Introduction

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Diphenhydramine or 2-(diphenylmethoxy)-N,N-dimethylamine (DPHM)² is a potent classical histamine H1-receptor antagonist. It has been widely used during human pregnancy for the treatment of nausea and vomiting, insomnia, allergic rhinitis, and common coughs and colds. However, information on the pharmacokinetics and metabolism of DPHM in the human fetus is lacking. Previous studies in our laboratory, using chronically instrumented pregnant sheep, have demonstrated that DPHM readily crosses the ovine placenta and is eliminated from the fetus via both placental and nonplacental pathways (Yoo et al., 1993; Kumar et al., 1997). Currently we are studying the comparative elimination and metabolic pathways involved in DPHM nonplacental clearance in adult and fetal sheep in vivo and in utero. This will shed some light on the extent of development and the in utero functional capacity of various drug-metabolizing enzyme systems in the late gestation fetal lamb as compared to adult sheep.

Diphenylmethoxyacetic acid (DPMA) and its amino acid conjugates are the major metabolites of DPHM in many species (dog, rhesus monkey, human) and account for ~40 to 60% of total DPHM elimination (Drach and Howell, 1968; Drach et al., 1970; Chang et al., 1974). We have also detected metabolism of DPHM to DPMA by adult as well as fetal sheep (Kumar et al., 1997). However, the quantitative importance of this pathway in overall DPHM elimination in adult and fetal sheep is not known because the pharmacokinetic characteristics of DPMA and the nature and extent of its secondary metabolism have not been examined in this species. In this study, we have determined the fractional contribution of DPMA formation toward maternal and fetal nonplacental DPHM clearance. As a prelude to achieving this objective, we have also examined detailed dispositional characteristics of the preformed (synthesized) as well as in vivo and in utero generated/formed DPMA in maternal and fetal sheep.

	Materials and Methods

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Animals and Surgical Preparation. A total of five pregnant sheep were used in these studies. All studies were approved by the University of British Columbia Animal Care Committee and the procedures performed on sheep conformed to the guidelines of the Canadian Council on Animal Care. The detailed surgical procedures used have been described previously (Rurak et al., 1988). Briefly, five pregnant Dorset-Suffolk crossbred ewes, with a maternal body weight of 82.4 ± 14.1 kg (mean ± S.D.), were surgically prepared between 118 and 129 days gestation (term ~145 days). Surgery was performed aseptically under halothane (1-2%) and nitrous oxide (60%) in oxygen anesthesia after induction with i.v. sodium pentothal (1 g) and intubation of the ewe. Polivinyl catheters (Dow Corning, Midland, MI) were implanted in both fetal femoral arteries and lateral tarsal veins and a maternal femoral artery and vein. Catheters were also implanted in the fetal trachea, amniotic cavity, and in three animals in the fetal urinary bladder (via a suprapubic incision). The catheters were tunneled s.c. and exteriorized via a small incision on the flank of the ewe and were stored in a denim pouch when not in use. All catheters were flushed daily with approximately 2 ml of sterile 0.9% sodium chloride containing 12 units of heparin per milliliter to maintain their patency. I.m. injections of ampicillin (500 mg) were given to the ewe on the day of surgery and for 3 days postoperatively. Ampicillin (500 mg) was also given via the amniotic cavity immediately after surgery and 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 (American Latex Corp., Sullivan, IN) was inserted via the urethra of the ewe and attached to a sterile polyvinyl bag for cumulative maternal urine collection.

Experimental Protocol. All experiments were conducted between 124 and 140 days gestation (term ~145 days). Two sets of experiments were carried out on all five pregnant sheep.

Maternal experiments. In three animals (E2174, E4227, and E4230), a 6-h maternal steady-state DPHM (DPHM hydrochloride, Sigma Chemical Co., St. Louis, MO) infusion at 670 µg/min, combined with initial 20.0 mg DPHM and 2.5 mg deuterium-labeled DPMA ([²H₁₀]-DPMA) boluses, was administered via the maternal femoral vein catheter. In the other two animals (E1225A and E303Y), the isotope labels on the compounds were reversed, i.e., deuterium-labeled DPHM ([²H₁₀]-DPHM) infusion (at 670 µg/min) was administered in combination with [²H₁₀]-DPHM (20.0 mg) and DPMA (2.5 mg) boluses.

