Disposition of valproic acid in maternal, fetal, and newborn sheep. II: metabolism and renal elimination.

Metabolism and renal excretion of valproic acid (VPA) were examined in maternal, fetal, and newborn sheep to identify the underlying reasons for the previously observed reduced VPA clearance in newborn lambs. Plasma and urine from VPA infusion studies in maternal, fetal, and newborn sheep were analyzed for VPA and its metabolites [VPA-glucuronide; beta-oxidation products: (E)-2-ene, (E)-3-ene, and 3-keto VPA; hydroxylated metabolites: 3-hydroxy, 4-hydroxy, and 5-hydroxy VPA (5-OH VPA); and 4-ene VPA, 4-keto VPA, 2-propylglutaric acid, and 2-propylsuccinic acid] using gas chromatography-mass spectrometry. All measured metabolites were detectable in maternal and fetal plasma, with 3-keto and 5-OH VPA being at higher concentrations in the fetus. Plasma concentrations of (E)-2-ene, (E)-3-ene, 3-keto, and 5-OH VPA were higher in the newborn compared with the mother, whereas those of the other metabolites were similar. A smaller percentage of the dose was excreted as VPA-glucuronide in newborn lamb urine (28.3 +/- 12.0%) compared with the mother (77.0 +/- 7.8%). Similarly, a lower fraction of the dose was excreted unchanged in newborn urine (11.0 +/- 5.8%) relative to the urine of the mother (19.3 +/- 5.8%); however, significantly larger percentages were excreted as (E)-2-ene (0.11 +/- 0.04 versus 0.02 +/- 0.01%), 3-keto (11.6 +/- 3.5 versus 1. 6 +/- 0.8%), 4-hydroxy (6.1 +/- 3.2 versus 2.3 +/- 1.3%), and 5-OH VPA (2.2 +/- 0.6 versus. 0.8 +/- 0.6%). The major reason for the reduced VPA elimination in newborn lambs appears to be impaired renal excretion and glucuronidation capacity. As a result, a larger fraction of the dose is channeled to beta-oxidation and hydroxylation pathways. The beta-oxidation activities are high at birth; this may explain the high plasma concentrations of (E)-2-ene and 3-keto VPA observed in newborn lambs and human newborns exposed to VPA.     

Dose-dependent pharmacokinetics and metabolism of valproic acid in newborn lambs and adult sheep.

Dose-dependent pharmacokinetics and metabolism of valproic acid (VPA) were studied in newborn and adult sheep to assess age-related differences in plasma protein binding and metabolic elimination. Newborn lambs received either a 10- (n = 8), 50- (n = 5), 100- (n = 4), or 250-mg/kg (n = 4) VPA i.v. bolus. Individual adult sheep (n = 5) received all four doses in a random order with an appropriate washout period between experiments. Unbound or metabolic clearance of VPA was significantly higher in adult sheep at the two lower doses when compared with lambs, and similar to the lambs at the two higher doses. Plasma protein binding was nonlinear at all doses. Estimates of binding capacity (B(max1)) at the saturable site were higher in adults (91.8 microg/ml) when compared with lambs (44.9 microg/ml), whereas the opposite trend was observed for binding affinity [K(d1) = 9.6 microg/ml (adult) versus 3.2 microg/ml (lambs)]. Characterization of developmental differences in overall VPA metabolic elimination involved fitting of unbound VPA plasma concentration data to a two-compartment model with Michaelis-Menten elimination. This resulted in similar in vivo estimates of apparent V(max) [445.0 microg/min/kg (adult) versus 429.9 microg/min/kg (lambs)]. However, apparent K(m) estimates appeared to be higher in lambs [30.0 microg/ml (adult) versus 69.6 microg/ml (lambs)]. Similar findings were obtained from in vivo estimates of V(max) and K(m) for VPA glucuronidation obtained from VPA-glucuronide metabolite urinary excretion data. Thus, it appears that age-related differences in metabolic clearance may be related to differences in the apparent in vivo K(m) as opposed to V(max) of VPA glucuronidation.     

Study of the placental transfer of cisapride in sheep. Plasma levels in the pregnant ewe, the fetus, and the lamb.

