Effect of folic acid on the pharmacokinetics of acutely administered phenytoin in pregnant and nonpregnant rats

The concentrations of both total and free phenytoin in the plasma of epileptic women tend to decrease during pregnancy, suggestive of a pregnancy-associated increase in the metabolic clearance of the drug. On the other hand, the metabolic clearance of free (unbound) phenytoin decreases during pregnancy in rats. One possible reason for this species difference is the routine dietary supplementation of folic acid in human pregnancy and the apparent ability of folic acid to lower phenytoin plasma concentrations even in nonpregnant humans. The purpose of this investigation was to determine the effect of treatment with folic acid on the pharmacokinetics of phenytoin in pregnant and female nonpregnant rats. In one experiment, the treated animals received folic acid in the drinking water, approximately 100-150 micrograms/kg/d, for 19 d. There was no apparent difference between the treated and untreated rats in the pharmacokinetics of a 10-mg/kg iv dose of phenytoin (which was administered to the pregnant rats on the 20th day of gestation), regardless of pregnancy status, In another experiment, pregnant and female nonpregnant rats received either folic acid, 400 micrograms/kg/d, or an equal volume of the solvent only, by gastric intubation for 19 d. The next day (which was the 20th day of gestation for the pregnant rats), the animals received an intravenous injection of phenytoin, 30 mg/kg. Again, pretreatment with folic acid had no apparent effect on the pharmacokinetics of phenytoin in both pregnant and nonpregnant rats. However, the results of this investigation confirm previous observations of dose-dependent phenytoin pharmacokinetics in rats and of decreased clearance of free phenytoin in late pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pregnancy on the pharmacokinetic disposition of two aromatic retinoids (etretinate and acitretin) in the rat. I. Intravenous studies

The influence of pregnancy on the disposition of two related aromatic retinoids (etretinate and its metabolite, acitretin) was evaluated in a rodent model. The plasma concentrations of etretinate and acitretin were monitored by a specific HPLC method following iv bolus doses to 17-day pregnant and nonpregnant Sprague-Dawley rats. The systemic clearance of etretinate was significantly lower in the pregnant rats compared to nonpregnant controls (129 vs. 185 ml/hr, respectively; p less than 0.05). This decrease was entirely due to a lower formation clearance of acitretin (acid) from etretinate (ester) in the pregnant animals (96 vs. 146 ml/hr; p less than 0.05). The in vitro plasma hydrolysis rate of etretinate was also lower in the pregnant animals. By contrast, the systemic clearance of acitretin was greater in the pregnant compared to the nonpregnant control animals (184 vs. 145 ml/hr, respectively; p less than 0.05). The apparent volumes of distribution for both retinoids were comparable in the pregnant and nonpregnant animals. Etretinate infusions in nonpregnant animals yielded systemic clearances (mean = 164 ml/hr) which were similar to those obtained for bolus dose experiments. Acitretin clearance increased (plasma levels decreased) following acitretin infusion to nonpregnant rats over the time course of the infusion. The results illustrate the marked effect of pregnancy on the disposition of these retinoids and suggest that acitretin may pose less of a teratogenic hazard than the parent compound etretinate.     

Kinetics of Phenytoin in Pregnant and Non-pregnant Rats

Abstract The plasma kinetics of phenytoin were studied in pregnant and nonpregnant rats after a single intravenous dose. The apparent volume of distribution was greater in the pregnant rats resulting in lower plasma concentrations in these rats within the first hour of injection. The beta half-life in pregnant rats (5.0 hours) was prolonged compared to the non-pregnant rats (2.2 hours). The plasma clearance values which take the altered volume of distribution into account, showed a smaller but still significant difference between the two groups of rats. This indicates a lower capacity of the pregnant rats to metabolize the drug.     

Microdialysis sampling of carbamazepine,phenytoin and phenobarbital in subcutaneous extracellular fluid and subdural cerebrospinal fluid in humans: an in vitro and in vivo study of adsorption to the sampling device

