Introduction

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The extent of fetal exposure to maternally administered drugs is determined by a number of factors, including physicochemical characteristics of the drug in question, permeability characteristics of the placenta and the length of time the drug is present in the maternal circulation (Reynolds and Knott, 1989; Rurak et al., 1991). During long-term administration with steady-state drug concentrations in the mother, two additional factors become important in determining fetal exposure: maternal and fetal plasma protein binding and fetal nonplacental clearance of the drug. Szeto et al. (1982a) have shown that under steady-state conditions, the fetal-to-maternal drug concentration ratio during maternal drug administration is determined by the maternal placental clearance divided by the sum of fetal placental and nonplacental clearances (i.e., C_fss/C_mss = CL_mf/[CL_fm + CL_fo]). Thus, when the fetus is able to eliminate the drug via nonplacental routes, fetal drug exposure is reduced.

Maternal and fetal placental and nonplacental clearances have been determined for a number of drugs in pregnant sheep. The most commonly used method is the two-compartment open model proposed by Szeto et al. (1982a). This technique involves paired maternal and fetal intravenous infusions of the drug to steady state and collection of paired maternal and fetal plasma samples. From the infusion rates of the drug and the maternal and fetal steady-state drug concentrations, maternal and fetal placental (i.e., bidirectional) and nonplacental clearance values can be estimated. To date, this method has been used with morphine and methadone (Szeto et al., 1982b), acetaminophen (Wang et al., 1986), metoclopramide (Riggs et al., 1990) and DPHM (Yoo et al., 1993). With the exception of acetaminophen, all of the compounds studied thus far have exhibited a higher value for CL_fm compared to CL_mf. This has also been found with labetalol using a different method to estimate maternal and fetal transplacental clearances (Yeleswaram et al., 1993). Given that the placental transfer of all these drugs appears to occur by passive diffusion and that CL_mf should equal CL_fm (see Discussion for further comment on this point), these findings are surprising. Yoo et al. (1993) reported that the magnitude of the CL_fm  CL_mf difference is linearly related to the fetal placental clearance. Of drugs studied to date, the greatest difference between fetal and maternal placental clearance occurs with the histamine antagonist DPHM, with the fetal clearance value being 3.7 times that determined in the ewe (Yoo et al., 1993). However, no explanation for this phenomenon appears to have been provided in the literature. The weight-normalized DPHM nonplacental clearance in the fetal lamb is also higher than the corresponding maternal value, but the routes of fetal nonplacental elimination have not been totally elucidated. This is also the case for other drugs that have been studied in pregnant sheep.

We consider that there could be at least two possible explanations for the higher value of fetal placental clearance. One relates to a methodological issue. Ideally, the two-compartment model experimental protocol should use simultaneous maternal and fetal drug infusions. However, this requires a stable isotope-labeled analog of the drug and an analysis method to quantify both forms of the drug when present together in biological fluids. This was not possible in previous studies, and time-separated maternal and fetal drug administration was carried out. Although the order of drug administration was randomized, it seems possible that with the rapid growth and maturation of the fetus in late gestation, the time separation of the maternal and fetal infusions could have artifactually affected the clearance estimates. The other possibility involves fetal hepatic first-pass uptake of a portion of the drug transferred to the fetus via placenta. Drug administered to the mother reaches the fetus via the umbilical vein (UV), and ~50% of UV flow enters the fetal liver before reaching the fetal systemic circulation (Holzman, 1984). If fetal hepatic drug uptake was significant, the fetal systemic availability and plasma concentration of maternally administered drug reaching the fetal systemic circulation would be reduced, and this would result in an underestimation of maternal placental clearance. Recently, however, we reported that there is no detectable hepatic first-pass uptake of DPHM from the UV blood in the fetal lamb in a study involving simultaneous UV and tarsal venous (TV) administration of unlabeled and deuterium-labeled DPHM and measurement of the two forms of drug in fetal systemic circulation (Tonn et al., 1996).

The overall objective of the current study was to determine the reason or reasons for the difference in maternal and fetal placental clearances and to elucidate the components of high fetal nonplacental clearance for DPHM. Because this drug exhibits the greatest CL_fm  CL_mf difference of all drugs studied in pregnant sheep, an explanation for this difference could also be relevant to other compounds. To achieve this objective, we reassessed maternal and fetal clearances of DPHM using simultaneous maternal and fetal infusions of unlabeled and stable isotope-labeled drug. We also reexamined fetal hepatic first-pass uptake of DPHM from the UV blood by measuring the fetal plasma concentrations of the labeled and unlabeled forms of a DPHM metabolite, DPMA, which has a very low placental permeability in sheep compared to the parent drug. Using these data, the contribution of fetal hepatic clearance to overall CL_fo was assessed; in addition, the maternal and fetal renal clearances of DPHM and DPMA were measured. Finally, we assessed the impact of fetal hepatic first-pass uptake of maternally derived DPHM on the estimates of maternal and fetal clearance parameters calculated using the two-compartment open model.

