ProstaglandinendoperoxideHsynthaseinhibitorsandother ...olsonlab/Prostaglandins Leukot Essent...

Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187

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*Correspondi

Alberta, 220 HM

780-492-2765; fa

E-mail addre

0952-3278/$ - see

doi:10.1016/j.ple

Prostaglandin endoperoxide H synthase inhibitors and othertocolytics in preterm labour

Bryan F. Mitchella, David M. Olsona,b,c,*aDepartment of Obstetrics and Gynaecology, Perinatal Research Centre, CIHR Group in Perinatal Health and Disease, University of Alberta,

220 HMRC, Edmonton, Alberta, Canada T6G2S2bDepartment of Pediatrics, University of Alberta, Edmonton, Alberta, CanadacDepartment of Physiology, University of Alberta, Edmonton, Alberta, Canada

Received 1 April 2003; accepted 1 April 2003

Abstract

Preterm delivery (o37 weeks of gestation) is the major obstetrical complication in developed countries, yet attempts to delay

labour and prolong pregnancy have largely been unsuccessful. One of the many reasons it is so difficult to prevent preterm birth is

that the nature of preterm labour changes as a function of gestational age, maternal lifestyle factors or infection, to list a few of the

reasons. The inhibitors of prostaglandin endoperoxide H synthase (PGHS), known as the Non-steroidal Antiinflammatory Drugs,

have been viewed with interest as tocolytics with promising effectiveness under most conditions of preterm labour. Three isoforms of

PGHS exist; the first two, PGHS-1 and -2, have been studied for their catalytic activity, X-ray crystallographic structure, and

physiological roles in the adult and the foetus. Mixed inhibitors and isoform-specific inhibitors of PGHS have been developed, and

their roles in delaying preterm labour are examined and compared to other tocolytics.

r 2003 Elsevier Ltd. All rights reserved.

Keywords: Preterm birth; Tocolytics; Prostaglandin; Cyclooxygenase; Prostaglandin endoperoxide H synthase; COX-1; COX-2; Fetus

1. Introduction

Preterm delivery (o37 weeks of gestation) is themajor obstetrical complication in developed countries[1]. It occurs in 5–10% of births and is associated with>75% of perinatal death and long-term infant dis-ability. Accordingly, a great deal of basic science andclinical research has focused on the regulation of uterinecontractions and prevention of preterm labour. Devel-opment and evaluation of drugs to stop uterinecontractions—called tocolytic agents—have been thesubjects of hundreds of research papers. Despite theseefforts, there is little indication that they have had anyfavourable impact on the clinical problem of pretermbirth. For example, in Canada, the incidence of pretermdelivery in 1997 was 7.1% [2]. This represents asignificant increase over the previous two decades. Three

ng author. Perinatal Research Centre, University of

RC, Edmonton, Alberta, Canada T6G 2S2. Tel.: +1-

x: +1-780-492-1308.

ss: [email protected] (D.M. Olson).

front matter r 2003 Elsevier Ltd. All rights reserved.

fa.2003.04.006

main factors explained the increasing incidence: betterascertainment due to better pregnancy dating throughuse of ultrasound; increasing frequency of iatrogenicdelivery prior to 37 weeks gestation for maternal orfoetal indications; increased incidence of multiplepregnancies mainly due to more frequent use of assistedreproductive technologies. The Canadian Perinatal

Health Report 2000 [3] issued by Health and WellnessCanada continues to emphasize that, ‘‘Preterm birthaccounts for 75–85% of all perinatal mortality inCanada and is an important determinant of neonataland infant morbidity, including neurodevelopmentalhandicap, chronic respiratory problems, infections andophthalmologic problems.’’ It is often thought thatdelaying preterm birth for those pregnancies less than 30weeks gestational age, which represents 1% of births [3],is most important and that if the pregnancy can achieve30 weeks, then the greatest concern is past. However,recent evidence showed that children at age 10, whowere born between 30 and 34 weeks, had significantlydiminished language development [4]. This demon-strates that prematurity at any gestational age can carry

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myometrial cell membrane

adenylate cyclase

cAMP

-agonists

cGMP

?? NO

phospho-lipase C

prostaglandinsoxytocin

Ca++

actinmyosin

MLCK

Fig. 1. The major stimulatory pathway for uterine contractions

involves membrane receptors coupled through G-proteins to phos-

pholipase C that, upon activation, leads to increased intracellular

Ca2+, activation of MLCK and muscle contraction. The predominant

inhibitory pathway also involves membrane receptor activation and G-

protein coupling but the second messenger system involves stimulation

of adenylyl cyclase and production of cAMP that inhibits increased

intracellular Ca2+ and inactivates MLCK. Nitric oxide may stimulate

B.F. Mitchell, D.M. Olson / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187168

risks. Therefore, the health, emotional and societalexpense of preterm birth is substantial, costing in thebillions of dollars each year [1]. The prevention ofpreterm birth is considered the most important perinatalchallenge facing industrialized countries [1,5].In this chapter, we will discuss the use of PGHS

inhibitors as potential treatment to prevent pretermbirth. We also shall briefly discuss the use of alternatetherapies that have been tested in randomized controlledtrials. Though the detailed experimental data describingthe role of prostaglandins (PGs) in parturition areprovided in other chapters of this volume, we will beginwith a brief overview of the physiology of uterinecontractility as necessary to understand the potentialmechanisms of action of the putative tocolytic agents.

guanylyl cyclase with subsequent increase in cGMP and inhibition of

MLCK or it may act through other undetermined pathways.

2. Uterine contractions

This subject will be addressed in greater detail inanother chapter in this volume. But it is necessary tobriefly review it here because therapies to delay pretermbirth are based upon what is known about thebiochemistry and physiology of birth. There are fiveseparate physiological events of parturition: membranerupture, cervical dilation, myometrial contractility,placental separation, and uterine involution [6]. Thus,although the uterus is the effector organ of labour, it isrecognized that the overall control of parturition is notlimited to changes in uterine contractility alone. Sinceuterine contractility is the most studied of the fivephysiological events of parturition, we will focus on it asthe endpoint of our discussion and a model for the otherphysiological processes.Throughout most of pregnancy, the uterus is in a

resting phase and is poorly responsive to manycontractile stimulants such as oxytocin. This phase ischaracterized by the long-lasting, low amplitude con-tractions referred to as ‘‘contractures’’ in sheep and‘‘Braxton-Hicks’’ contractions in women. Near the timeof parturition, the uterus leaves this ‘‘quiescence’’ phaseand enters the phase of ‘‘activation’’ [7]. During thistransition, the uterus attains increased concentrations ofreceptors to contractile stimulants and increases itscontent of gap junctions that provide rapid electricalcoupling between muscle cells, thus enabling the strong,coordinated contractions characteristic of active labour.With the appearance of appropriate stimulants, includ-ing PGs, oxytocin and likely many others, the uterusenters the ‘‘stimulation’’ phase with strong and regularcontractions that result in cervical effacement anddilation and descent of the foetus through the birthcanal eventuating in birth.As in other smooth muscle, the contractile force of the

myometrium is dependent on phosphorylation ofmyosin and subsequent shortening of the actin-myosin

filaments of the uterine muscle. This reaction isregulated by the enzyme, myosin light chain kinase(MLCK). There are three principal intracellular signal-ling pathways that may regulate MLCK and hence areimportant targets of current tocolytic drugs (Fig. 1). Thereceptors for PGF2a and for oxytocin are G-proteincoupled to membrane phospholipase C. Stimulation ofthese pathways lead to increased intracellular calciumthat activates MLCK resulting in uterine contractions.In contrast, another subset of G-protein coupledreceptors, of which the b-adrenergic agonist receptor isa representative example, are linked to adenylyl cyclase.Stimulation of these receptors will increase intracellularcAMP that in turn lowers calcium and inactivatesMLCK, thus inhibiting uterine contractions. In ananalogous manner, stimulation of guanylyl cyclase andgeneration of cGMP by nitric oxide donors may havethe same effect though there is much less data to supportthe physiologic relevance of this pathway in the uterus.Before describing tocolytic approaches to the arrest of

preterm labour, we will first describe briefly the synthesisof PGs, their relationship to labour at term and preterm,and how the nature of preterm labour changesduring gestation. This information will establishimportant perspectives upon which to review the dataon tocolytics.

