Maternal-amniotic-fetal distribution of macrolide antibiotics following intravenous, intramuscular,...

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BASIC SCIENCE: OBSTETRICS

Maternal-amniotic-fetal distribution of macrolide antibioticsfollowing intravenous, intramuscular, and intraamnioticadministration in late pregnant sheepJeffrey A. Keelan, PhD; Ilias Nitsos, PhD; Masatoshi Saito, MD, PhD; Gabrielle C. Musk, BVMS; Matthew W. Kemp, PhD;Matthew Timmins, PhD; Shaofu Li, BSc; Nobuo Yaegashi, MD, PhD; and John P. Newnham, MD

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OBJECTIVE: The objective of the study was to explore the maternal-fe-al pharmacokinetics of intraamniotic (IA), intravenous (IV), or intramus-ular (IM) administration of erythromycin or azithromycin in a pregnantheep model.

STUDY DESIGN: Pregnant ewes of 115-121 days’ gestation received aingle maternal IV infusion (5 mg/kg over 60 min), a single IM injection,r a single IA injection (3.2 mg/kg fetal weight) of either erythromycin

actobionate or azithromycin. Maternal/fetal blood and amniotic fluidAF) samples were collected across 48 h for macrolide assay by liquidhromatography and tandem mass spectrometry.

RESULTS: Maternal administration achieved therapeutic maternalplasma macrolide concentrations (�0.5 �g/mL) with low concentra-

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intraamniotic administration in late pregnant sheep. Am J Obstet Gynecol 2011;204:

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0002-9378/$36.00 • © 2011 Mosby, Inc. All rights reserved. • doi: 10.1016

even lower (�1.5% transfer). The IA administration achieved therapeu-tic concentrations in AF and sustained for 48 h, with poor maternal-fetaltransfer (�1% maternal, �0.3% fetal). Modest pharmacokinetic dif-ferences were evident between erythromycin and azithromycin.

CONCLUSION: Maternal macrolide administration achieves subthera-eutic concentrations in AF or fetal plasma, whereas a single IA injec-ion achieves therapeutic concentrations in AF but not in maternal-fetalirculations. Combined maternal and single IA administration of macro-ides may be a more effective regimen for treatment of intrauterine, butot fetal, infection.

ey words: antibiotics, intrauterine infection, pharmacokinetics,
heep, ureaplasma tions in AF equivalent to less than 7% transfer; fetal plasma levels were
Cite this article as: Keelan JA, Nitsos I, Saito M, et al. Maternal-amniotic-fetal distribution of macrolide antibiotics following intravenous, intramuscular, and
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Macrolide antibiotics such as eryth-romycin and azithromycin are

widely prescribed during pregnancy forthe treatment of a variety of microbialinfections; these drugs are perceived tobe effective, well tolerated, and free of se-rious maternal and fetal side effects.1-3

Erythromycin is a first-line antibioticprescribed for the treatment of Chlamydiatrochomatis and Group B streptococci in-fections and is the most frequently ad-ministered antibiotic for treatment of pre-mature prelabor rupture of membranes

From the School of Women’s and Infants’ HeMs Musk, and Mr Li) and School of MoleculaUniversity of Western Australia, Perth, WesteObstetrics and Gynecology, Tohoku University

Presented, in part, at the Annual Scientific ConfeInvestigation, Miami Beach, FL, March 15-19th,

Received Dec. 16, 2010; accepted Feb. 9, 2011

Reprints not available from the authors.

This study was supported in part by the WomenAustralia, and the National Health and Medical RAPP1010315).

