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Thiamphenicol (TAP) was administered as a single oral dose of 500 mg (8–9 mg/kg) to pregnant women about to undergo therapeutic abortions. The length of gestations varied from 7 to 12 weeks. The concentrations of TAP were determined by electron capture gas chromatography in maternal serum, placenta, amniotic fluid, and various embryonal/fetal tissues when abortion occurred 1.5 to 20 hr after drug administration. TAP was rapidly transferred across the placental membranes and was fairly evenly distributed in the tissues of the embryonal/fetal compartment. The placental concentrations of TAP were usually higher than the corresponding maternal serum levels (1.1 to 7-fold), suggesting some active role of the placenta in TAP uptake. This was confirmed by in vitro studies with immature and term placenta tissue. From a bathing medium containing 10 μg/ml (comparable to therapeutic blood levels) TAP was accumulated in the intracellular compartment to concentrations about 2.6 times those in the medium. This process was time and temperature dependent. Inspite of the low content of mitochondrial particles in human fetal liver the relatively few mitochondria present exhibited high protein synthesis in vitro which decreased with increasing gestational age. Mitochondrial protein synthesis was inhibited by TAP, and a 50% reduction of amino acid incorporation was observed at 10–15 μg TAP/ml. Such levels of the drug are required for successful chemotherapy. To ascertain such therapeutic drug levels, TAP is usually given at 3 g daily (divided, e.g., into three doses 1 g each). The data obtained for a single application of 0.5 g TAP suggest inhibition of mitochondrial protein synthesis during therapy. While the pharmacokinetic characteristics of TAP suggested that this compound might be well suited to treat intrauterine bacterial infections sensitive to TAP, the drug concentrations reached in human embryonal tissues in vivo are known to produce embryolethality in pregnant rats at comparable developmental stages. The findings reported here raise serious concerns about the suitability of TAP for repeated administration to pregnant women during the first trimester of gestation.

Methods have been developed for the determination of the benzodiazepine tranquilizer prazepam and its metabolites desmethyl diazepam, 3-hydroxy-prazepam and oxazepam by electron capture gas chromatography and selected ion monitoring with diazepam as the internal standard. The benzodiazepines were isolated from blood serum or homogenized tissue samples, either by extraction with ethyl acetate or on small Extrelut columns packed with porous silica. The concentrated extracts were directly injected into the gas chromatograph equipped with an electron capture detector. Following trimethylsilylation, analysis on a gas chromatography—mass spectrometry—computer system operated in the selected ion-monitoring mode was performed. Using 50–200 mg (μl) biological material, concentrations of prazepam and metabolites of 5 ng/g(ml) could be determined with signal-to-noise ratios of > 10. Using 1 g(ml) samples, the same signal-to-noise ratios were obtained with 1 ng/g(ml) concentrations. The methods developed were applied to the analysis of the displacental transfer of prazepam and desmethyl diazepam in early human pregnancy. Furthermore, prazepam metabolism in human fetal liver and cell cultures was studied.

In a prospective study, 22 children with recently manifested infantile spasms (18 patients with symptomatic and 4 with idiopathic infantile spasms) were treated with sodium valproate (VPA). Before VPA was instituted, a loading test was performed to exclude abnormal patterns of VPA metabolites by gas chromatography and mass spectroscopy of serum and urine. This test was repeated during VPA therapy; an abnormal pattern of VPA metabolites was not observed. VPA was started in increasing dosage until infantile spasms were controlled or a maximum dose of 100 mg/kg/day was reached. If VPA did not control seizures or at least reduce frequency significantly after a trial of 4-6 weeks, dexamathasone was added to VPA. If focal seizures occurred in association with localized epileptogenic EEG discharges, carbamazepine (CBZ) was added to VPA. After 4 weeks of VPA monotherapy, infantile spasms were controlled in 11 children. After 3 months of therapy, 16 children were free of seizures (14 patients VPA monotherapy), and 4 children had reduction of seizure frequency to less than 25%. VPA doses varied between 40 and 100 mg/kg/day (mean 74). The mean plasma concentration was 113 micrograms/ml (range 46-177). After 6 months of therapy, total seizure control was achieved in 20 of 22 patients (16 children VPA monotherapy). The mean observation time was 16 1/2 months (range 6-36 months). There were seven relapses in six children during the first 7 months of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

A detailed investigation of the plasma protein binding of carbamazepine (CBZ) and its major metabolite carbamazepine epoxide (CBZE) revealed that both compounds are more extensively bound to plasma proteins in the mother (% free fraction CBZ, 28.4 +/- 3.0; CBZE, 55.1 +/- 5.0) than in the fetus (% free fraction CBZ, 31.5 +/- 2.2; CBZE, 61.5 +/- 3.1) at the time of birth. Fetal and maternal protein binding is reduced compared to controls (% free fraction CBZ, 26.5 +/- 2.1; CBZE, 49.1 +/- 5.7). A correlation of the unbound fraction with various biochemical parameters suggested that among other factors, high bilirubin concentrations in the fetus could be responsible for the relatively increased free fraction of CBZ and CBZE compared to maternal values. As free fraction values of CBZ and CBZE are either the same (CBZ) or slightly higher (CBZE) in women at term compared to nonpregnant controls, a regular monitoring of maternal free fraction values seems unnecessary. For other anticonvulsive drugs, however, like diazepam and valproic acid, monitoring of both total plasma concentrations and free fractions is strongly recommended.

The anticonvulsant potency of 2-propyl-2-pentenoic acid (2-en-VPA; trans isomer), a major metabolite of the antiepileptic valproic acid (VPA), was evaluated in different animal models of epilepsy and compared with the respective data for VPA. Four models were used: the maximal electroshock seizure (MES) test in mice, the pentylenetetrazol seizure test in mice, gerbils with 'major' (generalized tonic-clonic) seizures in response to specific sensory stimulation, and rats with chronically recurring, spontaneous 'petit mal' seizures. The overall anticonvulsant profile of 2-en-VPA in these models compared favourably with that of VPA. Both drugs were considerably more potent to block seizures in epileptic rats and gerbils than in the traditional MES and pentylenetetrazol mouse models. As regards toxicity, no side-effects were observed with effective doses of 2-en-VPA in rats and gerbils, whereas in the doses necessary to block MES and pentylenetetrazol seizures in mice (200-300 mg/kg i.p.) 2-en-VPA was more sedative than VPA. LD50 values determined for both drugs were comparable. A major difference between 2-en-VPA and VPA was found with respect to embryotoxicity. Single doses of VPA administered to pregnant mice gave rise to significant teratogenic effects (exencephaly, embryolethality , growth retardation), whereas 2-en-VPA was not embryotoxic, even at extremely high doses (600 mg/kg). The data suggest that 2-en-VPA may be a valuable alternative antiepileptic drug.