	Methods

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Animals and Surgical Preparation

This study was approved by the University of British Columbia Animal Care Committee, and all procedures performed on the sheep conformed to the guidelines of the Canadian Council on Animal Care. Five pregnant Dorset Suffolk cross-bred ewes, with a maternal body weight of 70.5 ± 3.4 kg (mean ± S.E.M.), were surgically prepared at 119 to 127 days gestation (122 ± 1 day, term ~145 days). Surgery was performed aseptically under halothane (1-2%) and nitrous oxide (60%) anesthesia (balance O₂), after induction of anesthesia with intravenous sodium pentothal (1 g) and intubation of the ewe. Silicone rubber catheters (Dow Corning, Midland, MI) were implanted in FA and CA, common UV and lateral tarsal veins, trachea, urinary bladder (via a suprapubic incision) and the amniotic cavity. Electrodes (Cooper Corporation, Chatsworth, CA) were implanted biparietally on the dura to record the fetal electrocorticogram. In four animals, a transit-time 4SB blood flow transducer (Transonic Systems, Ithaca, NY) was placed around the common umbilical artery to measure umbilical blood flow. Catheters were also implanted in a maternal femoral artery (MA) and maternal femoral vein (MV). The catheters, electrodes and flow cables were tunneled subcutaneously to a small incision on the flank of the ewe where they exited. They were stored in a denim pouch when not in use. Each vascular catheter was flushed daily with ~2 ml of sterile 0.9% sodium chloride containing 12 units of heparin/ml to maintain catheter patency. Intramuscular injections of 500 mg of ampicillin and 80 mg of gentamicin were administered to the ewe on the day of surgery and for 3 days after surgery. Ampicillin (500 mg) and gentamicin (40 mg) were administered via the amniotic cavity immediately after surgery and then daily thereafter. After surgery, animals were kept in holding pens with other sheep and were given free access to food and water. The sheep were allowed to recover for 4 to 8 days before experimentation. On the morning of the experiment, a Foley bladder catheter was inserted via the urethra of the ewe and attached to a sterile polyvinyl bag for cumulative urine collection.

Experimental Protocol

Experiments were conducted at 125 to 133 days (128.8 ± 1.4 days) (term 145 days gestation). Before each experiment, DPHM · HCl (Sigma Chemical, St. Louis, MO) and [²H₁₀]DPHM · HCl (synthesized and purified in our laboratory; Tonn et al., 1993) were weighed to obtain the correct dose for administration. The weighed doses were dissolved in sterile 0.9% sodium chloride for injection and then filtered through a 0.22-µm nylon syringe filter (MSI, Westboro, MA) into a capped empty sterile injection vial.

Two types of experiments (studies 1 and 2) were carried out on this group of animals, as described below.

Study 1: Fetal isotope effect studies. These studies were conducted on two animals to check for the presence of any isotope effects in the disposition of [²H₁₀]DPHM and the metabolite [²H₁₀]DPMA compared with DPHM and DPMA. Equimolar doses of DPHM and [²H₁₀]DPHM were simultaneously administered via the TV catheter as a 2.0-mg loading dose followed immediately by a 90-min infusion (60 µg/min). Serial samples were collected at 5, 5, 15, 30, 45, 60, 75 and 90 min from the FA and CA (1.5 ml each) and MA (3.0 ml) catheters. Amniotic fluid and fetal urine samples (3.0 ml) were also collected at 5, 30, 60 and 90 min.

Study 2: Paired maternal-fetal infusions. Simultaneous infusions of DPHM and [²H₁₀]DPHM to the ewe and fetus, respectively, were carried out on all five sheep. In the two animals (animals E2241 and E2181) used for the isotope effect study, the paired maternal-fetal infusions were carried out 72 hr later. DPHM was administered as a 20-mg intravenous loading dose over 1.0 min, followed immediately by an infusion of 670 µg/min via the MV. Simultaneously, a 5.0-mg intravenous loading dose of [²H₁₀]DPHM was given via the TV over 1.0 min, followed by an infusion of the compound at 170 µg/min. Simultaneous blood samples were collected from the FA (1.5 ml) and MA (3.0 ml) catheters at 5, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min during the infusion and at 30, 60, 120, 180, 240 and 360 min and 8, 12, 18, 24, 30 and 40 hr after the infusion. FA samples (0.6 ml) were also collected at the same time intervals for blood gas analysis and measurement of glucose and lactate concentrations. CA and UV blood samples (1.5 ml) were collected at 5, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min during the infusion period. All fetal blood removed for sampling was replaced at intervals during the experiment by an equal volume of maternal blood obtained before the start of the experiment. Amniotic and tracheal fluid (3.0 ml) and maternal urine (10 ml) samples were obtained at 5, 60, 120, 180, 240, 300 and 360 min during the infusion and at 60, 120, 180, 240 and 360 min and 8, 12, 18, 24, 30 and 40 hr postinfusion.