The purpose of the study was to determine if binding of the drugs to the sampling equipment during microdialysis would influence the results for carbamazepine, phenytoin and phenobarbital. In vitro experiments with microdialysis catheters and separate parts of catheters were performed to estimate the degree of drug binding to the dialysis equipment. A mathematical model to calculate drug binding and recovery is proposed. In vivo protein unbound carbamazepine concentrations in subcutaneous extracellular fluid at different flow rates (6 patients), unbound carbamazepine (1 patient) and unbound phenobarbital (I patient) in subdural cerebrospinal fluid and subcutaneous extracellular fluid were estimated and the in vivo data were compared to the in vitro results and data generated by the mathematical model. Binding to the soft outlet polyurethane tubing was extensive and variable for phenytoin, which precluded in vivo testing, but limited and more predictable for carbamazepine and phenobarbital. None of the three compounds bound to the hard internaltubing. Phenytoin and phenobarbital did not bind to the dialysis membrane, while a small degree of binding may be present for carbamazepine. In vivo estimates of carbamazepine protein unbound subcutaneous extracellular concentrations by microdialysis, adjusted for binding to the plastic tubing, were 81% of protein unbound plasma concentrations. In single case studies, subdural cerebrospinal fluid and subcutaneous extracellular levels of carbamazepine and phenobarbital were similar and when corrected for binding to the plastic tubings they were also close to protein unbound plasma concentrations. Microdialysis can be used for reliable estimations of protein unbound carbamazepine and possibly phenobarbital concentrations when drug binding to the plastic tubing is considered. Reliable estimation of unbound phenytoin is not possible at present.     

Transfer of 2,4,5,2',4',5'-hexachlorobiphenyl across the in situ perfused guinea pig placenta

The transplacental crossover of 14C-2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) from the maternal circulation to the fetal side of the placenta was examined in intact fetuses and following the in situ perfusion of the guinea pig placenta. Fetal, late pregnant, and nonpregnant female guinea pig lipoprotein profiles and the association of 6-CB with these plasma constituents were also determined in vivo. Very low density lipoprotein (VLDL) concentrations were 10-fold higher in fetal than in maternal plasma, and the great majority of 6-CB which was transferred to intact fetuses became associated with this plasma fraction. 6-CB was found primarily in association with low density lipoproteins (LDL) in nonpregnant animals. In the late pregnant guinea pig, 6-CB became primarily associated with plasma protein in spite of circulating protein concentrations lower than those seen in the nonpregnant state. No differences in the levels of the three plasma lipoprotein classes were observed between pregnant and nonpregnant animals. It was found that an amount of 6-CB similar to that found in intact litter mates crossed the perfused placenta over the same time period. Despite the much higher VLDL concentrations on the fetal side of the placenta and the association of 6-CB with VLDL in intact fetuses, addition of 1,000 mg/dl VLDL to the 5.4% bovine serum albumin perfusion medium failed to influence the magnitude of 6-CB crossover. 6-CB crossover was influenced by protein concentration in the perfusion media in a concentration-dependent fashion. It is hypothesized that 6-CB and free fatty acids traverse the placenta and are retained by the fetus via similar mechanisms.     

Pharmacokinetics and renal handling of enprofylline in pregnant rats.

The pharmacokinetics and renal handling of enprofylline during pregnancy were investigated in Sprague-Dawley rats. Significant differences in the pharmacokinetic parameters of enprofylline were observed between nonpregnant rats and pregnant rats on the 20th day of gestation: volume of distribution was higher, and systemic clearance was lower in pregnant rats. Parameters obtained from rats at 7 days postpartum were the same as those obtained from nonpregnant rats. There were no significant differences in the fraction of urinary excretion of enprofylline between nonpregnant and pregnant rats. The protein binding of enprofylline in the plasma of pregnant rats was significantly lower than in nonpregnant rats, as a decrease in the albumin concentration consequentially reduced the binding capacity of enprofylline. The volume of distribution for unbound enprofylline in pregnant rats was not significantly different from nonpregnant rats, although a significant decrease was observed in pregnant rats in the systemic clearance for unbound enprofylline. In addition, the clearance ratio was lower in pregnant rats (2.8) when compared with nonpregnant rats (6.4). Pregnancy caused a decrease in the apparent maximum capacity of transport (Vmax) from 29.9 to 20.8 micrograms/min and in the Michaelis-Menten constant (KM) from 2.59 to 2.26 micrograms/ml, indicating that the tubular secretion ability of enprofylline becomes reduced during pregnancy. These results suggest that changes that occur in the plasma protein binding behavior and in renal handling as a result of pregnancy are primary factors influencing the disposition of enprofylline during pregnancy.     

Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.     