3. Prostaglandin synthesis

PGs are 20-carbon chain fatty acids that function aslocal hormones and are produced by all cells of thebody. Biologically active eicosanoids (PGs, leukotrienes,lipoxins, and other 20-carbon fatty acids) are formedfrom the polyunsaturated fatty acid, arachidonicacid (5,8,11,14-cis eicosatetraenoic acid). Arachidonicacid is a common constituent of phospholipids in all

ARTICLE IN PRESSB.F. Mitchell, D.M. Olson / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187 169

membranes within a cell, cholesteryl esters and triglycer-ides. The liberation of arachidonic acid from phospho-lipids is the initial step in the synthesis of PGs. This isaccomplished directly by the catalytic action of membersof the phospholipase A2 (PLA2) family of enzymes, orindirectly by the action of phospholipase C (PLC).The second step in PG synthesis is the oxygenation

and reduction of arachidonic acid to form an unstableintermediate endoperoxide. This step is catalyzed by PGendoperoxide H synthase (PGHS) or cyclooxygenase.Two isoforms of PGHS have been identified and wellstudied, PGHS-1 and -2. They are both homodimeric,haeme-containing, glycosylated proteins which catalyzetwo enzyme reactions, a cyclooxygenase reaction and aperoxidase reaction, forming, first, PGG2, which thenundergoes a two electron reduction to PGH2. Themature processed forms of PGHS-1 and -2 have a greatdeal of homology in their 576 and 587 amino acids,respectively, [8] between 60% and 65% identity betweenisoforms within a species and 85–90% sequence identityfor similar isoforms between species. The main excep-tions are six residues in from the C-terminal region ofPGHS-2, an extra 18 amino acids are inserted. The lastfour may facilitate binding to the nuclear and endo-plasmic reticulum membranes or the entire insertionmay mark the isoform for rapid proteolysis [9,10].PGHS-2 also lacks 17 amino acids that are present in theN-terminal region of PGHS-1. However, the amino acidresidues thought to be important for catalysis areconserved [11], and the two isoforms have about thesame affinity (Km) and capacity (Vmax) to convertarachidonic acid to PGH2 [12].The X-ray crystallographic forms of PGHS-1 and -2

are nearly superimposable. But it is the substitution ofvaline in PGHS-2 for isoleucine in PGHS-1 at positions434 and 523 (the residues in PGHS-2 are given theequivalent number as their counterparts in PGHS-1)that permits the design of inhibitors that are specific forPGHS-2 or -1. The smaller size of the Val 523 exposes aside-pocket off the main substrate channel in PGHS-2,which increases the volume of the PGHS-2 active site, afact that is exploited by specific inhibitors of PGHS-2.The longer side chain of isoleucine in PGHS-1 preventsaccess to this side pocket and thereby considerablylowers specificity of PGHS-1 inhibitors or non-selective non-steroidal antiinflammatory drugs(NSAIDs) [13–16].The single best characterized distinction between

PGHS-1 and PGHS-2 is their differential regulation ofexpression. PGHS-1 can be detected in most tissuesalthough not within all cells of a tissue and is thereforeconsidered to be ‘‘constitutive’’, or constantly expressed[17]. On the other hand, PGHS-2 is found at variablelevels in tissues and is expressed only in response tocytokines, growth factors, or tumour promoters [18],hence it is known as the ‘‘inducible’’ enzyme. PGHS-2 is

more highly concentrated on the nuclear envelope thanPGHS-1 [19], and oxygenates lower concentrations ofarachidonic acid (o1mM) more efficiently than PGHS-1 [20], suggesting that arachidonate can be streamedwithin a cell for preferential oxygenation by PGHS-2.A third distinct isoform of PGHS has been discov-

ered, PGHS-3 or COX-3 [21]. Along with a partialCOX-1 protein, it is derived from PGHS-1 but retainsintron 1 in its mRNA. It is found in the cerebral cortexof dogs followed by heart and in lesser amounts in othertissues including placenta and foetal tissues. COX-3 isinhibited more readily by acetaminophen than is PGHS-1, but aspirin and indomethacin are among its mostpotent inhibitors. Indeed, its presence was predicted bythe properties of acetaminophen, an NSAID with potentantipyretic and analgesic actions, but relatively littleantiinflammatory function, which was not characteristicof other NSAIDs that are better inhibitors of PGHS-1or -2 [22,23]. At this time there is no evidence whetherCOX-3 synthesizes PGs for the physiological processesof birth.The third enzymatic step of PG synthesis is the

conversion of PGH2 to one of the biologically activePGs (D2, E2, F2a, I2, or TXA2). It is presumed that mostindividual cell types contain primarily one isomerase,reductase, or synthase that converts the endoperoxide toone PG characteristic of that cell. Although researchershave examined this level of synthesis over the past 2years, there is little evidence to suggest that this step iseither rate limiting or regulated in terms of PG synthesisin the human foetal membranes. We believe there maybe some regulated isomerase or reductase activity in thedecidua, but further research is necessary in this regard.The PGs are now recognized as the ‘‘triggers’’ of

labour [24] because the myometrium contracts inresponse to exogenous PGs, in vivo and in vitro[25–28], PG synthetic enzymes (including PGHS-2mRNA in humans as we first showed) and levels inuterine tissues and fluids increase before or at the time oflabour [29–37], and inhibitors of PG synthesis decreaseuterine contractility, in vitro [38–40], and delay birthand prolong pregnancy, in vivo [24,41–44].

4. The changing nature of preterm labour

From a mechanistic point of view, we know very littleabout human preterm birth compared to what is knownabout term birth. It is clear, however, that preterm birthis different from term birth, and that preterm birth at 24weeks is different from preterm birth at 34 weeks. Oneexample of the differences between term and pretermbirth is the abundance or activity of PG synthetic ormetabolic enzymes and concentrations of maternalplasma and foetal amnion PGs. For instance, theconcentrations of tissue PGE2 and PGF2a in amnion

ARTICLE IN PRESSB.F. Mitchell, D.M. Olson / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187170

and placenta are significantly lower at preterm birththan at term birth [45]. This is due to the specific activityof PGHS, which we showed is considerably lower atpreterm labour than at term labour in human foetalmembranes. The specific activity in the human amnionrises from 672 to 2877 pg PGE2/mg protein/min inwomen delivered by cesarean section preterm but not inlabour to those following spontaneous preterm labour[46]. This contrasts to the term birth levels, which aremuch higher, rising from 1874 to 3976 pg PGE2/mgprotein/min in women delivered by elective cesareansection at term to those delivering spontaneously atterm. The situation in the chorion is similar except thatthe preterm levels rise proportionally less, from approxi-mately 972 to 2273 and from 1773 to 3272 (allincreases are Po0:05) at term labour [33–35]. Thesedata are confirmed by Sadovsky et al. [40] where theyshowed that although human amnion PGHS-2 proteinmass and PGE2 concentrations rise with labour atpreterm or term, the levels at preterm labour are equalto or lower than in non-labouring term tissues. Hencethe PG synthetic capacity of membranes is considerablylower at preterm birth than at term birth when there areno signs of infection.These foetal membrane PGs may interact with

receptors in the foetal membranes [47,48] or diffuse orbe transported to decidua or myometrium. However,high levels of chorionic prostaglandin 15-hydroxydehydrogenase (PGDH), the primary enzyme thatcatalyzes PGs into inactive metabolites, prevents intactPGs from the foetal or maternal compartments fromcrossing over to the other side [49–51]. In light ofinformation (below) regarding high decidual PG syn-thetic capacity throughout gestation, this metabolicbarrier may be acting opposite to conventional thought,that is it may prevent maternal PGs from interactingwith foetal membrane PG receptors which might lead toactivation of mediators, such as matrix metalloprotei-nases (MMPs) [52], that promote membrane rupture. Inpathological (infected) preterm birth or in about 15% ofidiopathic preterm births, however, the specific activityof PGDH in chorion decreases, potentially allowinggreater diffusion or transport of PGs across the chorionto facilitate myometrial contraction [53,54]. This capa-city for PGs to traverse the chorion intact under theseconditions still remains to be shown directly.The PGHS synthetic capacity in decidua throughout

gestation is different than in foetal membranes. NeitherPGHS-1 nor -2 mRNA abundance, enzyme activity orprotein concentrations change in decidua during gesta-tion or with labour onset in women [29,40] or inbaboons [55]. The specific activity of (total) PGHS isvery high (11173 pg PGE2/mg protein/min) in decidua,about 3- to 4-fold greater than in foetal membranes.Some controversy exists whether PG concentrations oroutput from the decidua increase with term labour onset

as some studies showed no changes [40,56] while otherssuggest that there is an increase in PG output fromdecidual cells with labour at term [57–60] which mayreflect changes in the specific PGE and F synthases. Onereport indicates there is no increase in PG output indecidua with preterm labour [40]. The human myome-trium does demonstrate an increase in PGHS-2 mRNAwith term labour onset [61], but another report [40]indicates that no increase in PGE2 output occurs withpreterm or term labour. However, the myometriumproduces mostly PGI2, which was not examined, butmore likely has an effect upon vascular tone than uterinecontractility [24].Prostaglandin synthesis changes in tissues obviously