Study 3: Simultaneous fetal UV and tarsal venous administration. In addition to the studies described above, data that we recently obtained using samples collected from eight animals used in our previous study of fetal DPHM hepatic first-pass uptake (Tonn et al., 1996) are reported here. As noted in the introduction, ~50% (range, 30-80%) of the UV flow traverses the fetal liver before reaching the fetal systemic circulation (Edelstone et al., 1978). Moreover, UV provides a major vascular input into the fetal liver, supplying ~93% and ~60% of the total blood flow to the left and the right and caudate lobes, respectively (Edelstone et al., 1978; Holzman, 1984). Thus, the drug present in UV could undergo a "partial" first-pass hepatic uptake before entering the fetal circulation if the fetal liver was sufficiently active in metabolizing the drug (fig. 1). The surgical preparation and experimental protocol used in these animals have been previously fully described (Tonn et al., 1996). The surgery was similar to that described above, with the exception that fetal and maternal bladder catheters were not used. To assess the fetal first-pass hepatic uptake for DPHM, we used simultaneous but separate bolus injections of [²H₁₀]DPHM and DPHM (5.0 mg each) via the common UV and TV (which drains directly into the inferior vena cava) or 90-min intravenous infusions of the compounds (60 µg/min each, preceded by a 2.0 mg intravenous bolus of each) via the same routes. This was coupled with collection of fetal arterial plasma for the measurement of [²H₁₀]DPHM and DPHM concentrations. In the present study, fetal plasma samples remaining from four of these fetal bolus and four of the infusion studies were used for the measurement of [²H₁₀]DPMA and DPMA concentrations.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Sketch of the fetal circulation showing the position of fetal liver and sites of drug administration for assessment of fetal first-pass hepatic drug uptake from UV (study 3).

Physiological Recording and Monitoring Procedures

From 24 hr before to 24 hr after the infusion period, fetal amniotic, tracheal and FA pressures, heart rate, electrocortical activity and urine production rate were continuously monitored. However, these data will be reported separately. In the animals with an implanted umbilical flow transducer, UV blood flow was measured with a Transonic model T201 transit-time flowmeter (Transonic Systems, Inc., Ithaca, NY). Fetal urine flow rate was estimated using a computer-controlled roller pump assembly developed in our laboratory. The fetal bladder catheter was allowed to drain by gravity into a sterile reservoir (10-ml syringe barrel) to which a disposable DTX transducer was connected. When the pressure in the reservoir increased above a preset level (usually 3 mm Hg) due to urine collection, the computer activated a roller pump (DIAS Ex154, DIAS Inc., Kalamazoo, MI), which pumped a calibrated volume of urine from the reservoir back to the amniotic cavity (via the amniotic catheter) during control periods. During the experimental period, the urine was collected into a sterile sample collection syringe, and at hourly intervals a 5-ml aliquot was taken, with the remainder of the urine returned to the amniotic cavity. The cumulative volume pumped/min, which equals fetal urine production/min, was stored on disk.

Blood pH, PO₂ and PCO₂ were measured using an IL 1306 pH/blood gas analyzer (Allied Instrumentation Laboratory, Milan, Italy). Blood O₂ saturation and hemoglobin concentration were determined using a Hemoximeter (Radiometer, Copenhagen, Denmark). Blood glucose and lactate concentrations were determined with a 2300 STAT plus glucose/lactate analyzer (Y.S.I. Inc., Yellow Springs, OH).

Plasma Protein Binding of DPMA in Fetal and Maternal Plasma

The plasma protein binding of DPMA was measured in vitro in fetal and maternal plasma using equilibrium dialysis as described by Tonn et al. (1992). Maternal and fetal plasma for these measurements was obtained from two additional sheep in our laboratory set up for other experiments, and the drug-free plasma was obtained on nonexperimental days.

Drug and Metabolite Analysis

The concentrations of DPHM and [²H₁₀]DPHM in all biological fluids collected were measured using a previously developed GC-MS assay capable of simultaneously measuring DPHM and [²H₁₀]DPHM (LOQ = 2 ng/ml each; Tonn et al., 1993). The concentrations of DPMA and its stable isotope labeled analog, [²H₁₀]DPMA, were measured simultaneously using another GC-MS method, which was also developed in our laboratory (LOQ = 2.5 ng/ml each; Tonn et al., 1995).

