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Keywords:

  • constriction of ductus arteriosus;
  • fetal toxicity;
  • human placental perfusion study;
  • nonsteroidal anti-inflammatory drug

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• The use of nonsteroidal anti-inflammatory drugs (NSAIDs) in full-term pregnant women leads to fetal or neonatal toxicity. However, the differences in fetal toxicity among NSAIDs remain to be fully investigated.

• Therefore, the ability to predict fetal toxicity of NSAIDs quantitatively at full-term pregnancy would be of clinical value.

WHAT THIS STUDY ADDS

• This study presents a novel approach to predict quantitatively the fetal risk of NSAIDs administered to the mother.

• This study shows that the fetal risk of diclofenac is higher than that of salicylic acid and antipyrine using the novel method.

• Human placental perfusion study and pharmacokinetic/pharmacodynamic analysis may provide basic data for predicting human fetal toxicity of drugs.

AIM The use of nonsteroidal anti-inflammatory drugs (NSAIDs) in full-term pregnant women leads to fetal or neonatal toxicity, such as constriction of the ductus arteriosus (DA) and persistent pulmonary hypertension in the newborn. The aim of this study was to predict quantitatively the fetal toxicity of three NSAIDs (antipyrine, salicylic acid and diclofenac) using the transplacental pharmacokinetic parameters obtained from our previous placental perfusion studies.

METHODS Human fetal plasma concentration profile after oral administration of each NSAID to the mother was estimated using the transplacental pharmacokinetic parameters and pharmacokinetic parameters in adult women. The fetal plasma concentration–response relationship for the three NSAIDs was estimated by pharmacokinetic/pharmacodynamic analysis of the results of previous studies investigating the effects of NSAIDs on the ratio of inner diameter of the DA to that of the pulmonary artery (DA/PA) in rats and the plasma concentration profiles of NSAIDs in pregnant rats.

RESULTS The risk of constriction of the DA was well predicted by the model. Mean DA/PA ratio after oral administration of diclofenac to the mother was estimated to be 39.0%, whereas both of the corresponding values after oral administration of antipyrine and salicylic acid were estimated to be 5.9%. These results suggest that the fetal risk of diclofenac is higher than those of salicylic acid and antipyrine.

CONCLUSIONS This study presents a novel approach to predict quantitatively the fetal risk of NSAIDs administered to the mother. Human placental perfusion study and pharmacokinetic/pharmacodynamic analysis may provide basic data for predicting human fetal toxicity of drugs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as antipyretics and analgesics in the field of gynaecology. When given to women during full-term pregnancy, diclofenac, an NSAID, readily permeates into fetal blood, inhibits the synthesis of prostaglandins and constricts the ductus arteriosus (DA), and may cause pulmonary hypertension and/or transient right-sided hypertrophic cardiomyopathy in the newborn [1, 2]. Salicylic acid leads to pulmonary hypertension in the newborn in a similar manner [3]. However, the differences in fetal toxicity among NSAIDs remain to be fully investigated. Therefore, the ability to predict fetal toxicity of NSAIDs quantitatively at full-term pregnancy would be of clinical value.

Fetal toxicity is considered to be determined by both the maternal-to-fetal transfer of drugs (pharmacokinetics; PK) and the intrinsic toxicity (pharmacodynamics; PD). While the latter, such as constriction of fetal DA, can be evaluated in rats [4, 5], prediction of PK is difficult, because maternal-to-fetal transfer of drugs is regulated by the placenta. Animal studies may not be helpful to predict human fetal PK because of interspecies differences in the structural and functional features of the placenta. We have developed a method to evaluate quantitatively transplacental pharmacokinetic (TP-PK) parameters between maternal or fetal blood and placenta by applying a novel PK model [6, 7] to the results of human placental perfusion study [8]. This method allows us to estimate the concentration profile of a drug in fetal blood from the maternal plasma profile.