lead to changes in PG concentrations in fluids, whichprovide more valuable information about PGs inrelation to the changing nature of preterm and termbirth. Evidence exists to suggest that amniotic fluid PGconcentrations may be relatively low at some pretermbirths, an observation that reflects the low syntheticcapacity of amnion at preterm birth without infection.Romero and Mitchell and others [62] showed thatamniotic fluid levels of PGE2 did not rise at pretermlabour compared to not-in-labour matched controls (themean values were actually lower), but PGF2a concentra-tions did increase from 252753 to 7317363 pg/ml,although this rise was not statistically significant. Onlywith infection-associated preterm labour (which occursin 30–40% of all preterm births) was there a significantincrease in amniotic fluid PG levels. During normalbirth maternal plasma levels of the PGF2a metaboliterise with cervical dilatation, increasing considerablyfrom late pregnancy not in labour (5978 pg/ml) to143732 pg/ml in early labour and 283755 pg/ml in latelabour. In contrast, the plasma levels were 63717 pg/mlin preterm labour without infection, which were notdifferent from term not-in-labour values [63,64].Another key association with preterm birth that

changes as gestation advances is the infection rate. Forinstance, 70% of spontaneous preterm birth p30 weeksgestational age is associated with intrauterine infection,whereas after 30 weeks a decreasing rate from 40% to30% (at term) of spontaneous birth is associated withinfection [65]. Pregnancies and births associated withinfection are characterized by increased levels ofcytokines (interleukin (IL)-1b, IL-6, IL-8, and tumournecrosis factor-a) in the foetal membranes [66], amnioticfluid [67–70], lower genital tract [71–73], and in thelower segment of the uterus [74]. Interestingly, normalpregnancies in the third trimester and term deliverieswithout infection are also associated with increasedlevels of IL-8 in myometrium [75] and increased levels ofIL-1b and IL-8 in the amnion and chorio-decidua [76].Furthermore, the synthetic capacities of PGs are stim-ulated by inflammatory agents [77–79] which leads toincreases in intrauterine fluid and tissue concentrations


of PGs and a decrease in PG metabolism [54]. It is cleartherefore, that the conditions associated with andresponsible for preterm birth change as gestationadvances. When infection is not present, preterm labouris characterized by very low PG synthetic capacities andconcentrations and very high metabolic capacities.Conversely, when infection is present, PG synthesisand levels are very high, and metabolism is decreased.This information suggests that inhibitors of PGHS mayhave variable effects upon PG synthesis and theprogression of preterm labour depending upon the timeand condition of gestation.In the situation of preterm labour without infection,

when PG levels are very low and at the levels of intactnon-labouring term pregnancies, the question derives ofhow do PGs lead to the initiation and maintenance oflabour? We have data in the mouse at preterm labourinduced by ovariectomy [80], ethanol [81] or lipopoly-saccharide [82] that uterine PGF2a concentrations donot increase above levels in age-matched pregnancy-intact non-labouring controls—a situation similar tohumans without infection. However, the uterine mRNAexpression for FP, the PGF2a receptor, increasessignificantly, suggesting that uterine sensitivity to PGsincreases rather than the absolute level of PGs. But thesedeliveries are dependent upon PG synthesis as admin-istration of the PGHS-2 inhibitor, NS-398, delayspreterm labour [83]. Thus the threshold for uterineresponsiveness to PGs is lowered. By extension, it ispossible that enhanced human uterine responsiveness tolow concentrations of PGs at preterm in the absence ofuterine infection may be responsible for the initiationand maintenance of labour.In light of the changing nature of PG synthesis and

metabolism at preterm labour at different times ingestation or when infection is present, it would be ofimmense value to researchers and clinicians to have acompilation of the known data regarding humanintrauterine and fluid PG concentrations and tissuesynthetic and metabolic capacities at preterm labourduring gestation in uninfected and infected pregnancies.The implications of this information base would directfuture research towards interesting, and likely new,research questions dealing with prediction of pretermlabour and mechanisms as well as the development andapplication of appropriate tocolysis to deal with thechanging nature of preterm labour. We call uponProfessors Murray Mitchell and Roberto Romero,who have the largest archives of data in this area, toconsider leading this effort.

5. History of tocolytic agents

There are several theoretical approaches that could betaken to prevent preterm delivery. Ideally, the process of

uterine activation could be stopped before the clinicalonset of labour. Unfortunately, there is no good methodof detecting uterine activation nor is there any knownmethod of preventing or arresting it in women, thoughprogesterone appears to be very effective in severalanimal models including sheep, rats and mice. Atpresent, tocolytic strategy is directed towards haltingor reversing the stimulation phase once labour isclinically apparent. Perhaps the lateness of this inter-vention in the process of parturition explains therelatively poor results obtained. As an introduction tothe use of PGHS inhibitors to arrest preterm labour, wewill first consider briefly the history of evaluation oftocolytic agents to establish important perspectives inwhich to review the data and then review the experienceusing other tocolytics.As noted, essentially all human trials of tocolytic

agents have evaluated drugs targeting the stimulationphase of labour. Interestingly, though the steroidprogesterone has been demonstrated to prevent uterineactivation and prevent parturition on several animalspecies, it has not been examined in pregnant women.This may be due to early data that concluded, on thebasis of maternal serum progesterone concentrations,that progesterone was not important in the mechanismof human parturition. It may also be secondary to thedifficulty in synthesizing a suitable preparation of thelipid soluble steroid suitable for pharmacologic pre-paration.The earliest tocolytic trials of the late 1960s and 1970s

evaluated the efficacy and safety of ethanol to inhibitpreterm labour [84]. This drug was thought to impairhypothalamic secretion of oxytocin and thus inhibit theprogress of labour, though the scientific support of thismechanism was sparse. The trials showed that intrave-nous ethanol treatment could delay parturition byseveral days with a very high incidence of unwantedmaternal side effects associated with acute alcoholintoxication. However, a retrospective analysis ofethanol-treated women revealed that, despite the pro-longation of pregnancy, the incidence of respiratorydistress syndrome and neonatal death was twice as highas in the control groups [85]. Ethanol is no longer usedas a tocolytic agent.The next class of tocolytic agents that were evaluated

was the b-adrenergic agonists. These drugs includedisoxuprine, ritodrine, salbutamol, terbutaline and hex-aprenaline. Literally dozens of randomized control trials(RCT) and many metaanalyses of these trials have beenperformed. There is consensus that these drugs willprolong pregnancy for at least 72 h but there is noevidence of improved perinatal outcome [86]. Unwantedmaternal and foetal side effects, including significantcardiovascular and metabolic complications, are veryfrequent. There are several reports of maternal deathsecondary to treatment with b-agonists, particularly

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Table 1

Proportion of women who present with preterm labour that is eligible

for a trial of tocolytic therapy

Study Number of patients

entered into study

Number of eligible

for tocolysis

Zlatnik et al. [47] 191 26 (16%)

NIH Collaborative study on

dexamethasone, 1981 [48]

7893 1130 (14.3%)

Canadian Investigators

Group, 1992 [49]

4782 2442 (50.9%)


when used in combination with glucocorticoid therapyto accelerate foetal pulmonary maturation. These deathshave usually been associated with pulmonary edemafrom excessive fluid retention and myocardial arrhyth-mia, often associated with hypokalemia. In Canada, themanufacturer voluntarily withdrew the only drug of thisclass that was approved for use as a tocolytic agent(ritodrine), hence its inclusion in the history section ofthis article. There is some continued use of these drugs inother countries.This history of tocolytic therapy brings into focus three

major issues. The first issue is that most tocolytic drugshave no specificity for uterine smooth muscle. If they areadministered in sufficient quantity to relax uterinesmooth muscle, it is highly likely that they also will relaxvascular and/or cardiac smooth muscle to produceprohibitive side effects, even serious maternal complica-tions. Most of these drugs freely cross the placenta andmay result in equally deleterious effects in the foetus. Thefirst lesson learned from the history of tocolytics is thatthese drugs can have significant negative maternal andfoetal consequences that are important to factor intoconsideration of the therapeutic index for these drugs.The second important issue concerns the interestingly

high success rate of placebo treatments that occur inthese trials. On average, the success rate in prolongingpregnancy for 48 h ranges from 40% to 80%. This canbe interpreted in two different ways. First, it may be thatthe diagnosis of labour was incorrect. The clinicaldiagnosis of labour usually requires both evidence ofuterine activity (contractions that are palpable andoccurring with a specified frequency for a specifiedduration of time) as well as evidence that the contrac-tions are effective (causing cervical effacement ordilation or associated with rupture of the membranes).Still, it is a very subjective diagnosis. Despite activecurrent research in this area, there is no test orcombination of tests to accurately determine thepresence of real labour as opposed to ‘‘false labour’’that will not eventuate in parturition even if untreated.Therefore, the surprisingly high efficacy of placebotreatment may be explained by a false diagnosis oflabour. This makes it essential to compare a proposedtocolytic agent to a placebo before concluding that it hasefficacy. In contrast, an alternate interpretation of thesedata is that the placebo treatment does have a real effectand that the majority of any success achieved bytocolytic drugs is by a mechanism that involves asignificant, physiological placebo effect.The third, and perhaps most important issue that the

early tocolytic trials bring out is the definition of anappropriate outcome measure. Essentially all trials haveutilized prolongation of pregnancy by 24 or 48 h or 7days. Others have measured birthweight or proportionsof babies achieving a specified gestational age. But theolder literature clearly indicated that these end-points

may be attained but the eventual outcome of the babymay not be improved or actually may be worse! Clearly,the only appropriate outcome measure is a decrease infoetal-infant mortality and morbidity, an endpoint thatfew trials have had the objective or ability to assess.There is no doubt that a healthy baby at 30 weeksgestation is preferable to a sick baby at 30 weeks plus 2days. If we fail to address appropriate outcomemeasures, we will repeat the historical mistakes ofcausing more harm than we do good with any drugsused in pregnant women.A final observation from previous tocolytic trials

relates to the proportion of women that might beappropriate for therapy to stop labour. In someinstances, treatment with tocolytic agents would befutile (advanced labour with cervical dilation >4 cm) oreven harmful (presence of infection or other evidence ofmaternal or foetal compromise). Indeed, experience hasshown that only a minority of women in preterm labourwill actually be candidates for attempted tocolysis(Table 1). Until we have more effective methods ofearly diagnosis or prevention of preterm labour, eventhe best tocolytic intervention may provide onlydisappointingly small returns.

