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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Objective To investigate the transplacental transfer of the macrolide antibiotics erythromycin, roxithromycin and azithromycin.

Methods Twenty-one term placentas were obtained with maternal consent immediately after delivery and a two-hour nonrecirculating perfusion of a single placental cotyledon was performed. Erythromycin (2 μg/mL), roxithromycin (2 μg/mL) and azithromycin (0.3 μg/mL) were infused to the maternal inflow at a constant rate, with antipyrine as a reference compound, and their appearance in the fetal circulation was followed. Drug concentrations were measured by high performance liquid chromatography for 120 min.

Results The mean transplacental transfers (TPTss) for erythromycin, roxithromycin and azithromycin were 3.0%, 4.3% and 2.6%, respectively, calculated as the ratio between the steady state concentrations in fetal venous and maternal arterial sides. Similar results were obtained when the TPT was calculated as the absolute amount of drug transferred across the placenta during 2-hour perfusion (TPTA). No significant differences were found among the three macrolides in TPTSS (P= 0.39) or TPTA (P= 0.35). The TPTSS of erythromycin, roxithromycin and azithromycin were 41%, 35% and 32% of the freely diffusable reference compound antipyrine, respectively. Steady state was reached in 60 minutes in each perfusion indicating sufficient perfusion time.

Conclusion The limited transplacental transfer of erythromycin, roxithromycin and azithromycin suggests compromised efficacy in the treatment of fetal infections. On the other hand, the placenta seems to produce an effective barrier reducing the fetal exposure when these three macrolides are used to treat maternal infections.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Macrolide antibiotics are effective in the treatment of bacterial infections of the upper and lower respiratory tract, skin and soft tissues, and the genital tract. In obstetric patients they are used for the treatment of endocervical chlamydial infections, mycoplasma pneumonia and hominis infections, and for intrapartum prophylaxis against group B streptococcal infections in mothers who are allergic to betalactam antibiotics1–5.

Erythromycin, the primary macrolide, has been widely used since the 1950 s in pregnant women. The newer macrolide derivatives, such as roxithromycin and azithromycin, have been developed to overcome the problems associated with erythromycin. The pharmacokinetic profiles of roxithromycin and azithromycin are characterised by reliable oral bioavailability, excellent tissue penetration and persistence, and longer half-life of elimination, which allows once or twice daily dosing6–8. Because roxithromycin and, especially, azithromycin are accumulated within the host cells, they may be more effective than erythromycin against intracellular pathogens, such as Mycobacteria, Legionella, Chlamydia, Listeria and Toxoplasma6–8.

Primary infection with Toxoplasma in pregnant women occurs all over the world with frequencies of 0.1–1%. In approximately 40% of cases, the fetus is infected9. The current therapy of choice for toxoplasmosis in pregnant women is the synergistic combination of pyrimethamine, sulphadiazine and spiramycin10. However, pyrimethamine and sulphadiazine may be associated with adverse pregnancy outcome, and therefore novel drugs need to be studied11,12. Due to its pharmacokinetic and antimicrobial characteristics, azithromycin has been suggested to have some advantages in the treatment of toxoplasmosis during pregnancy compared with other macrolide antibiotics11–13.

No teratogenic defects have been associated with the use of macrolide antibiotics during pregnancy5,14,15. Earlier clinical studies suggested that the transplacental transfer of erythromycin is limited, as the fetal blood levels were only 2% to 10% of those in maternal blood16–18. Because no previous studies have addressed the transplacental passage of roxithromycin and azithromycin, the aim of the present study was to investigate the transplacental transfer of the macrolide antibiotics erythromycin, roxithromycin and azithromycin by means of an in vitro perfusion of the human placental cotyledon.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Twenty-one placentas (seven for each macrolide) were obtained from normal pregnancies at term after vaginal or caesarean delivery. The placentas were normal, the mean weight (range) was 533 g (500–650), 539 g (460–600) and 521 g (450–600) for erythromycin, roxithromycin and azithromycin, respectively. All women were healthy and used no drugs during the late stage of their pregnancy, and verbal informed consent was obtained. The study protocol was approved by the joint Ethics Committee of the Faculty of Medicine, University of Turku and Turku University Central Hospital.