The placental transfer of cisapride, a new prokinetic agent, was studied in a sheep model. The pharmacokinetics of cisapride were studied in the lamb, the pregnant ewe, and the fetus by obtaining blood samples from chronically implanted arterial catheters. Comparable pharmacokinetic parameters were found in the lamb and the adult sheep: half-life, 1.39-1.83 hr; total plasma clearance, 1998-2160 ml/kg/hr; AUC, 92.6-100.1 ng.hr/ml. Cisapride plasma concentrations after continuous infusion were predicted correctly based on the parameters obtained after iv bolus. There was a materno-fetal transfer of cisapride following a single iv bolus administered to the mother. Cisapride crossed the placenta within 5 min and equilibrated with maternal plasma within 20 to 30 min after dosing. The average fetal-to-maternal plasma concentration ratio was 0.71. The amniotic fluid also contained measurable amounts of cisapride. The protein binding of cisapride in maternal and fetal plasma is 89.0% and 88.4%, respectively; the free fraction is 4 times larger than in humans. Cisapride crosses the ovine placental barrier. The sheep placenta is less permeable than the human placenta, but the higher free fraction of cisapride facilitates placental transfer.     

Ontogeny of valproic acid disposition and metabolism: a developmental study in postnatal lambs and adult sheep.

The ontogeny of valproic acid (VPA) disposition and metabolism was investigated in developing lambs and adult sheep (Dorset or Suffolk breed). Specifically, we wished to investigate the role of glucuronidation and beta-oxidation on VPA elimination during development. Catheters were implanted in a carotid artery, a jugular vein, and the urinary bladder in 10-day-old (10 d; n = 8), 1-month-old (1 M; n = 4), and 2-month-old lambs (2 M; n = 5). In adult sheep (n = 5), catheters were implanted in a femoral artery and vein. After the administration of a 10 mg/kg VPA i.v. bolus, serial blood samples and cumulative urine samples were collected for 36 h in the adult ewes and for 72 h in the lambs. Due to saturable protein binding, age-related differences in VPA clearance were more obvious when examining the total body clearance of unbound drug (Cl(u)tb). Mean Cl(u)tb increased significantly with age up to 2 months (10 d = 2.65 +/- 1.16 ml/min/kg; 1 M = 5.11 +/- 2.49 ml/min/kg; 2 M = 12.84 +/- 3.88 ml/min/kg) before decreasing to adult levels (7.73 +/- 2.64 ml/min/kg). Similarly, the urinary recovery of the major metabolite, VPA-glucuronide, was significantly less in 10 d lambs (29.2 +/- 16.0% of the dose) when compared with the adult and 2 M groups (both approximately 74% of the dose). No differences with age were observed in the portion of the dose excreted as the beta-oxidation metabolite, 2-n-propyl-3-oxopentanoic acid. The results suggest that alterations in Cl(u)tb with age may be attributable to postnatal development of enzymes involved in VPA glucuronidation.     

Placental transfer of ranitidine during steady-state infusions of maternal and fetal sheep

The placental transfer of ranitidine was studied at pharmacokinetic steady state in anesthetized, full-term, pregnant sheep. Ranitidine was administered to the ewe in three preparations and to the fetus in three other sheep. In all experiments, dose size was based on the combined maternal-fetal weight. Steady-state plasma levels were reached within 4 hr by using an initial intravenous bolus dose followed by continuous infusion. Following maternal dosage, the mean maternal (jugular vein) steady-state concentration (CMss) at 4 hr was 842 +/- 66 ng/ml (SEM), the mean fetal (carotid artery) steady-state concentration (CFss) was 26.5 +/- 4.2 ng/ml, and the mean fetal umbilical venous steady-state concentration was 28.9 +/- 3.5 ng/ml. Both the fetal and umbilical plasma concentrations were significantly less than the maternal plasma concentrations (p less than 0.01). With fetal dosage, mean CMss was 414 +/- 42 ng/ml at 4 hr and was significantly less than the mean CFss value at the same time, which was 6890 +/- 360 ng/ml (p less than 0.005). Ranitidine was not bound extensively to plasma proteins in the ewe or the fetus (range 12-55% bound). The reversal of the CMss/CFss gradient with the change from maternal to fetal administration and the low binding of the drug shows that the gradient following maternal dosage cannot be explained by ion-trapping or differential plasma protein binding. As active placental transport is considered unlikely, the low fetal plasma concentrations are probably due to the presence of significant fetal elimination of ranitidine. Furthermore, the substantial gradient between maternal and umbilical venous plasma concentrations suggests that placental elimination of ranitidine should also be considered.     