Plasma protein binding of drugs in pregnancy

The degree of binding to plasma proteins is an important determinant of drug disposition and response. Normal human pregnancy is associated with concentration of plasma proteins, free fatty acids and possibly other endogenous substances interfering with drug binding. The possibility of an associated change in plasma binding capacity therefore needs to be taken into consideration. Experimental studies conducted mostly in vitro have shown that the plasma protein binding of many (but not all) drugs is decreased during pregnancy, particularly during the last trimester. This phenomenon should be taken into account when interpreting serum concentrations of total (free + protein-bound) drug in clinical practice. Notable examples of drugs whose unbound fraction increases during pregnancy include diazepam, valproic acid, phenytoin, phenobarbitone, salicylic acid, pethidine, lignocaine, dexamethasone, sulphafurazole and propranolol. For many drugs, important differences have been demonstrated in the degree of protein binding between maternal and cord plasma. In some cases, this may provide an explanation for the finding of marked differences in total drug concentration between maternal and fetal plasma at the time of delivery.     

Plasma levels and protein binding of phenytoin during exercise in man: the effect of elevated free fatty acids

Five healthy subjects performed submaximal physical exercise approximately 20 h after a single oral dose of phenytoin (5 mg/kg of the sodium salt). The plasma levels of free fatty acids (FFA) increased 2- to 3-fold in the post-exercise period. In spite of this, the degree of plasma binding of phenytoin and its total concentration in plasma were unaffected. Thus FFA at the levels reached (1.5-2.9 mEq/l), did not displace phenytoin from its binding sites on albumin. Furthermore, during the FFA peak the plasma protein binding of warfarin, as measured in vitro, did not decrease as compared to the pre-exercise period. These findings contrast to previous observations in rats and dogs, where FFA caused a considerable displacement of warfarin and phenytoin at relatively low FFA/albumin molar ratios.     

Effect of anticonvulsant drugs on (35S)t-butylbicyclophosphorothionate binding in vitro and ex vivo

Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-gamma-vinyl GABA), we studied their abilities in vitro to displace (35S)t-butylbicyclophosphorothionate (35S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on 35S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 microM, P less than 0.001), ethosuximide (500 microM, P less than 0.001), and phenytoin (40 microM, P less than 0.001) decreased the specific 35S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 microM), the number of binding sites decreased and the binding affinity (P less than 0.05) in the cortex increased. Other anticonvulsants did not modulate 35S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied.     

Comparative distribution and embryotoxicity of acetylsalicylic acid in pregnant rats and rhesus monkeys.

Acetylsalicylic acid was given orally to pregnant rats on gestation Days 9–12 and to pregnant monkeys on Days 23–32 and to nonpregnant females of both species at doses of 100 and 150 mg/kg twice daily. Concentrations of salicylic acid were determined in maternal plasma, embryo, decidua, placenta, and chorionic and amniotic fluids at 1, 2, 4, 8, and 17 hr after the last dosage. Total salicylic acid concentrations in plasma after comparable doses were generally higher in rats than in monkeys and in nonpregnant than in pregnant rats, but concentrations varied in pregnant vs nonpregnant monkeys. Unbound salicylate in rat plasma ranged between 30 and 50% of the total plasma concentration and was closely paralleled by the concentration in the rat embryo. Unbound salicylate in monkey plasma was lower, ranging between 17 and 30% of the total plasma concentration and was to some degree paralleled by the concentration in the monkey embryo. The greater embryotoxicity of acetylsalicylic acid in the rat than in the monkey was correlated with higher concentrations and longer duration of concentrations in the respective embryos on a day-to-day basis.     

Effect of low doses of heparin on the plasma binding of phenytoin and prazosin in normal man

Summary The effect of low doses of heparin on the binding of phenytoin and prazosin to plasma proteins was evaluated in four normal subjects. Heparin activates the hydrolysis of triglycerides in plasma. The ensuing increase in non-esterified fatty acids (NEFA) was more marked in vitro than in vivo and increased the free fraction (FF) of phenytoin and prazosin in plasma. The higher FF caused a change in the plasma to whole blood ratio (P/B ratio) of both drugs. The changes in FF and P/B ratio after heparin were small, but could be of significance in pharmacokinetic studies.     

In vitro protein binding studies with BMS-204352: lack of protein binding displacement interaction in human serum.