6. PGHS inhibitors as tocolytic agents

6.1. The basis of PGHS inhibition

The use of non-steroidal antiinflammatory drugs(NSAIDs) extends back thousands of years when plantextracts containing salicylic acid were employed in thetreatment of pain, inflammation and fever [87–89]. Thepharmaceutical industry has long been involved in theproduction of NSAIDs; aspirin, which is derived fromthe acetylation of salicylic acid, was first introduced in1897. NSAIDs are the most commonly prescribed drugsin the world today [89]. However, it was not until 1971that John Vane discovered that aspirin and otherNSAIDs inhibit PG synthesis [87].These drugs owe their popularity to the fact they are

first choice analgesics and antiinflammatory agents forheadache and chronic inflammatory diseases including

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Table 2

Comparison of NSAIDs for selectivity towards PGHS-1 or PGHS-2

using the William Harvey Modified Whole Blood Assay

Drug IC80 COX-1

(mM)

IC80 COX-2

(mM)

Ratio IC80

COX-2/COX-1

Nonselective for COX-2

Aspirin 8.0 30 3.8

Indomethacin 0.46 2.0 4.3

Selective for COX-2

Meloxicam 22 2.0 0.091

Nimesulide 41 7.0 0.17

NS398 65 1.0 0.015

Celecoxib (Celebrex) 28 3.0 0.11

Rofecoxib (Vioxx) >100 5.0 o0.05

Modified from Warner et al. [95].

Comparisons were made with the concentration of NSAID that

achieved 80% inhibition of enzyme activity (IC80).

B.F. Mitchell, D.M. Olson / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187 173

rheumatoid disorders. Unfortunately, the first series ofcompounds produced in this class (e.g. aspirin, indo-methacin1) also led to severe gastric disorders, includingulceration, hemorrhage, and gastric damage, especiallyin chronic users. These problems are so bad, that about1% of chronic users develop ulcers or other complica-tions leading to 100,000 hospitalizations annually in theUSA [90] and 2000–2500 deaths each year in the UK[91,92]. The primary isoform in the gastro-intestinalsystem is PGHS-1, which produces PGs (e.g. prostacy-clin, PGI2) that have a cytoprotective role through themaintenance of mucosal blood flow and in promotingstem cell survival and proliferation of the epithelial cellsin the crypts of Lieberkuhn [93]. The original NSAIDsinhibited both PGHS-1 and -2 (Table 2).The next generation of NSAIDs was the result of

animal studies in which drugs were developed to combatinflammatory processes but minimize the gastric dis-orders. This led to the development of meloxicam,nimesulide, and NS-398. These have been marketed forapproximately 25 years, but it was not until theidentification of PGHS-2 that it was realized thesecompounds were specific inhibitors of PGHS-2 andmuch less specific inhibitors of PGHS-1 (Table 2) [13].After the identification of the structure of PGHS-1 and -2 by X-ray crystallography, which permitted structure-activity relationships to be established, the design of newPGHS-2 inhibitor drugs led to the approval of celecoxib(Celebrex) and rofecoxib (Vioxx) for chronic inflamma-tory diseases. Valdecoxib (Bextra) was approved for usein late 2001 by the US Food and Drug Administration,and JTE-522 by Japan Tobacco and RW Johnson is a

1The Recommended International Non-proprietary Name (rINN)

and the new British Approved Name (BAN) is indometacin. However,

we choose to retain the more commonly used (e.g. in Canadian

formularies), indomethacin, for this article.

candidate inhibitor [89]. All of the designer PGHS-2inhibitors are diaryl heterocycles, based upon the basicstructure of nimesulide (Fig. 2) [89].All NSAIDs compete with arachidonic acid for the

active site of PGHS, but three different kinetic modesexist for NSAID inhibition of PGHS: a rapid, reversiblebinding; a rapid, lower affinity reversible bindingfollowed by a time-dependent, higher affinity, slowlyreversible binding (presenting as a pseudoirreversiblemechanism); or a rapid, reversible binding followed bycovalent modification of serine 530 (e.g. acetylation byaspirin) [14–16,94]. The substitutions of valine forisoleucine in positions 523 and 434 create the sidepocket in the catalytic domain and also movesphenylalanine 518 aside, causing a 20% increase in thevolume of the binding site of NSAIDs for PGHS-2versus PGHS-1 and leading to a ‘‘tighter’’ binding ofspecific PGHS-2 inhibitors. Thus when the newerPGHS-2 inhibitors interact with the enzyme, the kineticsappear to be time-dependent and pseudoirreversible.Conversely, when specific PGHS-2 inhibitors interactwith PGHS-1, their kinetics are rapid, competitive, andreversible. The practical importance of these kineticsbecomes evident during in vivo inhibition of PGHS-1and -2. When PGHS-2 inhibitors are present in blood ortissues at sufficiently low levels that are below the half-maximal inhibitory concentrations (IC50) of PGHS-1,the enzymatic activity of PGHS-1 will barely be altered,while PGHS-2 is inactivated [14].This concept was tested and confirmed by Warner

et al. [95] who compared most of the available inhibitors(Table 2). They developed a new assay, known as theWilliam Harvey Modified Whole Blood Assay, whichtests inhibitors for PGHS-1 and -2 against TXB2 formedfrom platelets (PGHS-1 test) and PGE2 formed frominterleukin-1b-treated A549 cells in the presence ofwhole human blood (PGHS-2 test). Further, as mostinhibitors of PGHS-1 or -2 frequently do not haveparallel concentration-response curves, and it is moremeaningful to test compounds at concentrations whereinhibitors inactivate most of the enzymatic activity, theycompared the IC80 values of the inhibitors. Their resultsshow that some of the new and old PGHS-2 inhibitorsare 20 to >50-fold more selective for PGHS-2 thanPGHS-1 (rofecoxib and NS398), but not all are.Celecoxib, for instance, is only about 10-fold moreselective for PGHS-2, while the mixed action inhibitors,indomethacin and aspirin, are slightly more specific(p5-fold) for PGHS-1. Because NSAIDs are somewhatfat soluble and their distribution, metabolism andexcretion may vary in individuals due to a number offactors, we suggest that in all clinical trials usingNSAIDs for tocolysis, whole blood be withdrawn fromtreated mothers and tested for PGHS-1 inhibition(platelet production of TXB2) and PGHS-2 inhibition(endogenous leukocyte or cultured A549 cell production

ARTICLE IN PRESS

Fig. 3. The concentrations of indomethacin in fetal sheep plasma

(femoral artery (FA) and umbilical vein (UV)), urine (URINE) and

amniotic fluid (AMN) during and following a maternal infusion of

7.5 mg indomethacin/kg/min for 72 h, from Krishna et al. [97]. Note

that there is a relatively slow rate of loss of indomethacin from the

amniotic fluid following termination of the infusion. Kindly repro-

duced with the permission of Pharmaceutical Press.

Fig. 2. The chemical structures of representative PGHS inhibitors.


of PGE2) to ascertain the degree of selective [96] PGHSinhibition obtained. To date, none of the tocolytic trialsusing PGHS inhibitors have made these tests, whichcould lead to alterations in the dosages used and mighthave implications for maternal or foetal health, espe-cially since it may only be necessary to inhibit PGHS-2of maternal origin to delay preterm labour. Indeed, itmay only be necessary to lower PG levels marginally toachieve tocolysis sufficient for patient transfer to atertiary centre or to administer glucocorticoids.

