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1. The objective was to determine the ontogeny of stereoselective fluoxetine (FX) disposition in postnatal sheep from newborn to adulthood. 2. Catheters were implanted in a carotid artery and jugular vein. FX was administered intravenously, followed by serial arterial blood and cumulative urine collection. The concentrations of

To determine whether there are changes in blood gas and acid-base status with advancing gestation in the fetal lamb, similar to that reported in the human fetus, blood gas, acid-base, and blood metabolite values were measured in 447 control, arterial blood samples from 108 chronically instrumented fetal lambs between 103 and 146 days gestation. With advancing gestation, Po2, pH, O2saturation, and O2content fell significantly, while Pco2and hemoglobin concentration increased. Blood glucose and lactate concentrations were unchanged, although the lactate level increased with decreasing Po2, particularly when below ∼13 mmHg. Multiple linear regression indicated that increasing fetal number was associated with decreased Po2and glucose level and increased pH, HCO3−, base excess, and lactate concentration. Hemoglobin concentration was higher in female than male lambs. Overall, there was a linear relationship between glucose concentration and birth weight. It is concluded that in fetal lambs as in the human fetus, there are changes in blood gas and acid-base status with advancing gestation. This may be due to the decrease in fetal weight-normalized uterine and umbilical blood flows than occurs in these and other species as gestation proceeds. In addition, the reduced birth weight in twin and triplet lambs may be due to hypoglycemia rather than hypoxemia.

We used real-time ultrasound to measure motility and abdominal diameter in fetal lambs at weekly intervals for 30 minutes from 55 days to term (n = 8). Fetal body movement counts/min were relatively constant between 55 and ~90 days and declined progressively thereafter, a relationship best described by piecewise linear regression with 2 elements. The break point in the regression curves averaged 91.9 ± 5.2 days. The relationship between gestational age and abdominal diameter was also best described by piecewise linear regression. The break point of 113.1 ± 3.9 days was significantly greater than the movement break point. There was a significant linear relationship between the movement and abdominal break points, with the latter occurring 21.6 ± 6.6 days later. These results suggest that both fetal motility and growth may decrease in order to lower fetal O2 demands to match the progressive decline in fetal O2 delivery with advancing gestation.

Prenatal serotonin reuptake inhibitor exposure is common and neonatal outcomes vary greatly, often leading to confusion about whether to use or even continue antenatal use of these antidepressants. Importantly, some but not all infants are affected, which raises questions about how maternal drug metabolism contributes to fetal drug exposure. To address this key question, our paper reviews the role of key maternal, fetal, and placental pharmacokinetic, metabolic, and genetic factors that affect the extent of fetal drug exposure. Considering the role of these factors may further our understanding of variables that may assist in optimizing maternal psychopharmacotherapy during pregnancy and neonatal outcomes.

Prenatal exposure to stress and selective serotonin reuptake inhibitors (SSRIs) alter hypothalamic-pituitary-adrenal (HPA) stress reactivity in offspring, however, the effects of combined exposure to HPA activity in human infants is unknown.To examine HPA basal levels and stress responsiveness in 3-month olds with prenatal exposure to SSRIs.Salivary cortisol levels in infants of SSRI treated mothers (n=31, mean exposure 230.2+/-72.2 days) were compared with non-SSRI exposed (n=45) infants in response to a challenge (infant-controlled habituation task) and under basal conditions in the late afternoon/early evening. Mode of feeding, to account for possible postnatal drug exposure via breast milk, as well as measures of pre and postnatal maternal mood, were included as covariates.Lower post-stress cortisol levels were observed in non-SSRI exposed/non-breastfed infants compared with non-SSRI exposed infants who were breastfed at 3 months of age. Stress reactivity patterns among SSRI exposed infants did not differ with mode of feeding. The cortisol reactivity slope (CRS) was significantly lower among non-SSRI exposed non-breastfed infants compared with non-SSRI exposed breastfed infants. Early evening basal cortisol levels were lower in SSRI exposed infants than in non-SSRI exposed infants, controlling for maternal mood and mode of feeding. Postnatal SSRI exposure (infant SSRI drug levels) via breast milk was not associated with stress or basal cortisol levels. Total cortisol, reflected by the AUC measure, did not differ significantly between exposure groups.Prenatal SSRI exposure altered HPA stress response patterns and reduced early evening basal cortisol levels. Stress challenge HPA response differences only became apparent when the moderating effect of method of feeding was accounted for. These findings suggest an early "programming" effect of antenatal maternal mood, prenatal SSRI exposure and postnatal maternal care giving on the HPA system.

Meloxicam (MEL), a cyclooxygenase (COX)-2 inhibitor, decreases prostaglandin production and blocks preterm labor in sheep. The objective of this study is to investigate MEL dosage regimens on COX-1, COX-2, and prostaglandin dehydrogenase (PGDH) expression in ovine intrauterine and fetal tissues. Animals in preterm labor received maternal infusions of saline or MEL at maintained high or graded doses (study 1) or acute graded doses (study 2). MEL blocked preterm labor. In study 1, MEL decreased COX-2 expression in the endometrium, myometrium, and amnion but not placenta or fetal tissues. In study 2, COX-2 expression was unchanged. COX-1/PGDH expression was unaffected. While MEL is an effective tocolytic, reductions in COX-2 protein occurred only with maintained MEL exposure. MEL effects are tissue specific and do not affect COX-1 or PGDH expression. Maternal MEL does not affect fetal COX expression in the sheep, possibly contributing to its lack of fetal side effects.

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.

The purpose of this study was to examine the disposition of diphenhydramine (DPHM) across the ovine blood-brain barrier (BBB). In six adult sheep, we characterized the central nervous system (CNS) pharmacokinetics of DPHM in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) using microdialysis in two experiments. In the first experiment, DPHM was administered via a five-step i.v. infusion (1.5, 5.5, 9.5, 13.5, and 17.5 microg/kg/min; 7 h per step). Average steady-state CNS/total plasma concentration ratios (i.e., [CNS]/[total plasma]) for steps 1 to 5 ranged from 0.4 to 0.5. However, average steady-state [CNS]/[free plasma] ratios ranged from 2 to 3, suggesting active transport of DPHM into the CNS. Plasma protein binding averaged 86.1 +/- 2.3% (mean +/- S.D.) and was not altered with increasing drug dose. Plasma, CSF, and ECF demonstrated biexponential pharmacokinetics with terminal elimination half-lives (t1/2beta) of 10.8 +/- 5.4, 3.6 +/- 1.0, and 5.3 +/- 4.2 h, respectively. The bulk flow of CSF and transport-mediated efflux of DPHM may explain the observed higher CNS clearances. In the second experiment, DPHM was coadministered with propranolol (PRN) to examine its effect on blood-brain CSF and blood-brain ECF DPHM relationships. Plasma total DPHM concentration decreased by 12.8 +/- 6.3% during PRN, whereas ECF and CSF concentrations increased (88.1 +/- 45.4 and 91.6 +/- 34.3%, respectively). This increase may be due to the inhibitory effect of PRN on a transporter-mediated efflux mechanism for DPHM brain elimination.