Local anaesthetic agents, both of the amide type (e.g., lidocaine, bupivacaine, etidocaine) and of the ester type (e.g., 2-chloroprocaine) are widely used to relieve pain in obstetric and gynaecological practice. The pharmacokinetics of these compounds are discussed in this review, with particular emphasis on the fetal exposure and its relationship to adverse effects on the fetus. 2-Chloroprocaine is rapidly hydrolyzed by esterases, and only traces of this compound reach the fetus, even following multiple injections, suggesting safety for the fetus. The main disadvantage of this compound is the short duration of action (0.5-1 h). The amide-type agents are active for longer time periods (up to several hours). The fetal/maternal total concentration ratios were approximately 0.3 for bupivacaine and etidocaine, 0.5 for lidocaine, 0.7 for mepivacaine, and 1 for prilocaine. The low ratios of bupivacaine and etidocaine result from extensive binding (90%) of these drugs to maternal alpha1-acid glycoprotein which exceeds corresponding fetal protein binding (50%). These low fetal/maternal total concentration ratios cannot be equated with fetal safety, because fetal side effects are better related to the free drug levels. Since the amide-type anaesthetics are weak bases, fetal acidosis will increase the maternal/fetal pH gradient and will result in accumulation of free drug in the fetus and possible fetal side effects. Addition of epinephrine to the injection solution reduces the maternal blood levels of the amide-type compounds, but apparently not the fetal levels to the same extent. Because of possible unwanted effects of epinephrine (decreased uterine blood flow), the addition of this compound is not generally accepted to be of advantage. Paracervical blockade may result in elevated fetal blood levels (possibly by transarterial diffusion into uterine arteries) and possibly fetal bradycardia.

A fully automated reversed-phase high-performance liquid chromatography (HPLC) procedure with precolumn switching is presented which allows quantitative assay of 13-cis-retinoic acid and all-trans-retinoic acid, their 4-oxo-metabolites and retinol in plasma, amniotic fluid and tissue homogenates. A binary gradient system allowed baseline separation of the retinoids within 15 minutes. Sample preparation was kept simple in order to minimize degradation and isomerization of the unstable substances and required only the addition of isopropanol, freezing in liquid nitrogen, and centrifugation. Overall recovery was quantitative allowing for external standardization. Calibration curves were linear in mouse plasma, amniotic fluid and human serum albumin solution (r < 0.99) and in mouse embryo homogenate (r < 0.98) over a concentration range of 4 orders of magnitude. The detection limit was 2 ng/ml or g with a sample size of only 0.1 ml or 0.1 g. Coefficients of variation of the assay were less than 5% (w...

Nicotine and cotinine concentrations were measured in placental tissue during the first trimester, in amniotic fluid during the second trimester and in placental tissue and fetal serum at birth. These values were compared to the corresponding serum concentrations of the smoking mothers. Nicotine concentrations in the placentas (range 3.3-28 ng/g), in amniotic fluid (range 1.5-23 ng/ml) and in fetal serum (range 0.5-25 ng/ml) were all higher than the corresponding maternal serum values: amniotic fluid/maternal vein serum concentration ratio 1.54 +/- (SD) 0.27 (n = 23; week 16-24 of gestation), umbilical vein serum/maternal vein serum ratio 1.12 +/- 0.30 (n = 26; at birth); placental tissue/maternal vein serum ratio 2.58 +/- 1.30 (n = 17; at birth); these ratios were between 1.2 and 5 during week 10 of gestation (n = 3). The ratios did not depend on the time between the last cigarette smoked and sampling. Significant correlations were found between nicotine concentrations in amniotic fluid and maternal serum (r = 0.88), between fetal and maternal serum levels (r = 0.88) and between placental and maternal serum levels (r = 0.52). Cotinine concentrations in placental tissue (range 10-131 ng/g), amniotic fluid (range 5-188 ng/ml) and fetal serum (range 15-233 ng/ml) were lower than or similar to corresponding maternal serum levels. Our results indicate that the human fetus is exposed to higher nicotine concentrations that the smoking mothers.

Cadmium concentrations were measured by flameless atomic absorption spectrometry in blood and mature milk of 15 nonsmoking and 56 smoking mothers during the nursing period. Both blood and milk concentrations increased with increasing cigarette consumption. The median blood and milk concentrations in nonsmokers were 0.54 and 0.07 microgram/l, respectively; these values rose to 1.54 and 0.16 micrograms/l in blood and milk of mothers smoking more than 20 cigarettes per day. Milk concentrations of cadmium were approximately 10% of corresponding blood concentrations. The cadmium exposure of infants nursed by nonsmoking as well as by smoking mothers was far below the exposure of formula-fed infants or the provisional acceptable weekly intake level set by the WHO.

Experiments on teratogenic effects of stable isotopes from our own and other laboratories are evaluated. In the first series of investigations, the enrichment of the stable isotope /sup 13/C derived from U-/sup 13/C-glucose was studied in mouse embryos at various stages of development, including limb buds in organ culture. Preimplantation mouse embryos incubated in vitro in /sup 13/C-enriched medium for 48 hours showed normal development during subsequent differentiation in vitro and also in vivo after embryo transfer to faster mothers. These embryos were 15% to 20% enriched in /sup 13/C. Administration of U-13-C-glucose to pregnant mice during organogenesis led to an increase of the absolute /sup 13/C content of the embryo for several days after the end of isotope administration, whereas the enrichment in maternal tissue decreased. No alterations of embryonic development were detected due to stable isotope enrichment. Development of cultured mouse limb buds was unaffected by incubation with 82 mol% U-/sup 13/C-glucose as judged from morphologic and biochemical criteria. The second part of the article describes the value of deuterium-labeled drugs as probes into the mechanism of activation of teratogenic metabolites. A comparison of the pharmacokinetics as well as the teratogenicity between cyclophosphamide and some specific deuterium-labeled analoguesmore » showed that the isotope effect observed can be related to a particular metabolic pathway crucial for teratogenic activation by this drug.« less