(PPROM) based on the findings of theORACLE I trial.4

Azithromycin, a second-generationmacrolide, is less widely used in preg-nancy, although it has a longer durationof action and half-life, greater tissue pen-etration, reduced adverse effects, and awider microbial coverage. Azithromycinis effective against many organisms ofsignificance in pregnancy such as Staph-ylococci, Streptococci, Chlamydia, Hae-

ophilus, Legionella, Listeria, Nisseriae,oxoplasma, Mycoplasma, and Plasmo-

(Drs Keelan, Nitsos, Kemp, and Newnham,d Chemical Sciences (Dr Timmins), Theustralia, Australia, and the Department ofdai, Miyagi, Japan (Drs Saito and Yaegashi).

ce of the Society for Gynecologic1.

d Infants Research Foundation, Westernarch Council of Australia (project Grant

/j.ajog.2011.02.035

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ium spp. Because of its preferential ac-umulation in tissues, it has improvedfficacy against intracellular pathogensuch as Mycoplasma and Ureaplasmapp.

In light of the widely accepted role ofntrauterine infection and inflammationn the aetiology of preterm labor andPROM, antimicrobial therapy in preg-ancy has been evaluated with the goal ofeducing rates of preterm birth and neo-atal morbidity and mortality.2,5-7 A re-ent metaanalysis of maternal macrol-de administration following PPROMound statistically significant reductionsn chorioamnionitis, preterm birth, andeonatal morbidity.8 However, antibi-

otic treatment has been shown to have, atbest, only modest benefits on pretermdelivery rates and neonatal outcomeswhen administered to women at risk ofpreterm birth2 or women presentingwith preterm birth.6-7,9

This relative lack of success is consist-ent with strong evidence that systemic

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1.e2 American Journal of Obstetrics & Gynecology MONTH 2011

largely ineffective in eradicating intrauter-ine Ureaplasma infection,10 although re-

orts of more successful outcomes do ex-st.11,12 Ureaplasmas and Mycoplasmas arehe microorganisms most frequently iso-ated from human amniotic fluid (AF)13

and the placenta14-16 and are strongly asso-ciated with an intrauterine inflammatoryresponse17,18 and preterm birth.19 Accord-ingly, their eradication from the amnioticcavity is likely an essential prerequisite fordevelopment of an effective antimicrobialregimen. The efficacy of an intraamniotic(IA) route of antibiotic administration hasnot yet been evaluated and reported.

In our experimental sheep model ofintrauterine Ureaplasma colonization dur-ing pregnancy, we have recently shownthat maternal intramuscular (IM) eryth-romycin administration does not eradi-cate Ureaplasma infection from the am-niotic fluid, chorioamnion, umbilicalcord, or the fetal lung.20 However, the

iodistribution of macrolide antibioticsdministered maternally in this modelas not yet been described. The inabilityf maternal systemic erythromycin ad-inistration to combat intrauterine Urea-

lasma colonization is likely to be attribut-ble to poor transplacental passage.

In the perfused human placentaodel, the transfer rate of macrolides is

-4%.21 Sampling and analysis of mater-al and fetal plasma at the time of thera-eutic abortion22 or elective cesareanection at term23 suggests that the degree

of erythromycin passage to the fetus isboth low and inconsistent. Transplacen-tal passage of erythromycin may be lim-ited by the presence of drug efflux pumpsin the placenta and fetal membranes.24,25

However, a small study performed inpregnant women at term reported thatthe transfer of macrolides from maternalcirculation to amniotic fluid was consid-erable (reaching 30-50% of maternalplasma concentrations) and that the pla-centa accumulated antibiotics and main-tained high concentrations for severaldays after administration.26

The aim of the present study, there-fore, was to compare the pharmacoki-netics of maternal vs IA administrationof erythromycin and azithromycin inthe pregnant ovine model to deter-

FIGURE 1Erythromycin concentrations in maternal plasma, AF, andfetal plasma after maternal IV, IM, or IA administration

Concentrations of erythromycin in maternal plasma, AF, and fetal plasma following maternal A, IVadministration, B, IM administration, or C, IA administration. Data shown are the mean � SD (n � 5animals).AF, amniotic fluid; IA, intraamniotic; IM, intramuscular; IV, intravenous.

Keelan. Macrolide pharmacokinetics in pregnancy. Am J Obstet Gynecol 2011.

mine the best antibiotic and route of

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www.AJOG.org Basic Science: Obstetrics Research

administration for pharmacologicaltreatment of intrauterine Ureaplasmainfection.