Aims To compare the disposition of fluoxetine and norfluoxetine enanantiomers in the mother, foetus and infant. Methods Blood from pregnant women taking fluoxetine (n = 9), during pregnancy was sampled in the third trimester and at delivery (maternal and cord venous blood), and from the infants 48 h after delivery. The subset of these women who were breastfeeding, plus additional subjects recruited in the postpartum period, were studied further, and maternal and infant blood, and breast milk was sampled between 6 days and 11 months (n = 23). Drug and metabolite concentrations were measured using gas chromatography/mass spectrometry or liquid chromatography, tandem mass spectrometry. Results There was a high correlation between maternal and foetal (cord blood) fluoxetine and norfluoxetine enantiomers (r2−0.9), the mean foetal/maternal ratios (95% confidence intervals) being 0.91 (0.61, 1.02) and 1.04 (0.93, 1.05), for fluoxetine and norfluoxetine, respectively. In 2 day old infants exposed to the drug in utero, the fluoxetine and norfluoxetine plasma concentrations were the same as in cord blood at delivery. Over the next 2 months, the plasma concentrations in the infants fell progressively. Stereoselective disposition of both the drug and metabolite in the mother, foetus, infant and breast milk was observed. The S : R ratios in the foetus and newborn (∼3) were significantly higher than in the serum (∼2) or breast milk (∼1.9) of the mothers, resulting in greater exposure of the foetus and infants to the biologically active enantiomers, particularly S-norfluoxetine. Conclusions Foetal and infant exposure to fluoxetine and norfluoxetine is enhanced by their stereoselective disposition in the mother, foetus, breast milk and infant. Increased exposure may also result from decreased metabolism of the drug in the foetus and neonate.

Depression during pregnancy is frequently treated with the selective serotonin reuptake inhibitor (SSRI), fluoxetine (FX), commonly known as Prozac (Eli Lilly & Co, Indianapolis, IN). FX potentiates serotoninergic neurotransmission and serotonin has been implicated in the regulation of circadian rhythms. We have therefore investigated the effect of chronic administration of FX on maternal and fetal circadian rhythms in sheep. Following an initial bolus dose of 70 mg FX, an 8-day continuous infusion of FX (n = 11, 98.5 μg/kg • d) was performed. Controls (n = 13) were treated with sterile water vehicle only. Maternal and fetal plasma melatonin and prolactin concentrations were determined every 3 hours for 24 hours and then every 6 hours for 24 hours beginning on the fourth day of infusion. FX treatment did not alter either the basal or circadian rhythms of either maternal or fetal plasma melatonin and prolactin concentrations. Fetal cardiovascular and behavioral state parameters were measured continuously. While the incidence of low-voltage (LV) electrocortical (ECOG) activity was significantly reduced in fetuses in the FX group, there was no effect of FX on the diurnal rhythms in fetal arterial pressure, heart rate, breathing movements, or behavioral state. These results show that maternal FX treatment does not result in significant alterations in maternal and fetal hormonal and behavioral circadian rhythms.

Objective. In this prospective study, we examined biobehavioral responses to acute procedural pain at 2 months of age in infants with prenatal and postnatal selective serotonin reuptake inhibitor (SSRI) medication exposure. Based on previous findings showing reduced pain responses in newborns after prenatal exposure, we hypothesized that altered pain reactivity would also be found at 2 months of age.Methods. Facial action (Neonatal Facial Coding System) and cardiac autonomic reactivity derived from the respiratory activity and heart rate variability (HRV) responses to a painful event (heel-lance) were compared between 3 groups of infants: (1) infants with prenatal SSRI exposure alone (n = 11; fluoxetine, n = 2; paroxetine, n = 9); (2) infants with prenatal and postnatal SSRI (via breast milk) exposure (total n = 30; fluoxetine, n = 6; paroxetine, n = 20; sertraline, n = 4); and (3) control infants (n = 22; nonexposed) during baseline, lance, and recovery periods. Measures of maternal mood and drug levels were also obtained, and Bayley Scales of Infant Development-II were administered at ages 2 and 8 months.Results. Facial action increased in all groups immediately after the lance but was significantly lower in the pSE group during the lance period. HR among infants in the pSE and ppSE groups was significantly lower during recovery. Using measures of HRV and the transfer relationship between heart rate and respiration, exposed infants had a greater return of parasympathetic cardiac modulation in the recovery period, whereas a sustained sympathetic response continued in control infants. Although postnatal exposure via breast milk was extremely low when infant drug levels could be detected in ppSE infants, changes in HR and HRV from lance to recovery were greater compared among infants with levels too low to be quantified. Neither maternal mood nor the presence of clonazepam influenced pain responses.Conclusions. Blunted facial-action responses were observed among infants with prenatal SSRI exposure alone, whereas both prenatal and postnatal exposure was associated with reduced parasympathetic withdrawal and increased parasympathetic cardiac modulation during recovery after an acute noxious event. These findings are consistent with patterns of pain reactivity observed in the newborn period in the same cohort. Given that postnatal exposure via breast milk was extremely low and altered biobehavioral pain reactivity was not associated with levels of maternal reports of depression, these data suggest possible sustained neurobehavioral outcomes beyond the newborn period. This is the first study of pain reactivity in infants with prenatal and postnatal SSRI exposure, and our findings were limited by the lack of a depressed nonmedicated control group, small sample size, and understanding of infant behaviors associated with pain reactivity that could have also have been influenced by prenatal SSRI exposure. The developmental and clinical implications of our findings remain unclear, and the mechanisms that may have altered 5-hydroxytryptamine-mediated pain modulation in infants after SSRI exposure remain to be studied. Treating maternal depression with antidepressants during and after pregnancy and promoting breastfeeding in this setting should remain a key goal for all clinicians. Additional study is needed to understand the long-term effects of prenatal and early postnatal SSRI exposure.

Depression during pregnancy is frequently treated with the selective serotonin reuptake inhibitor, fluoxetine (FX). FX increases serotonergic neurotransmission and serotonin plays a role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have therefore investigated the effect of chronic administration of FX to the pregnant ewe on the maternal and fetal HPA axes. Nineteen late-gestation sheep were surgically prepared for chronic study of the fetus. FX (n = 7, 98.5 microg/kg/d) or sterile water (control, n = 8) was administered to the ewe for 8 d by constant rate i.v. infusion with an initial FX bolus dose of 70 mg. Maternal and fetal plasma ACTH and cortisol concentrations were determined at 0700 h each day. Maternal plasma ACTH concentrations fell on infusion d 2, but no changes were observed in maternal plasma cortisol concentrations. Fetal plasma ACTH concentrations increased on infusion d 7, and fetal plasma cortisol concentrations increased on infusion d 6, 7, and 8 in the FX group. In addition, the regression coefficient for the relationship between fetal ACTH and cortisol levels was significantly greater in the FX group compared with the control group. Thus, maternal FX treatment increased fetal plasma cortisol concentration. These results are of particular interest in the context that exposure of the fetus to excess glucocorticoids at critical windows during development has been shown to increase the risk of poor health outcomes in later life.