The rate of metabolism as well as the metabolic profiles of the enantiomers and the racemate of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine (EM 12), a teratogenic thalidomide analogue, have been investigated in vitro and in the marmoset monkey in vivo. The half-life of racemic EM 12 and of the two enantiomers in vitro (phosphate buffer pH 7.4, 37 degrees C) were all in the same range (12 h). Two major hydrolysis products were found which were formed via amide cleavage of the piperidinedione ring of the molecule (EM 27 and EM 356). Their concentrations were similar. In contrast, EM 356 was the main metabolite of EM 12 present in the urine of marmoset monkeys following single i.p. or p.o. doses of 5 mg/kg b.w. About twice as much EM 356 was produced after administration of R-EM 12 than after administration of S-EM 12. The concentration ratio of the metabolites obtained after p.o. and i.p. administration of the substances were in the same range. Separation of the EM 12 enantiomers present in urine suggests that considerable racemisation took place in vivo, although at a slower rate than in vitro: about 25% of the respective optical antipodes were present 5 h after administration of the R-enantiomer and 7.5 h after administration of the S-enantiomer (compared to 1.5 h in vitro). Our results indicate that racemic EM 12 as well as its enantiomers are chemically and metabolically more stable than thalidomide; however, extensive racemisation occurs both in vivo and in vitro. The metabolism and renal excretion of the enantiomers of EM 12 in the marmoset monkey were shown to be stereoselective.

A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.

Neural tube defects were induced dose-dependently by single injections of the anticonvulsant drug valproic acid (VPA) as sodium salt in mice on gestational day 8. Folinic acid (5-CHO-THF) coadministration by i.p. injection or by a constant rate infusion via osmotic minipumps, implanted s.c., significantly reduced the exencephaly rates using a randomized double-blind experimental procedure. 5-CHO-THF supplementation cut the exencephaly rates into half even at high maternal plasma levels of VPA (p less than 0.005, chi 2-test); resorption rates were not affected. The VPA plasma kinetics were not changed by any of the application regimens of 5-CHO-THF. The investigation of the folate metabolite pattern (determined by HPLC) showed that 5-CHO-THF and 5-methyl-tetrahydrofolic acid (5-CH3-THF) were the main metabolites in untreated mice. After supplementation with 5-CHO-THF, only the concentrations of this folate vitamer were increased in the plasma from 0.3 microgram/ml (normal) to 0.6 or 1.9 micrograms/ml (after injection of 3 x 1 mg/kg or 3 X 4 mg/kg) and to 4.2 micrograms/ml (after infusion via osmotic minipumps). Our results indicate that VPA-induced exencephaly in mice combined with the investigation of the plasma levels of VPA and the different folate metabolites could be an appropriate animal model to study protective effects of folates on the occurrence of neural tube defects.

The transfer of valproic acid (VPA, 2-propylpentanoic acid) and its unsaturated active metabolite (2-en, 2-propyl-2-pentenoic acid) from plasma to liver has been studied in the mouse between 2 min and 6 h following oral administration of 50 mg of the sodium salts per kg body weight. Transfer of both compounds was extremely rapid. Liver concentrations of VPA were higher than those in plasma, while liver concentrations of 2-en were lower than those in plasma. The low hepatic levels of 2-en may be explained by extensive plasma protein binding of this metabolite. The liver/plasma concentration ratios were concentration-dependent, indicating the presence of active transport mechanisms and/or saturation of plasma protein binding. Our results indicate that 2-en should be further studied in regard to its potential for the induction of liver toxicity. The desirable low level of 2-en reached in the liver, seen together with previously observed favourable-anticonvulsant profile and low teratogenicity, would indicate that this compound may be a valuable alternative antiepileptic agent.

Isolated hepatocytes of human fetuses (week 9–20 of gestation) were prepared by digestion of liver slices with Dispase l and repeated centrifugations. These cells metabolized drugs by first order kinetics for at least 4 h and retained their drug metabolizing enzyme activities for more than 24 h. Using cultures of human fetal liver slices (week 5–20 of gestation) first order kinetics of drug metabolism could be measured for up to 3 days. These findings correlate well with concomitant morphological studies. Electron microscopy revealed that the liver cells of human fetuses (week 9–20 of gestation) contained numerous cavities of the rough endoplasmic reticulum (ER) and only single membranes of the smooth ER. The isolated liver cells maintained their morphological integrity for up to 24 h in culture. Sections derived from liver slices cultured for up to 3 days did not show any significant morphological alterations, except for some swellings of mitochondria, vacuolisations and fat inclusions that occurred in the sinus endothelia and blood cells. A gradual disintegration began on day 4, and after 7 days intact cells could no longer be demonstrated. The metabolism of the benzodiazepine drugs prazepam, diazepam and medazepam as well as diphenylhydantoin, hexobarbital and halothane were measured in these systems. Both the isolated hepatocyte and liver organ cultures should be useful to study whether a relationship exists between the emryotoxicity of certain drugs and their metabolism in the human fetus.

The antiepileptic drug valproic acid (VPA) was administered via four different routes in the mouse during gestational stages sensitive for interference with neural tube defect formation: a single oral intubation or injection, sc or ip, on Day 8, or infusion via subcutaneously implanted osmotic minipumps from Day 7 1/2 to 8 1/2 of gestation. Embryotoxicity was evaluated on Day 18 (incidence of exencephaly, embryolethality and fetal weight retardation). Oral intubation of VPA resulted in significantly lower peak concentrations of VPA as well as lower embryotoxicity as compared to sc and ip administration. The metabolites of the beta-, omega- and omega-1 oxidation pathways were present in both maternal serum and gestational tissues in very low concentrations (usually less than 2% of corresponding VPA levels). Infusion of VPA via osmotic minipumps (lower steady-state VPA levels as compared to peak levels following injection of VPA) resulted in embryolethality and fetal weight retardation, but little exencephaly. The metabolic pattern was similar in all four administration experiments. Phenobarbital pretreatment of the dams (previously shown to reduce VPA serum concentrations and induce the omega- and omega-1 oxidation pathways) reduced the embryotoxicity of VPA. These results suggest that VPA embryotoxicity is mediated by the parent drug, and not one of the metabolites considered in this study.

A simple procedure for the quantitative analysis of trifluoroacetic acid (TFA) in urine and serum from patients narcotized with halothane is described. This involves addition of sodium hydroxide to the body fluid, evaporation of the aqueous phase and esterification of TFA in concentrated sulphuric acid with 2,2,2-trichloroethanol. The gaseous phases above the reaction mixture were then analyzed by gas chromatography with a nickel-63 electron-capture detector. The detection limit was 1 microgram of TFA per mililitre of body fluid (200 microgram of body fluid are analysed) and the relative standard deviation was +/-6%. Patients treated with ethrane, another commercial anaesthetic, did not produce any detectable TFA.