The aim of this study was to predict quantitatively the fetal toxicity (constriction of the fetal DA) after maternal administration of NSAIDs by means of a PK/PD approach using TP-PK parameters estimated from our human placental perfusion study, pharmacokinetic data in humans and fetal toxicity estimated in rats.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

Figure 1 shows a schematic representation of the PK/PD approach to estimate fetal toxicity of NSAIDs in humans in this study.

image

Figure 1. Schematic representation for estimation of fetal toxicity of NSAIDs in humans from animal and in vitro data

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Pharmacokinetic parameters in women (I)

A conventional one- or two-compartment model with first-order absorption was fitted to the plasma concentration profiles after single oral administration of diclofenac (50 mg), salicylic acid (300 mg) or antipyrine (600 mg) to healthy women [9–11] by using a nonlinear least-squares program (MLAB; Civilized Software, Bethesda, MD, USA) to obtain pharmacokinetic parameters.

Transplacental pharmacokinetic parameters in humans (II)

Transplacental pharmacokinetic parameters for diclofenac, salicylic acid and antipyrine were adopted from our previous reports (Table 1) [6, 7].

Table 1.  Parameters used to simulate the fetal plasma concentration profiles of diclofenac, salicylic acid and antipyrine Thumbnail image of

Estimation of fetal plasma concentration profile in humans (III)

Figure 2 shows a developed hybrid physiologically based pharmacokinetic model to describe the transfer of a drug between mother and placenta or placenta and fetus, based on our published pharmacokinetic model [6]. The concentration profile in fetal plasma after repetitive oral administration of a usual dose of each NSAID [dosing interval, τ (h): diclofenac = 12, salicylic acid, antipyrine = 8] to the mother was estimated with the developed model and TP-PK parameters using the maternal plasma concentration profile derived from pharmacokinetics parameters in healthy females as an input function. It was assumed that the fetus has no metabolic capability and that the fetal distribution volume based on bodyweight is equal to that of the mother (V=Vd× 3/60; maternal weight, 60 kg; fetal weight, 3 kg).

image

Figure 2. Physiologically based hybrid pharmacokinetic model of NSAIDs to explain the transplacental and fetal kinetics. The transplacental and fetal profiles were calculated based on the ‘hybrid’ model using the maternal plasma profiles as input functions. Abbreviations are as follows: Cp,m, maternal plasma concentration (diclofenac, ng ml−1; and salicylic acid and antipyrine, µg ml−1); ka, absorption rate constant (h−1); F, bioavailability (diclofenac, 0.54; salicylic acid, 0.68; and antipyrine, 1); D, dosage (diclofenac, 50 000 000 ng; salicylic acid, 300 000 µg; and antipyrine, 600 000 µg); Vd, maternal distribution volume (ml); Cm, drug concentration in intervillous compartment (diclofenac, ng ml−1; and salicylic acid and antipyrine, µg ml−1); Cf, drug concentration in fetal venous compartment (diclofenac, ng ml−1; and salicylic acid and antipyrine, µg ml−1); Xp, drug amount in placental compartment (diclofenac, ng per placenta; and salicylic acid and antipyrine, µg per placenta); K1, K4, influx clearance in vivo (ml h−1 per placenta); k2, k3, first-order rate constant (h−1); ks, elimination rate constant (h−1); Qm, maternal flow rate (ml h−1 per placenta); Qf, fetal flow rate (ml h−1 per placenta); Vm, intervillous volume in placental tissue (113 ml per placenta); Vf, fetal vascular volume in placental tissue (24 ml per placenta); and V, fetal distribution volume (ml). In the case of antipyrine, rapid equilibration between placental tissue and the fetal intravascular space was assumed

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Dose–response relationship of acute toxicity in pregnant rats (IV)

The dose dependence of the constriction of DA, as assessed in terms of the ratio of its inner diameter to that of the pulmonary artery (PA), 4 h after administration of diclofenac, salicylic acid or antipyrine was taken from the literature [4, 5]. As the maximal constriction of DA was observed at 4–8 h after the administration of NSAIDs in rats, we used the data at 4 h.