Background: Selective serotonin reuptake inhibitor antidepressants (SSRIs) and benzodiazepines are frequently used to treat maternal depression and anxiety disorders during pregnancy. Recent reports suggest that prenatal SSRI exposure is associated with a neonatal discontinuation syndrome. It remains unclear whether these symptoms are directly related to SSRI exposure alone or are due to concurrent pharmacologic factors. Also, this study explores relationships between neonatal outcomes and medication levels during pregnancy, at delivery, and in the newborn period. Method: This study sought to compare newborn behavior following second and third trimester exposure to either single-agent SSRIs (group 1) or SSRIs combined with clonazepam (group 2). A prospective cohort of mothers and their infants (N = 46) who had received SSRI medication alone or in combination with clonazepam were studied from June 1996 through June 2000 and compared with a nonexposed control group (N = 23). Infants were assessed in the newborn period for signs suggestive of a discontinuation syndrome. Maternal drug levels were measured during the pregnancy and at delivery. Infant drug levels from cord blood and at day 2 of life were also obtained. Results: Overall, 30% of the exposed infants (groups 1 and 2, N = 14) showed symptoms of transient poor neonatal adaptation compared with 9% (N = 2) of control infants. In group 1, 25% had symptoms (fluoxetine N = 3; paroxetine N = 3; sertraline N = 1) and in group 2. 39% of infants had symptoms (paroxetine with clonazepam, N = 7). Symptoms were typically mild respiratory distress and, less commonly, hypotonia. Symptoms were self limited and not associated with other neonatal conditions. When paroxetine was combined with clonazepam, infants with symptoms had significantly elevated paroxetine levels when compared with similarly exposed infants without symptoms (p

We examined the stereoselective disposition of fluoxetine (FX) and its metabolite norfluoxetine (NFX) in five pregnant sheep. Racemic FX was administered i.v. to the ewe (50 mg) and the fetus (10 mg) on separate occasions. Maternal and fetal blood, maternal urine, and fetal amniotic and tracheal fluid samples were collected for 72 h. FX and NFX isomers were quantified by gas chromatography-mass spectrometry. They rapidly crossed the placenta [maternal to fetal area under the plasma concentration versus time curve (AUC) ratios 0.59 and 0.65, respectively]. There was significant FX stereoselectivity with S/R FX AUC ratios averaging 1.65 +/- 0.33 and 1.73 +/- 0.29 in ewe and fetus, respectively, after maternal dosing. The maternal clearance and volume of distribution were also higher for (R)-fluoxetine than for (S)-fluoxetine. FX, NFX, and their glucuronides were present in maternal urine but accounted for only 3.4% of maternal drug elimination. In contrast, NFX was not detected in the fetus after fetal FX administration, which is consistent with the absence of measurable fetal nonplacental clearance of the drug and the lack of NFX formation in fetal hepatic microsomal incubations. There was also no fetal production of FX and NFX glucuronides in vivo. Both FX and NFX were extensively and stereoselectively bound in maternal and fetal plasma, with the free fraction S/R FX ratio averaging 0.46 +/- 0.06 and 0.58 +/- 0.10 in ewe and fetus, respectively. Thus, FX exhibits extensive stereoselective disposition, which is likely due to differential plasma protein binding of the FX isomers, and there is no detectable fetal formation of NFX, FX, and NFX glucuronides.

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) μg 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 ∼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 μL 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.

Selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines are frequently used to treat maternal depression during pregnancy, however the effect of increased serotonin (5HT) and gamma-amino-butyric acid (GABA) agonists in the fetal human brain remains unknown. 5HT and GABA are active during fetal neurologic growth and play early roles in pain modulation, therefore, if prolonged prenatal exposure alters neurodevelopment this may become evident in altered neonatal pain responses. To examine biologic and behavioral effects of prenatal exposure, neonatal responses to acute pain (phenylketonuria heel lance) in infants with prolonged prenatal exposure were examined. Facial action (Neonatal Facial Coding System) and cardiac autonomic reactivity derived from the relationship between respiratory activity and short term variations of heart rate (HRV) were compared between 22 infants with SSRI exposure (SE) [fluoxetine (n = 7), paroxetine (n = 11), sertraline (n = 4)]; 16 infants exposed to SSRIs and clonazepam (SE+) [paroxetine (n = 14), fluoxetine (n = 2)]; and 23 nonexposed infants during baseline, lance, and recovery periods of a heel lance. Length of maternal SSRI use did not vary significantly between exposure groups-[mean (range)] SE:SE+ 183 (31-281):141 (54-282) d (p > 0.05). Infants exposed to SE and SE+ displayed significantly less facial activity to heel lance than control infants. Mean HR increased with lance, but was significantly lower in SE infants during recovery. Using measures of HRV and the transfer relationship between heart rate and respiration, SSRI infants had a greater return of parasympathetic cardiac modulation in the recovery period, whereas a sustained sympathetic response continued in the control group. Prolonged prenatal SSRI exposure appears to be associated with reduced behavioral pain responses and increased parasympathetic cardiac modulation in recovery following an acute neonatal noxious event. Possible 5HT-mediated pain inhibition, pharmacologic factors and the developmental course remain to be studied.

The effects of 15-50% fetal hemorrhage (at approximately 1%/min) were studied in 13 pregnant ewes at 130-135 days of gestation for up to 5 days posthemorrhage. The upper limit of acute blood loss appears to be approximately 45%, and the rate of restoration of blood volume decreases with the severity of hemorrhage, particularly with hemorrhage > 30-40%. The restoration of fetal blood volume was due primarily to the restoration of plasma volume; in the animals subject to 40-45% blood loss (n = 9), red cell mass was still only 69.1 +/- 3.9% of the prehemorrhage value at day 5 posthemorrhage. There appear to be two phases in the restoration of plasma: 1) plasma volume was restored by 2 h posthemorrhage and 2) the restoration of plasma protein mass occurred primarily from 2 to 24 h. There was a significant correlation between blood volume and plasma protein mass. However, the regression line for the posthemorrhage days was shifted significantly upward in relation to that for the hemorrhage day because of a significant rise of plasma protein concentration. This may be important for the maintenance of blood volume after hemorrhage. Finally, fetal growth rate was determined by comparing fetal weight estimated in utero (from blood volume) with birth weight in 12 nonhemorrhaged control fetuses and in the 9 fetuses subject to 40-45% hemorrhage. The average rate of growth per day was 1.57 +/- 0.34% and -1.82 +/- 1.02%, respectively. The latter value is not significantly different from zero, suggesting that the acute blood loss impaired fetal growth.