6.2. Maternal-foetal distribution of PGHS inhibitors

The best study examining the distribution andpharmacodynamics of indomethacin in the mother andfoetus during and following chronic administration tothe mother is from the University of British Columbiausing sheep as the experimental animal (Fig. 3) [97].Indomethacin was infused at the rate of 7.5 mg/kg/minfor 3 days, and it was observed that only 5.271.1%crossed the placenta to the foetus. This rate was low anddue to the polarity of the compound and the perme-ability characteristics of the sheep’s epitheliochorialplacenta. However, the inhibitor lingered in the foetusfor much longer than it did in the mother followingcessation of infusion for the same reason—it crossedback over the placenta to the mother slowly, especiallyas maternal clearance had to precede it, so it coulddiffuse down its concentration gradient. The concentra-tions in the amniotic fluid were about 10% those in thefoetal plasma; foetal renal clearance was extremely low(3.871.1 ml/min/kg); most (70%) of the glucuronidatedindomethacin was found in the foetal urine, suggestingfoetal rather than maternal metabolism; and none wasdetected in the foetal tracheal fluid leaving the placentaas the only means of total body clearance of the drug. Inprevious studies the foetal to maternal concentrationratio of indomethacin in sheep was 0.28 [98] or 0.04 [99].The human, with a hemochorial placenta, has a much

higher foetal to maternal concentration ratio of 1.0[100]. This is consistent with the higher permeability ofhydrophobic agents in this type of placenta [101], andwhile it should lead to more rapid clearance of the drugfrom the foetus, it also implies a more rapid build-up ofthe inhibitor. When a PGHS inhibitor can cross theplacenta as easily as it seems possible in humans, oraccumulates for a considerable time as indomethacindoes in the sheep foetus, it becomes important tounderstand more about the effects of the drug on thephysiology of the foetus.

6.3. PGs, PGHS, and PGHS inhibitors in foetal

development and physiology

PGs play roles in the organogenesis and physiology ofthe foetus and newborn. Their actions on organogenesis


include effects on cell proliferation in several organs[102,103], both the mediation and stimulation of growthfactor actions [104–107], the regulation of angiogenesis[108,109], the progression of pulmonary alveolarization[110] and the control of renal nephrogenesis [111–114].Their effects on cell and systemic physiology include thetransport of free fatty acids from pulmonary fibroblaststo epithelial cells [115], regulation of lung liquidsecretion [116,117], the secretion of lung surfactant[118,119], the enhancement of intestinal enzyme activ-ities [120], central effects on febrile, respiratory andcardiovascular responses [121] and the regulation ofvascular tone in the lungs [122], kidney [123,124], braincerebral microvasculature [125], the small intestine, andthe ductus arteriosus [126–128]. They stimulate ACTHrelease from the foetal pituitary and cortisol releasefrom the adrenal gland [129] and they control urineformation [124,130].Evidence suggests a developmental regulation of

PGHS activity. The conversion of arachidonic acid toPGE2 in foetal lung tissue increases with developmentalage up to the adult in sheep, suggesting an increase inthe activity of PGHS [131–133]. The data from rat smallintestine are similar, in that conversion of arachidonicacid to PGs increased from one week old newborns toadults, and in the human, the PGE2 content of gastricsecretions increases with age in newborns 35 weeks ofgestational age and older. There are species differencesas to which isoform of PGHS is the dominant andregulated form in the developing lung. In the foetallamb, Brannon and Shaul and their colleagues publishedstudies indicating that PGHS-1 mRNA increasessignificantly in pulmonary artery smooth muscle,endothelial, and airway epithelial cells [134,135],whereas Asano et al. [136] described PGHS-2 as thedominant and constitutive form in cultured human lung

1st 2nd0

0.5

1

1.5

2

1st 2nd 3rd Post-Natal0

0.5

1

1.5

2


1

2

3

4

1st 2nd0

0.5

1

1.5

2

Nor

mal

ized

PGH

S-2

mR

NA

Nor

mal

ized

PGH

S-1

mR

NA

Fetal Lung Fetaa

ab abb

aab ab

c

aab

Trimester T

Fig. 4. The relative abundance of PGHS-1 and PGHS-2 mRNA in human f

days). Note the remarkable similarity of increasing PGHS-2 mRNA abund

significant differences (ANOVA, Tukey’s test, Po0:05).

epithelial cells. We have reported that PGHS-1 mRNAlevels in human foetal tissues obtained from approxi-mately day 50 of gestation to normal term and into thefirst 9 days of the newborn period are unchanged inkidney and intestine and decrease in lung with increasingdevelopmental age whereas PGHS-2 mRNA levelsincrease from the first to the third trimester in thesetissues and further in the newborn period in the lung andkidney (Fig. 4) [137]. This pattern mimics that forPGHS-2 in the human foetal membranes [34]. Similarincreases in PGHS-2 mRNA expression are noted infoetal lambs from day 105 to 135 in hippocampus and inthe sensory-motor cortex, but not in the cerebellum,where expression was very low [138]. Interestingly,PGHS-2 mRNA is the predominant isoform expressedin the cortex and cerebral microvasculature of thenewborn pig, but levels are at their peak in the newbornand decline in juvenile animals. It is very intriguing tonote that the human data and much of the animal data,although scarce, suggest that the increase in PGHS geneexpression is most dramatic in the third trimester andreaches a peak in the early newborn. In some tissues,expression will decrease in older newborns, and in othersit may continue to increase to adulthood.Many of the roles of PGs in foetal development and

physiology have been identified by studies utilizing genedeletion (knockout) for PGHS-2 in mice, which indicatethat renal nephric dysgenesis, cardiomyopathies, andperitonitis occur in the newborns that normally dieshortly after birth [111,139]. It is suggested that loss ofPGHS-2 results in a postnatal maturation arrest in thesubcapsular nephrogenic zone of the kidney. There wereno remarkable foetal or neonatal effects with PGHS-1gene deletion [140].The use of PGHS inhibitors is another experimental

tool used to discern the function of PGs in foetal tissues.


0.5

1

1.5

2

3rd Post-Natal


0.5

1

1.5

2

2.5

3rd Post-Natal

l Kidney Fetal Intestine

ab

c

a

ab

c bc

rimester Trimester

etal tissues throughout gestation and into the newborn period (up to 9

ance in all three fetal tissues in late gestation. Different letters denote


In newborns of mothers receiving indomethacin duringpregnancy, the absence of foetal PGs leads to anincreased risk for intraventricular hemorrhage, necrotiz-ing enterocolitis, either closure of the ductus arteriosus,or a patent ductus if pulmonary vascular resistance istoo high, loss of renal urine formation and a subsequentdecrease in amniotic fluid volume and its consequencesupon pulmonary development [123,141,142]. BothPGHS-1 and -2 are present in the ductus of the foetalsheep, but PGHS-2 is found primarily in the endothelialcells lining the lumen. Use of NS398 produces the largestcontraction of the ductus, in vitro, more so than thePGHS-1 inhibitor, valeryl salicylate, or indomethacin.Similar results were also obtained in vivo [126,143].Other studies show that in utero exposure to indo-methacin causes vasoconstriction, hypoxia and ulti-mately death of the smooth muscle cells in the media ofthe foetal sheep ductus arteriosus, which may lead topatent ductus in the newborn [144].Additionally, indomethacin infused to the foetal

sheep causes a mild respiratory acidosis and hypoxemiaand decreased blood flow in other vascular beds,including the cerebral hemispheres and other brainregions [145]. Another well established aspect of foetalindomethacin exposure and decreased PG production isa stimulation of foetal breathing movements [145–147].Foetal exposure to indomethacin leads to major changesin organ blood flow patterns [148], increased suscept-ibility to hypoxic pulmonary vasoconstriction [149] anda direct inhibition of pulmonary alveolarization [126].Administration of the PGHS-2 inhibitor, meloxicam, tofoetal sheep in preterm labour depressed the hypotha-lamic-pituitary-adrenal axis as evidenced by the low-ering of the plasma concentrations of ACTH andcortisol [43]. The acute administration of indomethacindid not decrease renal urine formation when adminis-tered to ewes and foetuses in one study [116], but it diddecrease urine formation in another study when infusedto the foetus over an 8-h period [150]. A PGHS-2specific inhibitor, SC58236, administered to neonatalrats at the time of nephrogenesis, blocked nephrondevelopment [112]. Thus it is clear that PGs are essentialfor development and function in a number of foetalorgans.One of the newest considerations is the role of PGHS

in the foetal response to stress. We examined the mRNAexpression of renal PGHS-2 and the PGE2 receptors,EP2 and EP4, to placental restriction in pregnant ewes[151]. The reduction in the total mass of the placentathroughout pregnancy led to growth restricted foetusesand hypoxemia. It also increased the mRNA abundancefor PGHS-2 and EP2 in the foetal kidney. An inversecorrelation developed between pO2 and PGHS-2mRNA, and it appeared that the capacity of the foetalkidney to synthesize and respond to PGs in thesestressed conditions could direct the expression of renin

as there was a direct correlation between both EP2 andEP4 mRNA and renin mRNA. The effect of PGHSinhibitors on the hypoxemic or growth-restricted foetushas not been explored.