The dose–response relationship was described by Equation 1.

  • image(1)

where E, γ, D and ED50 represent the DA/PA inner diameter ratio, Hill's coefficient, dose of NSAIDs to a pregnant rat and dose that gives half-maximal constriction of fetal DA/PA inner diameter ratio, respectively.

The observed ratios of inner diameters were fitted to Equation 1 by using a nonlinear least-squares program (MLAB) to obtain γ and ED50.

Relationship between maternal dose and fetal unbound plasma concentrations in rats (V)

Fetal plasma concentration of NSAID 4 h after maternal intravenous administration of salicylic acid (10 mg kg−1) or antipyrine (18.8 mg kg−1) to pregnant rats was 28.6 or 19.0 µg ml−1, respectively [12, 13]. The unbound plasma concentrations of salicylic acid in rat fetus were obtained from the unbound fraction in rat fetal plasma (0.413) [12], while that of antipyrine was calculated from the unbound fraction in human fetal plasma (0.869) [14], because that in rat fetal plasma was not available.

For diclofenac, PK data in pregnant rats were not available. Therefore, we assumed that the unbound concentration in rat fetal plasma is equivalent to that in male rats given the corresponding dose of diclofenac orally [15].

Concentration–response relationship in rat fetus (VI)

We converted the estimated dose–response curve (IV) into a concentration–response curve by using the relationship between maternal dose and fetal unbound plasma concentrations in rats 4 h after administration of each NSAID (V). We also assumed that the relationship between the maternal dose and fetal plasma concentration is linear in each dose range (diclofenac, 0–100 mg kg−1; salicylic acid, 0–1000 mg kg−1; and antipyrine, 0–1000 mg kg−1).

Then, Equation 1 can be rewritten as follows.

  • image(2)

where C and EC50 represent unbound fetal plasma concentration 4 h after administration and unbound fetal plasma concentration that gives half-maximal constriction of fetal DA/PA inner diameter ratio, respectively.

Prediction of human fetal toxicity profiles (VII)

We estimated the time profiles of DA/PA inner diameter ratio in the human fetus after administration of NSAIDs to the mother by using the estimated plasma concentration profiles in the human fetus (III) and the concentration–response curve in rats (VI), based on the assumption that there is no interspecies difference in the concentration response. The unbound fraction of each drug in human fetal plasma was taken from the literature and used for the estimation (diclofenac, 0.006 [16]; salicylic acid, 0.457 [17]; and antipyrine, 0.869 [14]).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

Pharmacokinetic parameters in women (I)

The calculated pharmacokinetic parameters of diclofenac, salicylic acid and antipyrine in healthy adult women are shown in Table 1.

Estimation of fetal plasma concentration profile in humans (III)

The estimated concentration profiles of NSAIDs in human fetal plasma during repetitive oral administration are shown in Figure 3.

image

Figure 3. Estimation of plasma NSAIDs concentrations in human fetus. (A) Repeated oral administration of 50 mg of diclofenac twice a day to the mother. (B) Repeated oral administration of 300 mg of salicylic acid three times a day to the mother. (C) Repeated oral administration of 600 mg of antipyrine three times a day to the mother. Dotted and continuous lines represent the maternal and fetal plasma drug concentrations, respectively

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The maximal fetal plasma concentrations of diclofenac, salicylic acid and antipyrine at a steady state were estimated to be approximately 0.1, 3.5 and 0.75 times the maternal concentration, respectively. The fetal-to-maternal concentration ratio for diclofenac is markedly lower than that for the other two drugs (Figure 3).