Diphenhydramine disposition in the sheep maternal-placental-fetal unit: determinants of plasma drug concentrations in the mother and the fetus

The objective of this study was to identify the important factors that determine plasma concentrations of diphenhydramine (DPHM) in the mother and the fetus after maternal as well as fetal steady-state drug administration. Inter-relationships were evaluated between maternal and fetal placental and nonplacental clearances, plasma protein binding, and steady-state plasma concentrations of DPHM among data obtained from 18 pregnant sheep during late gestation. The major determinant of plasma DPHM concentrations in the mother after maternal as well as fetal administration appears to be maternal plasma protein binding and maternal nonplacental clearance. In contrast, the major determinant of fetal plasma DPHM concentrations after maternal drug administration was the extent of fetal first-pass hepatic drug uptake from the umbilical vein. However, after fetal drug administration, the fetal plasma concentrations were related to the extent of fetal plasma protein binding and fetal placental and nonplacental clearances. The index of fetal-to-maternal placental drug transfer after fetal drug administration (steady-state maternal-to-fetal plasma concentration ratio) was related to steady-state fetal plasma unbound fraction and fetal placental and nonplacental clearance. However, this index was not related to the magnitude of the factors operating on the maternal side of the placenta such as maternal plasma protein binding and maternal nonplacental clearance. This might indicate a lack of complete equilibration of the unbound drug concentrations on the two sides of the placenta at the exchange site.     

Fetal and maternal placental and nonplacental clearances of metoclopramide in chronically instrumented pregnant sheep

The placental and nonplacental clearances of metoclopramide were studied in nine chronically instrumented, near-term pregnant sheep using a two-compartment open model. Metoclopramide was administered to the ewe and fetus on separate occasions as an initial iv bolus loading dose followed by a constant-rate infusion, with steady-state maternal and fetal plasma concentrations being obtained by 45 min. Following the maternal infusions, metoclopramide reached average steady-state concentrations of 50.0 +/- 20.2 ng/mL in the ewe and 27.1 +/- 8.6 ng/mL in the fetus, with a mean fetal-to-maternal concentration ratio of 0.57 +/- 0.14. The ability of the fetus to eliminate metoclopramide by nonplacental routes appears to be responsible for this ratio being less than unity, rather than differential protein binding and ion-trapping effects. Mean steady-state concentrations were 13.8 +/- 4.5 and 253.7 +/- 92.1 ng/mL in the ewe and fetus, respectively, after fetal drug administration. Metoclopramide was bound significantly less to fetal (39.5 +/- 8.9%) than to maternal (49.5 +/- 7.9%) plasma proteins, with values similar to that reported for humans (approximately 40%). Clearance of metoclopramide across the placenta from the fetus to the ewe (6.2 +/- 2.4 L/h/kg) was significantly greater than that in the reverse direction (4.3 +/- 1.3 L/h/kg) and accounted for approximately 80% of total fetal drug elimination. This may be explained by the higher percentage of fetal cardiac output to the placenta and the flow-limited transfer of this compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breast cancer resistance protein 1 limits fetal distribution of nitrofurantoin in the pregnant mouse.