An improved method for the quantitative determination of valproic acid (VPA) and sixteen of its metabolites has been developed using gas chromatography-mass spectrometry with selected-ion monitoring. The method is applicable to serum or urine and all metabolites are measured in a single chromatographic run of 29.5 min. Ions selected for quantitative purposes were the characteristic [M-57]+ ions of the tert.-butyldimethylsilyl (tBDMS) derivatives. The method utilizes heptadeuterated VPA as well as six heptadeuterated metabolites as internal standards [i.e. 2-[2H7]propyl-2-pentenoic acid (2-ene[2H7]VPA), 2-[2H7]propyl-4-pentenoic acid (4-ene[2H7]VPA), 2-[2H7]propyl-3-oxopentanoic acid (3-keto[2H7]VPA), 2-[2H7]propyl-4-oxopentanoic acid (4-keto[2H7]VPA), 2-[2H7]propyl-3-hydroxypentanoic acid (3-OH[2H7]VPA), 2-[2H7]propyl-5-hydroxypentanoic acid (5-OH[2H7]VPA)]. The method demonstrates very good accuracy and precision over a large range of concentrations for VPA and all metabolites measured in both human and sheep biological fluids. The assay was applied to the analysis of VPA and metabolites in serum and urine samples collected from three non-pregnant ewes following intravenous bolus administration of a mixture of VPA and [13C4]VPA. Sheep were observed to produce measurable quantities of the majority of metabolites found in humans, with the notable exception of the di-unsaturated compounds (i.e. 2,3'-diene VPA and 2,4-diene VPA). The pharmacokinetics and metabolism of VPA and [13C4]VPA appear to be equivalent in the sheep model. The elimination half-life of VPA and [13C4]VPA in the ewe were estimated to be approximately 3.5 +/- 0.4 and 3.2 +/- 0.4 h, respectively.

Background There have been contradictory reports on the risks of persistent pulmonary hypertension (PPHN) in infants exposed to SSRIs in utero. However, there has been no assessment of fetal pulmonary arterial dynamics in such pregnancies. Aims and subjects To measure fetal right pulmonary artery (RPA) variables using Doppler ultrasound at 36 weeks gestation in fetuses of mothers taking SSRI antidepressants (n = 23) and in a control, normal pregnancy group (n = 35). Outcome measures At 36 weeks gestation, Doppler ultrasound estimates of Pulsatility Index (PI), Resistance Index (RI), vessel diameter, peak systolic velocity, mean velocity and volume flow were obtained from the fetal right pulmonary artery in a morning session (~ 0830), before the SSRI mothers took their daily drug dose and in an afternoon session (~ 1300). Venous blood was drawn at 5 time points across the day (~ 08:30 AM, ~ 10:30 AM, ~ 13:00 PM, ~ 13:45 PM, and ~ 15:00 PM) from the SSRI treated mothers for measurement of plasma SSRI concentration using high performance liquid chromatography tandem mass spectrometry. Results There were no differences in the RPA Doppler measures between the control and SSRI-exposed fetuses. However 8 of the 23 latter fetuses experience transient respiratory difficulties at birth and, in these RPA flow was significantly higher than in the SSR-exposed fetuses without respiratory problems. There were, however, no differences in RPA PI and RI between the 2 groups. Conclusions In SSRI-exposed infants with transient postnatal respiratory difficulties, fetal RPA flow in increased, likely due to partial constriction of the ductus arteriosus. However, this was not associated with PPHN.

Prenatal selective serotonin reuptake inhibitor (SSRI) exposure increases the risk for adverse neonatal behavioral outcomes; although it is unknown whether altered brain function is present before birth. We investigated fetal vascular and heart rate changes at 36-wk gestation in SSRI-treated women with mood disorders (n = 29) [exposed (EXP)] and controls (n = 45) [non-EXP (NEXP)]. Fetal middle cerebral artery (MCA) flow parameters and heart rate characteristics were obtained during pre-SSRI dose morning and postdose afternoon sessions. Maternal mood and cord Hb and hematocrit were measured. Basal fetal heart rate (fHR) did not differ between groups or across the day. The fHR short- and long-term variations, accelerations, and duration of high variability episodes remained lower and did not change across the day in EXP, whereas all increased significantly in NEXP. In both groups, MCA flow velocity and volume flow increased significantly across the day. EXP MCA pulsatility index was significantly lower, as was MCA cross-sectional area. EXP cord Hb and hematocrit were significantly increased. Prenatal SSRI exposure reduced fetal MCA flow resistance and fHR variability, before and after an SSRI dose, controlling for maternal mood. These changes and the SSRI-related increased red cell indices suggest possible fetal hypoxia.

UDP glucuronosyltransferases (UGTs) are important for the metabolism of many drugs. To understand the ontogeny of these enzymes in sheep ( Ovis aries ), knowledge of their expression levels at different developmental stages is important. cDNA sequences of six UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) were cloned in sheep liver by rapid amplification of cDNA ends. The mRNA and protein levels of the enzymes in sheep liver (adult n = 4; newborn n = 3; fetus n = 3) were determined using PCR and Western blots. mRNA levels in adult were very high followed by newborn and then fetus. However, no protein bands were detected on the Western blots of sheep hepatic microsomal proteins using several different antibodies against human UGTs. The effect of antenatal glucocorticoid administration was also studied by infusion of cortisol (0.45 mg/h; 80 h) to another group of fetuses (n = 5). This did not result in any significant changes in fetal mRNA levels. However, the correlation observed between the UGT enzymes and hepatocyte nuclear factor 4α (HNF4α) suggests a possible regulatory role for this transcription factor in sheep. We postulate that fetal and newborn lambs may have a reduced ability to metabolize drugs that are substrates of these enzymes.

The objective was to compare gestation length in chronically instrumented (laboratory) pregnant sheep (n = 131) and in the breeding flock (n = 476) that provided the experimental sheep. In the breeding flock, gestation length was normally distributed and varied between 141 and 151 days (mean = 147 ± 0.1 days). In the laboratory sheep, gestation length varied between 128 and 151 days (mean = 142 ± 1 day), and was bimodal, with 35.9% delivering preterm (

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)

Studies were conducted to characterize the pharmacokinetics of diphenhydramine in nonpregnant ewes after iv administration of 25-, 50-, 100-, and 200-mg doses of diphenhydramine hydrochloride on a crossover basis. Plasma drug concentration versus time data exhibited multiexponential characteristics. The initial distribution half-life increased from 5 to 9 min and the elimination half-life from 34 to 68 min as the dose was increased. There was also an increase in the volume of distribution (from 3 to 6 L/kg) with increasing dose. The elimination half-life and the volume of distribution after a 200-mg dose were significantly greater than after a 25-mg dose. There was, however, a linear increase in AUC0 infinity as dose was increased. The average total body clearance (approximately 5 L/h/kg) remained unchanged regardless of dose. The free fraction of diphenhydramine determined by equilibrium dialysis averaged 0.229 +/- 0.080, and the extent of drug binding to plasma protein was independent of the drug concentrations encountered (30-780 ng/mL) in the nonpregnant sheep in vivo. Concentration-independent binding of the drug was also confirmed by in vitro binding studies over the drug concentration range 10-2000 ng/mL. Therefore, it appears that changes in the volume of distribution are likely to be a result of changes in tissue uptake or binding of the drug as a function of dose.