Fetal experiments. Similar to the maternal experiments above, in three animals (E2174, E4227, and E4230), a 6-h fetal steady-state DPHM infusion at 170 µg/min, combined with initial 5.0 mg DPHM and 0.85 mg [²H₁₀]-DPMA boluses, was administered via the fetal lateral tarsal vein catheter. In the other two animals (E1225A and E303Y), [²H₁₀]-DPHM infusion (at 170 µg/min) was administered in combination with [²H₁₀]-DPHM (5.0 mg) and DPMA (0.85 mg) boluses.

Physiological Recording and Monitoring Procedures. Fetal 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). Glucose and lactate concentrations in fetal blood were determined with a 2300 STAT plus glucose/lactate analyzer (Y.S.I. Inc., Yellow Springs, OH).

Fetal and Maternal Plasma Protein Binding of DPHM and [²H₁₀]-DPHM. The plasma protein binding/unbound fraction of DPHM (or [²H₁₀]DPHM) was measured ex vivo in pooled fetal and maternal steady-state plasma samples using an equilibrium dialysis procedure, as described by Yoo et al. (1993).

Drug and Metabolite Analysis. The concentrations of DPHM, [²H₁₀]-DPHM, DPMA, and [²H₁₀]-DPMA in all biological fluids were measured using previously developed gas-chromatographic-mass spectrometric analytical methods (Tonn et al., 1993, 1995). The linear calibration range of these assays is 2.0 to 200.0 ng/ml for DPHM and [²H₁₀]-DPHM and 2.5 to 250.0 ng/ml for DPMA and [²H₁₀]-DPMA. The inter- and intraday coefficients of variation are <20% at the limit of quantitation and <10% at all other concentrations (Tonn et al., 1993, 1995).

Pharmacokinetic Analyses. The maternal and fetal steady-state arterial plasma DPHM and [²H₁₀]-DPHM concentration data were treated according to a two-compartment open model to calculate the placental and nonplacental clearances of DPHM (or [²H₁₀]-DPHM) in the ewe and fetus. This model assumes steady-state plasma concentrations and drug elimination from both the maternal and fetal compartments. The equations to estimate placental and nonplacental clearance parameters have been described previously (Szeto et al., 1982).

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Statistical Analysis. All data are reported as the mean ± S.D. The achievement of steady-state for DPHM (or [²H₁₀]-DPHM) concentrations in maternal and fetal plasma was established according to two criteria: 1) the slope of plasma concentration versus time curve should not be significantly different from zero and 2) the coefficient of variation of the measured concentrations should be <15%. 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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The average maternal body weight was 82.4 ± 14.1 kg and estimated fetal body weights on the day of maternal and fetal experiments were 3.16 ± 0.41 kg and 2.92 ± 0.22 kg, respectively. During the maternal experiments, the control period fetal femoral arterial pH, Po₂, Pco₂, O₂ saturation, and hemoglobin, glucose, and lactate concentrations were 7.371 ± 0.017, 20.6 ± 1.8, and 50.2 ± 3.3 mm Hg, 42.6 ± 7.0%, 11.3 ± 0.6 g/dl, 0.80 ± 0.21, and 1.15 ± 0.18 mM, respectively. Likewise, during fetal administration, the control values for these variables were 7.344 ± 0.031, 24.8 ± 5.6, and 53.1 ± 2.9 mm Hg, 55.0 ± 8.4%, 10.3 ± 1.3 g/dl, 0.78 ± 0.29, and 1.56 ± 0.65 mM, respectively. There were no consistent changes in any of these variables during the course of experiments.

Maternal-Fetal Steady-State Plasma Drug Concentrations and Unbound Fractions and Placental and Nonplacental Clearance Estimates. Table 1 presents gestational age (GA) of the animals on the day of experiment and maternal and fetal DPHM clearance (total, placental, and nonplacental clearances) data calculated using the two-compartment pharmacokinetic model of the maternal-fetal unit (Szeto et al., 1982).