Measurement of thiamphenicol transfer to the rat embryo during organogenesis was performed as one step of the determination of possible drug embryotoxicity in man. Extrapolation of data on animal embryotoxicity to man will only become possible when data on the toxicokinetic properties of the substance under investigation are available for both animal and man. 1) Thiamphenicol, given between day 11.5 and 14 of rat gestation, rapidly reached the embryo; 4–6 h after single i.v. or s.c. injection, embryonic and maternal thiamphenicol levels became equal and decreased from that time on at the same rate. 2) No evidence was found for development of a placental barrier with increasing placental function. The same dose applied at different developmental stages yielded the same embryonic drug concentrations. 3) Elimination via kidney (unchanged) or bile (glucuronide), may become rate-limiting for thiamphenicol excretion. Doses exceeding 50 mg/kg (i.v.) or approx. 100 mg/kg (s.c.) yielded thiamphenicol levels higher than those expected from linear dose-concentration relationships. 4) Drug concentrations (> 3–5 μg/g wet weight) obtained with dosing regimens (> 100 mg/kg/day) used for experimental induction of embryolethality in rats are equal to those necessary for inhibition of mitochondrial protein synthesis in vitro and to those necessary for treatment of bacterial infections in man.

A number of metabolites of the anticonvulsant drug valproic acid (VPA) as well as related substances were tested in regard to their teratogenicity in the mouse following single sc injections of 600 mg/kg on Day 8 of gestation. VPA was highly teratogenic at this dose level and over 60% of live fetuses had neural tube defects (exencephaly). Homologous compounds with shorter or longer alkyl chains were less teratogenic. Substitution of the α-H atoms in related branched carboxylic acids by methyl or ethyl groups abolished the teratogenic response. Introduction of a double bond in the ω-position of VPA (4-en-VPA) did not change the teratogenicity of VPA, while a ω-2 double bond (2-en-VPA) abolished teratogenicity. The other VPA metabolites tested as well as two straight-chain acids (n-octanoic acid and 4-pentenoic acid) and the two clinically used substances valpromide (valproic acid amide) and ethosuximide did not induce neural tube defects, although some of them induced slightly increased resorption rates and fetal weight retardation. The serum protein binding capacities of the various compounds did not correlate with the teratogenic response. Also the concentrations reached in the gestational material did not predict the teratogenicity of the substances tested. Our results indicate that the teratogenicity of the class of compounds studied represents a more specific effect than the anticonvulsant activity which could lead to the development of alternative antiepileptic drugs with low embryotoxic potential.

Summary: Levels of the antiepileptic drug valproate (VPA) and five of its active metabolites (2-en-VPA, 3-keto-VPA, and 3-,4-, and 5-hydroxy-VPA) were determined by gas chromatography-mass spectrometry in cerebrospinal fluid (CSF) of 15 epileptic children undergoing chronic treatment with VPA. In eight of these children, total and free drug and metabolite concentrations in plasma were also measured. All VPA metabolites present in plasma could also be detected in CSF, although concentrations were substantially'lower than those of the parent compound. CSF concentrations of VPA and most of its metabolites were positively correlated with total and free concentrations in plasma. However, concentrations in CSF were always significantly lower than free plasma concentrations, which may be explained by asymmetric transport at the blood-CSF barrier. The data on low CSF levels of VPA metabolites do not exclude the possibility that accumulation of active metabolite(s) may occur in certain brain areas during chronic treatment of epileptic patients with VPA.

The placental transfer of primidone and its metabolites phenobarbital, phenylethylmalondiamide (PEMA) and p-hydroxyphenobarbital (free and conjugated) has been investigated at birth in 14 epileptic women who had been treated with primidone throughout pregnancy. All drugs studied were found in similar concentrations in maternal and cord blood. In seven of the newborns the pharmacokinetics of these drugs were studied during the first postnatal weeks. Primidone and phenobarbital were eliminated with mean half-lives of 23±10 h and 113±40 h, respectively, PEMA with 35±6 h. In some neonates the serum concentrations of phenobarbital and PEMA increased during the first few days due to their formation by neonatal primidone metabolism. Some babies showed a biphasic elimination pattern with elimination rates increasing after a few days. Although half-lives varied greatly, they corresponded well with renal clearance values and aminopyrine demethylase activities as measured by13CO2-exhalation from13C-labelled aminopyrine. Two newborns whose mothers had been treated with phenytoin in addition to primidone, showed half-lives, renal clearance values and aminopyrine demethylase activities well within the corresponding ranges for adults, thus demonstrating prenatal induction. Newborns whose mothers had been treated with valproate as comedication, did not exhibit elevated excretion rates as compared to newborns of mothers who were treated with primidone alone. Withdrawal symptoms developed in two newborns at times when primidone had been essentially excreted, and in spite of the presence of elevated phenobarbital and PEMA levels. All drugs studied were also present in mothers' milk. During breast feeding, drugs ingested with the milk contributed to the neonate's blood levels, particularly in the case of phenobarbital.

Interspecies differences in regard to the teratogenicity of drugs can be the result of differing pharmacokinetic processes that determine the crucial concentration-time relationships in the embryo. Maternal absorption, as well as distribution, of the drugs does not usually show great species differences. The first-pass effect after oral application is often more pronounced in animals than man (e.g., valproic acid, 13-cis-retinoic acid), although in some cases the reverse was found (e.g., hydrolysis of valpromide). Existing differences can be adjusted by appropriate choice of the administration route and measurements of drug levels. Many variables determine the placental transfer of drugs: developmental stage, type of placenta, properties of the drug. Even closely related drugs (e.g., retinoids) may differ greatly in regard to placental transfer. Maternal protein binding is an important determinant of placental transfer, since only the free concentration in maternal plasma can equilibrate with the embryo during organogenesis; this parameter differs greatly across species (e.g., valproic acid: five times higher free fractions in mouse and hamster than in monkey and man). The metabolic pattern has not yet been demonstrated to be a major cause of species differences, although recent evidence on phenytoin and thalidomide support the hypothesis that some species differences can be the result of differing activation/deactivation pathways. Laboratory animals usually have a much higher rate of drug elimination than man. Drastic drug level fluctuations are therefore present during teratogenicity testing in animals, but not to the same degree in human therapy. It must, therefore, be investigated if peak concentrations (such as for valproic acid and possibly caffeine) or the area under the concentration-time curve (AUC) (such as for cyclophosphamide and possibly retinoids) correlate with the teratogenic response. Only then is a rational and scientific basis for interspecies comparison possible. It is concluded that the prediction of the human response based on animal studies can be improved by consideration of the appropriate pharmacokinetic determinants.