The efflux transporter, the breast cancer resistance protein (BCRP), is most abundantly expressed in the apical membrane of the placental syncytiotrophoblasts, indicating that it could play an important role in protecting the fetus by limiting xenobiotic/drug penetration across the placental barrier. In the present study, we examined whether Bcrp1, the murine homolog of human BCRP, limits fetal distribution of the model BCRP/Bcrp1 substrate, nitrofurantoin (NFT), in the pregnant mouse. NFT was administered i.v. to FVB wild-type and Bcrp1(-/-) pregnant mice. The maternal plasma samples and fetuses were collected at various times (5-60 min) after drug administration. The NFT concentrations in the maternal plasma samples and homogenates of fetal tissues were determined by a high-performance liquid chromatography/UV assay. Although the maternal plasma area under the concentration-time curve (AUC) of NFT in the Bcrp1(-/-) pregnant mice (97.4 +/- 10.0 microg . min/ml plasma) was only slightly (but significantly) higher than that in the wild-type pregnant mice (78.4 +/- 6.0 microg . min/ml plasma), the fetal AUC of NFT in the Bcrp1(-/-) pregnant mice (1493.0 +/- 235.3 ng . min/g of fetus) was approximately 5 times greater than that in the wild-type pregnant mice (298.6 +/- 77.4 ng . min/g of fetus). These results clearly suggest that Bcrp1 significantly limits fetal distribution of NFT in the pregnant mouse, but has only a minor effect on the systemic clearance of the drug.     

Fetal disposition of delta 9-tetrahydrocannabinol (THC) during late pregnancy in the rhesus monkey

Three late-term (Gestational Days 146-151) rhesus monkeys were given 0.3 mg/kg delta 9-tetrahydrocannabinol (THC) intravenously via the maternal radial vein to quantify the placental transfer and fetal disposition of THC, the major psychoactive component of marijuana. Simultaneous blood samples were obtained from a maternal uterine vein and an intraplacental artery at 0, 3, 15, 30, 45, 60, 90, 120, and 180 min after dosing using an intraplacental cannulation technique. Samples of fetal plasma, spleen, testis, lung, brain, liver, bile, kidney, adrenals, thymus, and placenta were obtained at 180 min postdose. Samples were analyzed for THC and a major metabolite, 11-nor-9-carboxy-THC (11-nor), by radioimmunoassay (RIA). Peak plasma THC values were obtained 3 and 15 min after dosing in the mother (1419 ng/ml) and fetus (83 ng/ml), respectively. By 3 hr, maternal and fetal plasma THC levels were equal (37 ng/ml). Maternal plasma was sampled beyond 180 min at 24, 48, 72, and 96 hr postdose, times at which THC and 11-nor concentrations were either near or at the lower limit of sensitivity for the RIA (2 ng/ml). While maternal plasma 11-nor levels peaked at 1 hr (44 ng/ml), almost no 11-nor was detected in fetal plasma (less than 5 ng/ml). Fetal tissue concentrations of THC were 53 +/- 6 ng/g (SE) for brain and 114 +/- 10 ng/g for liver, while 11-nor was undetectable in placenta, fetal liver, and fetal brain. These data demonstrate that THC rapidly crosses the placenta and enters the fetus. The lack of 11-nor in fetal plasma and tissues suggests that this metabolite does not readily cross the placenta and that the fetus does not readily metabolize THC to 11-nor at this stage of development. A portion of this data was presented at the 1985 meeting of The American Society of Primatologists and at the 1986 meeting of The Teratology Society.     

Pharmacokinetics and renal excretion of diphenhydramine and its metabolites, diphenylmethoxyacetic acid and diphenhydramine-N-oxide, in developing lambs