Pharmacokinetic Analysis

Study 2: Paired maternal-fetal infusions. The fetal and maternal plasma concentration-vs.-time data from this study were fit separately to a two-compartment open model with elimination occurring from the central compartment (Gibaldi and Perrier, 1982). The data were fit using ADAPT II pharmacokinetic modeling program and a maximum likelihood fitting algorithm [variance model for Cp: Var = *(Cp) + , where  and  are estimated variance parameters] (D'Argenio and Schumitzky, 1992). Plasma concentration at time 0 (Cp₀) was extrapolated from the fitted equation.

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

Study 3: Simultaneous fetal UV and TV administration: For these fetal hepatic first-pass experiments, fetal systemic availability of DPHM (or [²H₁₀]DPHM) after UV administration was calculated from the parent drug data as previously described (Tonn et al. , 1996):

(11)

where respective AUC values refer to AUC of parent drug after UV or TV administration.

(12)

(13)

0-

Statistical analysis. All values are reported as the mean ± S.E.M. The achievement of steady state was determined using three groups of mean concentration values (i.e., 150 and 180, 240 and 270, and 330 and 360 min) with a repeated-measures analysis of variance (Zar, 1984). A paired t test was used to test for differences between fetal and maternal pharmacokinetic parameters. The significance level was P < .05 in all cases. Fetal weight in utero at the time of experimentation was estimated from the weight at birth and the time interval between the experiment and birth (Koong et al., 1975).

	Results

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Study 1: Fetal Isotope Effect Studies

In two experiments, DPHM and [²H₁₀]DPHM were simultaneously infused via the TV for 90 min to determine possible differences in the disposition of two forms of the drug. The overall mean steady-state plasma concentrations of DPHM and [²H₁₀]DPHM in the two animals were 183.6 ± 30.9 and 182.4 ± 30.3 ng/ml, respectively. For DPMA and [²H₁₀]DPMA, steady-state concentrations were not achieved during the infusion, but there were no apparent differences between the AUCs for DPMA (1598.2 ng.min/ml) and [²H₁₀]DPMA (1429.5 ng.min/ml). In addition, no differences were observed in the concentrations of DPHM and [²H₁₀]DPHM in maternal plasma (10.4 ± 0.5 vs. 11.0 ± 0.5 ng/ml), AUCs in amniotic fluid (1054.8 vs. 1101.0 ng.min/ml) and fetal urinary concentrations at the end (90 min) of the infusion (2944.0 vs. 2874.3 ng/ml). Overall, the data do not indicate any significant isotope effect in the disposition of labeled forms of DPHM and DPMA.

Study 2: Paired Maternal/Fetal Infusions

The five experiments involving the simultaneous 6-hr infusions of DPHM and [²H₁₀]DPHM to ewe and fetus, respectively, were carried out at 125 to 133 days gestation (128.8 ± 1.4 days). Estimated fetal weight was 2.46 ± 0.09 kg. During the control period, the fetal femoral arterial values for pH, PO₂, PCO₂, O₂ saturation, hemoglobin, glucose and lactate concentrations were 7.36 ± 0.02, 22.6 ± 1.65 mm Hg, 47.3 ± 0.5 mm Hg, 55.3 ± 23.8%, 10.0 ± 0.3 g/dl, 0.98 ± 0.09 mM and 0.70 ± 0.11 mM, respectively. There were no consistent changes in any of these variables during or after the infusion period. Likewise umbilical blood flow (281 ± 39 ml/min/kg, n = 3) was not consistently altered during the experiment.

Maternal and fetal plasma DPHM and [²H₁₀]DPHM concentrations and placental and nonplacental clearance values. The average plasma concentrations of DPHM and [²H₁₀]DPHM in MA and FA plasma are illustrated in figure 2. They reached a plateau at ~120 min, and there were no statistical differences between the plasma concentrations at 150 and 180, 240 and 270, and 330 and 360 min, suggesting the achievement of steady state by 150 min. Thus, mean steady-state concentration values used for subsequent calculations were taken from 150 to 360 min. The mean steady-state concentrations of DPHM were 260.8 ± 18.9 and 45.6 ± 17.4 ng/ml in MA and FA plasma, respectively, whereas the mean concentrations of [²H₁₀]DPHM in the same vessels were 44.6 ± 5.8 and 244.0 ± 42.4 ng/ml, respectively. The total MA and FA steady-state concentrations of DPHM (i.e., labeled and unlabeled DPHM) were 305.4 ± 24.4 and 289.6 ± 57.6 ng/ml. In the four fetuses in which there was a functional CA catheter, the mean concentrations of [²H₁₀]DPHM (infused via the tarsal vein) were 203.7 ± 29.9 and 186.1 ± 27.0 ng/ml in FA and CA plasma, respectively. The mean FA-CA concentration difference of 17.5 ± 6.1 ng/ml was significantly different from 0. In contrast, the concentrations of DPHM (infused to the ewe) in FA and CA plasma averaged 27.2 ± 4.4 and 26.6 ± 4.3 ng/ml in these four animals and were not significantly different. As in previous studies (Rurak et al., 1991), there was accumulation of DPHM (both labeled and unlabeled) in fetal lung and amniotic fluids. The average drug concentration ratio between lung fluid and FA plasma was 4.0 ± 1.7 for DPHM and 4.5 ± 1.6 for [²H₁₀]DPHM, whereas the corresponding ratios in amniotic fluid (i.e., amniotic fluid/FA) were 0.6 ± 0.2 and 0.8 ± 0.2 for labeled and unlabeled drug, respectively. After the infusion, concentrations of DPHM and [²H₁₀]DPHM in all fluids declined rapidly with a terminal elimination half-life in plasma of 70.5 ± 6.9 and 51.8 ± 7.2 min in the ewe and fetus, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Plasma concentrations of DPHM and [2H10]DPHM in MA and FA plasma during and after simultaneous intravenous infusions of DPHM (670 µg/min) to the ewe and of [2H10]DPHM (170 µg/min) to the fetus. , Maternal DPHM; , maternal [2H10]DPHM; , fetal DPHM; , fetal [2H10]DPHM.