A method for the quantitative determination of primidone and its metabolites phenobarbital, phenylethylmalondiamide (PEMA) and hydroxyphenobarbital (free and conjugated) in serum, urine, saliva, breast milk and tissue has been developed. Following the addition of the methyl analogues of primidone, phenobarbital and PEMA as internal standards and of saturated ammonium sulphate, the samples (5--100 microliter) were extracted twice with ethyl acetate--benzene (20:80). The extracts were divided into two equal portions; one portion was ethylated by Greeley's method for the analysis of primidone, phenobarbital and hydroxy-phenobarbital, while the other was trimethylsilylated for the analysis of primidone and PEMA. A gas chromatographic--mass spectrometric system was used for the analysis of the derivatized extracts. Linear calibration curves were obtained in the concentration range studied (between 100 ng/ml and 30 microgram/ml). The recoveries of the drugs were between 80 and 93%. The relative standard deviations were between 3.2 and 5.9% (100-microliter serum samples containing 1 microgram/ml of the drugs). The lower detection limits were found to be between 1.4 and 3.7 ng/ml using serum samples of 100 microliter. These methods have been applied to the study of the placental transfer and neonatal disposition of primidone and its metabolites in the human.

Thirty-two metabolites and analogues of the antiepileptic drug valproic acid (2-propylpentanoic acid; VPA) were tested for anticonvulsant and toxic effects in mice, in an attempt to find out if any of these compounds were superior to valproic acid. Valproic acid and ethosuximide, another clinically established antiepileptic drug, were included in these studies for comparison. After intraperitoneal administration, the anticonvulsant potency of the various drugs was determined in three Scizure tests: the threshold for maximal electroconvulsions, the maximal electroshock Scizure test and Scizures induced by subcutaneous injection of pentylenetetrazol. For the most potent compounds, median minimal neurotoxic doses (TD50 s ) and LD50 s (after i.p. and i.v. injection) were determined. Valpramide, the primary amide of valproic acid, proved to be the most potent compound in the three Scizure tests, used, being 2–5 times as potent as valproic acid, but valpramide was also considerably more sedative and toxic than valproic acid or ethosuximide. Of the metabolites of valproic acid tested, the unsaturated compounds 4-en-valproic acid (4-en-VPA) and the trans-isomer of 2-en-valproic acid (2-en-VPA) were most potent and, depending on the Scizure test used, reached 60–100% of the efficacy of the parent drug. Both metabolites had LD50, values which were similar or greater than those of valproic acid but they were more sedative than the parent compound. Analogues of valproic acid with shorter side-chain lengths were only weakly active as anticonvulsants, whereas elongation of the side-chains led to increases in anticonvulsant potency ut also in sedative/hypnotic side effects and toxicity, Non-branched monocarboxylic acids and cyclic compounds in which the side-chains have been closed to a ring were inactive or only weakly active. However, addition of a methyl group in position 1 at the ring of cyclohexanoic acid markedly increased the anticonvulsant potency without altering LD50, values. Similarly, introduction of an additional branching with a methyl group at C2 of analogues of valproic acid led to considerable enhancement of anticonvulsant effectiveness. Although these methyl-substituted compounds were more sedative than valproic acid, they seem to be interesting tools with regard to the structural prerequisites of anticonvulsant, toxic and teratogenic effects of branched fatty acids.

The plasma pharmacokinetics of the enantiomers of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine (EM 12) and the racemic mixture of this substance were investigated in Callithrix jacchus, a thalidomide-sensitive primate. Single doses of 5 mg/kg body wt were administered orally or intraperitoneally. Maximum plasma concentrations were reached 1 h after administration of the enantiomers, and 3 h after application of the racemate. The mean plasma elimination half-life was in the range of 5 h for the enantiomers, as well as for the racemic mixture, although there was a tendency toward slower elimination and higher plasma AUC values of the S-enantiomer: thus, after administration of the (greater than 99%) pure enantiomers, the plasma AUC value of the administered S-enantiomer was found to be more than one-third higher than that of the administered R-enantiomer. Racemisation of the R- and the S-form of EM 12 occurred both in vitro (phosphate buffer, pH 7.4, 37 degrees C) and in vivo. The maximum plasma concentrations of the antipodes produced via chiral inversion were between 13% and 21%; the plasma AUC values of the resulting antipodes were between 24% and 30% of the corresponding values of total EM 12. The plasma pharmacokinetic data, including the extent of the chiral inversion obtained after p.o. and i.p. application of the substances, were in the same range. The results indicate that both enantiomers racemise with appreciable rates; this may be expected to complicate the interpretation of studies designed to evaluate stereoselective differences with respect to teratological activities of EM 12 and related substances such as thalidomide.

A single dose of 500 mg thiamphenicol (TAP) was given to 30 women at various times prior to interruption of pregnancy for social and/or medical reasons during the first trimester (curettage, hysterotomy, or prostaglandin induction). Concentrations of TAP were then determined in maternal serum, placental, and fetal tissues by a gas chromatographic method using electron capture detection. Similar, but sometimes significantly higher, concentrations of TAP were found in placental and fetal tissues as compared to maternal serum (data expressed as μg/ml or μg/g).

13-cis-Retinoic acid (isotretinoin) is teratogenic in humans at therapeutic doses (0.5-1.5 mg/kg) but only marginally teratogenic in the mouse at a high dose of 100 mg/kg. Previous results explained why the cis isomer of retinoic acid was much less teratogenic than the trans isomer in mice. It was found that the placental transfer of all-trans retinoic acid to the mouse embryo was far greater than that of the 13-cis isomer. Since our previous study had been performed with exceedingly high doses (100 mg/kg) of 13-cis-retinoic acid and all-trans-retinoic acid, we have now performed additional experiments with 10-fold lower doses. Studies were also done with the main metabolites of the two retinoids (the 4-oxo-derivatives) to elucidate the metabolism, pharmacokinetics, and teratogenicity of each single compound. It was shown that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid were extremely teratogenic, whereas their corresponding cis isomers caused only 2% cleft palate. Embryonic exposure to the trans isomers was likewise higher than that to the cis isomers, as shown by the far higher embryonic peak concentrations and by the 30-fold higher areas under the concentration-time curve values reached for the trans isomers compared with the cis isomers. At 8 hr, embryo/maternal plasma ratios were higher than 1 after administration of the all-trans compounds. Concentrations found in the placenta and yolk sac were higher for the trans forms than for the cis forms. We propose a model for a facilitated transport of the all-trans forms to the developing embryo and suggest that the conversion to the trans isomer and trans metabolite could play a major role in the teratogenicity of 13-cis-retinoic acid in humans.