The developmental disposition of diphenhydramine (DPHM) and its metabolites, diphenylmethoxyacetic acid (DPMA) and DPHM-N-oxide (DPHMNO), was investigated in postnatal lambs. Lambs received a DPHM intravenous (iv) bolus 15 days (Group A; n = 5) or 2 months (Group B; n = 6) after birth. Total body clearance of DPHM in postnatal lambs (Group A = 138.7 +/- 80.5 mL/min/kg; Group B = 165.7 +/- 51.3 mL/min/kg) was similar to the nonplacental clearance values (i.e., the component of fetal total body clearance that is not due to elimination via the placenta) estimated for fetal lamb (116.3 +/- 49. 6 mL/min/kg), and significantly greater than estimates in adult sheep (38.5 +/- 12.3 mL/min/kg). In addition, Group A DPHM renal clearance (CL(r), >1.80 +/- 1.24 mL/min/kg) was similar to that of the fetus (2.06 +/- 0.24 mL/min/kg), and significantly greater than that for Group B (0.26 +/- 0.17 mL/min/kg) and the adult (0.012 +/- 0.005 mL/min/kg). In contrast, similar to the fetal situation, postnatal DPMA CL(r) (Group A = 0.02 +/- 0.02 mL/min/kg; Group B = 0.05 +/- 0.01 mL/min/kg) was significantly less than adult values (0. 53 +/- 0.19 mL/min/kg). Because DPMA is not sequentially metabolized in sheep, the lower CL(r) in postnatal lambs results in longer apparent elimination half-lives of this metabolite (Group A = 90.4 +/- 32.2 h; Group B = 13.13 +/- 11.0 h) compared with that in the adult (2.9 +/- 1.6 h). No age-related difference in DPHMNO CL(R) was observed. Alterations in the CL(r) of DPHM and DPMA are likely related to differences in the rate of development of mechanisms (i.e. , tubular secretion and reabsorption and glomerular filtration rate) involved in the urinary drug excretion of organic acids and bases.     

Comparative formation, distribution, and elimination kinetics of diphenylmethoxyacetic acid (a diphenhydramine metabolite) in maternal and fetal sheep.

Deamination to diphenylmethoxyacetic acid (DPMA) is the major route of diphenhydramine (DPHM) clearance in many species. In this study, we assessed the contribution of this pathway to nonplacental DPHM elimination and disposition of DPMA in maternal and fetal sheep. Paired maternal-fetal experiments were conducted in five chronically catheterized pregnant sheep (124-140 days gestation) with an appropriate washout period in between. Both maternal and fetal dosing experiments involved administration of an i.v bolus of deuterium-labeled DPMA ([2H10]-DPMA) combined with a 6-h infusion of DPHM (or a bolus of unlabeled DPMA with an infusion of deuterium-labeled DPHM). Maternal and fetal arterial plasma and urine samples were collected and analyzed for DPMA, [2H10]-DPMA, DPHM, and deuterium-labeled DPHM concentrations using gas chromatography-mass spectrometry. The preformed DPMA (or [2H10]-DPMA) had a substantially lower clearance (maternal: 0.55 +/- 0.18 versus 40.9 +/- 14.0 ml/min/kg; fetal: 0.37 +/- 0.11 versus 285. 6 +/- 122.2 ml/min/kg) and steady-state volume of distribution (Vdss, maternal: 0.10 +/- 0.02 versus 2.1 +/- 1.1 l/kg; fetal: 0.40 +/- 0. 06 versus 13.1 +/- 3.1 l/kg) as compared with the parent drug. The contribution of DPMA formation to maternal and fetal DPHM nonplacental clearance in vivo was 1.78 +/- 2.12% and 0.87 +/- 0.56%, respectively, indicating that DPMA formation is not a major route of DPHM clearance in fetal or maternal sheep. The recoveries of DPMA (or [2H10]-DPMA) in maternal urine were 88.0 +/- 6.5 and 92.1 +/- 7. 4% of the administered dose during maternal and fetal dosing experiments, respectively. Thus, this metabolite does not appear to be secondarily metabolized in fetal or maternal sheep. These findings are in contrast to other species (dog, rhesus monkey, human) where DPMA and its conjugates constitute approximately 40 to 60% of the total DPHM metabolites.     

Placental transfer of diphenhydramine in chronically instrumented pregnant sheep

To study the placental transfer and pharmacokinetics of the H1 receptor blocker, diphenhydramine [2-(diphenylmethoxy)-N,N-dimethylethylamine], 100 mg of the drug was administered to four pregnant sheep (122-129 d gestation) by intravenous injection through catheters chronically implanted in the ewe and fetus. Rapid placental transfer occurred, with peak fetal plasma concentrations occurring within 5 min after injection. The fetal-maternal ratio of the area under the plasma concentration versus time curves averaged 0.85, indicating significant fetal exposure to the drug. The average apparent terminal elimination half-life in the ewe (52 min) was not significantly different from that obtained in the fetus (46 min). The maternal total body clearance was 3.6 L X h-1 X kg-1, and the volume of distribution at steady state was 3.2 L/kg. In summary, this study demonstrates rapid and extensive placental transfer of diphenhydramine after maternal drug administration. Since placental permeability to lipid-soluble compounds does not differ greatly in different species, it is likely that a similar situation exists in humans.     