Fetal and maternal DPMA and [²H₁₀]DPMA plasma concentrations. A mean plasma concentration-vs.-time plot of DPMA and [²H₁₀]DPMA in MA and FA plasma is shown in figure 3. Although concentrations of DPHM and [²H₁₀]DPHM reached steady state at ~120 min from the start of the infusion (fig. 2), the plasma levels of DPMA and [²H₁₀]DPMA did not reach steady state during the entire duration of infusion and continued to increase for 30 to 120 min after infusion. At all time points during the infusion period, the concentration of [²H₁₀]DPMA was higher in the fetus than in the mother, whereas for the unlabeled metabolite the situation was reversed. The peak concentrations of DPMA in maternal and fetal plasma averaged 137.4 ± 18.5 and 92.8 ± 16.8 ng/ml, respectively, whereas the peak maternal and fetal plasma concentrations of [²H₁₀]DPMA were 28.7 ± 4.3 and 135.0 ± 20.1 ng/ml, respectively. The maternal-fetal concentration differences for both forms of the metabolite were significantly different from 0. The time at which the peak levels occurred postinfusion was 18.0 ± 7.3 min for both labeled and unlabeled DPMA in the ewe, whereas in the fetus the value was 87.0 ± 14.5 min. After the peak, the fetal metabolite levels declined much more slowly than in the ewe. The elimination half-life of the metabolite in the fetus and ewe (determined by simultaneous fitting of the parent drug and metabolite data) was 15.2 ± 2.5 and 3.0 ± 0.2 hr, respectively. These values are significantly different. In the two animals with functional UV catheters, the extraction ratio of [²H₁₀]DPMA across the fetal side of the placenta averaged 0.06 ± 0.01. This value is not significantly different from 0 but is different from the umbilical extraction ratio for [²H₁₀]DPHM given above. Finally, DPMA or [²H₁₀]DPMA metabolites were never detected in amniotic or fetal lung fluid.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   MA and FA plasma concentrations of DPMA and [2H10]DPMA during and after simultaneous intravenous infusions of DPHM (670 µg/min) to the ewe and of [2H10]DPHM (170 µg/min) to the fetus. , Maternal DPMA; , maternal [2H10]DPMA; , fetal DPMA; , fetal [2H10]DPMA.

Fetal and maternal renal elimination of DPHM, [²H₁₀]DPHM, DPMA and [²H₁₀]DPMA. The renal clearances of DPHM, [²H₁₀]DPHM, DPMA and [²H₁₀]DPMA in the ewe and fetus are given in table 3. DPMA and [²H₁₀]DPMA were both present in adult urine during and after the infusion. In contrast, only very small quantities were detected in fetal urine. The cumulative excretion plot for DPHM and [²H₁₀]DPHM in maternal and fetal urine clearly shows a plateau after the infusion (fig. 4A), whereas the metabolite appears to be near a plateau only at the end of the experimental protocol (fig. 4B). Thus, cumulative excretion of the metabolite may have been somewhat underestimated. The weight-corrected renal clearance of DPHM was ~200-fold less in adult sheep compared with fetal lambs, whereas the opposite situation existed for the metabolite (relative renal clearance ~50-fold greater in mother than in fetus). The contribution of renal DPHM clearance to maternal nonplacental clearance was 0.025 ± 0.011%, whereas in the fetus the contribution was 2.22 ± 0.42% (table 3). These values are significantly different. The percentage of total maternal DPHM and fetal [²H₁₀]DPHM dose excreted as DPMA and [²H₁₀]DPMA, respectively, averaged 0.95 ± 0.07% in the ewe and 0.014 ± 0.011% in the fetus. These values are also significantly different from each other.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Cumulative urinary excretion of (A) DPHM in maternal urine () and [2H10]DPHM in fetal urine () and (B) DPMA in maternal urine () and [2H10]DPMA in fetal urine () during and after simultaneous intravenous infusions of DPHM (670 µg/min) to the ewe and of [2H10]DPHM (170 µg/min) to the fetus.