A new method for determining the amino acid sequence of polypeptides consists in initial partial hydrolysis to yield a complex mixture of oligopeptides. After derivatization to enhance its volatility, the mixture is analyzed by combined gas chromatography and mass spectrometry. The sequence of the polypeptide is established by a computer from the identified oligopeptides. So far polypeptides having up to 40 amino acids have been analyzed by this method. The advantages and disadvantages of the new method compared with the stepwise procedure of the Edman degradation are considered. Since the two methods are based on fundamentally different principles they may prove to be complementary.

Novel drug reservoirs are described which, after their implantation under mouse skin, continuously released organic liquids such as the anti-epileptic drug valproic acid at preselected rates for periods up to several weeks. The liquids, which were filled into the reservoirs, diffused through silastic membranes. The area and thickness of these membranes determined the administered dose. Administration of the anti-epileptic drug valproic acid at various selected doses resulted in persistent drug concentrations spanning from subtherapeutic to toxic levels. The drug reservoirs were easily refillable in situ which greatly extended the duration of the experiment. The dose administered could be determined within 5-15 per cent (rel. S.D.). It is suggested that the maintenance of persistent drug levels in small laboratory animals may be an appropriate model for the pharmacological and toxicological study of those compounds with short half-lives and high clearance rates in these species.

Human therapeutic valproic acid (VPA) levels could be maintained in the mouse for a period of 1 week by constant rate application via subcutaneously implanted osmotic minipumps. Also, the concentrations of VPA metabolites observed in mouse plasma were similar to those seen in human plasma. The drug application could be prolonged by replacing exhausted pumps with freshly-filled devices. Removal of the implanted pumps and measurement of the decay of the drug levels revealed that the half-life of the main plasma metabolite 2-en(2-propyl-2-pentenoic acid) exceeded that of VPA. This result was confirmed by constant-rate application of this metabolite; the plasma clearance of 2-en (as calculated from the steady-state levels observed) was found to be lower than that of VPA. Brain levels of VPA and 2-en during steady-state were 3–10 per cent of corresponding plasma levels. The blood-brain kinetics of 2-en following administration of VPA were similar to those observed following application of 2-en itself. VPA was cleared faster from the brain than from the plasma, while 2-en was more persistent in the brain than in the plasma. Our results indicate that controlled, constant-rate application of drugs such as VPA, via implantable osmotic minipumps, may be a valuable procedure for a number of pharmacological and toxicological studies, particularly where persistent drug levels must be maintained for extended time periods.

A method has been developed for the simultaneous quantitative determination of valproic acid (2-propylpentanoic acid) and its metabolites 2-propyl-2-pentenoic acid (trans), 2-propyl-3-pentenoic acid (trans), 2-propyl-4-pentenoic acid, 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, 5-hydroxy-2-propylpentanoic acid, 3-oxo-2-propyl-pentanoic acid, and and 2-propylglutaric acid. All compounds were extracted at pH 5.0 with ethyl acetate. The concentrated extracts were trimethylsilylated and the resulting mixtures analyzed by a gas chromatography-mass spectrometry-computer system operated in the selected ion monitoring mode. Linear calibration curves were obtained in the concentration ranges studied (0.1-20 microgram/ml for metabolites, 0.1-150 microgram/ml for valproic acid. The recoveries of the drugs were between 92 and 97%. The relative standard deviations were between 3.9 and 8.1% (analysis of multiple 10-microliter samples of patient urine). The lower detection limits were found to be between 2.8 and 18 ng/ml using 200-microliter serum samples. The derivatized extracts were stable for at least one week. Applications of the method described include studies of placental transfer for valproic acid and metabolites in the human, the elimination of these substances by the neonate, their transfer via mother's milk, and their levels in mouse brain.

The protein binding (expressed as percent free drug fraction) of the antiepileptic drug valproic acid (VPA) was studied in 65 fetus-mother pairs from weeks 13 to week 41 of gestation. The fetal free fractions (expressed as percent of total concentrations) of VPA were exceedingly high (greater than 50%) during weeks 13 to 16 of gestation; these values decreased to 20% by week 20 and further decreased gradually to 10% at term. There was a highly significant negative correlation between free VPA fractions and fetal albumin concentrations. Maternal free fractions of VPA gradually increased from 10% during early pregnancy to 20% at term. The free fractions of VPA were significantly higher in mothers who had received oxytocin. Thus, protein binding in the fetus exceeded that of the mother at term, whereas the converse was true during early gestation. These results agree with previous in vivo findings. It is likely that the free concentrations in the mother determine the drug effects and toxicity in both the mother and the fetus. Intermittent drug administration--particularly of large single doses--could result in a transient increase of the free concentrations of VPA, particularly because of the strong concentration dependence of VPA protein binding. Increased free fractions can also be expected from increased concentrations of displacing agents of endogenous or exogenous origin (other drugs).

The anticonvulsant activity of valproic acid (VPA) was determined in amygdala-kindled rats after single and repeated (total of 7 injections given 3 times per day) administration of 200 mg/kg i.p. After a single injection, VPA significantly reduced the severity and duration of the kindled seizures and increased the duration of after-discharges recorded from the stimulated amygdala, but only 12% of the animals were totally protected from seizures. The percentage of animals totally protected increased to 88% after 7 doses. This pronounced increase in the anticonvulsant activity was not related to alterations in the plasma concentrations of VPA or its major active metabolites. Furthermore, determination of VPA and its metabolites in the substantia nigra after a single and repeated administration yielded the same data, again indicating that the increase in the anticonvulsant activity was not due to drug accumulation. In contrast to the marked increase in the anticonvulsant efficacy of VPA during short-term treatment, wet-dog shake behaviour induced by a single injection of the drug was significantly attenuated after repeated dosing, indicating that the anticonvulsant effect of VPA and the wet-dog shake behaviour induced by the drug were not mediated by the same mechanism. This was substantiated by experiments with one of the major metabolites of VPA in rat plasma, trans-2-en-VPA, which had approximately the same anticonvulsant efficacy as VPA but did not induce wet-dog shakes in rats.

A method to prepare isolated hepatocytes from the small amounts of liver or liver fragments obtained from the human fetus following therapeutic abortion in early pregnancy is described. The hepatocytes have been characterized by electron microscopy and by their ability to dealkylate the benzodiazepine drug prazepam. The preparation of hepatocytes from a liver sample obtained from a kidney transplant donor is described and discussed.