The central nervous system (CNS) pharmacokinetics of the H(1) receptor antagonist diphenhydramine (DPHM) were studied in 100- and 120-day-old fetuses, 10- and 30-day-old newborn lambs, and adult sheep using in vivo microdialysis. DPHM was administered i.v. at five infusion rates, with each step lasting 7 h. In all ages, cerebrospinal fluid (CSF) and extracellular fluid (ECF) concentrations were very similar to each other, which suggests that DPHM between these two compartments is transferred by passive diffusion. In addition, the brain-to-plasma concentration ratios were >or=3 in all age groups, suggesting the existence of a transport process for DPHM into the brain. Both brain and plasma DPHM concentrations increased in a linear fashion over the dose range studied. However, the ECF/unbound plasma and CSF/unbound plasma DPHM concentration ratios were significantly higher in the fetus and lambs (approximately 5 to 6) than in the adult (approximately 3). The factors f(CSF) and f(ECF), the ratios of DPHM areas under the curves (AUCs) in CSF and ECF to the plasma DPHM AUC, respectively, decreased with age, indicating that DPHM is more efficiently removed from the brain with increasing age. The extent of plasma protein binding of the drug increased with age. This study provides evidence for a transporter-mediated mechanism for the influx of DPHM into the brain and also for an efflux transporter for the drug, whose activity increases with age. Moreover, the higher brain DPHM levels in the fetus and lamb compared with the adult may explain the greater CNS effects of the drug at these ages.

Objective Preterm birth occurs in 5% to 10% of all pregnancies and is associated with considerable neonatal mortality and morbidity. Effective and safe drugs to prevent preterm labor are not currently available. We have hypothesized that the nonsteroidal anti-inflammatory drug meloxicam, a more selective cyclooxygenase-2 inhibitor will successfully inhibit labor but avoid the complications associated with inhibition of cyclooxygenase-1. Study design Preterm labor was induced in chronically catheterized sheep by RU486 administration. Animals were then randomized to receive maternal infusions of saline (n = 5) or meloxicam (n = 4) for 48 hours or until delivery when the animals were killed and tissues and blood samples collected. Results Maternal infusion of meloxicam inhibited uterine contractions, increasing contraction duration, and attenuating frequency and amplitude. Saline-treated animals progressed to delivery. Administration of meloxicam was not associated with any change in fetal or maternal blood gas status, osmolality, arterial pressure, heart rate, or fetal blood flows. Conclusion Meloxicam may represent a potentially safe and effective tocolytic agent.

Women are at greatest risk of suffering from depression during the childbearing years and thus may either become pregnant while taking an antidepressant or may require a prescription for one during pregnancy. The antidepressant fluoxetine (FX) is a selective serotonin reuptake inhibitor (SSRI), which increases serotonin neurotransmission. Serotonin is involved in the regulation of a variety of physiological systems, including the sleep–wake cycle, circadian rhythms and the hypothalamic–pituitary–adrenal axis. Each of these systems also plays an important role in fetal development. Compared with other antidepressant drugs, the SSRIs, such as FX, have fewer side effects. Because of this, they are now frequently prescribed, especially during pregnancy. Clinical studies suggest poor neonatal outcome after exposure to FX in utero. Recent studies in the sheep fetus describe the physiological effects of in utero exposure to FX with an 8 day infusion during late gestation in the sheep. This is a useful model for determining the effects of FX on fetal physiology. The fetus can be studied for weeks in its normal intrauterine environment with serial sampling of blood, thus permitting detailed studies of drug disposition in both mother and fetus combined with monitoring of fetal behavioural state and cardiovascular function. Fluoxetine causes an acute increase in plasma serotonin levels, leading to a transient reduction in uterine blood flow. This, in turn, reduces the delivery of oxygen and nutrients to the fetus, thereby presenting a mechanism for reducing growth and/or eliciting preterm delivery. Moreover, because FX crosses the placenta, the fetus is exposed directly to FX, as well as to the effects of the drug on the mother. Fluoxetine increases high-voltage/non-rapid eye movement behavioural state in the fetus after both acute and chronic exposure and, thus, may interfere with normal fetal neurodevelopment. Fluoxetine also alters hypothalamic function in the adult and increases the magnitude of the prepartum rise in fetal cortisol concentrations in sheep. Fetal FX exposure does not alter fetal circadian rhythms in melatonin or prolactin. Studies of the effects of FX exposure on fetal development in the sheep are important in defining possible physiological mechanisms that explain human clinical studies of birth outcomes after FX exposure. To date, there have been insufficient longer-term follow-up studies in any precocial species of offspring exposed to SSRIs in utero. Thus, further investigation of the long-term consequences of in utero exposure to FX and other SSRIs, as well as the mechanisms involved, are required for a complete understanding of the impact of these agents on development. This should involve studies in both humans and appropriate animal models.

To examine fetal (FHR) and neonatal heart rate patterns following use of common oral antihypertensives in pregnancy.A systematic review of randomized controlled trials (RCTs), observational studies (N/= 6 women), and animal studies. Data were abstracted (two reviewers) to determine relative risk (RR) (or risk difference (RD) for low event rates) and 95% CI.Eighteen RCTs (1858 women), one controlled observational study (N = 22), and seven case series (N = 117) were reviewed. Most hypertension was pregnancy-induced (N = 14 studies). The FHR was assessed by cardiotocogram (CTG) (N = 17 studies (visual interpretation); 1 study (computerized CTG), or umbilical artery velocimetry (N = 4). Four studies examined neonatal heart rate. In placebo-controlled RCTs (N = 192 women), adverse FHR effects did not differ between groups [9/101 (drugs) vs. 7/91 (placebo); RD 0.02, 95% CI (- 0.06, 0.11); chi2 = 1.02]. In six drug vs. drug RCTs (295 women), adverse FHR effects did not differ between groups [29/144 (methyldopa) vs. 42/151 (other drugs); RR 0.72, 95% CI (0.49, 1.07); chi2 = 0.69]. In one labetalol vs. placebo trial, neonatal bradycardia did not differ between groups [4/70 (labetalol) vs. 4/74 (placebo); OR 1.06, 95% CI (0.26, 4.39)], while in three drug vs. drug RCTs, neonatal bradycardia was not observed (0/24 vs. 0/26).Available data are inadequate to conclude whether oral methyldopa, labetalol, nifedipine, or hydralazine adversely affect fetal or neonatal heart rate and pattern. Until definitive data are available, FHR changes cannot be reliably attributed to drug effect, but may be due to progression of the underlying maternal or placental disease.

Hypoxemia is known to induce various physiological changes which can result in alteration in drug pharmacokinetics. To examine the effect of acute moderate hypoxemia on metoclopramide (MCP) pharmacokinetics, a continuous 14-hour infusion of MCP during a normoxemic, hypoxemic and subsequent normoxemic period was conducted in eight adult sheep. Arterial blood and urine samples were collected to examine the effects on the pharmacokinetics of MCP and its deethylated metabolites. MCP and its mono- and di-deethylated metabolites were quantitated using a GC/MS method. Steady-state concentrations of MCP were achieved in each of the three periods. During hypoxemia, MCP plasma steady-state concentration increased significantly from 50.72 +/- 1.06 to 63.62 +/- 1.79 ng/mL, and later decreased to 55.83 +/- 1.15 ng/mL during the post-hypoxemic recovery period. Total body clearance (CL(TB)) of MCP was significantly decreased from 274.2 +/- 48.0 L/h to 205.40 +/- 28.2 L/h during hypoxemia, and later restored to 245.8 +/- 44.2 L/h during the post-hypoxemic period. Plasma mono-deethylated MCP concentration (32.78 +/- 1.73 ng/mL) also increased, compared to the control group (21.20 +/- 1.39 ng/mL), during hypoxemia and subsequent normoxemic period. Renal excretion of MCP and its metabolites was also decreased during hypoxemia, while urine flow was increased with a concomitant decrease in urine osmolality. Thus, the results indicate that acute moderate hypoxemia affects MCP pharmacokinetics.