Maternal-Fetal Arterial Plasma AUC Ratios of the Parent Drug, the Preformed Metabolite, and the In Vivo Generated Metabolite. Table 2 presents different AUC ratios for the parent drug and preformed and in vivo formed metabolite in maternal and fetal arterial plasma. The ratio of FA plasma to maternal femoral arterial (MA) plasma of the formed metabolite AUCs after maternal drug administration (2.97 ± 0.82) was significantly higher than the FA/MA ratio of preformed metabolite AUCs after maternal metabolite administration (0.41 ± 0.21; paired t test, p < .005). Although the MA/FA ratio of the formed metabolite AUCs after fetal drug administration (0.25 ± 0.35) was higher than the MA/FA ratio of preformed metabolite AUCs after fetal metabolite administration (0.02 ± 0.02) in all the individual animals, the difference between the means was not statistically significant. Also, the AUC ratio of the formed metabolite to the parent drug in FA after maternal drug administration (21.5 ± 26.4) was higher compared with the corresponding ratio after fetal drug administration (2.32 ± 1.16) in all the individual animals; however the difference between means was only near statistical significance (paired t test, p = .08). Similarly, the AUC ratio of the formed metabolite to the parent drug in MA after fetal drug administration (2.69 ± 3.87) was higher compared with the corresponding ratio after maternal drug administration (1.13 ± 1.44) in all the individual animals; however, the difference between means was not statistically significant (paired t test, p > .05).

Comparative Maternal-Fetal Pharmacokinetics of the Parent Drug, the Preformed Metabolite, and the In Vivo Generated Metabolite. Figure 1 shows the typical plasma concentration-time profiles of the parent drug, the preformed metabolite, and the in vivo generated metabolite in maternal and fetal arterial plasma during separate maternal (Fig. 1A) and fetal (Fig. 1B) administration experiments in E303Y.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Representative plasma concentration-time profiles of the parent drug, the preformed metabolite, and the in vivo generated metabolite in maternal and fetal plasma of E303Y. Figure 1, A and B, are the data from separate maternal and fetal administration experiments, respectively. In both experiments, unlabeled DPMA was administered as the preformed metabolite in combination with [2H10]-DPHM. The [2H10]-DPMA is thus the in vivo generated metabolite.

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Renal Elimination of Parent Drug, Preformed Metabolite, and in vivo Generated Metabolite in Mother and Fetus. Figure 2 shows typical cumulative excretion profiles of the parent drug, the preformed metabolite, and the in vivo generated metabolite in maternal and fetal urine during separate maternal (Fig. 2A) and fetal (Fig. 2B) administration experiments in E303Y.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Representative cumulative renal excretion profiles of the parent drug, the preformed metabolite, and the in vivo generated metabolite in maternal and fetal urine of E303Y. Figure 2, A and B, are the data from separate maternal and fetal administration experiments, respectively. In both experiments, unlabeled DPMA was administered as the preformed metabolite in combination with [2H10]-DPHM. The [2H10]-DPMA is thus the in vivo generated metabolite. Figure 2B shows that even after fetal administration negligible amounts of the preformed as well as the in vivo generated metabolite are excreted in fetal urine in comparison to maternal urine.

	Discussion

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Although considerable in vitro data are available on the expression and activity of human fetal drug-metabolizing enzymes (Pacifici et al., 1982; Perucca, 1987; Burchell et al., 1989; Krauer and Dayer, 1991; Jacqz-Aigrain and Cresteil, 1992; Hakkola et al., 1994; Cazeneuve et al., 1994; Shimada et al., 1996), the in utero role of these enzymes in drug metabolism and the resulting effects on fetal drug and metabolite exposure cannot be examined in detail due to obvious practical limitations. We and others have used chronically instrumented pregnant sheep to study the extent of in utero functional development of fetal metabolism of a number of drugs such as propranolol (Mihaly et al., 1982), labetalol (Yeleswaram et al., 1993), ritodrine (Wright et al., 1992), acetaminophen (Wang et al., 1986), morphine (Olsen et al., 1988), and valproic acid (S.K., K.W.R., and D.W.R., unpublished data).