Differential transplacental binding of valproic acid: influence of free fatty acids

The unbound fraction of valproic acid (VPA) was found to be significantly lower in cord serum (6.0 +/- 0.8%) than in maternal serum collected before oxytocin (12.2 +/- 2.7%) or after delivery (9.9 +/- 2.3%). The difference was probably due to the concentration of free fatty acids (acting as displacing agents) being higher in maternal serum. The transplacental binding gradient explains the clinical observation that total VPA levels at delivery are higher in the newborn than in the mother.     

Diphenhydramine disposition in the sheep maternal-placental-fetal unit: gestational age, plasma drug protein binding, and umbilical blood flow effects on clearance.

The objective of this study was to examine the interrelationships between maternal and fetal plasma drug protein binding, umbilical blood flow (Q(um)), gestational age (GA), and maternal-fetal diphenhydramine (DPHM) clearances in chronically instrumented pregnant sheep. Maternal and fetal DPHM placental (CL(mf) and CL(fm), respectively) and nonplacental (CL(mo) and CL(fo), respectively) clearances and steady-state plasma protein binding were determined in 18 pregnant sheep at 124 to 140 days' gestation (term, approximately 145 days). The data demonstrated a highly significant fall of approximately 66% in CL(fm) and a decreasing trend in CL(fo) ( approximately 47%) over the GA range studied. However, no such relationships existed between GA and CL(mf) or CL(mo). Concomitant with this was a decrease in fetal DPHM plasma unbound fraction with GA, with no such change being evident in the mother. Both CL(mo) and CL(fo) were related to the respective DPHM plasma unbound fraction. A strong relationship also existed between fetal plasma unbound fraction and CL(fm). Thus, the decrease in fetal unbound fraction of DPHM during gestation could contribute to the fall in CL(fm), and possibly CL(fo). However, over the GA range studied, fetal DPHM free fraction decreased by approximately 47%, whereas CL(fm) fell by approximately 66%. Because fetal unbound fraction and CL(fm) are linearly related, the GA-associated fall in unbound fraction appears to be insufficient to account for the entire decline in CL(fm). In separate studies in pregnant sheep, we observed a approximately 40% fall in weight-normalized Q(um) between 125 and 137 days' gestation. Because CL(fm) for DPHM is similar to that of flow-limited compounds (e.g., ethanol, antipyrine), this decrease in Q(um) may also contribute to the GA-related fall in CL(fm).     

Clearance and disposition of indometacin in chronically instrumented fetal lambs following a 3-day continuous intravenous infusion

Indometacin is used in pregnancy for the treatment of premature labour, but there are limited data on the disposition of the drug in the fetus. In order to elucidate fetal indometacin pharmacokinetics at plasma levels and duration comparable with those occurring with use of the drug for tocolysis in humans, indometacin was administered at doses of 1.9 (low dose, LD; n = 5) or 7.5 (high dose, HD; n = 9) microg min(-1) to steady state over a 3-day period in chronically instrumented fetal lambs. Indometacin concentrations in biological fluid samples were analysed by a sensitive capillary gas chromatography-electron capture detection method. The mean steady-state fetal arterial plasma indometacin concentrations were 68.6+/-16.5 ng mL(-1) in the LD infusion and 230.3+/-28.8 ng mL(-1) in the HD infusion. Indometacin concentrations in amniotic fluid were approximately 10% of those in fetal plasma, and below assay detection limits in tracheal fluid. Total body clearance (TBC) in the LD and HD infusions were not different and the overall mean was 11.3+/-1.2 mL min(-1) kg(-1). In the 11 experiments where paired fetal arterial and umbilical venous samples were collected, the extraction of indometacin across the placenta averaged only 5.2+/-1.1%, indicating low placental permeability to the drug in sheep. However, fetal placental clearance (CLpl) of indometacin (10.0+/-2.5 mL min(-1) kg(-1), n = 10) averaged 115.1+/-41.2% of TBC in these animals and the calculated value for fetal non-placental clearance (0.6+/-2.8 mL min(-1) kg(-1)) was not significantly different from zero. Fetal renal clearance of intact indometacin (3.8+/-1.1 microL min(-1) kg(-1); n = 12) was also very low. However, treatment of fetal urine with glucuronidase indicated the presence of glucuronide conjugates and these comprised 69.9+/-8.2% of the total drug concentration (i.e. intact+conjugated) in urine. Thus, the fetal lamb appears to be able to glucuronidate indometacin, but the contribution of this and other non-placental routes to overall fetal elimination of the drug appear minimal. CLpl of the drug is also low owing to the physicochemical properties of indometacin (high polarity) and the permeability characteristics of the sheep placenta.     