Study 3: Simultaneous Fetal UV and TV Administration

Figure 5 illustrates the concentration-vs.-time plots for unlabeled and labeled forms of DPHM and DPMA measured from samples obtained in our previous study of fetal hepatic first-pass DPHM uptake after UV administration (Tonn et al., 1996). Figure 5A shows the data from a bolus experiment in which [²H₁₀]DPHM was administered via the UV, whereas figure 5B shows the data from an infusion study in which unlabeled DPHM was infused via the umbilical route. In both experiments, there were no consistent differences in the fetal arterial concentrations of DPHM and [²H₁₀]DPHM. In contrast, the plasma concentration of the form of DPMA derived from the drug administered via the UV was consistently higher than that derived from drug given via the TV. Table 4 gives the FA plasma AUC values for labeled and unlabeled DPHM and DPMA. The estimates of fetal first-pass hepatic extraction ratio based on intact drug concentrations were obtained using equations 11 and 12, and the mean value of 0.06 ± 0.06 is not significantly different from 0, as reported previously (Tonn et al., 1996). In contrast, the estimates of fetal hepatic extraction ratio obtained using the AUC values for DPMA and [²H₁₀]DPMA and equation 13 indicated significant drug uptake by the fetal liver. The mean extraction ratio was 0.44 ± 0.05, and this was significantly lower than the value of 0.71 ± 0.07 obtained above in the paired maternal-fetal infusion protocol (table 2).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A, Representative FA plasma concentrations of DPHM (), [2H10]DPHM (), DPMA () and [2H10]DPMA () after simultaneous intravenous bolus administration of DPHM and [2H10]DPHM (5 mg each) to the fetus (E#989). DPHM was given via the fetal TV, and [2H10]DPHM was given via the UV. B, Representative FA plasma concentrations of DPHM (), [2H10]DPHM (), DPMA () and [2H10]DPMA () after simultaneous steady-state infusion of DPHM and [2H10]DPHM (60 µg/min each) to the fetus (E#2164). DPHM was infused via the fetal UV, and [2H10]DPHM was infused via the fetal TV.

Fetal and Maternal Plasma Protein Binding of DPMA

The time required to reach equilibrium for the determination of the binding of DPMA was 8 hr. No significant volume shifts were associated with this equilibrium time, and no nonspecific binding of DPMA to the equilibrium dialysis cell and the membrane could be detected. The metabolite was highly bound in both maternal and fetal plasma, with the percent bound averaging 99.4 ± 0.01% and 98.9 ± 0.07%, respectively. The free fraction in adult plasma (0.006 ± 0.002) was significantly less than that in fetal plasma (0.010 ± 0.001).

	Discussion

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Use of Stable Isotope-Labeled Compounds to Study Maternal-Fetal Drug Disposition

The current study appears to be the first in which a stable isotope-labeled drug analog has been used to study maternal-fetal drug disposition in pregnant sheep. The use of stable isotope-labeled compounds provides several advantages in studies of drug disposition during pregnancy, particularly in situations in which the simultaneous administration of the drug via two routes is to be used. As noted in the introduction, our primary reason for using this methodology in the current study was to eliminate the potential confounding effects of fetal growth and maturation that occur over the period between time-separated maternal and fetal drug infusions. This approach also reduces the overall duration of the experiment. This is an important factor in studies involving chronically instrumented pregnant animals in which there is a finite time window available for each preparation and, hence, shorter experiments allow for additional studies to be conducted on the same animal. However, it is important that the labeled and unlabeled forms of the drug be biologically equivalent (Baillie, 1981). If this is not so, the labeled drug could display different dispositional characteristics compared with the unlabeled drug and thus be of limited use in pharmacokinetic studies (Baillie, 1981). Consequently, it was first necessary to determine whether such an "isotope effect" existed for [²H₁₀]DPHM in fetal sheep (study 1). The data obtained on the concentrations of DPHM, [²H₁₀]DPHM, DPMA and [²H₁₀]DPMA during and after simultaneous infusion of unlabeled and labeled parent drug at equivalent rates via the TV suggest that the isotope effects with [²H₁₀]DPHM are negligible or absent in pregnant sheep in terms of both the pharmacokinetics of the parent drug and the formation and disposition of [²H₁₀]DPMA. The results are consistent with our earlier data obtained in studies involving bolus drug administration in fetal and nonpregnant sheep (Tonn et al., 1996).