MATERIALS AND METHODSSurgical procedures andantibiotic administrationAll experimental procedures describedin this study were approved by the Ani-mal Ethics Committee of the Universityof Western Australia. Pregnant ewesbearing single fetuses underwent asepticsurgery at 110 days’ gestation (term �150 days) for maternal and fetal cathe-terization. Ewes were premedicated withIM acetylpromazine (0.02 mg/kg) andbuprenorphine (0.01 mg/kg, IM) ap-proximately 30 minutes prior to induc-tion of anesthesia with intravenous (IV)midazolam (0.25 mg/kg) and ketamine(5 mg/kg).

After induction of anesthesia, the tra-chea was intubated, and anesthesia wasmaintained with 1.5-2% isoflurane in100% O2 delivered with intermittent pos-itive pressure ventilation. Using aseptictechniques, a paramedian abdominal inci-sion was made, the pregnant uterus wasidentified, and the fetal head palpated andthen delivered through a uterine incision.Polyvinyl catheters containing heparinizedsaline (50 IU/mL) were inserted into a fetalcarotid artery and amniotic fluid cavity.The fetus was administered antibiotics(100 mg engemycin [oxytetracyclin], IM)and returned to the uterus. After closure ofthe uterine incision, catheters were exteri-orised through a small incision in the ewe’sflank and the abdominal incision wasclosed in 2 layers.

Polyvinyl catheters containing hepa-rinized saline (50 IU/mL) were then in-serted into the maternal carotid arteryand jugular vein. The catheters were se-cured to the maternal skin in 3 places andpositioned behind the ewe’s neck. Theewe was administered antibiotics (400mg engemycin, IM) and a fentanyl patch(Duragesic 75 �g/h; Alza Corp., Moun-ain View, CA) was applied to the mater-al skin for postsurgery analgesia. Ewesere allowed to recover in individualoor pens with free access to food andater and were monitored daily for at

east 5 days before experimentation.

At 115 days’ gestation, chronicallyatheterized pregnant ewes (53.5-64.4g) were randomly selected to receivene of the following: (1) a single mater-al IV infusion of erythromycin lactobi-nate (5 mg/kg maternal weight; n � 5)

over 60 minutes in 100 mL of saline; (2)single maternal IV infusion of azithro-mycin (5 mg/kg maternal weight, n � 5);3) a single maternal IM injection ofrythromycin lactobionate (5 mg/kg ma-ernal weight, n � 5) (note: an IM azi-

FIGURE 2Azithromycin concentrations in mafetal plasma after maternal IV or IA

Concentrations of azithromycin in maternal plasdministration or B, IA administration. Data show

AF, amniotic fluid; IA, intraamniotic; IV, intravenous.

Keelan. Macrolide pharmacokinetics in pregnancy. Am J Obs

thromycin administration arm was not

MONTH 2011 Ame

incorporated because clinically this anti-biotic is not given IM); or a single IA in-jection of (4) erythromycin lactobionateor (5) azithromycin (3.2 mg/kg esti-mated fetal weight, n � 5).

At this gestational age, fetal weight wasestimated to be 2.4 kg and amniotic fluidvolume 250 mL. Maternal (5 mL) and fetal(2 mL) arterial blood and amniotic fluid (2mL) samples were collected into heparin-ized tubes 15 minutes before and immedi-ately prior to the administration of the

nal plasma, AF, anddministration

, AF, and fetal plasma following maternal A, IVre the mean � SD (n � 5 animals).