Probenecid-associated alterations in valproic acid pharmacokinetics in rats: can in vivo disposition of valproate glucuronide be predicted from in vitro formation data?

Previous investigations have suggested that probenecid (PRB) alters the in vivo disposition of valproic acid (VPA), perhaps by inhibiting hepatic formation of valproate glucuronide (VG). Because VPA and PRB bind moderately to plasma proteins, protein binding also is a potential locus of interaction. The purpose of this investigation was to determine whether in vitro systems could accurately predict PRB-associated perturbations in the hepatobiliary disposition of VPA and VG in vivo. VPA and PRB were coadministered to rats for 60 min at various infusion rates to examine steady-state VPA disposition. PRB did not alter the binding of VPA in serum or hepatic cytosol. However, PRB decreased the apparent intrinsic clearance of VPA (1.81 +/- 0.58 versus 1.23 +/- 0.23 ml/min; P =.025) by competitively inhibiting VPA elimination. In a separate study, rat hepatic S9 fractions were incubated with VPA (7.2-721 microg/ml) and PRB (0-2850 microg/ml). VG formation (V(max) = 0.80 +/- 0.06 microg/min/mg of protein; K(m) = 173 +/- 28.8 microg/ml) was impaired by PRB in a competitive manner (K(i) = 876 +/- 559 microg/ml), consistent with the in vivo data. Despite inhibition of phase II metabolism of VPA to VG by PRB, the VG biliary excretion rate at similar unbound VPA concentrations in hepatic cytosol was not lower in PRB-treated rats. These results indicate that VG disposition in the presence of PRB cannot be predicted accurately based solely on in vitro inhibition of glucuronidation and emphasize the complexity of processes associated with the hepatobiliary system.     

The contribution of fetal metabolism to the disposition of morphine.

The contribution of fetal metabolism to drug disposition in pregnancy is poorly understood. With maternal administration of morphine, like many drugs, steady-state concentrations in fetal plasma are less than in maternal plasma. The contribution of fetal metabolism to this difference is unknown. Morphine was used as a model drug to test the hypothesis that fetal metabolism contributes significantly to drug clearance by the fetus. Infusions of morphine, morphine-3-beta-glucuronide (M3G), and morphine-6-beta-glucuronide (M6G) were administered to the fetal baboon. Plasma concentrations of drug and metabolite obtained near steady state were measured by high-performance liquid chromatography. During morphine infusion, morphine, M3G, and M6G concentrations rose linearly with dose. M3G concentrations exceeded M6G by 20-fold. Mean +/- S.D. clearances of morphine, M3G, and M6G from the fetus were 69 +/- 17, 2.3 +/- 0.60, and 1.6 +/- 0.24 ml x min(-1), respectively. Clearances seemed to be dose-independent. The mean +/- S.D. fraction of morphine dose metabolized was 32 +/- 5.5%. This converts to a fetal metabolic clearance of 22 +/- 6.5 ml x min(-1). In conclusion, one third of the elimination of morphine from the fetal baboon is attributable to metabolism, one third to passive placental transfer, and one third undefined. Furthermore, there is no evidence for saturation of metabolism. Fetal metabolism is surprisingly high compared with in vitro estimates of metabolism and morphine clearance in human infants. For morphine, fetal drug metabolism accounts for half the difference between fetal and maternal plasma concentrations.     