6.4. Effects of PGHS inhibitors on adult physiology and

health

As mentioned earlier, the chronic administration offirst generation (mixed) PGHS inhibitors caused severegastric disturbances that led to death in some cases. Thismotivated the design of second and third generationdrugs to treat chronic inflammation but reduce theunwanted gastric effects. The new third generationPGHS-2 inhibitors, celecoxib and roficoxib, based uponthe structure activity relationship of the X-ray crystal-lographic image of PGHS-2, have vastly improved thegastrointestinal safety and tolerability of treatingchronic arthritis [152–154]. Further, they may be ableto play effective roles in treating intestinal polyposis,colorectal carcinogenesis, and Alzheimer’s disease [155].Questions are being raised, however, regarding

possible hazards in the use of the new PGHS-2-specificinhibitors. Their inhibition of PGI2 synthesis may leadto increased prothrombic activity, and caution should beexercised in patients at risk of cardiovascular complica-tions. The possibility of altered renal function andwound healing are also concerns [154–156]. Indeed, acontroversial retrospective study published recentlyhighlighted increased risks for developing a thromboticcardiovascular event with rofecoxib versus naproxen(which has inhibitory activity for both PGHS-1 and -2)[157,158]. (For more about the debate, read the Lettersto the Editor in JAMA 286:2808-2813, 2001 and theCMAJ 167:739-741, 2002.) Also, traditional NSAIDscause renal sodium retention and reduced glomerularfiltration rate leading potentially to peripheral edema,increased blood pressure, weight gain, and congestiveheart failure. Several recent reviews of studies indicatethat neither rofecoxib nor celecoxib have effectsdifferent than the older NSAIDs, implying they maylead to renal failure [159–163]. Clearly, close monitoringof maternal as well as foetal effects of specific PGHS-2inhibitors is warranted during tests of their tocolyticpotential.

6.5. Indomethacin as a tocolytic

Given the rapidly accumulating evidence that PGs areinvolved in the mechanism of parturition, it is notsurprising that PGHS inhibitors were proposed early onas potentially useful tocolytic agents. Three RCTs havecompared indomethacin, a non-specific PGHS inhibitor,to placebo [164–166]. If a meta analyses is done, there isa highly significant prolongation of pregnancy achievedwith indomethacin (Table 3). On first glance, it appears

ARTICLE IN PRESS

Table 3

Meta analysis of the three randomized trials comparing the efficacy of

indomethacin to placebo

Study Indomethacin Placebo R.R. (95% CI)a

Niebyl [165] (10) 3/15 10/15 0.30 (0.10, 0.88)

Zuckerman [164] (9) 1/18 14/18 0.07 (0.01, 0.48)

Panter [166] (11) 3/16 8/18 0.42 (0.13, 1.32)

Total 7/49 32/51 0.43 (0.30, 0.63)

Outcome measure: Delivery within 48 h.aRelative risk (Fisher’s Exact test) (95% CI).

B.F. Mitchell, D.M. Olson / Prostaglandins, Leukotrienes and Essential Fatty Acids 70 (2004) 167–187 177

that this drug would be a good choice for tocolytictherapy in women. It should be noted however, thatthere was no evidence in single trials or in the metaanalyses of any benefit to the baby. Additionally, duringthe 1970s and 1980s, it was becoming clear thatinhibition of prostaglandin synthesis could have seriousnegative consequences in the foetus. This is due to theimportance of foetal PGs in development of the kidney,foetal hemostasis and foetal vascular regulation, includ-ing maintenance of vasodilation of the ductus arteriosusand regulation of splanchnic and cerebral blood flow.Thus, in the later trials, it was important to determinewhether any of these potential adverse effects wereoccurring in the foetus of women treated with indo-methacin.In 1991, a RCT was reported comparing indometha-

cin to b-agonist therapy and specific foetal side effectswere measured [167]. Though indomethacin treatmentwas equally efficacious to the b-agonists, it wasaccompanied by some disturbing foetal consequences.Three of the 22 indomethacin-treated foetuses hadneonatal courses complicated by primary pulmonaryhypertension, a possible consequence of foetal constric-tion of the ductus arteriosus. Two others had apersistent patent ductus arteriosus that required medicaltreatment for closure. No such incidents occurred in theb-agonist treated babies. Even more alarming was aretrospective report in 1993 comparing outcomes inbabies treated with indomethacin at o30 weeks gesta-tion compared to babies matched for gestational age buttreated with other tocolytic drugs [142]. The indometha-cin-treated group had a significantly increased incidenceof necrotizing enterocolitis, severe intraventricularhemorrhage and persistent patent ductus arteriosusrequiring surgical ligation. They also had a higherserum creatinine level that would be compatible with atoxic effect of indomethacin on the foetal kidneys. Themost recent placebo-controlled RCT for indomethacin,reported in 1999, was designed specifically to assess thesafety as well as efficacy of the drug [166]. Unfortu-nately, enrolment was difficult and the study wasterminated after achievement of only 10–15% of thecalculated sample size. Even with these small numbers,

increased flow velocity was measured in the ductusarteriosus indicating ductal constriction after indo-methacin in 2 of 19 foetuses compared to none in thecontrols. Further, there was decreased urine output inthe first 24 h in 6 if the 12 indomethacin-treated babies inwhom this was measured. This was statistically greaterthan the 2 of 13 in the placebo-treated group. Therewere no differences in urine output at 48 h of age.Several investigators have confirmed the effects of

indomethacin on foetal renal function and ductal flowvelocity. A decrease in amniotic fluid volume iscommonly seen in women treated with indomethacin,probably reflecting a decrease in foetal urine output[168]. Indeed, indomethacin has been proposed as atreatment for polyhydramnios, though most cliniciansfeel the potentially toxic effects of the drug on thedeveloping kidneys outweigh any potential benefit. Thesame consideration should be given to the use ofindomethacin as a tocolytic agent. The effects ofindomethacin on ductal constriction appear to bedependent on gestational age [141,169]. Increased flowvelocity is more easily measured at later gestational agesbut it is clear that it occurs even in foetuses between 24and 30 weeks gestation. Although the changes in urineflow and ductal flow velocity appear to be transient andreverse after several hours or days, this does notguarantee that they are harmless. Recent intenseresearch in the area of ‘‘foetal programming’’ hasprovided strong support for the concept that transientdisturbances in foetal physiology may stimulate com-pensatory mechanisms that provide short-term benefitto the foetus but may persist to become disadvantageousin later life [170].

6.6. Isoform selective PGHS inhibitors

It is perhaps unfortunate that all the early studies withPGHS inhibitors in pregnancy were performed usingindomethacin. This is a non-selective drug that inhibitsboth PGHS-1 and PGHS-2. It has a broad range of well-described side effects and it is interesting that its use wassuperceded by other NSAIDs in most clinical conditionsexcept preterm labour. It is possible that other PGHSinhibitors may not be associated with the extent of foetalside effects noted above for indomethacin.With the advent of PGHS-2-selective inhibitors, there

was hope that these drugs would provide tocolyticefficacy with a less worrisome profile of foetal sideeffects. Indeed the first report of maintenance ofpregnancy using a PGHS-2 inhibitor, nimesulide, in asingle case study where no changes in the amniotic fluidindex or ductal pulsitility index were evident, didpromote this optimism [171]. This hope is temperedsomewhat by the findings that the tissue distribution ofPGHS isoforms is quite variable among species. Forexample, PGHS-2 inhibitors cause ductal constriction in


the sheep foetus [143] but this was not observed in thepig foetus [172]. There is very little informationregarding human foetal distribution of PGHS isoforms,although the data available [137] indicate a significantincrease in PGHS-2 mRNA expression in the thirdtrimester in lung, kidney and intestine. A recentlyreported RCT comparing celecoxib with indomethacinin 24 pregnant women at 24–34 weeks gestationconfirmed the increased foetal ductus flow velocity anddiminished amniotic fluid index with indomethacin butobserved no such changes with celecoxib [173]. Thetocolytic efficacy of the drugs was not reported, as thetrial was not designed to assess this. However, ananecdotal report suggests that maternal consumption ofPGHS-2 specific inhibitors can be associated withirreversible renal failure in the neonate [174], and twostudies since then confirm that nimesulide treatmentleads to foetal oligohydramnios [175,176].To date, there are no large RCTs to evaluate the

efficacy and safety of PGHS isoform-specific inhibitors.There remains hope that a member of this class oftocolytic drugs will provide a high therapeutic index thatmay reduce mortality and morbidity resulting frompreterm birth.

7. Alternative tocolytic agents

Because of potential toxicity and debatable therapeu-tic efficacy, many clinicians elect not to use indometha-cin or other PGHS inhibitors for treatment of pretermlabour. The purpose of this section is to provide a verybrief synopsis of the alternative classes of drugs that arebeing used as tocolytic agents.

7.1. b-adrenergic agonists

Perhaps the best clinical trials in this area of researchhave been performed to evaluate this group of drugs.The most recent Cochrane Library systematic reviewindicates evidence of prolongation of pregnancy for atleast 72 h [86]. However, despite enrollment of hundredsof pregnant women in these clinical trials, there is noconvincing evidence of improved perinatal outcome forthe baby. Unfortunately, even though the drugs are veryspecific inhibitors for the b2 subclass of receptors, thesereceptors are also present on vascular smooth muscle.As noted previously, cardiovascular and metabolic sideeffects limit the usefulness of these drugs as tocolyticagents. Additionally, tachyphylaxis develops withinseveral hours and the patients become resistant to theeffects of the inhibitors. Though this class of drugsremains the ‘‘gold standard’’ for tocolytic agents withrespect to evidence-based use, their clinical use appearsto be diminishing steadily.