The major route of DPHM elimination in many species is its metabolism to DPMA, which is subsequently conjugated to amino acids such as glycine and glutamine (Drach and Howell, 1968; Drach et al., 1970; Chang et al., 1974). The aim of the present study was to examine the comparative kinetics of DPHM metabolism to DPMA in maternal and fetal sheep and the resulting effects on fetal exposure to this metabolite. The above aim required knowledge of the distribution and elimination characteristics of the parent drug as well as the metabolite in the mother and the fetus. However, the kinetics of drug metabolites formed after parent drug administration are complex because metabolite formation, distribution, and elimination occur concurrently and their kinetics is difficult to resolve. Furthermore, estimation of the total contribution of a particular metabolic pathway is much more difficult in the fetus where the formed metabolites may either be excreted into multiple number of fluid compartments that can not be cumulatively sampled (e.g., amniotic and tracheal fluids, fetal urine) or may cross the placenta and mix with those formed in the maternal circulation.

One approach to examine the kinetics of metabolite distribution and elimination is the i.v. administration of the preformed (synthesized) metabolite (Kaplan et al., 1970, 1973; Boxenbaum and Riegelman, 1976; Patel et al., 1978; Lai et al., 1979; Cobby et al., 1978). These data may then be combined with the drug and metabolite data after parent drug administration and the kinetics of metabolite formation can be estimated. However, this approach can underestimate the amount of drug converted to a particular metabolite if the metabolite undergoes rapid sequential metabolism in the liver before its egress into the circulation (Pang and Gillette, 1979). In such situations, the preformed metabolite should be administered via the portal venous route to more closely simulate the secondary hepatic metabolism and subsequent systemic availability of the metabolite formed from the parent drug (Pang et al., 1979). In our studies, however, it was acceptable to administer the preformed metabolite via the i.v. route due to a lack of any detectable secondary metabolism of DPMA in either maternal or fetal sheep (see below). This considerably simplified our surgical procedure in the fetus where implanting and maintaining chronic portal venous catheters is a difficult task.

Maternal and fetal total drug clearance includes a significant but variable contribution from placental drug elimination (~1-10% in the mother versus ~40-75% in the fetus; Yoo et al., 1993; Kumar et al., 1997; Table 1). Thus, a comparison of the exact capacity of a particular metabolic pathway in the mother and the fetus requires the estimation of their drug clearance via nonplacental routes. The estimation of maternal and fetal placental and nonplacental clearances in turn requires separate maternal and fetal steady-state drug administration (Szeto et al., 1982). This, combined with the essential study of maternal and fetal preformed metabolite kinetics (see above), meant that four experiments had to be conducted in each pregnant sheep preparation in our study. Given the duration of each experiment (96 h), this was difficult to accomplish in the limited time window (~2 weeks) available for experimentation in these late gestation animals before term. Also, rapid fetal growth and physiological alterations during late gestation may confound the results of experiments conducted over a prolonged time period. Hence, we utilized a protocol where either a combination of the unlabeled parent drug and the labeled metabolite (DPHM and [²H₁₀]-DPMA) or the labeled drug and the unlabeled metabolite ([²H₁₀]-DPHM and DPMA) was administered to the mother or the fetus in two separate experiments. The assumption made in this protocol is that the preformed metabolite does not alter the pharmacokinetics of the parent drug and vice versa. The parent drug clearances in these animals were similar to those obtained in our earlier studies where the preformed metabolite was not administered (Yoo et al., 1993; Kumar et al., 1997). In addition, the plasma concentrations, AUCs, and the amount of formed DPMA recovered in maternal urine after parent drug administration to the mother was similar in this and our previous study (Kumar et al., 1997). These observations appear to support the above assumptions.