Clinical depression is diagnosed in 5-15% of women during pregnancy, increasing the risk of negative outcomes. Fluoxetine (FX), a selective serotonin reuptake inhibitor, is prescribed during pregnancy. In adults, FX alters sleep patterns with single doses decreasing total sleep time and rapid eye movement sleep. The effects of FX on sleep in the fetus are unknown. However, 5-hydroxytryptophan, the precursor of serotonin, has been reported to prolong high-voltage (HV) electrocortical (ECoG) activity and increase the incidence of fetal breathing movements (FBM) in the sheep fetus. We hypothesize that FX exposure will decrease the incidence of LV ECoG in the fetus. Twenty-one pregnant sheep were surgically prepared for chronic study of blood gases, ECoG activity, eye movements and FBM. After 3 days of recovery, ewes received a 70-mg bolus i.v. infusion of FX or sterile water followed by continuous infusion at a rate of 0.036 mg/min for 8 days. The incidence of low-voltage (LV) ECoG decreased from 54+/-4% on the preinfusion day to 45+/-5% on infusion day 1 in the FX group and remained decreased throughout the infusion period. In addition, the incidence of both eye movements and FBM was decreased on infusion day 1 compared to preinfusion day in the FX group. HV ECoG increased from 39+/-3% on preinfusion day to 68+/-14% on FX infusion day 1 and remained elevated throughout the infusion period. These data show that maternal FX administration alters fetal behavioural state.

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([2H10]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [2H10]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-microliter aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [2H10]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [2H10]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [2H10]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [2H10]DPMA in biological samples obtained from sheep (e.g. plasma and urine).

Cytochrome P450 (CYP) enzymes are important for the metabolism of many drugs. While there is information on their identity and ontogeny in humans and rodents, similar data in sheep are lacking. In the present study, cDNA sequences of several CYP enzymes (CYP2A6, CYP2C19, CYP2D6) were cloned by rapid amplification of cDNA ends. In adult, newborn and fetal sheep the mRNA and protein levels of these CYPs and the regulatory factor, hepatic nuclear factor 4α (HNF4α) were determined in liver samples using real-time PCR and western blotting. The effect of antenatal glucocorticoid on these enzymes was also studied by i.v. infusion of cortisol (0.45 mg h–1; 80 h) to another group of fetuses. The mRNA and protein levels of the CYPs and HNF4α were low or absent in the fetus, followed by increasing levels in the newborn and adult. Fetal cortisol administration significantly increased the mRNA and protein levels of CYP2D6. Moreover, the correlation observed between the CYP and HNF4α mRNA levels suggests a possible regulatory role for this transcription factor. The findings suggest that fetal and newborn lambs have a low ability to metabolise drugs that are substrates of these enzymes, and that this ability increases with advancing postnatal age, similar to the situation in humans.

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.

This report describes both the synthesis of a stable isotope analog of the H1 receptor antagonist diphenhydramine (DPHM), and the simultaneous quantitation of DPHM and a deuterated stable isotope analog of DPHM, viz. (2H10)DPHM in biological fluids from the chronically instrumented pregnant ewe. (2H10)DPHM was synthesized and purified, and both its structure and purity were verified. Biological samples were prepared for analysis using liquid-liquid extraction prior to capillary gas chromatography/mass spectrometry. The method employed electron impact ionization with selective ion monitoring of ions with m/z 165 for DPHM and m/z 173 for (2H10)DPHM. The minimal quantifiable concentration of DPHM and (2H10)DPHM from a 1.0 ml sample was 2.0 ng ml-1 in fetal and maternal plasma, fetal tracheal fluid and amniotic fluid. The method was validated from 2.0 ng ml-1 to 200.0 ng ml-1 for both DPHM and (2H10)DPHM in plasma, fetal tracheal fluid and amniotic fluid. Differences in the disposition between DPHM and (2H10)DPHM were not apparent during a control experiment in which both labeled and unlabeled DPHM were administered to a chronically instrumented fetal lamb. This method provides the required sensitivity and selectivity for the simultaneous quantitation of unlabeled and labeled DPHM during pharmacokinetic experiments conducted in near-term pregnant sheep.

A chiral high-performance liquid chromatographic assay for the separation of the four stereoisomers of labetalol, an antihypertensive, in biological fluids has been developed. Baseline separation of the isomers was achieved using an alpha 1-acid glycoprotein stationary phase. No interference from endogenous substances was observed following extraction from various biological fluids obtained from pregnant (ewe and fetus) and non-pregnant sheep. The concentration of the individual isomers of labetalol was determined by first measuring the total concentration of racemic labetalol obtained from an achiral assay followed by reassay of each sample by the chiral method after which, by using the estimate of the percentage of each individual isomer, the individual concentration of each of the four isomers was determined. The mobile phase was 0.02 M phosphate buffer containing 0.015 M tetrabutylammonium phosphate. The pH of the mobile phase was adjusted to 7.10. The detector was set at an excitation wavelength of 230 nm and emission wavelength of 400 nm to monitor the nascent fluorescence intensity of the isomers of labetalol. The limit of detection of the individual isomers was 0.15 ng (0.6 ng of injected racemic labetalol). The assay was linear over the range 0.6-15.0 ng of labetalol (injected) with the intra- and inter-day mean coefficients of variation being less than 9.0 and 6.0%, respectively. Application of the assay in the study of pharmacokinetics of the stereoisomers of labetalol in sheep following administration of racemic labetalol has been demonstrated.

Pharmacokinetic studies of the histamine H1-receptor antagonist diphenhydramine were conducted in eight chronically instrumented pregnant sheep at 126-138 days of gestation. Diphenhydramine was administered by simultaneous intravenous bolus injection and infusion to steady state given 48 h apart, to the ewe and the fetus on separate occasions. Average steady-state drug concentration in plasma after maternal infusion was 212.1 +/- 67.8 ng/mL in the mother and 36.3 +/- 14.4 ng/mL in the fetus, resulting in a fetal-to-maternal concentration ratio of 0.19 +/- 0.10. Following fetal infusions, maternal and fetal steady-state drug concentrations were 31.1 +/- 11.6 and 447.6 +/- 185.2 ng/mL, respectively. The free fraction of diphenhydramine determined in the fetus (0.277 +/- 0.087) was significantly greater than that in the mother (0.141 +/- 0.079). Transplacental and nonplacental clearances were calculated at steady state according to a general two-compartment open model, with drug elimination occurring from both compartments. The total fetal clearance (472.7 +/- 215.7 mL/min) was relatively small compared with the total maternal clearance (3426.1 +/- 905.8 mL/min). The transplacental clearance from fetus to mother (264.4 +/- 138.7 mL/min) was approximately threefold higher than that from mother to fetus (82.4 +/- 40.5 mL/min). Maternal nonplacental clearance (3343.8 +/- 890.7 mL/min) accounted for 97.8 +/- 1.1% of the maternal total clearance, whereas fetal nonplacental clearance (208.4 +/- 80.4 mL/min) accounted for 45.1 +/- 4.7% of the fetal total clearance. It is concluded that in the fetus both the transplacental and nonplacental pathways are important for drug elimination.