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preceding text; after the infusion/injec-tions, samples were taken at 1, 2, 4, 8, 12,24, and 48 hours. Arterial PO2 (PaO2),PCO2 (PaCO2), O2 saturation (SaO2), pH(pHa), and electrolytes were measuredwith a RapidLab 1265 blood gas analyzer(Siemens, Munich, Germany). Fetal andmaternal plasma and amniotic fluid sam-ples were collected and stored at �80°C

rior to quantification of the macrolide

FIGURE 3Macrolide pharmacokinetic profilesor AF following maternal or IA adm

Comparison of macrolide pharmacokinetic profileor IA administration. Concentrations in other comshown. Data shown are the mean � SD (n � 5AF, amniotic fluid; IA, intraamniotic.

Keelan. Macrolide pharmacokinetics in pregnancy. Am J Obs

ntibiotic concentrations.

1.e4 American Journal of Obstetrics & Gynecology

Sample extraction and analysisCalibration standards, consisting of a se-ries of 5 10-fold serial dilutions of eryth-romycin and azithromycin antibiotics(4000 to 0.04 ng/mL), ovine plasma/am-niotic fluid samples, and quality controls(all 0.1 mL) were extracted with 300 �Lmethanol by vortexing for 30 secondsand mixing on an orbital shaker for 10minutes after addition of internal stan-

maternal plasmastration

A, maternal plasma or B, AF following maternalrtments are not visible in these plots so are notmals).

ynecol 2011.

dard (0.5 ng roxithromycin). Quality

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control samples (200 ng/mL in plasma oramniotic fluid) were run with each assaybatch.

After centrifugation (3200 � g, 20inutes), extracts were transferred to

lass tubes and dried with gentle heating30oC) under nitrogen. Dried extractsere resuspended in 100 �L methanol

and filtered through 0.22 �m celluloseacetate centrifuge tube filters (CorningInc, Corning, NY); 0.5 �L was injectedinto an Agilent 1200-series capillary liq-uid chromatography system coupled toan Agilent 6340 Ion Trap mass spec-trometer (Agilent Technologies, PaloAlto, CA). Methanol blanks were in-jected after every 11 sample injections.Separation was achieved using reverse-phase chromatography on a 3.5 �m Agi-ent Eclipse XDB-C18 column (2.1 �50 mm); a gradient of 0-75% acetoni-rile in 0.1% formic acid over 7 minutesas employed, followed by a 1 minute

ecovery wash at 100 % acetonitrile.Macrolide MRM transitions wereonitored in positive mode as follows:

zithromycin (748.9-591.0), erythromy-in (733.9-576.6), and roxithromycin837.0-679.0); elution times were 2.9,.1, and 5.9 minutes, respectively. Allass to charge ratio peaks were manu-

lly integrated using 6300 Series Ionrap liquid chromatography and mass

pectrometry software 6.1.Integrated signals from samples, con-

rols, and standards were blank cor-ected, expressed as a ratio of the internaltandard, and log transformed. A linearurve fit was applied to the calibrationurve and this used to derive the concen-ration of the samples. Extraction effi-iency for each sample was calculated byeference to the signal from a givenmount of roxithromycin internal stan-ard extracted and processed as per stan-ards and samples. The limit of quanti-ation was defined as 5 SD above theackground signal.

Statistical and pharmacokineticanalysisMacrolide concentration data from 5 an-imals per time point were grouped andthe mean and SDs calculated. These datawere subject to pharmacokinetic analysis

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gramme PKSolver.27 Maternal pharma-okinetic data were fitted using a 2 com-artment model, whereas the data fromA administration were best described by

noncompartmental model. Plasmand amniotic fluid control values from-6 assays were determined to derivehe interassay coefficient of variationCV).

RESULTSThe azithromycin and erythromycin as-says had a limit of quantitation of lessthan 0.1 and less than 0.3 �g/L, respec-tively. Mean extraction efficiency was70.75%. Interassay CVs for erythromy-cin in maternal plasma and AF controlsamples were 14.7% and 19.4%, respec-tively (n � 6). Azithromycin interassayCVs for plasma and AF samples (n � 4)were 6.2% and 5.7%, respectively.