7.2. Magnesium sulfate

This interesting drug is perhaps the most commonlyused tocolytic agent in North America. The putativemechanism of action for this divalent cation involvescompetition with Ca2+ for entry into the cell throughcalcium channels and for activation of MLCK bybinding with calmodulin. Its common use is interestingbecause a meta analysis of the two randomized placebocontrol studies that have evaluated the drug found thatit is no more efficacious than the placebo [177,178]. Onthe other hand however, several trials have found itsefficacy to be comparable to b-agonists, indomethacinor calcium channel blockers [179]. Although highinfusion rates can lead to serious neural, respiratory orcardiac toxicity due to excessive concentrations ofmagnesium, this is not a common problem with lowinfusion rates and the drug has a lower side effect profilethan most other tocolytic agents. Though it is widelydebated whether or not magnesium sulphate has anytocolytic efficacy, a population-based study from North-ern California in 1995 suggested that it might have otherbeneficial effects [180]. In a study of more than 150,000children born between 1983 and 1985, investigatorsfound a 70–90% reduction in the rate of cerebral palsyin children exposed to prenatal magnesium sulphate.This was not found for other tocolytic agents and wasnot explained by concomitant administration of gluco-corticoids. This finding undoubtedly added to thepopularity of magnesium sulphate as a tocolytic drugof choice. However, further controversy followed.A large RCT designed to determine prospectively the

potential neuroprotective effects of magnesium (theMAGnet trial) was terminated after the first interimanalysis revealed a marked increase in the death rate ofchildren treated with magnesium sulphate [181]. Thereport of these results in 1997 documented that 9 of the75 foetuses receiving magnesium had died in the firstyear of life compared to only 1 of 75 in the controlgroup. This 12% overall pediatric mortality rate withmagnesium sulphate clearly would prohibit its use.Additionally, in the largest placebo control trial ofmagnesium sulphate, there were 7 total pediatric deathsin the 76 magnesium-treated group compared to 1 of 80placebo treated controls. This increased the sense ofalarm. However, careful review of the causes of death inthe 16 deaths from these two studies reveals no patternthat could biologically be explained by magnesiumtoxicity. Four babies had multiple congenital abnorm-alities. A set of twins died from twin–twin transfusionsyndrome. Two babies were asphyxiated at birth andtwo others were born with birthweight o900 g and diedof complications of prematurity. One died fromplacental abruption several weeks after magnesiumhad been given. Interestingly, 5 infants from theMAGnet study died of respiratory arrest or sudden


infant death syndrome following discharge from hospi-tal, at ages 16–260 days. These results stimulated areanalysis of the Northern California data to ensure thatthe population-based study had not been biased byexclusion of infants that received magnesium sulphateand subsequently died [182]. The reanalysis excluded thelikelihood of this type of bias and reaffirmed the largeneuroprotective effect (75–90%) associated with the useof magnesium sulphate with respect to the incidence ofcerebral palsy. In the 5 years since publication of theMAGnet data, there have been no other reports ofunusually high pediatric mortality rates with magnesiumsulphate. It seems unlikely that a mortality rate of>10% would go unnoticed with a drug having suchcommon usage as magnesium sulphate. Additionally, inthe large multi-centered RCT evaluating the use ofmagnesium sulphate for prevention of eclampsia (the‘‘Magpie’’ trial), more than 1200 mothers o34 weeksgestation were given magnesium sulphate with nosignificant increase in perinatal mortality rate prior todischarge from hospital compared to placebo-treatedcontrols [183]. At present, two large RCTs are addres-sing the neuroprotective effects of prophylactic magne-sium. Unfortunately, these trials are using much lowerdoses of magnesium than would be used in tocolytictherapy. Therefore, the question of magnesium toxicityremains unclear. The continued use of magnesiumsulphate suggests that many clinicians have consideredthe data as a ‘‘worrisome statistical aberration’’ but areawaiting further evidence before abandoning use of thisdrug.

7.3. Calcium channel blockers

Increased intracellular Ca2+ is essential for uterinecontractions. Since this increase relies partly on influx ofCa2+ into the cell, there is good rationale for the use ofcalcium channel blockers (nifedipine and nicardipine) toinhibit uterine contractions. However, as with mosttocolytic agents, they have no specificity for the uterusand side effects from relaxation of vascular smoothmuscle are likely to restrict use of effective concentra-tions. There have been no studies comparing these drugsto a placebo and therefore, like magnesium sulphate, nogood evidence of efficacy. As with magnesium sulphate,several trials have compared them to b-agonists andhave found them to be comparable, usually with fewerside effects [184]. Though still experimental, their use asa tocolytic agent seems to be increasing, particularly as areplacement for b-agonists.

7.4. Nitric oxide donors

There is a theoretic role for nitric oxide in regulationof uterine activity. In rats, the inducible form of nitricoxide synthase is present in the endometrium and

appears to decrease at the time of uterine activation[185]. A similar situation may be present in the pregnanthuman uterus [186]. Therefore, augmentation of nitricoxide production may provide a physiological methodto induce myometrial relaxation. The current dogma isthat this gaseous molecule stimulates guanylyl cyclase toproduce cGMP that leads to inactivation of MLCK.However, it has recently been reported that nitric oxidemay act in other, yet unknown mechanisms to inducesmooth muscle relaxation [187,188].The efficacy of nitric oxide donors (glyceryl trinitrate

patches) has been evaluated in one pilot study wherethey were found to be no different from placebo patches[189]. In another study, nitric oxide (nitroglycerinetablets) was shown to have no greater uterine relaxationactivity than a placebo, even at doses of nitroglycerinethat caused a measurable decrease in blood pressure[190]. These data suggest that this class of tocolyticagents also will be severely restricted by non-specificcardiovascular side effects.

7.5. Oxytocin antagonists

Over the past two decades, a large number ofanalogues of the neuropeptide hormones oxytocin andvasopressin have been developed. Some of theseanalogues act as agonists and some as antagonists.Some are very selective for either the oxytocin or thevasopressin receptor but many will bind to bothreceptors [191]. Since there is considerable evidence thatoxytocin plays a role in the initiation or propagation ofhuman labour, there is rationale for using oxytocinantagonists to treat preterm labour. Unlike othertocolytics, there is hope that this class of drugs mayhave specific uterine effects since the major site ofoxytocin receptors is in the uterus and myoepithelialcells of the breast. Though there has been suggestionthat oxytocin may have some vasoactive properties, ithas been shown that it has no vasoconstriction orvasorelaxation effects when directly applied to mesen-teric or uterine vessels from non-pregnant or pregnantrats [192]. The only oxytocin antagonist that has beenevaluated in a clinical trial (atosiban) has considerablecross-reactivity with the vasopressin receptor, whichmay give rise to undesirable side effects [193].In a large RCT comparing atosiban to a variety of b-

agonists, the oxytocin antagonist was found to beequally as efficacious as the b-agonists and much bettertolerated by pregnant women [194]. A large, randomizedplacebo control trial also has been performed [195].Unfortunately, the results are difficult to assess. Therandomization was conducted in blocks to distributeregistration equally within the various participatingcenters. Unfortunately, there was no intentional strati-fication for gestational age and, in the final analysis ofthe 491 women enrolled, there was a biased distribution

ARTICLE IN PRESS

Table 4

Proportion of women in preterm labour completing a course of

glucocorticoids therapy in randomized control trials comparing a

tocolytic agent to placebo

Canadian

Investigators Study

(ritodrine) (%)

Panter et al.

(indomethacin)

(%)

Romero et al.

(atosiban) (%)

Delayed delivery >48h

Tocolytic 78.6� 81� 67�

Placebo 64.6 56 56

Completed glucocorticoids

Tocolytic 34.6 81 48

Placebo 36.0 83 53

�Po0.05


according to gestational age with the atosiban grouphaving significantly more women at lower gestationalages (o26 weeks). At >28 weeks gestation, atosibanwas superior to placebo at prolonging gestation by>48 h. However, in common with essentially all otherliterature on tocolysis, there was no significant effect onneonatal outcome. Indeed, at o26 weeks gestation,there was a significantly increased infant mortality ratein the atosiban-treated foetuses. Almost certainly, thiswas due to the unbalanced inclusion of very pretermfoetuses in the two groups. In the atosiban group, 10babies died of the 27 that were o26 weeks gestation atadmission compared to the 0 of 16 in the placebo group.In the atosiban-related deaths at this gestational age, 7of the 10 were o24 weeks gestation with birthweighto650 g. Thus, the safety of atosiban is unclear at verylow gestational ages. Future developments in this fieldhopefully will yield more specific antagonists with animproved therapeutic index.