Calculation of rat fetal concentration–response relationship from fetal toxicity study and pharmacokinetic study in pregnant rats (IV, V and VI)

The ED50 values for fetal toxicity (DA/PA inner diameter ratios) 4 h after administration of diclofenac, salicylic acid or antipyrine to pregnant rats were estimated to be 1.19, 391 and 455 mg kg−1, respectively (Figure 4 and Table 2).

image

Figure 4. Dose- and concentration-dependent decrease in fetal ductus arteriosus (DA)/pulmonary artery (PA) inner diameter ratios by NSAIDs in pregnant rats. (A) diclofenac, (B) salicylic acid, (C) antipyrine. Filled circles represent the observed data obtained from the published data [4, 5]. The lines represent the model fit

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Table 2.  The ED50 and estimated EC50 values of diclofenac, salicylic acid and antipyrine for constriction of fetal ductus arteriosus in rats
 ED50 (mg kg−1)γPharmacokinetic parametersfp, fetusCp, free (4 h) (µg ml−1)Estimated EC50 (µg ml−1)
Dose (mg kg−1)Cp, total (4 h) (µg ml−1)
  1. Abbreviations are as follows: ED50, dose eliciting half-maximal constriction of DA; γ, Hill's coefficient; Cp, total plasma concentration in rat fetus 4 h after administration; fp, fetus, plasma free fraction in rat fetus; Cp, free, free plasma concentration in rat fetus 4 h after administration; EC50, free plasma concentration eliciting half-maximal constriction of DA.

Diclofenac1.19 [4]0.41550.206 [15]0.028 [15]5.77 × 10−31.37 × 10−3
Salicylic acid391 [5]11028.6 [12]0.413 [12]11.8459
Antipyrine455 [5]118.819.0 [13]0.869 [14]16.5399

The EC50 values of diclofenac, salicylic acid and antipyrine were estimated to be 1.37 × 10−3 (4.63 nM), 459 (3.33 mM) and 399 µg ml−1 (2.12 mM), respectively (Figure 4 and Table 2).

Prediction of human fetal toxicity profiles (VII)

Figure 5 shows the estimated time profiles of DA/PA inner diameter ratio after administration of NSAIDs to the mother (Figure 5, continuous line). Average DA/PA inner diameter ratio in the case of diclofenac was estimated to be 39.0% and was higher than that in the case of antipyrine (5.9%) or salicylic acid (5.9%). Even if a threefold shift in the concentration–response relationship is assumed (dotted and dashed lines), the results are essentially not affected.

image

Figure 5. Prediction of human fetal toxicity (ductus arteriosus (DA) and pulmonary artery (PA) inner diameter ratios) of NSAIDs. (A) Repeated oral administration of 50 mg of diclofenac twice a day to the mother. (B) Repeated oral administration of 300 mg of salicylic acid three times a day to the mother. (C) Repeated oral administration of 600 mg of antipyrine three times a day to the mother. Dotted and dashed lines represent the profile estimated with threefold and one-third-fold differences in EC50 values used for calculation. A grey band represents the profile estimated with one-half- to twofold differences in unbound fetal plasma concentration

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

Prediction of fetal plasma concentration profiles

In this study, we first attempted to estimate the fetal plasma concentration profiles from the maternal plasma concentration profiles of NSAIDs based on TP-PK parameters obtained from our previous perfusion study, on the assumption that the fetus has no metabolic capability. This assumption is considered to be plausible for the following reasons. First, the expression levels of cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) isozymes in the fetus are much smaller than in the mother [18]. CYP3A7 is predominantly expressed in fetal liver, while the expression level of CYP3A4 is low [19]. While the enzymatic activities of CYP2C9 and CYP2C19 in the fetus increase towards delivery, the activities are only 30% of those in the mother even at delivery [20]. Second, this assumption may avoid false-negative fetal risk assessment.

