treatment for idiopathic preterm labor: results of a randomized, double blinded, placebo controlled trial. Am J Obstet Gynecol 154: 98-103, 1986.
MINOCHA A, GALLIGAN JJ: Erythromycin inhibits contractions of nerve muscle preparations of the guinea pig small intestine. J Pharmacol Exp Ther 257: 1248-1252, 1991.
NISSAN A, MAUDLEJ N, BEGLAIBTER N, HASKEL Y, FREUND HR, HANANI M: A direct inhibitory effect of erythromycin on rat urinary bladder smooth muscle. J Urol 161: 1006-1009, 1991.
OMURA S, TSUZUKI K, SUNAZUKA T, MARUI S, TOYODA H, INATOMI N, ITOH Z: Macrolides with gastrointestinal motor stimulating activity. J Med Chem 30: 1941-1943, 1987.
PEETERS TG, MATTHIJS G, DEPOORTERE I, CACHET T, HOOGMARTENS J, VANTRAPPEN G: Erythromycin is a motilin receptor agonist. Am J Physiol 257: 470-474, 1989.
PERITI P, MAZZEI T: Clarithromycin: pharmacokinetic and pharmacodynamic interrelationships and dosage regimen.
J Chemother 11: 11-27, 1999.
PHILLIPE M: Neomycin inhibition of hormone stimulated smooth muscle contractions in myometrial tissue. Biochem Biophys Res Commun 1205: 245-250, 1994.
ROMERO R, SIBAI B, CARITIS S, PAUL R, DEPP R, ROSEN M, KLEBANOFF M, SABO V, EVANS J, THOM E: Antibiotic treatment of preterm labour with intact membranes: a multicenter, randomized, double-blinded,
placebo-controlled trial. Am J Obstet Gynecol 169: 764-774, 1993.
Clarithromycin Inhibits Contractions of Isolated Human Myometrium 245
SMITH GN, WALKER MC, McGRATH MJ: Randomised, double-blind, placebo controlled pilot study assessing nitroglycerin as a tocolytic. Br J Obstet Gynaecol 106: 736-739, 1999.
SANBORN BM: Hormones and calcium: mechanisms controlling uterine smooth muscle contractile activity. The Litchfield Lecture. Exp Physiol 86: 223-837, 2001.
TAMAOKI J, TAGAYA E, SAKAI A, KONNO K: Effects of macrolide antibiotics on neurally mediated contraction of human isolated bronchus. J Allergy Clin Immunol 95: 853-859, 1995.
TRUJILLO MM, AUSİNA P, SAVINEAU JP, MARTHAN R, STRIPPOLI G, ADVENIER C, PINTO FM, CANDENAS ML: Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the
estrogen-primed rat myometrium. Life Sci 66: 2441-2453, 2000.
WEBER FHJR, RICHARDS RD, MCCALLUM RW: Erythromycin: a motilin agonist and gastrointestinal prokinetic agent. Am J Gastroenterol 88: 485-490, 1993.
WRAY S: Uterine contraction and physiological mechanisms of modulation. Am J Physiol 264: C1-C18, 1993.
ZARA G, QIN XY, PILOT M: Response of the human gastrointestinal tract to erythromycin. J Gastro Motil 3: 26-31,
Reprint requests
Dr. Ahmet AYAR, Firat University, Faculty of Medicine (TIP FAK), Department of Pharmacology, Tr-23119, Elazig,
Turkey, Fax: + 90 424 237 91 38, e-mail: aayar@firat.edu.tr

Source: http://www.harvardapparatusregen.com/media/harvard/pdf/TD03UN_1.pdf