A rapid and sensitive microbore high-performance liquid chromatographic (HPLC) assay is reported for the quantitation of labetalol, an anti-hypertensive agent, in small volumes (250 microliters) of biological fluids (viz., maternal plasma, fetal plasma, amniotic fluid and fetal tracheal fluid) obtained from the chronically instrumented pregnant sheep. Labetalol was extracted from the samples using ethyl acetate and then partitioned into dilute phosphoric acid. Chromatography was performed on a microbore HPLC system using a 2.1 mm I.D. C18 column and detection was accomplished by a low-dispersion fluorescence detector designed for trace analysis. The drug was well separated from endogenous substances in all biological fluids sampled. The calibration curves were linear for all fluids over the range of study with mean coefficients of variation consistently below 5%. Quantitation was possible down to approximately 30 pg of labetalol injected (approximately 1.6 ng/ml in plasma using 250 microliters).

A sensitive and selective gas chromatographic assay method employing splitless injection, fused-silica capillary columns and electron-capture detection is reported for the quantitation of the tocolytic drug, ritodrine, in a variety of biological fluids obtained from the pregnant ewe and fetus. This method has improved sensitivity and selectivity over previously published assay procedures. A 25 m x 0.31 mm I.D., cross-linked 5% phenylmethylsilicone, fused-silica capillary column was employed for all analyses. Linearity of response was observed over the range 2.5-75 ng of ritodrine base per 0.05-0.5 ml of biological fluid, representing approximately 1-75 pg at the detector. The coefficient of variation was less than 10% over the range 2.5-75 ng of added ritodrine. The minimum quantifiable amount is approximately 2.5 ng from a 0.5-ml biological fluid sample. Applicability of this method to biological fluids, obtained from ovine subjects, is demonstrated by the analysis of samples obtained during the course of ritodrine placental transfer studies.

The present study used actigraphy to monitor rest–activity cycles in lambs. We employed an Actiwatch Activity Monitor, which was secured on the lamb’s neck in 13 term lambs and six preterm lambs. Activity measurements began on the day of delivery and lasted for 7.3 ± 0.7 days. All lambs exhibited bouts of activity, lasting from ~2 to 60 min, separated by periods of inactivity of about equal duration. There was a progressive increase in the frequency and intensity of activity bouts with age, and a decrease in duration. In relation to postnatal age, preterm lambs had a significantly lower frequency and intensity of activity bouts compared with term lambs and significantly longer mean active bout duration. However, in relation to post-conceptual age, preterm animals were less active at birth, but thereafter the trajectory for activity development was steeper compared with the term lambs. These differences between term and preterm lambs may be due to several factors including differences in: (1) the lengths of time the two groups spent in utero and as neonates as a proportion of the perinatal period, which could influence the rate of muscle and bone growth; (2) prenatal and postnatal hormonal profiles; and (3) maternal care. We also found differences in postnatal motility in male and female lambs, with the trajectory of activity increasing in males at Days 4–5, which could be due, in part at least, to sex differences in both prenatal and postnatal hormonal profiles.

The kinetics of metoclopramide and the effects of haemodialysis on metoclopramide kinetics were examined in eight uraemic subjects 1 h and 24 h prior to the onset of dialysis. In spite of the relatively minor contribution of renal clearance to total body clearance in normals, metoclopramide kinetics were substantially altered in uraemia. The total body clearance was decreased by 2-4 fold, terminal elimination half-life proportionately increased, while the volume of distribution appeared to be unaffected compared with that previously demonstrated in normal healthy subjects. Haemodialysis does not appear to be effective in removing metoclopramide from the body and metoclopramide clearance subsequent to dialysis is unaltered. The kinetic parameters in the uraemic subjects are not significantly different between drug administrations 1 or 24 h prior to the time of onset of haemodialysis. Following kidney transplantation, in one subject, there appeared to be a rapid return to apparently normal kinetics from the uraemic state.

The pharmacokinetics of metoclopramide was studied in chronically instrumented pregnant and nonpregnant sheep. Metoclopramide was administered to the ewe by intravenous bolus injections (on a crossover basis) of 10, 20, and 40 mg, with an additional 80‐mg dose to the nonpregnant animals. Transfer of the drug to the fetus was rapid with significant concentrations in fetal plasma 1 min after maternal dosing. The ratio of fetal‐to‐maternal area under the plasma concentration–time curves averaged 0.74, indicating significant fetal exposure to the drug. Maternal metoclopramide administration resulted in minimal fetal effects, with no change in arterial pressure, heart rate, or arterial pH or Pco2, and only a small (∼ 1.8 mm Hg) transient decline in Po2. Plasma concentrations in maternal and fetal plasma in most animals were best described by a biexponential equation with rapid distribution and elimination phases. The terminal elimination half‐lives in maternal and fetal plasma averaged 71.3 and 86.8 min, respectively, with fetal half‐life being significantly longer. The number of fetuses present had no consistent effects on either maternal or fetal pharmacokinetic parameters. Total body clearance and volume of distribution averaged 3.5 L/h/kg and 5.8 L/kg, respectively, in the pregnant ewe, and 4.5 L/h/kg and 6.9 L/kg, respectively, in the nonpregnant animals. The terminal elimination half-life in the nonpregnant ewes averaged 67.5 min. Pharmacokinetic parameters were compared in the pregnant and nonpregnant ewes at the 10-, 20-, and 40-mg doses, and no significant differences were observed in the distribution or elimination rate constants, elimination half-life, or volume of distribution. Total body clearance in the nonpregnant animals, however, was 22% higher than that calculated in the pregnant animals. Metoclopramide was observed to follow linear or dose-independent kinetics in both the pregnant and nonpregnant ewe over the 4–8-fold dose range studied.