The biodistribution profiles of themacrolide antibiotics in the 3 compart-ments (maternal plasma, AF, and fetalplasma) following IV, IM, and IA ad-ministration are shown in Figures 1and 2 using a log concentration axis tollow data from all compartments to beisplayed on the same graph. Figure 3ompares the profiles achieved of the 2ntibiotics on a linear axis. The phar-acokinetic parameters for IM and IVaternal macrolide administration are

isted in Table 1, whereas the parame-ers for IA administration are shown inable 2.

Erythromycin pharmacokineticsMaternal administration of erythromy-cin achieved peak maternal plasma anti-biotic concentrations within an hour, ac-companied by markedly attenuated risein AF and fetal plasma concentrations(Figure 1, A and B). Delivery by IV in-usion resulted in a greater maternallasma maximum concentration (Cmax)han IM administration, but concentra-ions declined less rapidly with IM ad-

inistration, such that the area underhe curve (AUC) was considerablyreater with IM administration (3.97 vs.227 �g/mL per hour) and the initial

half-life (t½�) was approximately 5times longer (2.7 hours vs. 0.35 hours)(Table 1 and Figure 3, A). Maternal
plasma concentrations following IV ad- (
ministration exceeded the estimated min-imal inhibitory concentration (MIC) forUreaplasma (0.5 �g/mL) only at the 1 hourime point, whereas with IM administra-ion MIC levels were sustained for approx-mately 4 hours (Figure 3, A).

Transfer from maternal circulation toF was negligible (�1% at peak mater-al levels), failing to achieve therapeuticoncentrations at any time point. Thextent of transfer to AF was greatest fol-owing IV administration, reaching lev-ls of 0.025 �g/mL at 2 hours, althoughith IM administration the rise in AF

evels was more gradual and sustained

TABLE 1Maternal plasma macrolide pharm

Parameter UnitErythrIM

A �g/mL 1.15...................................................................................................................

Alpha 1/hour 0.25...................................................................................................................

B �g/mL 0.04...................................................................................................................

Beta 1/hour 0.25...................................................................................................................

Dose mg 284.9...................................................................................................................

k10 1/hour 0.25...................................................................................................................

k12 1/hour 0.00...................................................................................................................

k21 1/hour 0.25...................................................................................................................

t� Hour 2.67...................................................................................................................

t� Hour 2.67...................................................................................................................

V/F (mg)/(�g/mL) 276.8...................................................................................................................

CL/F (mg)/(�g/mL)per hour

71.8

...................................................................................................................

V2/F (mg)/(�g/mL) 0.0...................................................................................................................

CL2/F (mg)/(�g/mL)per hour

0.0

...................................................................................................................

Tmax Hour 1.21...................................................................................................................

Cmax �g/mL 0.75...................................................................................................................

AUC0-t �g/mL per hour 3.97...................................................................................................................

AUC0-inf �g/mL per hour 3.97...................................................................................................................

AUMC �g/mL perhour2

17.41

...................................................................................................................

MRT Hour 4.39...................................................................................................................

Vss �g/(ng/mL)...................................................................................................................

Two compartment analysis of maternal plasma macrolide phmg/kg maternal weight).AUC, area under the curve; AUMC, area under the first mometration reached; F, bioavailability (fraction of dose absorbed)apparent volume of distribution; V2/F, apparent peripheral vol

Keelan. Macrolide pharmacokinetics in pregnancy. Am J O

0.011 �g/mL at 24 hours). Fetal plasma a

MONTH 2011 Ame

oncentrations were even lower than AFevels (less than 0.004 �g/mL), repre-enting less than 1% of maternal concen-rations during the first 8 hours of thexperiment.

Intraamniotic erythromycin adminis-ration (Figure 1, C) achieved much

higher concentrations than maternal ad-ministration (Cmax 8.7 �g/mL) with a faronger half-life (t½� 27.6 h). As a result,he AUC was 100-fold greater than thatbserved in maternal plasma followingaternal IV administration (Table 2),

nd therapeutic concentrations wereaintained in AF for at least 48 hours

kinetic parameters

ycin Erythromycin AzithromycinIV IV

3.075 1.218..................................................................................................................