Study 2: Paired Maternal-Fetal Infusions

DPHM and [²H₁₀]DPHM plasma concentrations in the ewe and fetus. The average total fetal plasma concentration of DPHM achieved in the current experiments (i.e., DPHM + [²H₁₀]DPHM = ~289 ng/ml) lies between the two plasma concentrations achieved in the time-separated maternal (~36 ng/ml) and fetal infusions (~450 ng/ml) in our previous study (Yoo et al., 1993). In the ewe, the total drug concentration in the current study (305 ng/ml) exceeds the levels achieved in the previous maternal (212 ng/ml) and fetal (31 ng/ml) infusions. Similar to our previous findings with simultaneous infusion of unlabeled and labeled DPHM via the UV and TV (Tonn et al., 1996), we did not find any evidence for preferential delivery of maternally derived drug to the fetal brain and other upper body structures. This is, however, the case with O₂, glucose and other nutrients that are present in higher concentrations in the fetal ascending aorta compared with the descending aorta (Charlton and Johengen, 1984). This feature is thought to be due to the preferential distribution of umbilical venous blood to the upper body (including the brain) (Edelstone and Rudolph, 1979). In contrast, in the current study, the CA-FA concentration difference for DPHM (administered to the ewe) was not significantly different from 0, whereas with [²H₁₀]DPHM (infused via the TV), there was a positive FA-CA difference (i.e., opposite to the situation with endogenous compounds). As we previously discussed (Tonn et al., 1996), this difference may be due to uptake of DPHM by the fetal lung, with a resulting greater dilution of the drug with venous return in the left heart (which primarily supplies the upper body) compared to the right heart (which primarily supplies the lower body, see fig. 1).

Placental and nonplacental clearances of DPHM in fetal and maternal sheep. The maternal and fetal clearance values obtained for DPHM in the current study are similar to those we previously determined (Yoo et al., 1993). In particular, we confirmed that fetal placental clearance of the drug is much higher than the maternal placental clearance and that the weight-normalized fetal nonplacental clearance is also greater than that in the ewe. In the current study, CL_fm was 5.4-fold higher than CL_mf, whereas our previous estimates were 3.7 times higher based on total drug concentrations and 1.6 times higher based on free drug (Yoo et al., 1993). The fetal transplacental and nonplacental clearance values for DPHM are the highest of any drug examined in pregnant sheep; this is also the case with the CL_fm  CL_mf difference. Given the overall agreement between the results of our two studies, we conclude that this latter difference is not an artifactual result of the time-separated maternal and fetal infusions in previous studies that used the two-compartment open model.

Maternal and fetal plasma concentrations of DPMA and [²H₁₀]DPMA. In humans, monkeys and dogs, DPHM is thought to be metabolized via two sequential N-demethylation steps followed by deamination to DPMA. This DPMA metabolite and its conjugates are the major urinary metabolites of DPHM in these species (Chang et al., 1974; Drach et al., 1970; Drach and Howell, 1968; Glazko et al., 1974). DPMA is also present in the urine and plasma of nonpregnant ewes after DPHM administration (Tonn et al., 1995). In the present study, DPMA and [²H₁₀]DPMA were detected in both maternal and fetal plasma during and after the simultaneous infusions of DPHM and [²H₁₀]DPHM to the ewe and fetus, respectively. The consistently higher concentrations of the labeled metabolite in fetal plasma compared with those in the mother during the infusion period provide strong evidence for its formation in the fetus. The presence of DPMA in the fetus and [²H₁₀]DPMA in the ewe could be the result of two processes: (1) placental transfer of DPHM to the fetus and [²H₁₀]DPHM to the ewe, with subsequent formation of the unlabeled and labeled metabolites in fetal and maternal compartments, respectively; and (2) fetal-to-maternal transfer of [²H₁₀]DPMA and maternal-to-fetal transfer of DPMA. However, it seems unlikely that the latter process could be of much importance because the minimal umbilical extraction ratio of [²H₁₀]DPMA (~0, see above) and the long fetal elimination half-lives of the labeled and unlabeled metabolite (~15 hr) suggest limited transfer across the ovine placenta. This is perhaps due to a greater polarity of the metabolite compared with DPHM and its high degree of plasma protein binding (~99%). However, the larger maternal ratio for AUC_[²H10]DPMA/AUC_[²H10]DPHM (0.96 ± 0.12) compared to AUC_DPMA/AUC_DPHM (0.62 ± 0.07) suggests that at least a portion of the labeled metabolite that is formed in the fetus is transferred to the mother. The long half-life of the metabolite in the fetus also suggests that the elimination pathways for this metabolite are not as well developed in the fetus as they are in the ewe. We have, in fact, obtained recent data from studies involving fetal bolus administration of DPMA that indicate that virtually all of the administered dose ultimately appears in maternal urine over the ensuing 96 hr (Kumar et al., 1996). This confirms that both fetus and ewe have no detectable ability to metabolize DPMA and that the metabolite can cross the sheep placenta, albeit at a very slow rate. In other species, the contribution of DPMA formation to overall DPHM elimination is high (40-60%; Chang et al., 1974; Drach et al., 1970; Drach and Howell, 1968; Glazko et al., 1974). However, our recent studies in fetal and adult sheep suggest a much lower value (~1%, Kumar et al., 1995, 1996).