The in vitro protein binding of diazepam and its major plasma metabolite, N-desmethyldiazepam (DMD), was measured in women at full term of pregnancy, in mixed cord plasma (fetal plasma), and in control subjects. The free fraction of both drugs was determined by ultrafiltration at 37°. The mean free fraction of diazepam was 0.023 ± 0.0043 in control subjects and rose to 0.040 ± 0.0071 in women at term, whereas in their fetuses the free fraction was 0.021 ± 0.0057. The unbound fraction of DMD was 0.030 ± 0.0084 in control subjects, 0.052 ± 0.015 in women at term, and 0.035 ± 0.010 in fetal plasma. The free fractions of both compounds differed in women at term and in their fetuses. The higher free fraction of the two drugs in maternal plasma than in cord plasma may be explained by elevated maternal concentrations of free fatty acids (and triglycerides), which may act as displacing agents in maternal plasma. The higher binding of these drugs in fetal plasma than in maternal plasma may explain their cumulation in vivo and may be responsible for the frequently observed adverse effects of maternal diazepam treatment on the newborn infant. Clinical Pharmacology and Therapeutics (1983) 34, 220–226; doi:10.1038/clpt.1983.156

In vitro experiments using whole rat embryo cultures show that all -trans retinoic acid (all- trans RA) administered at low concentrations (30 ng/ml culture medium) is 10 times more active than 13-cis retinoic acid (13-cis RA) and 3 times more active when administered at high concentrations (1000 ng/ml culture medium). Morphological investigation of the embryos shows that both substances directly influence embryonic development in an identical manner. Isomerisation products of the administered compounds (all-trans RA from 13-cis RA and vice versa) were detected by HPLC both in the culture medium and the embryo. Correlation of embryonic retinoid concentration with the observed effects led us to suggest that the isomerisation to all- trans RA is crucial in regard to 13-cis RA-induced abnormal embryonic development. A 100% effect can be induced in vitro with very low amounts of all- trans RA (7.2 ng/g) in the embryo.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to be efficiently transferred to mouse neonates and offspring by lactating mothers. During the first 2 postnatal weeks the pups received doses of TCDD via the milk which were, on a body weight basis, similar to those which had been administered prenatally to their mothers. The distribution of TCDD in the offspring (high in liver, low in other tissues) was similar to that found in the maternal organism. Maternal TCDD levels rapidly decreased during the lactation period while tissue levels in the nursing pups increased, resulting in offspring tissue levels which greatly exceeded those of their mothers at the respective 3-week periods after birth. The postnatal development of pups from mothers treated on days 14-17 of gestation and nursed by untreated foster mothers was studied. Postnatal mortality was increased. Surviving animals did not exhibit visible signs of abnormal development, although the reduced number of pups per litter may have contributed to this apparently normal development. In small rodents excretion into milk constituted a major pathway for the elimination of maternal TCDD. Whether the same holds true for man is still unknown, but the measurement of TCDD levels in breast milk may be an appropriate and practical method for the assessment of human exposure to this substance.

The association of fetal and neonatal distress, birth measurements, major malformations, and minor anomalies was studied prospectively in 14 infants of women with epilepsy who were receiving valproic acid (VPA) monotherapy and in 12 infants of women with epilepsy who were receiving VPA in combination with other anticonvulsant drugs. Comparison was made with 26 matched-pair controls and 116 controls from a larger study of antiepileptic drugs. During the first trimester, total VPA serum concentrations were well above therapeutic levels (100 to 184 micrograms/ml) in two women receiving high VPA doses (2000 and 1500 mg daily). Although dosage remained the same, serum concentrations decreased during pregnancy to therapeutic levels (33.9 to 57.0 micrograms/ml). The VPA percent free fraction increased in the third trimester and was threefold higher at birth. Almost half of the infants exposed to VPA monotherapy were distressed during labor, and 28% had low Apgar scores. Fetal and neonatal distress may be caused by the high VPA percent free fraction during labor and at birth. Mean body measurements at birth after VPA monotherapy were comparable to those in the matched control group, but were reduced in the group of infants receiving VPA combination therapy. Four infants exposed to VPA monotherapy were born with major malformations. The median number of minor anomalies was four times higher in infants whose mothers received VPA alone or VPA combination therapy than in controls. Seven infants had a pattern of craniofacial and digital anomalies that was distinctly different from that observed after in utero exposure to other anticonvulsant medications. The occurrence of major malformations and the number of minor anomalies may be dose related.

A total of 35 newborns whose mothers had been treated with either primidone (PMD), phenobarbital (PB) or a combination of one of these drugs with other antiepileptic drugs were included in this study. Fetal/maternal serum concentration ratios at birth, milk/serum concentration ratios and neonatal half-lives were determined for PMD, PB and phenylethylmalondiamide (PEMA). Steady-state serum levels of PMD in 2 nursed infants were 2.5 and 0.7 micrograms/ml, respectively, for PEMA values of 1.4 and 0.4 micrograms/ml. PB steady-state concentrations ranged between 2.0 and 13.0 micrograms/ml (6 infants). Maternal PB serum protein binding did not change during and after pregnancy. Neonatal free-fraction values at birth were similar to maternal values: 63.2 +/- 17.2% (n = 11). In the postnatal period, however, PB free-fraction values rose to more than 90% in some infants. In one case, neonatal free concentrations of PB were even higher during the 1st week after birth than the corresponding maternal values. Symptoms of sedation were observed in these neonates for which elevated free-fraction values of PB could be responsible. Behavior problems, such as withdrawal symptoms, were observed in neonates who eliminated PMD with short half-lives, while other infants with longer PMD half-lives or slower elimination because of nursing showed no such symptoms.

Fourteen epileptic women treated with primidone, either alone or in combination with other antiepileptic drugs, were studied prospectively during their pregnancy. Plasma levels of primidone and its metabolites were monitored and correlated to findings in the offspring. Maternal serum concentrations of primidone and metabolites were generally low during pregnancy. The levels of its main metabolites--phenobarbital and PEMA--were found to drop within the first month of pregnancy in two cases. The plasma concentrations remained low until birth and rose sharply thereafter. The phenobarbital/primidone ratio (mean 0.84) and PEMA/primidone ratio (mean 0.56) in pregnant patients were found to be lower than in non-pregnant patients, except when primidone was given in combination with phenytoin in which case the expected phenobarbital/primidone (mean 2.5) and PEMA/primidone (mean 1.5) ratios were found. A ventricular septal defect was found in one of the offspring of the fourteen mothers and five children had microcephaly. There was a high incidence of poor somatic development with dystrophy (n=3) and short stature (n=2). Head circumferences (n=8), lengths (n=4) and/or weights (n=8) were below the 10th percentile in a number of children. Four children showed marked facial dysmorphy. Our preliminary data suggest that primidone intake during pregnancy may be important in the pathogenesis of minor anomalies and in the induction of poor somatic development.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.