Presence of drug metabolites in the fetal circulation may result from their maternal-to-fetal placental transfer or metabolite formation by the fetus itself. The evidence of in utero fetal formation of the DPMA metabolite is provided by the various AUC ratios presented in Table 2. If the fetal ability to form a particular metabolite is absent or negligible, the FA/MA AUC ratios of the preformed and the formed metabolite during maternal parent drug and preformed metabolite administration should be similar (because in this case the maternal-to-fetal placental transfer of the metabolite will be the only determining factor of this ratio). A higher FA/MA ratio of the formed metabolite (2.97 ± 0.82) compared to the preformed metabolite (0.41 ± 0.21) in our study suggests additional fetal formation of this metabolite from the drug transferred to the fetus via the placenta. Analogously, after fetal drug and preformed metabolite administration, a higher (but not statistically significant due to high interanimal variability) MA/FA ratio of the formed metabolite (0.25 ± 0.35) compared with the preformed metabolite (0.02 ± 0.02) indicates additional maternal formation of this metabolite from the drug transferred in the fetal-to-maternal direction.

Anatomically, the fetal liver is located midway between the placenta and rest of the fetal circulatory system (Battaglia and Meschia, 1988). The umbilical vein drains the fetal placental vasculature and supplies the fetus with oxygen, nutrients, and drugs transferred from the maternal circulation via the placenta. Approximately 30 to 70% of the umbilical blood flow passes through the fetal liver before reaching the fetal circulation; the rest bypasses the liver directly to the inferior vena cava via the ductus venosus shunt (Edelstone et al., 1978). We have reported previously that the fetal liver can metabolize a significant but variable (due to high variability in ductus venosus shunt fraction) proportion of DPHM present in the umbilical vein in a first-pass manner (Kumar et al., 1997). This leads to a greater fetal hepatic metabolism and metabolite formation from the drug transferred to the fetus via the placenta than when it is directly administered i.v. to the fetus. This factor, combined with some maternal-to-fetal placental transfer of the metabolite formed in the mother, leads to higher (but again not statistically significant due to high interanimal variability) AUC ratios of the formed metabolite-to-the parent drug in fetal arterial plasma after maternal drug administration as compared with when the drug is given directly to the fetus (21.5 ± 26.4 versus 2.32 ± 1.16; Table 2). Similar to the FA ratios above, but to a lesser extent, the MA ratio of the formed metabolite-to-the parent drug after fetal administration was greater in all the individual animals compared with that after maternal drug administration (2.69 ± 3.87 versus 1.13 ± 1.44; Table 2). However, the only possible explanation for this phenomenon appears to be some fetal-to-maternal placental transfer of the metabolite formed in the fetal circulation after fetal drug administration, thus leading to an increase in the above ratio.

There are significant differences in the disposition of the parent drug and the preformed DPMA (or [²H₁₀]-DPMA) metabolite both in the mother and the fetus. In particular, the CL_tb and Vd_ss of the metabolite are much lower compared with the parent drug. In fact, the Vd_ss of the preformed metabolite is only about 2 and 3 times the blood volume in the mother and the fetus, respectively. This indicates very limited tissue distribution of this compound, which could be partly related to its very high plasma protein binding (> 99%; Kumar et al., 1997) and low lipophilicity (octanol/pH 7.4 phosphate buffer partition coefficient = 0.29; S.K., K.W.R., and D.W.R., unpublished data). Also, the T_1/2 and MRT of the metabolite are longer compared with the parent drug in both the mother and particularly in the fetus. A very long fetal T_1/2 and MRT of the metabolite suggest that although the fetal lamb has the ability to form DPMA from DPHM, the mechanisms involved in DPMA elimination are not fully developed. The placental transfer of DPMA appears to be extremely slow and limited because relatively low C_max values in FA and MA occur at least 3 to 5 h after maternal and fetal preformed metabolite i.v. bolus administration, respectively. Slow placental transfer of this metabolite is partly responsible for its slow elimination from the fetus (see below). Thus, at least in this situation, fetal metabolism of the drug actually leads to a considerably increased fetal exposure to the metabolite due to its "trapping" in the fetal circulation.