The placentas were transported in ice to the perfusion laboratory within 30 minutes after the delivery. Heparinised 0.9% NaCl solution was injected into the umbilical artery, a good cotyledon was chosen and the corresponding distal branches from the chorionic artery and vein were cannulated. The criteria for selecting a good cotyledon have been characterised by Schneider et al.19: about 4 to 6 cm in diameter; preferably supplied by a single chorionic artery and vein; and with the decidual plate intact. The cotyledon was cut out from the placenta and placed on a metal plate with the maternal side upward over a plexiglas cone into which the maternal effluent was allowed to drain. Four butterfly needles were placed 2–3 mm below the maternal surface in the intervillous space; the cannulated fetal (chorionic) artery was also connected to the perfusion system. The perfusion was started with the flow rate of 3 mL/minute on the fetal side and 10 mL/minute on the maternal side20–22. The perfusate on both sides was Krebs-Ringer bicarbonate buffer (Sigma Chemical Co, St. Louis, Missouri, USA). The medium was gassed with a 95% oxygen and 5% carbon dioxide mixture at 37°C throughout the perfusion.

Antipyrine (80 μg/mL), which is known to diffuse passively through the placenta, was used as a reference compound to detect possible failure in each individual perfusion. A blood gas analysis (pH, pO2 and pCO2; Type PHA 927 b Radiometer AS, Copenhagen, Denmark) of samples of fetal arterial and venous perfusates was performed to test the viability of the placental tissue at the beginning and end of each experiment. In addition, the integrity of the preparation was assessed by monitoring the stability of the fetal perfusion pressures (Multi-Pen Recorder, Rikadenki, Tokyo, Japan).

After a stabilisation period of 30 minutes (i.e. perfusion with the perfusion medium and antipyrine only), the cotyledons were perfused for two hours with either erythromycin succinate (n= 7; Orion Pharma, Finland), roxithromycin (n= 7; Leiras, Finland) or azithromycin (n= 7; Pfizer, Ireland) added to the maternal side perfusate. Drug concentrations in perfusate were chosen to correspond to the therapeutic concentrations of orally administered drug; 2 μg/mL, 2 μg/mL and 0.3 μg/mL, respectively. All experiments were conducted in an open (nonrecirculating) system. The perfusion method, as shown in Fig. 1, was a modification of that reported by Schneider et al.19. Samples of perfusates were obtained from the fetal venous cannula and from the maternal effluent reservoir every 10 minutes until 120 minutes. Each sample was centrifuged (2000 rpm, 5 minutes) to remove cellular debris and stored at −20°C until analysis. The rejection rate of perfused placentas in our study was 30% (30 placentas perfused and 21 included in the analyses). The discarded placentas did not fulfil the criteria for validity.

image

Figure 1. Schematic presentation of the applied perfusion model.

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Erythromycin, roxithromycin and azithromycin concentrations in the perfusate were determined by high performance liquid chromatography with electrochemical detection after necessary modifications23,24. The lower limits of quantitation were 50 ng/mL for erythromycin, 10 ng/mL for roxithromycin and 1 ng/mL for azithromycin. The inter-assay coefficients of variation for the low and high control perfusate samples were 7.9% and 4.7% (mean 0.24 μg/mL and 13.5 μg/mL), respectively, for roxithromycin; 7.8% and 6.1% (mean 33.8 ng/mL and 114.7 ng/mL), respectively, for azithromycin; and 9.5% (mean 0.40 μg/mL) for erythromycin.

Antipyrine concentrations were measured spectrophotometrically, as described earlier25. The interassay precision was evaluated by using the control perfusate sample containing 40 μg/mL antipyrine, the coefficient of variation of which was 13.4%.

Data analysis

The time to reach steady state concentration (tss) was estimated graphically for each perfusion with antipyrine and each macrolide. The concentration at steady state (Css) was thereafter calculated as the mean of the measured concentrations beyond tss. The transplacental transfer percentages (TPTss) were calculated using the following equation:

  • image

where CSS(fetal) = steady state concentration in fetal venous outflow, FFR = steady state fetal flow rate (3 mL/min), C(ma) = concentration in maternal arterial inflow, MFR = steady state maternal flow rate (10 mL/min).