The antiepileptic drug valproic acid is a putative human teratogen as suggested by retrospective studies (Robert and Rosa 1983; Lindhout and Meinardi 1984), a number of case reports (reviewed in Tein and McGregor 1985) and prospective investigations (Koch et al. 1983; Jager-Roman et al. 1986; Nau et al. 1981a). The use of thus drug for seizure control during pregnancy was shown to result in an increased incidence of neural tube defects, in particular spina bifida aperta in addition to numerous other malformations, particularly of the face and the skeletal structures. Clinical doses range between 10–40 mg/kg/day administered as a single daily dose (Rowan et al. 1981) or in divided doses (Gjerloff et al. 1984). The therapeutic range is said to be between 50–100 µg/ml, although peak drug levels may be much higher: Rowan et al. (1981) reported peak levels of 180 µg/ml in a patient treated with a once-a-day dosing regimen. We have recently measured peak levels of 120 and 184µg/ml in two epileptic patients during the first trimester of pregnancy (Jager-Roman et al. 1986).

(1) Following the administration of the highly toxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to pregnant mice, exceedingly low concentrations of this substance were found in the embryo and fetus between gestational days 11 and 18 (between 0.04 and 0.14% of the maternal dose/g tissue). (2) Tissue levels of tCDD in embryos on days 9 and 10 exceeded those of later gestational stages. (3) TCDD levels in fetal livers were 2--4 times higher than those in other fetal organs. (4) Maternal liver contained the highest concentrations of TCDD (4 -10% of the dose/g). A comparison of the results obtained following oral, s.c. and i.p. applications indicated that there was no major first pass effect involved following oral administration. (5) Placenta and other extrahepatic maternal organs exhibited TCDD levels, which were approximately 1, embryos and features 2 orders of magnitude lower than maternal liver concentrations. (6) Good correlation was found between the doses applied (5, 12.5 and 25 microgram/kg) and the tissue levels observed. (7) A single dose of 25 microgram/kg given on day 10 or 5 doses of 5 microgram/kg, 1/day from days 7-11, resulted in higher levels of TCDD in embryos on day 13 than did a singly dose of 25 microgram/kg on day 7; cleft palate was induced at different rates by these treatment schedules.

The dosage-regimen-dependent teratogenicity as well as plasma and tissue levels of the antiepileptic drug valproic acid (VPA) were studied in the mouse by comparing various injection regimens and infusion of the drug via implanted osmotic minipumps. Concentrations of 225-248 micrograms VPA/ml maternal plasma (about 2X above the therapeutic concentration range) and 70-75 micrograms VPA/g gestational material (on gestation Day 8) resulted in a significant incidence of neural tube defects (exencephaly in the mouse). Similar effects were produced if those concentrations were reached several times after multiple injections or by steady-state application via implanted pumps. A single injection was less effective than multiple injections, although drug accumulation did not occur. The doses (or area under the concentration-time curve values) did not correlate with the teratogenic response of the different administration regimens: much higher (factor 10) doses were needed with the infusion regimen to produce exencephaly rates comparable to those obtained with the injection regimen. The pattern of embryotoxicity was also schedule dependent: steady-state concentrations produced predominantly embryolethality and fetal weight retardation, while intermittent injections produced a high incidence of exencephaly (up to 60% of live fetuses). The dose of VPA (and the areas under the concentration-time curves) correlated with the embryolethality and fetal weight retardation of the drug, while the peak or steady-state concentrations reached in mother and gestational material correlated with the incidence of neural tube defects.

The exposure of infants to nicotine via milk of smoking mothers or via inhaled side-stream smoke ("passive smoking") was evaluated. Newborn infants nursed by smoking mothers and unexposed to passive smoking showed measurable serum concentrations of nicotine (0.2 to 1.6 ng/ml) and its main metabolite, cotinine (5 to 30 ng/ml), and also excreted measurable amounts of nicotine and cotinine in their urine: the ratio of nanograms of nicotine/milligrams of creatinine (N/C ratio) ranged from 5.0 to 110 (median 14), and the corresponding ratio of nanograms of cotinine/milligrams of creatinine (C/C ratio) from 10 to 555 (median 110). Infants of the same age nursed by nonsmoking mothers did not excrete measurable amounts of the two substances except in one case. Older and non-breast-fed infants exposed only to passive smoking had N/C ratios in the range of 4.7 to 218 (median 35) and C/C ratios in the range of 117 to 780 (median 327 ng/mg). Infants exposed to passive smoking and to smoke via breast milk had N/C ratios in the range of 3.0 to 42 (median 12) and C/C ratios in the range of 225 to 870 (median 550). The significant serum concentrations and urinary excretion rates of nicotine in the breast-fed infants of smoking mothers suggest that nursing contributes to the nicotine exposure of these neonates. In older infants, the wide variation of cotinine excretion values did not allow separate evaluation of the two exposure routes.

We present a fully automated high-pressure liquid chromatographic (HPLC) assay for simultaneous quantitation of five antiepileptic drugs [ethosuximide (ES), primidone, phenobarbital, phenytoin, and carbamazepine] and their major metabolites [phenylethylmalondiamide (PEMA), carbamazepine-10,11-dihydro-10,11-diol (CBZD), and carbamazepine-10,11-epoxide (CBZE)] in serum samples without sample pretreatment. Two other metabolites, p-hydroxyphenobarbital (HPB) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), can be separated as well. Interference with serum constituents prevents a quantitative determination. Serum samples are injected onto a pre-column, the biological matrix is eluted with water, and the compounds of interest are subsequently separated on an analytical column. The anticonvulsants are eluted at 60 degrees C with a mobile phase containing acetonitrile:water (21:80 by vol) at a flow rate of 1.0-2.0 ml/min (flow-step gradient). The eluted compounds are monitored at 200 nm. Each analysis requires about 30 min. Analytical recoveries varied from 90 to 98%. Within-day coefficients of variation (CVs) ranged from 0.9 to 4.9%, between-day CVs from 2.8 to 7.6%. The lower limit of detection for all drugs is 0.2 microgram/ml serum, with a sample size of 100 microliters. At therapeutic concentrations we achieved baseline separation for all eight compounds, and there was no interference from other drugs.