8. Glucocorticoid therapy in preterm labour

The main clinical relevance of a 48-h endpoint fordetermining success with a tocolytic agent is derivedfrom the fact that this time interval is thought to beoptimal for effectiveness of glucocorticoids to acceleratematuration of the foetal lung. Prenatal administrationof glucocorticoids is perhaps the most solid, evidence-based therapy for management of preterm labour [196].However, the bulk of that evidence is now 15–30 yearsold. Recent research has raised concerns about potentialnegative effects of foetal glucocorticoids [197]. In animalmodels, and with some supporting evidence in humans,glucocorticoid therapy may stimulate maturation at theexpense of permanent growth restriction [198]. Reduc-tion in the numbers of fully functional cells could haveobvious detrimental effects on many organ systemsthroughout development and in the adult. This hasstimulated a second look at this form of treatment [199].At the present time, most guideline-producing agenciescontinue to recommend a single course of therapy butare hesitant to recommend repeated doses. Clearly, thisis an area where results from future research maysignificantly change recommended practices.A great deal has changed in the management of the

preterm newborn in the last two or three decades. Theincidence and severity of respiratory distress syndromehas decreased markedly, due in large part to therapywith artificial surfactant. Though the original surfactantRCTs suggested that glucocorticoid therapy still offeredsome benefits to infants who were treated withsurfactant, these trials were designed to evaluate theefficacy of the surfactant preparation [200,201]. Theanalyses regarding glucocorticoid therapy were post hocretrospective reviews that would not constitute first class

evidence. Furthermore, these data are now a decade old.It seems logical to suggest that with earlier and moreaggressive post-natal therapy with artificial surfactant,the value of prenatal glucocorticoids may be relativelyless. If so, this may well influence the assessment of costsand benefits when contemplating tocolytic therapy.Even if we were to accept that glucocorticoids

provided a favourable risk–benefit ratio for the foetus,it is an assumption that treatment would be facilitatedby the use of tocolytic drugs, even those that haveefficacy for delaying birth by 48 h. This assumption maywell be false. Three RCTs evaluating efficacy oftocolytic drugs have evaluated glucocorticoid therapyas a secondary outcome measure. In all three (Table 4),the treatment was significantly better than the placebocontrol for prolonging pregnancy >48 h. However, inall three, the placebo-treated patients actually had ahigher (though not statistically different) rate ofcompleting glucocorticoid therapy.

9. Future considerations

The work reviewed has generated several considera-tions of the use of PGHS inhibitors, or other agents thatdelay preterm labour, that future research shouldaddress. Several suggestions for research on PGHSinhibitors or other aspects of the prostaglandin synth-esis-receptor system are discussed in the followingparagraphs.The optimal period (or the wrong period) of gestation

or foetal development to use PGHS inhibitors should beinvestigated. The acute affects on the foetus may bedifferent at 24 weeks than they are at 34 weeks. Forexample, renal development or function or ductalfunction may have differing sensitivities to PGHSinhibitors during foetal development so that there aretimes during gestation when certain PGHS inhibitorsare safer or carry less risk. In contrast, the foetalprostaglandin synthesis-receptor system responds to


stress as demonstrated during hypoxia in the foetalsheep kidney [151], therefore inhibiting it may not be inthe best interest of the foetus at that time, even if themother is in preterm labour. Knowing the degree offoetal stress and its responses to it may alter therapeutictreatment for prevention of preterm labour. But thereare few data in these areas and more research isnecessary to make informed decisions.The long-term effects of foetal exposure to PGHS

inhibitors have not been studied. There are no data onthe effects of foetal renal exposure to PGHS inhibitorson adult blood pressure regulation or adult renalfunction even though it is recognized that PGHS-2knockout foetuses have nephric dysgenesis and rarelysurvive beyond the newborn period [111, 139]. Thisknowledge increases the likelihood that disruption ofnephrogenesis by PGHS inhibitors may lead to long-term consequences involving renal function or bloodpressure regulation.An assessment of how the risk of preterm labour is

managed may focus attention on developing tocolyticsfor specific needs, for instance a ‘‘stabilization tocolytic’’to arrest uterine contraction for transport of mother to atertiary centre or for assessment of maternal and foetalhealth versus a ‘‘long-term tocolytic’’ for delay of labourand maintenance of pregnancy for more than 48 h.Trials should be conducted testing the effectiveness of

combination therapies such as those recently reported insheep in preterm labour that received both nimesuldeand atosiban to block PGHS-2 activity and the oxytocinreceptor, respectively [41]. Such combinations maysufficiently suppress the mechanisms of preterm labourso that lower concentrations of PGHS inhibitors can beadministered, thereby avoiding or diminishing some oftheir side effects.Novel efforts to modify or block components of the

prostaglandin synthesis-receptor system should be ex-plored. Reviews and studies have pointed out the effectsof n-3 fatty acid diets in extending the length ofgestation [202, 203]. Animal diets high in dihomo-g-linolenic acid (DHA), an n-3 fatty acid substrate forPGHS, lead to lower levels of the biologically active PGsof the 2 series. Faroese women with diets high in n-3fatty acids have longer gestations and their babies havehigher birthweights [204], and when pregnant womenconsumed a fish-oil supplement, gestation was extendedby 4 days on average [205]. Women at risk of pretermbirth may benefit from diets high in n-3 fatty acids, as asubsequent study showed that fish oil supplementationreduced the risk of a recurrent preterm delivery from33% to 21% in a large European multicentre trial [206].Recently, we reported that tyrphostins, inhibitors of

tyrosine kinases, block LPS-induced preterm labour inmice [207]. We determined several years ago thattyrosine phosphorylation mechanisms were associatedwith enhanced expression of PGHS-2 mRNA in

cultured human amnion cells [208], and that tyrphostinsinhibited PGHS-2 mRNA expression [209]. Manysignalling pathways employ tyrosine phosphorylation,including cytokine pathways, and tyrphostins are notspecific for PGHS-2 signalling, so more investigation isrequired to learn how they specifically block pretermlabour.Changes in expression levels of the PGF2a receptor

(FP) are associated with uterine activation and pretermbirth in mice [80,81], thus research exploring therole and antagonism of the FP receptor in pretermlabour should prove fruitful. Indeed, recently an FPreceptor antagonist, THG113, has been developed byTheratechnologies in Montreal that effectively delayspreterm labour in mice [210]. This peptide antagonistonly slightly (1%) crosses the placenta, is rapidlymetabolized, and has no known side effects in mice.In collaboration with Dr. Jon Hirst in Melbourne, wenow have the first evidence in sheep that THG113also decreases uterine electromyographic activity anddelays preterm labour induced by RU486, the pro-gesterone receptor antagonist. Antagonizing FP actionin women may be suitable for short-term tocolysis, suchas during transport or assessment as suggested above,for administration of glucocorticoids, or perhaps forlonger-term delay of labour and maintenance ofpregnancy.

10. Summary and conclusions

Preterm delivery remains the most important obste-trical problem of the developed world. A relatively smallproportion of women in preterm labour are candidatesfor attempted tocolytic therapy. Though the data arefew and the quality of the studies diverse, there is someevidence to suggest that PGHS inhibitors can prolonggestation. However, there is considerable concernregarding the safety of these drugs for the foetus.Though there is hope that more isoform-selective PGHSinhibitors will offer an improved therapeutic index,there currently are no data to evaluate them in womenwith preterm labour. A host of other tocolytic agentshas been evaluated but none shown to have anybeneficial effects of neonatal outcome. The concept ofprolonging gestation for 48 h to permit administrationof glucocorticoids to accelerate foetal pulmonarymaturation urgently needs to be re-addressed in thecontext of current neonatal care.There is agreement that a very important factor in

neonatal outcome with preterm birth is that deliveryoccurs in a centre with immediately available expertisein neonatology. No studies have assessed the efficacy oftocolytic drugs to facilitate successful transport ofwomen in suspected preterm labour from outlying areasto regional centres with expertise in neonatology.


An accurate and clinically useful method to diagnoselabour remains elusive. It is not surprising then that weknow so little about treatment of preterm labour. It isvery possible that the many RCTs evaluating tocolytictherapy are demonstrating a strong placebo effect. Atthe present time, this placebo effect may represent themanagement plan with the best therapeutic index—acombination of good efficacy with a low rate ofacceptable side effects.Our hope for the future is that research into the

regulation of parturition will open new avenues ofprevention. Our current strategy of arresting or rever-sing the stimulation phase of labour may not yield anybetter results than we now have. The breakthrough maycome with further understanding of and the ability torecognize and control the process of uterine activation.New generations of PGHS inhibitors may play a centralrole in these developments. However, at present thereare grave doubts about the value of any type of tocolytictherapy and perhaps the primary consideration shouldbe, ‘‘First, do no harm’’.

Acknowledgements

Work from the authors’ laboratories was supportedby the Canadian Institutes of Health Research, theAlberta Heritage Foundation for Medical Research, andthe Molly Towell Perinatal Research Foundation. Thecontributions of Mr. Dean Zaragoza and Ms. SheilaMcManus are gratefully acknowledged.

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