BMS-204352, a maxi-K channel opener, is currently under development for the treatment of stroke. Protein binding of BMS-204352 was determined in sera from several species, namely, rat, monkey, dog, and human. Data indicated that the compound was shown to be highly protein bound in serum from all species (ca. 99.6%). In order to test for the potential for drug-drug interactions and competitive displacement of BMS-204352 by diazepam, phenytoin, propranolol, and warfarin, in vitro experiments were performed using spiked human serum and ex vivo human plasma samples. Protein binding was determined using equilibrium dialysis for 4 h at maximal therapeutic concentrations for each drug alone or in appropriate combination in spiked serum samples. Ex vivo samples from a clinical BMS-204352 study (0, 1, and 24 h) were dialyzed separately after addition of diazepam, phenytoin, propranolol, or warfarin. Drug content in biological matrices was measured for radioactivity using liquid scintillation counting. Results indicated that (1) addition of diazepam, phenytoin, propranolol, or warfarin did not alter the free fraction of BMS-204352; (2) BMS-204352 did not displace diazepam, phenytoin, propranolol, or warfarin from their protein binding sites, and (3) comparison of ex vivo plasma samples after BMS-204352 dosing indicated no impact of BMS-204352 and/or its metabolites on the free fraction of diazepam, phenytoin, propranolol, or warfarin. In conclusion, the potential for a drug-drug interaction due to alterations in protein binding with BMS-204352 is unlikely.     

Hepatic extraction of endogenous inhibitors of drug binding to serum protein in the pregnant rat

Significant decreases in the serum protein binding of a fluorescent dye, 1-anilino-8-naphthalenesulfonate (ANS), and salicylic acid (SA) were observed in pregnant rats compared to that in nonpregnant (control) rats. A significant difference in the serum protein binding of ANS and SA between serum samples taken at the hepatic vein and portal vein or femoral artery was also observed in the pregnant rats, while such a sampling site difference in the serum protein binding was not observed in the control rats. In the pregnant rats the affinity of ANS binding to the primary binding site in the serum from the hepatic vein was approximately 70% higher than that in the case of serum from the portal vein. The hepatic extraction of nonesterified fatty acids (NEFA) was also determined, and the extraction ratios in the control and pregnant rats were 0.55 and 0.31 respectively. We concluded from these findings and other evidence that certain endogenous inhibitors of drug binding to serum protein (such as NEFA), which increase during pregnancy, were extracted efficiently by the liver and that the difference in the serum protein binding of ANS and SA between serum samples taken at the hepatic and portal veins or femoral artery in the pregnant rats may be explained by the hepatic extraction of endogenous inhibitors. Our present results support the previous finding by Chou et. al. [Int. J. Pharm. 18, 217 (1984)] that in pregnant rats the serum protein binding of phenytoin is greater in the hepatic vein than that in the femoral vein.     

Prediction of in vivo hepatic clearance from in vitro data using cryopreserved human hepatocytes

1. Cryopreserved human hepatocytes were used to predict in vivo hepatic clearance (CL hepatic) from estimates of in vitro intrinsic clearance (). 2. was estimated for phenytoin, valproic acid, carbamazepine, theophylline, quinidine and procainamide after their addition to hepatocytes suspended either in human serum or in serum-free media. was estimated from in vitro concentration versus time data fitted to a monoexponential decay model. was estimated from concentrations measured at four time points and from just two-point measures, namely the initial concentration (C 0) and the final concentration measurement (C last). 3. Predicted CL hepatic was within twofold of reported in vivo values of CL hepatic for all substrates. Moreover, predictions were not significantly different whether derived from hepatocytes suspended in serum or in serum-free medium. 4. Two-point estimates of were just as accurate in predicting CL hepatic as were multipoint estimates of. 5. Although the data set was limited, the findings suggest that the measurement of the disappearance of xenobiotics from serum or serum-free media in which primary human hepatocytes have been suspended provides a physiologically relevant estimate of hepatic clearance that can be employed early in the drug development process to eliminate xenobiotics with unacceptable clearances.     

Effects of salicylate on maternal and fetal phenytoin pharmacokinetics in rats

The effects of salicylate on maternal and fetal phenytoin pharmacokinetics were investigated in 19-day pregnant Sprague-Dawley rats after a 5 mg/kg bolus injection of 14C-phenytoin was given with and without (control) prior salicylate (75 mg/kg) treatment. Maternal plasma and fetal whole body samples were obtained at various times after the phenytoin bolus and evaluated simultaneously using a three-compartment maternal-fetal model. An increase in maternal plasma phenytoin clearance, central compartment volume, and over-all apparent volume of distribution with no change in the terminal first order hybrid disposition rate constant (beta) was observed in the salicylate-treated rats. These dispositional changes were consistent with the elevations in serum free or unbound phenytoin concentrations observed in vitro in the presence of salicylate. The maternal-to-fetal clearance of phenytoin was faster in the rats given sodium salicylate. However, the maternal-to-fetal (k13) as well as fetal-to-maternal (k31) phenytoin transfer rate constants were not altered by the salicylate treatment. Additionally, no changes in the apparent fetal phenytoin volume of distribution or area under the fetal phenytoin concentration-time curves were seen. Although the maternal pharmacokinetics of phenytoin were altered by sodium salicylate co-administration, the extent of fetal exposure to phenytoin did not change.     