1.964 2.284..................................................................................................................

0.247 0.154..................................................................................................................

0.374 0.0269..................................................................................................................

282.9 273.5..................................................................................................................

1.4916 0.2187..................................................................................................................

0.3540 1.8112..................................................................................................................

0.4921 0.2806..................................................................................................................

0.3529 0.3035..................................................................................................................

1.8546 25.797..................................................................................................................

85.2 199.3..................................................................................................................

127.0 43.6

..................................................................................................................

61.2 1286.2..................................................................................................................

30.1 360.9

..................................................................................................................

1.0..................................................................................................................

1.552 0.631..................................................................................................................

2.227 4.674..................................................................................................................

2.227 6.277..................................................................................................................

2.567 213,990

..................................................................................................................

1.15 34.1..................................................................................................................

146.4 1485.5..................................................................................................................

cokinetic parameters following IV or IM administration (5

rve; CL/F, apparent clearance; Cmax, maximum concen-T, mean residence time; Tmax, timepoint at Cmax; V/F,of distribution; Vss, volume of distribution at steady state.

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of transfer from AF to fetal plasma wasvery poor, however, with peak levels (ap-proximately 0.017 �g/mL) at 8 h afterinjection well below therapeutic concen-trations (Figure 1, C). Concentrations inmaternal plasma were even lower, barelyreaching 0.001 �g/mL.

Azithromycin pharmacokineticsAzithromycin pharmacokinetic profiles(Figure 2) were broadly similar to those

f erythromycin, although there wereome differences (Figure 3). MaternalV administration achieved transient (1our) therapeutic concentrations (�0.5

�g/mL) in maternal plasma. Apprecia-ble levels were subsequently measureablein AF and increased with time, reaching0.036 �g/mL at 24 hours, equivalent toapproximately 50% of maternal concen-trations at that time point (Figure 2, A).

uch lower azithromycin concentra-ions were measured in fetal plasma�0.002 �g/mL).

The IA azithromycin injection re-ulted in extremely high AF levels (�18

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TABLE 2Amniotic fluid macrolide pharmaco

Parameter Unit

Dose mg...................................................................................................................

Lambda_z 1/hour...................................................................................................................

t hour...................................................................................................................

Clast obs/Cmax...................................................................................................................

Vzobs (mg)/(�g/mL)...................................................................................................................

Clobs (mg)/(�g/mL) per hour...................................................................................................................

Tmax hour...................................................................................................................

Cmax �g/mL...................................................................................................................

C0 �g/mL...................................................................................................................

AUC0-t �g/mL per hour...................................................................................................................

AUC0-inf_obs �g/mL per hour...................................................................................................................

AUMC0-inf_obs �g/mL per hour2

...................................................................................................................

MRT0-inf_obs hour...................................................................................................................

Vssobs (�g)/(ng/mL)...................................................................................................................

Noncompartmental analysis of AF macrolide pharmacokineticAUC, area under the curve; AUMC, area under the first momconcentration; C0, concentration at time zero; MRT, mean rvolume of distribution at steady state; Vz, volume of distributio

Keelan. Macrolide pharmacokinetics in pregnancy. Am J O

time (t½� 17.5 hours); surprisingly, the g


rate of clearance of azithromycin fromAF was faster than that of erythromycin(Table 2 and Figure 3, B), although theAUCs were fairly similar because of thehigher starting concentrations of azi-thromycin (Table 2). AF-administeredazithromycin appeared to show a muchgreater degree of transfer to maternalplasma than fetal plasma (approximately100-fold) in contrast to the IA erythro-mycin pharmacokinetic profile (Figure2). Concentrations of azithromycin inmaternal blood equilibrated at around0.03-0.04 �g/mL for much of the first 12hours after IA administration (approxi-mately 30% of AF levels), whereas in fe-tal blood, levels did not exceed 0.004�g/mL.