Kinetics of valproic acid glucuronidation: evidence for in vivo autoactivation.

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (approximately 79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (V(max)(app)), the substrate concentration resulting in 50% of V(max)(app) (S(50)(app)), and Hill coefficient (n) of 2.10 +/- 0.75 micromol/min/kg, 117 +/- 56 microM, and 1.34 +/- 0.14, respectively. Comparable estimates of V(max)(app) (2.63 +/- 0.33 micromol/min/kg), S(50)(app) (118 +/- 53 microM), and n (2.06 +/- 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.     

Disposition of the diastereoisomers quinine and quinidine in the ovine fetus

The disposition of the diastereoisomers quinine and quinidine was investigated in the near-term pregnant ewe. Five sheep were administered quinine and quinidine separately in random order by a combination of bolus and 30-h iv infusion. On a subsequent occasion, four of the five sheep were also administered the two drugs simultaneously. After separate dosage, systemic clearance of quinine tended to be greater than that of quinidine (714 +/- 299 versus 422 +/- 146 mL/min, p = 0.08). Maternal renal clearance exhibited no stereoselectivity and represented less than 2% of total clearance. Simultaneous administration did not alter the disposition of either drug in the mother. After separate dosage, fetal total concentrations (Cf) of quinine and quinidine were substantially lower than maternal total concentrations, as reflected in Cf:Cm ratios of 0.15 +/- 0.06 versus 0.10 +/- 0.08, respectively. Similarly, fetal unbound concentrations (Cfu) were substantially lower than maternal unbound concentrations (Cmu; Cfu/Cmu = 0.46 +/- 0.09 for quinine and 0.23 +/- 0.09 for quinidine). This indicates the presence of fetal elimination of both isomers. Fetal renal clearances of quinine and quinidine were similar (0.34 +/- 0.24 mL/min versus 0.38 +/- 0.24 mL/min) and less than that of endogenous creatinine, indicating the absence of net renal tubular secretion. After simultaneous dosage of quinine and quinidine, Cf:Cm (0.48 +/- 0.24 and 0.31 +/- 0.19, respectively) and Cfu:Cmu (0.73 +/- 0.14 and 0.52 +/- 0.20, respectively) were greater than for separate dosages. Fetal renal clearance of both drugs was unchanged, suggesting that the higher Cfu:Cmu ratios after simultaneous dosage were due to mutual inhibition of the fetal metabolism of these drugs.     

Kinetic analysis of the transport of salicylic acid, a nonsteroidal anti-inflammatory drug, across human placenta.

The aim of this study was to develop a pharmacokinetic model to describe the transplacental transfer of drugs, based on the human placental perfusion study. The maternal and fetal sides of human placentas were perfused with salicylic acid together with antipyrine, a passive diffusion marker. The drug concentration in the placental tissue was determined at the end of perfusion. A compartment model consisting of maternal space, fetal intravascular space, and placental tissue was fitted to the observed concentration profiles of salicylic acid in the maternal and fetal effluents. The developed model could adequately explain the concentration profiles of salicylic acid in the effluents with influx clearances from maternal and fetal perfusates to placental tissue of 0.0407 and 0.0813 ml/min/g cotyledon and efflux rate constants from placental tissue to maternal and fetal perfusates (k2 and k3) of 0.0238 and 0.176 min(-1), respectively. The kinetics of antipyrine was adequately described by assuming rapid equilibrium between fetal perfusate and placental tissue compartments. The influx plasma clearance from the maternal side (K'1) in humans was estimated by taking into account the protein binding. The K'1/k2 value of salicylic acid was 1.07 ml/g cotyledon and was larger than that of antipyrine (0.642 ml/g cotyledon). We evaluated the transplacental transfer kinetics of salicylic acid by human placental perfusion study with various perfusion protocols. Based on the data obtained, we developed a pharmacokinetic model, which should enable us to estimate the influx profile of drugs into umbilical arterial blood from the maternal plasma concentration profile.