For diclofenac, the fetal peak plasma concentration was estimated to be only one-tenth of the maternal value, even with the assumption of no metabolic activity in the fetus (Figure 3). In contrast, fetal plasma concentrations of salicylic acid and antipyrine were estimated to be approximately 3.5 and 0.75 times the maternal concentrations, respectively (Figure 3). Therefore, the maternal-to-fetal concentration ratios of these NSAIDs are predicted to be in the order salicylic acid > antipyrine > diclofenac.

It is assumed that log(D× MW−0.5) value of a drug, where D and MW represent lipophilicity and molecular weight of a drug, respectively, is used as an index of the membrane permeability in cases where no specific transport proteins are involved in the membrane transport. The log(D× MW−0.5) values of diclofenac, salicylic acid and antipyrine in physiological conditions (pH 7.4) were calculated as −0.11, −2.93 and −0.87, respectively. There was a discrepancy between the order of the log(D× MW−0.5) values of the NSAIDs and that of the predicted maternal-to-fetal concentration ratios of these NSAIDs. This discrepancy implies that the placental transport of the NSAIDs is mediated by specific transport system(s). Among them, maternal-to-placental uptake of salicylic acid may be mediated via a specific transport system, at least in part. Indeed, we have shown in BeWo cells, a human placental choriocarcinoma cell line, that a specific transport system mediates the uptake of salicylic acid together with protons [21]. Monocarboxylate transporter 4, which is a known to be a proton-dependent transporter and transports l-lactic acid as a substrate, is highly expressed on the maternal side of human placental trophoblasts [22].

Prediction of human fetal toxicity

In the present study, we quantitatively predicted the fetal risk of NSAIDs given to the mother by integrating TP-PK data from our human placental perfusion study and PD data obtained in rats. As a result, the clinical risk of constriction of the DA by the three NSAIDs was calculated to be diclofenac (39.0%) > antipyrine (5.9%) = salicylic acid (5.9%), suggesting that diclofenac has the highest risk of fetal toxicity (as assessed in terms of constriction of the DA), even though the maternal-to-fetal transfer of diclofenac was estimated to be lowest among them. This is because diclofenac has a much more potent DA-constricting activity than antipyrine or salicylic acid [5]. This estimation is consistent with the clinical observation that many more cases of DA constriction are reported for diclofenac than for the other drugs [1, 23, 24].

We also assumed, in this model analysis, that the concentration–response relationship for constriction of the DA in humans is equivalent to that in rats. This assumption seems reasonable, because the response is mediated by prostaglandin E2 receptor [25], for which the amino acid sequence homology between rats and humans is as high as 82% [26]. There are no data for comparison of the activity against prostaglandin E2 receptor between rats and humans. However, even if a threefold shift in the concentration–response relationship is assumed, the fetal risk of diclofenac is still estimated to be higher than that of salicylic acid or antipyrine.

It is known that pharmacokinetic changes during pregnancy, such as increasing the apparent volume of distribution, decreasing the plasma concentrations and increasing the half-life [18], are induced by the increase in plasma volume. Since there have been no data for pregnant women, we used PK parameters estimated from adult nonpregnant women to simulate the fetal plasma concentration profiles (Table 1).

In order to validate the present approach, it is required to compare the simulated profiles with the observed umbilical plasma concentration at delivery in pregnant woman taking NSAIDs. However, no data for umbilical plasma concentration profiles of these three NSAIDs have been reported in the literature.

When the present approach to estimate the fetal plasma concentration profiles is further validated, it is expected to be a useful tool for drug discovery for drugs targeting the fetus or drugs that can potentially be used at full-term pregnancy.

Conclusion

In conclusion, we present a novel approach to predict quantitatively the fetal toxicity in humans after drug administration to the mother, using TP-PK parameters estimated from study of placental perfusion and PD data for fetal toxicity obtained from experimental animals.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES

This study was supported in part by grants from Japan Human Sciences Foundation and Japan Society for the Promotion of Science (JSPS).

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Competing Interests
  8. Acknowledgments
  9. REFERENCES
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