The half-life of azithromycin in AFwas less than that of erythromycin, 17.5vs 27.6 hours (Table 2), such that by 48hours after injection, the levels of the 2antibiotics were similar despite the largerCmax values at 1 hour achieved with azi-hromycin administration (Table 2 andigure 3, B). Azithromycin had a far

etic parameters

Erythromycin AzithromycinIA IA

7.68 7.68..................................................................................................................

0.0251 0.0397..................................................................................................................

27.57 17.46..................................................................................................................

0.2595 0.1247..................................................................................................................

1.00 0.53..................................................................................................................

0.03 0.02..................................................................................................................

1 1..................................................................................................................

8.690 18.930..................................................................................................................

0.0001 0.0553..................................................................................................................

216.6 309.8..................................................................................................................

306.3 369.3..................................................................................................................

11799.3 9245.5..................................................................................................................

38.5 25.03..................................................................................................................

1.0 0.52..................................................................................................................

r IA bolus injection (3.2 mg/kg fetal weight).curve; C, concentration; Cl, clearance; Cmax, maximumnce time; Tmax, timepoint at Cmax; Vss[obs], observedring terminal phase.

et Gynecol 2011.

reater accumulation in tissues than

MONTH 2011

rythromycin as reflected by their re-pective volumes of distribution (Vss):485 vs 147 �g/(ng/mL).

COMMENTTo effectively eradicate an intrauterineinfection, administration of antibioticsmust achieve therapeutic concentrationswithin the amniotic, maternal, and fetalcompartments. The MIC for erythromy-cin/azithromycin ranges from 0.01 togreater than 100 �g/mL according to or-ganism and method of determination; aconcentration of about 0.5-1 �g/mL is

eeded to achieve a MIC for nonresistantreaplasma spp.28

In the present study, although thera-peutic macrolide concentrations werereached briefly in maternal plasma fol-lowing maternal IM or IV administra-tion, neither route achieved effectiveconcentrations in AF or fetal plasma.This supports findings from severalstudies in humans that suggest that theextent of transfer of macrolides frommaternal to fetal plasma is low.21,26

Passive diffusion of molecules greaterthan 500 Daltons across the placenta isknown to be minimal (macrolides havea molecular weight of �700 Daltons);

oreover, the placenta expresses a num-er of active efflux pumps, at least onef which recognizes erythromycin andould likely act to minimize uptake and

ransfer across the placenta.25 Althoughno studies have been carried out to dateon transfer of macrolides across the fetalmembranes, we have shown that effluxproteins are also expressed in these tis-sues24 and would predict that rates oftransfer from maternal blood to AF andvice versa would also be low.

The low levels of macrolides in the fe-tal circulation after IA treatment sug-gests that these antibiotics are poorly ab-sorbed by the immature fetal gut29

because the fetus swallows AF in largeamounts and macrolide concentrationswere relatively high for an extended pe-riod of time after IA delivery. This repre-sents a significant hurdle to achievingtherapeutic fetal antibiotic levels. It re-mains to be explored whether fetal gutabsorption may be improved in utero

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Previous data exist on macrolidepharmacokinetics from studies in bothpregnant women30 and nonpregnantheep.31 Goudah et al,31 studying IVrythromycin administration to lactat-ng, nonpregnant ewes, reported a t½� of

0.4 hour, t½� of 4.5 hours, and an AUCf 7.91 �g/mL per hour. Our data (t½� of.35 hours, t½� of 1.85 hours, AUC 2.23

�g/mL per hour), suggest that maternalerythromycin clearance is increased inpregnancy, as might be predicted.

Salman et al30 recently presented datan azithromycin pharmacokinetics afterral administration in pregnant woment½� of 0.88 hours, t½� of 20.7 hours,UC 28.7 �g/mL per hour). Our com-arable data (t½� of 0.30 hours, t½� of5.8 hours, AUC 6.28 �g/mL per hour)

are suggestive of reduced maternal expo-sure in the pregnant sheep due to in-creased clearance. Although there are noprior reports of erythromycin clearancerates from AF, Ramsey et al26 reportedhat concentrations of azithromycin inF following maternal oral administra-

ion reached approximately 150 �g/mLand steadily declined, with a similar con-centration profile to that shown in Fig-ure 3, B. Hence, although there is notcomplete agreement between the phar-macokinetic data from all 3 studies, thedata are reasonably consistent with re-spect to overall picture.