The transplacental transfer was also evaluated by comparing the absolute amount of macrolide infused into placenta (AMA) with the amount of drug found in fetal venous outflow (AFV) during the 2-hour perfusion (TPTA= AFV/AMA× 100). The amount of drug on each side was calculated as the area under the drug concentration versus time curve (AUC (0–2 h)) multiplied by the corresponding flow.

The transplacental transfer index (TI) (i.e. the ratio of transplacental transfer between macrolide and antipyrine) was calculated using the following equation:

  • image

Recovery (RC) (i.e. the amount of drug found in the fetal and in the maternal effluent perfusates in relation to what was perfused through the cotyledon) was calculated using the following formula:

  • image

All results are expressed as mean (SD); tss is given as median (range). The statistical analyses were performed using unpaired two-tailed t test (blood gas analysis parameters and fetal artery pressure), one-way ANOVA with Tukey's test for post hoc analyses (transplacental transfers, transfer index, area under the curve and recovery) and Kruskal-Wallis test (tss). Level of significance was set at P < 0.05. The statistical program Systat for Windows, Version 5.0 (Systat Inc, Evanston, Indiana, USA) was used for all analyses.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

The pH of the perfusate and the fetal artery perfusion pressure are given in Table 1. For each macrolide and each individual perfusion, both pH and fetal artery pressure remained stable during the two hour perfusion period. The values remained within the physiological range and no statistically significant differences were found between the values at the beginning and at the end of the perfusion. A slight decrease in antipyrine transfer towards the end of perfusion was noticed in experiments with the roxithromycin (Fig. 2 b). The antipyrine concentrations remained stable during the erythromycin and azithromycin perfusions (Fig. 2 a and 2 c).

Table 1.  Viability of the placentas expressed by pH and perfusion pressure in fetal artery measured at the beginning and at the end of each perfusion. Values are given as mean (SD)
 Start of perfusionEnd of perfusion
 Fetal arteryFetal veinFetal arteryFetal vein
Erythromycin (n= 7)    
 Arterial blood pH7.4 (0.05)7.3 (0.12)7.5 (0.15)7.3 (0.05)
 Pressure (mmHg)23.2 (13.7) 25.6 (15.8) 
Roxithromycin (n= 7)    
 Arterial blood pH7.5 (0.08)7.3 (0.12)7.5 (0.08)7.4 (0.17)
 Pressure (mmHg)15.7 (4.7) 14.6 (5.4) 
Azithromycin (n= 7)    
 Arterial blood pH7.5 (0.06)7.4 (0.09)7.5 (0.05)7.5 (0.06)
 Pressure (mmHg)20.7 (4.0) 17.8 (3.7) 
image

Figure 2. The transplacental transfer percentages of antipyrine (l) and: (a) erythromycin; (b) roxithromycin; (c) azithromycin (t). The data are mean for seven placentas; error bars have been omitted for clarity.

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The transplacental transfer of macrolides was found to be relatively low. The TPTss for erythromycin, roxithromycin and azithromycin were 3.0%, 4.3% and 2.6%, respectively (Table 2, Fig. 2). Similarly, the TPTA were 2.5% for erythromycin, 4.3% for roxithromycin and 2.6% for azithromycin. No significant differences were found among the three macrolides in TPTSS (P= 0.39) or TPTA (P= 0.35).

Table 2.  Pharmacokinetic parameters of erythromycin, roxithromycin and azithromycin. Values are given as mean (SD) or median [range]. CSS= concentration at steady state; tSS= time to reach steady state concentration; AUCFV(0–120 min)= area under the drug concentration versus time curve; TPTSS= transplacental transfer calculated as the ratio between concentrations in fetal venous outflow and maternal arterial inflow in steady state; TPTA= transplacental transfer calculated by comparing the absolute amount of drug infused into the placenta with the amount of drug found in fetal venous outflow; TI = transplacental transfer index.
 Erythromycin (n= 7)Roxithromycin (n= 7)Azithromycin (n= 7)
tSS (min)30 [20, 60]40 [10, 60]30 [20, 60]
CSS(μg/mL)0.14(0.05)0.21 (0.12)0.026 (0.013)
AUCFV(0.120 min) (μg/mL × min)11.8(4.0)21.5(13.7)2.5(1.40)
TPTSS (%)3.0 (2.6)4.3(2.5)2.6(1.6)
TPTA (%)2.5 (2.2)3.7 (2.2)2.1(1.4)
TI0.41 (0.31)0.35 (0.17)0.32 (0.19)