The MRT of the in vivo formed metabolite is longer than that of the preformed metabolite in both the mother and the fetus, suggesting differences in the disposition of in vivo generated and exogenously administered metabolite. This could be related to the presence of a diffusional barrier to the egress of the highly polar DPMA metabolite from within the cell (hepatocyte) into the systemic circulation after its formation from the parent drug. This can lead to an overall slower diffusion-limited elimination and a longer residence time of the formed metabolite as compared with the preformed metabolite (De Lannoy and Pang, 1986; Schwab et al., 1990). In such instances, it is important to utilize mass balance approaches (e.g., eq. 1) rather than the rate-constant based pharmacokinetic modeling to calculate the quantitative importance of a particular metabolic pathway in total drug clearance using preformed metabolite administration.

The contribution of DPMA formation to nonplacental DPHM clearance was not statistically different in the mother and the fetus and was typically ~1% in all but one animal (Table 3). These data indicate that although the DPMA formation pathway is almost equally functional in the mother and fetus, this is not a major route of DPHM clearance in sheep. This is in contrast with many other species where DPMA and its amino acid conjugates account for ~40 to 60% of total DPHM metabolites (Drach and Howell, 1968; Drach et al., 1970; Chang et al., 1974).

Significant differences also exist in the renal handling of the parent drug and the DPMA metabolite in the mother and the fetus. Renal elimination of the parent drug in the mother accounted for <0.5% of its total body clearance. In contrast, the CL_r of preformed DPMA accounted for 88.8 ± 6.4% (Tables 3 and 4) of its total maternal clearance. Hence, almost the entire maternal i.v. dose was eventually recovered unchanged in maternal urine (88.0 ± 6.5%; Table 4), indicating that DPMA is not secondarily metabolized in maternal sheep. This is also a species difference in sheep compared to the monkey, dog, and human where significant amounts of DPMA are recovered in urine as its glycine, glutamine, or an uncharacterized conjugate (Drach and Howell, 1968; Drach et al., 1970; Chang et al., 1974). Due to the lack of sequential metabolism of DPMA, the amount of in vivo formed DPMA recovered in maternal urine as a percentage of the parent drug dose was similar to the percent contribution of this pathway to maternal nonplacental DPHM clearance (1.60 ± 1.86 versus 1.78 ± 2.12%, respectively; Tables 3 and 4). Similar to the mother, the excretion of the unchanged DPHM in fetal urine also accounted for <0.5% of the total fetal dose. In contrast to the mother, however, only 1.79 ± 2.08% of the total fetal i.v. dose of the preformed metabolite was recovered in fetal urine due to a very low fetal renal clearance of this metabolite (Table 4). This is presumably related to a lack of any significant renal tubular secretion of many organic acid compounds such as para-aminohippurate (Elbourne et al., 1990), acetaminophen, and morphine glucuronides (Wang et al., 1986; Olsen et al., 1988), valproic acid (S.K., K.W.R., and D.W.R., unpublished data), and indomethacin (Krishna et al., 1995) in the late-gestation fetal lamb. Instead, almost all of the fetal i.v. dose of this metabolite (92.1 ± 7.4%; Table 4) underwent fetal-to-maternal placental transfer and was eventually recovered in maternal urine over the ensuing 96-h sampling period. The lack of any significant fetal renal elimination and slow placental transfer of this metabolite appears responsible for its prolonged T_1/2 in the fetus.

In summary, using an approach based on the simultaneous administration of differently labeled parent drug and metabolite to the mother and the fetus, we have shown that the contribution of DPMA formation to DPHM nonplacental elimination in the maternal and the fetal sheep is relatively similar and is typically ~0.5 to 1%. Hence, this is a minor pathway in overall DPHM elimination in this species. The DPMA metabolite is not sequentially metabolized in fetal or adult sheep. It is eliminated solely via the renal pathway in the mother and via the placenta (and eventually in maternal urine) in the fetus. These findings are in contrast to other species where DPMA and its amino acid conjugates account for ~40 to 60% of total DPHM metabolites.