Disposition of levo-[3H]Cocaine in pregnant and nonpregnant mice

levo-[³H]Cocaine (10 mg/kg free base) was injected ip to pregnant and nonpregnant mice. Concentrations of cocaine were examined at 15 min and 1,3, and 6 hr. In all tissues, peak concentrations were attained at 15 min. In pregnant mice, the concentrations in decreasing order were: uterus, placenta, spleen, kidney, fat, liver, lung, brain, spinal cord, heart, muscle, eye, and plasma; the 15-min tissue/plasma concentration ratio for the brain was 12.4 and varied from 3 to 68 for other tissues. Fetal brain and liver, in comparison with maternal organs, accumulated much smaller amounts. In several maternal organs, cocaine declined to low concentrations at 3 hr and to negligible levels at 6 hr. The 15-min tissue concentrations in nonpregnant compared to pregnant females were significantly lower in several organs; differences were less marked at 1 and 3 hr. During the first hour, excretion of total radioactivity was higher in the urine of nonpregnant than pregnant females. Cocaine was extensively metabolized since unchanged cocaine levels as percentage of total urinary radioactivity at 1 hr were 7.8 (pregnant) and 1.5 (nonpregnant). SKF 525-A (50 mg/kg ip; 1 hr prior to cocaine) caused marked tissue elevation of unchanged cocaine at 15 min in nonpregnant and at 15 min and 3 hr in pregnant animals. These data suggest that pregnancy alters pharmacokinetics of cocaine.     

Associating in vitro target binding and in vivo CNS occupancy of serotonin reuptake inhibitors in rats: the role of free drug concentrations

The present study aimed at investigating the theory that free (unbound) active site concentrations are the best predictors of target binding of compounds blocking the serotonin transporter (Sert) in the central nervous system (CNS). Thirteen serotonin reuptake inhibitors were evaluated for their Sert-binding affinities in vitro and in vivo in rats together with their unbound fractions in plasma and brain. Cortical Sert occupancy was used in vivo to acquire EC??-estimates from total plasma, free plasma, whole brain, and free brain concentrations after acute drug administration. The in vitro-in vivo Sert occupancy analyses showed that the best correlation was achieved when unbound brain concentrations were employed. Unbound brain concentrations also provided a better correlation when compared with unbound plasma concentrations, which could be related to lack of equilibrium between plasma and brain at time of measurements or involvement of active brain efflux processes. In addition, brain-free fractions were shown to be directly correlated to the lipophilicity of the compounds. These data emphasize the use and impact of applying free fraction data in assessment of pharmacological in vitro-in vivo correlations and demonstrates its use to validate in vivo Sert occupancy as pharmacodynamic marker for serotonin reuptake inhibitors in rats.     

Bioavailability and pharmacokinetics of phenytoin during pregnancy

Summary Five epileptic women needing to commence phenytoin therapy during pregnancy received a single intravenous and a single oral dose of phenytoin several days apart before starting regular intake of the drug. Plasma phenytoin concentration — time data were analysed by three different pharmacokinetic techniques. However assessed, the mean oral bioavailability of the drug proved to be about 90% of the intravenous bioavailability. This finding makes it unlikely that impaired bioavailability accounts for the increase in oral phenytoin dosage necessary in pregnancy to maintain plasma phenytoin concentrations at pre-pregnancy values. Phenytoin clearance in the pregnant subjects was approximately double the published values for phenytoin clearance in nonpregnant persons. This suggests that increased (metabolic) clearance accounts for the increased phenytoin dosage requirement of pregnancy.     

Effects of penicillins on binding of phenytoin to plasma proteins in vitro and in vivo

Effects of penicillins on the binding of phenytoin to plasma proteins were examined in vitro and in vivo. The results from in vitro studies showed that the penicillins including oxacillin and dicloxacillin were effective in displacing phenytoin from its binding sites. In vivo, the total phenytoin concentration in serum decreased during penicillin administration, while the free phenytoin concentration increased. As a result, penicillins caused a significant increase in the apparent volume of distribution and in the total body clearance of phenytoin. These results can be explained on the basis of the displacement of phenytoin from its plasma protein binding site by penicillins.