The TPT of erythromycin, roxithromycin and azithromycin were 41%, 35% and 32%, respectively, of that of freely diffusable antipyrine (Table 2). There were no statistically significant differences in TI among the three macrolides (P= 0.79). The median time of perfusion to reach the steady state was 30 minutes for erythromycin and azithromycin, and 40 minutes for roxithromycin. In all perfusions steady state was reached by 60 minutes.

The recovery of erythromycin was slightly lower (60%) compared with roxithromycin (90%) and azithromycin (81%). However, this finding did not reach statistical significance (P= 0.23).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

All the macrolides studied, erythromycin, roxithromycin and azithromycin, transfer across the placenta. However, the placental transfer is with < 5% relatively low. Our results are in accordance with the earlier clinical results of studies on erythromycin16–18. After multiple oral doses, the concentration of erythromycin in the blood of aborted fetuses was only about 2% of the concentration measured in the mother at the time of the abortion16. No earlier reports, clinical or experimental, exist on the transplacental passage of roxithromycin or azithromycin. The low placental transfer of the drugs studied also was evident as calculated as the absolute amount of drug transferred across the placenta during the 2-hour perfusion (TPTA). The similarity of the transfers calculated with TPTA and TPTSS reflects the fact that steady state in the fetal venous outflow was rapidly achieved with all macrolides studied. Therefore, the 2-hour perfusion seems to be adequate to study the placental transfer of macrolides.

Macrolide antibiotics can be used in the treatment of various maternal infections during pregnancy, but because of the poor placental transfer these antibiotics seem not to be the drug of choice in treating the infected fetus. On the other hand, possible adverse effects in the fetus are avoidable while treating pure maternal infections, like respiratory infections or cervicitis caused by Chlamydia. Theoretically, the macrolides studied, especially azithromycin, may be good candidates to treat pregnancy-related toxoplasmosis where the placenta serves as source of parasites and the fetus may be protected by stopping the infection in the placenta.

Macrolides typically accumulate in tissues, with azithromycin having a tissue to serum concentration rate several-fold higher than other macrolides7. Therefore, the present finding of 80% recovery of azithromycin (compared with the 60% recovery of erythromycin) indicates a surprisingly low affinity of this drug to placental tissue. However, it should be stressed that the macrolide concentrations in placental tissue were not measured in the present study. Further, since it has been shown that azithromycin is transported inside the leukocytes to the infection focus, the tissue distribution of this drug is likely to be different with infected placenta26.

The present experiments were performed employing a noncirculating perfusion model. This model was chosen to produce a constant rate of availability of the study drugs to the maternal artery simulating the pharmacokinetic features that are present when steady state is reached in chronic oral drug treatment of the mother. Infusion with clinically relevant concentrations of the three macrolides to the maternal inflow in this experimental model resulted in steady state concentrations in fetal venous outflow that are below or at the lower limits of the therapeutic range for many clinically important pathogens7,27,28. After entering the fetus the drug is distributed to the fetal tissues and the resulting tissue concentrations are likely to be very low, although accumulation in some tissues may exist. This finding further suggests that the clinical efficacy of erythromycin, roxithromycin or azithromycin may be compromised in the treatment of intrauterine fetal infections caused by pathogens that are generally sensitive to these drugs. On the other hand, they may offer an effective tool against maternal infections with minimal fetal exposure.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

The limited transplacental transfer of erythromycin, roxithromycin and azithromycin suggests compromised efficacy in the treatment of fetal infections. On the other hand, the placenta seems to produce an effective barrier reducing the fetal exposure when these three macrolides are used to treat maternal infections.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References
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