To examine the effects of vasopressin on fetal oxygenation the hormone was infused intravenously for 1 h (1.4–3.5 mU∙min−1∙kg fetal weight−1) to chronically catheterized fetal lambs in utero (113–137 days gestation). Arterial pressure rose (48.3 to 59.6 mmHg) (1 mmHg = 133.322 Pa) and heart rate fell (185.3 to 141.0 beats/min) during the infusion. There was a significant increase in fetal arterial [Formula: see text] (20.0 to 23. 1 mmHg) and significant declines in pH (7.414 to 7.381) and base excess. Umbilical blood flow rose, and the percentage increase in flow (23%) was identical to the proportional rise in arterial pressure. Accompanying the rise in umbilical blood flow was a rise in umbilical oxygen delivery. But as there was no change in fetal oxygen consumption, fractional oxygen extraction by the fetus fell significantly (0.31 to 0.25). These data indicate that the vasopressin-induced rise in fetal vascular [Formula: see text] results from an increase in umbilical oxygen delivery and concomitant fall in fractional extraction. Fetal vasopressin levels are greatly elevated during hypoxia, and under conditions of reduced oxygen supply, the effects of the hormone on umbilical oxygen delivery and vascular [Formula: see text] could have definite survival value.

We examineo the effect of sustained hypoxemia with progressive acidemia on pituitary-adrenal endocrine function (adrenocorticotropic hormone, cortisol) and on adrenal blood flow in fetal sheep. Hypoxemia was induced by the maternal sheep breathing a gas mixture containing 9% oxygen, with 3% carbon dioxide added. Induced hypoxemia resulted in a progressive fetal metabolic acidosis but with little change in maternal pH. During induced hypoxemia there was little change in maternal plasma adrenocorticotropic hormone or cortisol level. Fetal adrenocorticotropic hormone and cortisol increased to peak values within 2.8 hours of induced hypoxia but by 7.2 hours had begun to fall to values that were not significantly different from those at 1.4 hours. Fetal adrenal blood flow (microsphere technique) also increased significantly and remained elevated throughout the duration (7.2 hours) of hypoxemia. The maximum fetal adrenal blood flow achieved during hypoxemia was significantly correlated with the basal (prehypoxemia) flow to the adrenals. We conclude that the changes in fetal adrenocorticotropic hormone, cortisol, and adrenal blood fiow seen in short-term hypoxemia are reproduced during sustained hypoxemia with acidemia. Furthermore, the noted rise in the fetal adrenocorticotropic hormone level may be an important factor contributing to the increase in adrenal blood flow during hypoxemia.

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.

The pharmacokinetic characteristics of the antiemetic drug metoclopramide and the antihistamine diphenhydramine have been determined in a chronically catheterized pregnant sheep preparation. Metoclopramide and diphenhydramine were administered by separate maternal and fetal intravenous infusions to a steady state as well as by maternal intravenous bolus dosing. Drug concentrations in the maternal and fetal plasma and the amniotic and tracheal fluids were measured by means of capillary gas-liquid chromatographic assay techniques. Both metoclopramide and diphenhydramine were excreted into tracheal fluid in substantial quantities. Tracheal metoclopramide concentrations were found to exceed fetal plasma levels by about fifteenfold while diphenhydramine attained maximal excretion in tracheal fluid of about five times that seen in fetal plasma. Drug levels were observed to accumulate slowly in amniotic fluid and eventually to exceed tracheal concentrations. The markedly elevated concentrations of these drugs in fetal lung fluid suggests that the fetal lung may be an important route of drug distribution, elimination, and excretion. (Am J Obstet Gynecol 1987;157:1286-91.)

In seven pregnant ewes with catheters chronically implanted in the fetus, real-time ultrasound observations of fetal breathing and body movements were correlated with direct measurements of fetal arterial and tracheal pressures, heart rate, and intrauterine pressure. There was excellent correlation between the ultrasound record of fetal breathing and recordings of intratracheal pressure changes even when breath amplitude was low or frequently was high. Fetal body movements (stretches, rolls, kicks) were observed during both breathing and nonbreathing periods; vigorous movements were accompanied by rapid deflections of the amniotic and tracheal pressure traces. Movement was frequently accompanied by transient increases or decreases in heart rate. The results indicate that real-time ultrasound is an accurate method of observation of fetal movement in pregnant sheep and is particularly valuable when combined with direct measurements of fetal physiologic parameters.

To estimate fetal oxygen extraction in the human, the inverse, linear relationship between umbilical arterial Po 2 and oxygen extraction was calculated from measurements made on cord blood samples obtained after vaginal (n = 12) and cesarean (n = 16) delivery and used to predict oxygen extraction in utero. Comparisons with similar data from fetal lambs in utero and during labor and delivery indicate that extraction in the human and sheep fetus is 47% and 27%, respectively, at Pa o 2 = 20 mm Hg. For the human fetus to have extraction similar to that in the fetal lamb, vascular Po 2 would have to be substantially higher. This would lead to a higher umbilical venous oxygen content in the human, compensating for the lower umbilical blood flow and yielding a rate of oxygen delivery comparable with that observed in the fetal sheep. These data indicate that there may be significant quantitative differences in the fetal oxygen delivery systems in the human and sheep.

To determine whether anesthesia lowers fetal oxygen consumption, sodium pentobarbital (10 mg/kg) was given intravenously to seven chronically instrumented pregnant ewes (123 to 144 days' gestation). Oxygen consumption fell by 23% in association with a rise in fetal vascular Po 2 . Fetal breathing movements were abolished for 50.5 minutes, while the number of fetal heart rate accelerations fell by 80% in the first 30 minutes after pentobarbital injection. It is concluded that anesthesia reduces fetal oxygen consumption, probably by abolishing skeletal muscle activity, and perhaps also by reducing cerebral metabolic rate.

In seven experiments on five chronically monitored fetal lambs (114 to 136 days' gestation), oxygen consumption, umbilical blood flow, and blood gases were measured before and after temporary paralysis of the fetus by the intravenous injection of gallamine. In two of the experiments, breathing movements were absent in the control, pre-gallamine period, and no change in oxygen consumption (Vo 2 ) was observed after the drug. In the other five experiments, breathing activity was present in the control period. After gallamine,Vo 2 fell by 17%, as a result of a decline in umbilical oxygen extraction; there was no change in umbilical blood flow. In all experiments, umbilical arterial and venousPo 2 values rose after paralysis, by 14% and 18%, respectively. The changes inPo 2 values are probably secondary to the fall in fetal oxygen consumption. The data indicate that fetal skeletal muscle activity, in the form of breathing activity and perhaps gross body movements, contributes significantly to total fetal oxygen demands. Conversely, cessation of fetal activity in adverse situations, when oxygen supplies are limited, could aid in maximizing the delivery of the available oxygen to vital organs, such as the heart and brain.

Twenty-three patients undergoing general anaesthesia for Caesarian section for healthy term pregnancies were entered into a double blind study using metoclopramide (MCP) and a normal saline placebo. Of these patients, eight received intravenous metoclopramide, 12 a normal saline placebo and three were lost to clinical follow-up. The maternal gastric volumes were measured and maternal and foetal MCP plasma concentrations were determined by gas-liquid chromatography. The Neurological and Adaptive Capacity Score tests of Amiel, Barrier and Schnider (NACS) were used to attempt evaluation of neonatal responses to MCP. Maternal gastric volume was significantly lower (p less than 0.05) in the treated patients. There were no marked differences in Apgar scores, cardiovascular parameters or neurobehavioural scores between the treated and untreated groups of neonates. At no time were the foetal metoclopramide plasma concentrations observed to exceed maternal values.