Erythromycin and azithromycin havebeen reported to cross the placenta withsimilarly low levels of efficiency.21 Our

ata agree and would suggest that anyifferences between the 2 are minor and

acking biological significance. However,t is possible that the increased transferf azithromycin across the fetal mem-ranes into maternal plasma might haveome clinical advantages. We confirmed

any previous findings that azithromy-in has a longer half-life than erythromy-in in maternal plasma (25.7 vs 1.9 h)32

and also that azithromycin had a 10-foldgreater degree of tissue accumulationthan erythromycin. However, we unex-pectedly found that erythromycin hasthe longer residence time in amnioticfluid (27.6 vs 17.5 h), which could reflectenhanced uptake of azithromycin into
intrauterine tissues and/or increased t
transmembrane transfer to maternalplasma.

In light of the minor pharmacokineticdifferences between the 2 macrolides, wewould propose that azithromycin ad-ministration should have therapeuticadvantages over erythromycin in thepresent context because of its wider mi-crobial coverage, more extensive tissueuptake characteristics and improved sideeffect profile.

Although the ovine placenta differsstructurally from the human placenta ina number of important ways,33 the preg-nant ewe remains a useful model to studyplacental function/transport and fetalgrowth and development.34 Our datasuggest that maternal administration ofmacrolides is unlikely to be effective indelivering antibiotics at therapeutic con-centrations to the fetus or AF (the site ofprimary infection). This is a likely expla-nation for the limited efficacy of mater-nal macrolide therapy in treating intra-uterine and fetal infection.

Importantly, our data also show thatthe IA route of administration exhibits avery different pharmacokinetic profilefrom maternal IV or IM administration,with amniotic concentrations stayingabove the MIC for at least 2 days after abolus injection. Clinically these findingsare important and encouraging becausethey suggest that a single IA injection of amacrolide antibiotic could be effective ineradicating an IA infection of a suscepti-ble organism such as Ureaplasma par-vum. Ideally, IA injection would alsoachieve effective concentrations in thefetal circulation and maternal blood.However, our findings suggest that this isvery unlikely, at least for a relatively re-sistant organism such as Ureaplasmaparvum (the MIC for other microorgan-isms can be as low as 10-30 ng/mL26).

The transfer of macrolides out of AF ap-ears to be very inefficient, particularly inhe maternal direction, although this doesave the benefit of extending the residenceime. As a consequence, we postulate thato achieve therapeutic concentrations in

aternal and amniotic compartments, aombination of maternal and IA adminis-ration will be necessary. Such a regimenhould eradicate the organisms from ma-
ernal blood and AF and also deliver the
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ntibiotic to both faces of the fetal mem-ranes to prevent reinfection from reser-oirs within the membranes. However, al-hough some antibiotic will reach fetallasma via a combined maternal-amnioticelivery approach, such a regimen woulde unlikely to achieve concentrations suf-cient to effectively treat fetal infection.Further research is required to deter-ine whether attainment of therapeuticacrolide levels in the mother and AF

but not the fetus) is effective in treatingntrauterine infection at early gestationalges when secondary prevention of pre-erm labor is feasible. Alternative treat-

ent strategies need to be devised to im-rove fetal macrolide delivery; the sheepodel is ideal for this purpose. Success-

ully addressing these issues should leado the development of protocols for theffective treatment of intrauterine infec-ion and prevention of preterm birth. f

ACKNOWLEDGMENTSThe assistance of the staff of the Large AnimalFacility (The University of Western Australia) andour commercial sheep suppliers, Sara and An-drew Ritchie (Darkan, Western Australia) aregratefully acknowledged.

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