Fetal and maternal renal elimination of DPHM, [²H₁₀]DPHM, DPMA and [²H₁₀]DPMA. There appear to be significant differences in the renal excretion of both DPHM and DPMA between adult and fetal sheep. The renal clearance of DPHM in the pregnant ewe (0.012 ± 0.005 ml/min/kg) is significantly less than the reported GFR in adult sheep (~2.4 ml/min/kg; Hill and Lumbers, 1988). This suggests that a portion of the filtered and/or secreted load of DPHM is reabsorbed in the renal proximal tubule. As a consequence, the renal clearance of DPHM in maternal sheep contributes <0.1% of the total body clearance. In contrast, the fetal renal excretion of DPHM (~2.22 ± 0.42 ml/min/kg) exceeds reported values for fetal GFR (~1 ml/min/kg; Hill and Lumbers, 1988). This indicates fetal renal tubular secretion of DPHM in addition to glomerular filtration of the drug. This finding is similar to the results of previous studies on the renal excretion of various organic cations in the late-gestational fetal lamb (cimetidine, ranitidine, meperidine and tetraethylammonium), all of which have renal clearance values that are greater than the GFR (Czuba et al., 1990; Elbourne et al., 1990; Mihaly et al., 1983; Szeto et al., 1979). In contrast to these amine compounds, the fetal renal clearance of DPMA (0.013 ± 0.010 ml/min/kg) was not significantly different from 0. There also is limited fetal renal tubular secretion of para-aminohippurate, the glucuronide and sulfate conjugates of acetaminophen and morphine glucuronide (Elbourne et al., 1990; Olsen et al., 1988; Wang et al., 1986). Also, we recently found the same phenomenon with valproic acid⁵ and indomethacin (Krishna et al., 1995).

Studies 2 and 3: Fetal Hepatic Uptake and Metabolism of DPHM

Evidence of fetal hepatic first-pass DPHM uptake from UV. In a previous study (Tonn et al., 1996), we examined the fetal hepatic first-pass uptake of DPHM after UV drug administration after both bolus and constant-rate intravenous infusions and found no evidence for a first-pass effect. In contrast, a substantial (>90%) hepatic presystemic elimination of the drug was observed with mesenteric venous administration in adult sheep. However, the results from the fetal experiments did not completely rule out the involvement of fetal liver in DPHM metabolism/DPMA formation, and we recently demonstrated formation of the DPMA metabolite when fetal hepatic microsomal preparations are incubated with DPHM.⁶ The data from study 2 on DPMA/DPHM (8.20 ± 1.62) and [²H₁₀]DPMA/[²H₁₀]DPHM AUC (2.24 ± 0.53) ratios in the fetus (table 2) indicate that more of the maternally derived form of the drug (reaching the fetus via the UV and hence undergoing a "partial" fetal hepatic first-pass) is converted to the metabolite than is the form administered directly to the fetus. These AUC ratios clearly demonstrate that the fetal liver is involved in the metabolism and nonplacental clearance of the drug. With equation 8 and the AUC ratios from the current study, fetal hepatic first-pass extraction of DPHM present in UV blood averaged 0.71 ± 0.07. However, with the DPHM, [²H₁₀]DPHM, DPMA and [²H₁₀]DPMA concentrations measured in samples from our previous umbilical hepatic first-pass experiments (study 3) and equation 13 (analogous to equation 8), a mean value of 0.44 ± 0.05 was obtained. We believe that the higher estimate of this parameter obtained in the paired infusion study (study 2) is due to maternal-to-fetal transfer of a portion of the maternally formed DPMA to artifactually increase the fetal AUC_DPMA/AUC_DPHM ratio. In the fetal hepatic first-pass study (study 3), both labeled and unlabeled DPHM were administered to the fetus, so that maternal-to-fetal transfer of intact drug or metabolite was unlikely. Thus, the mean value of 0.44 for fetal hepatic DPHM extraction estimated from direct fetal UV administration is probably more accurate.

Impact of fetal hepatic drug uptake on two-compartment model estimates of maternal and fetal clearances. The placental clearance values calculated using the two-compartment model are the "fundamental" clearances of the maternal-placental-fetal system and can be used to estimate the maximum possible rate of drug transfer across the placenta (under given conditions of blood flow and protein binding) assuming sink conditions on the other side. These clearance parameters are thus reflective of the true placental permeability of the drug in question. This is in contrast to the net rate of placental drug flux, which depends only on the rate of nonplacental drug elimination on the other side of placenta.