Developmental toxicity and pharmacokinetics of oral and intravenous phenytoin in the rat

Reproductive Toxicology, Vol. 4, pp. 191-202, 1990

0890-6238/90 $3.00 + .00 Copyright© 1990PergamonPressplc

Printed in the U.S.A.

DEVELOPMENTAL TOXICITY AND PHARMACOKINETICS OF ORAL AND INTRAVENOUS PHENYTOIN IN THE RAT JEFFREY R. ROWLAND, PAMELA E. BtNKERD,and ANDREW G. HENDRICKX California Primate Research Center, University of California, Davis, California Abstract -- Correlations between oral and intravenous (i.v.) doses of phenytoin, maternal plasma levels, and subsequent developmental toxicity were examined in the Sprague--Dawley rat. Oral administration of 150 to 1500 mg/kg and i.v. administration of 25 to 100 mg/kg phenytoin from gestational days (GD) 8 to 17 resulted in a dose-dependent increase in maternal death and toxicity [impaired motor function, decreased maternal weight gain (oral dose only)], embryolethality, and intrauterine growth retardation, in addition to significant increases in craniofacial (1125 mg/kg oral; 75 mg/kg i.v.) and urogenital (1125 mg/kg oral) malformations. Pharmacokinetic sampling in oral and i.v. groups on GD 8-9 and 16-17 revealed significant increases in maternal drug exposure over the treatment period, as evidenced by 2- to 3-fold increases in total plasma phenytoin (bound + free) half-life, area under the concentration curve, peak concentration (oral dose only), and decreases in clearance. These findings emphasize the importance of pharmacokinetics in the evaluation of phenytoin-induced developmental toxicity. Key Words: Phenytoin; Sprague-Dawley; rat; developmental toxicity; pharmacokinetics; oral; intravenous; plasma.

solubility, phenytoin particle size, variation in type of dosing vehicle, and variable absorption of phenytoin across the intestinal mucosa (19) often lead to inconsistent correlations between dose, plasma concentrations, and developmental toxicity. Following intramuscular (i.m.) and subcutaneous (s.c.) dosing, similar dose/ plasma concentration inconsistencies arise due to precipitation and limited absorption of phenytoin at the injection site, while i.p. injection has been associated with increased embryonic:maternal exposure ratios and subsequent increases in embryolethality and teratogenicity (9,20-22). An attempt to minimize these confounding factors, with direct measurement of maternal plasma phenytoin levels, represents a more dependable approach when making intraspecies and interspecies comparisons concerning developmental toxicity. In this investigation, oral administration was chosen because it is the preferred clinical route, while a second means of dosing (i.v.) was selected in an attempt to circumvent the variable absorption difficulties encountered with the previously discussed methods of administration. Therefore, the current study was initiated 1) to examine the maternal disposition of oral and i.v. administered phenytoin at the beginning and end of organogenesis, and 2) to compare subsequent developmental toxicity of oral and i.v. doses of phenytoin in relation to the pharmacokinetic data.

INTRODUCTION

Epidemiologic and clinical studies suggest an approximate 2- to 3-fold increase in malformations in neonates exposed to phenytoin during the prenatal period (1,2). Rather than major malformations, a pattern of subtle developmental changes consisting of facial dsymorphogenesis, phalangeal hypoplasia, and growth and mental retardation characterize the fetal hydantoin syndrome (3). In order to further evaluate the developmental toxicity of phenytoin, pharmacokinetic analysis has become essential. In epileptic women taking phenytoin during pregnancy, periodic pharmacokinetic sampling is necessary in order to maintain phenytoin levels in the therapeutic plasma range of 10 to 20 ~g/mL (4,5). These therapeutic levels are necessary to control seizure activity, while minimizing maternal toxicity and the suspected risk of phenytoin-induced developmental toxicity. Although developmentally toxic "threshold" levels still remain uncertain, a positive correlation between increasing maternal plasma levels and the frequency of congenital malformations has been demonstrated (6,7). Numerous studies have demonstrated the developmentally toxic effects of phenytoin in mice (8-14), rats (15-17), and rabbits (18). Limited pharmacokinetic data, however, are available at the exposure levels used in these studies. With oral dosing, factors such as limited

MATERIAL AND METHODS

Address correspondence to: Andrew G. Hendrickx, Ph.D., California Primate Research Center, University of California, Davis, California 95616. Received 26 June 1989; Revision received 5 September 1989; Accepted 15 September 1989.

Teratology Sprague-Dawley rats (Bantin and Kingman, Fremont, CA) were housed in individual cages under 191

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Table 1. Reproductive outcome in rats treated with phenytoin during organogenesis: oral dosing Dose (mg/kg/day) 0 150 375 750 1125 1500

Pregnant animals (n)

Maternal deaths (n)

Maternal wt _gain (g)a (X ± SD)b

R esorptions (X % ±- SD)b

Fetal death (X % --- SD)b

8 (4)c 9(5) 8 8 8 (6) 4

0 0 0 0 2 3

11 ----- 16 --4 Z 17 --56 ± 264 --73 ----- 23 a --74 ----- 12a --51

14----- 17 8 ± 22 4 ± 4 2 ----- 5 38 ± 49 100

0 0 2± 5 3----- 9 14 ± 32

a(GD 20 - GD 8) weight minus gravid uterine weight. bValues are means of litter values. CNumber of dams (n) used in pharmacokinetic sampling. dp <~ 0.05.

Syracuse, NY: 80 to 100 mg/kg, i.m.) and xylazine (Rompun, Miles Laboratories, Elkhart, IN: 8 to 10 mg/kg, i.m.) for placement of carotid (oral/i.v. blood sampling) and jugular (i.v. dosing) catheters. For i.v. dosing, phenytoin was administered at levels of 25, 50, 75, and 100 mg/kg daily on GD 8 through 17. The volume of phenytoin (50 mg/mL, Dilantin TM , phenytoin sodium injection, USP, Parke-Davis, Morris Plains, N J) was adjusted to achieve the appropriate dosage for each group (0.1 to 0.4 mL) and was administered over a 1 min period. The i.v. controls received 1.0 mL/kg of 0.9% sterile saline (Invenex Laboratories, Chicago Falls, OH). To verify phenytoin concentration, samples were stored at 0 °C and analyzed by fluorescence polarization immunoassay (FPI) using the Abbott TDx TM drug analyzer (TDx TM Phenytoin, Abbott Laboratories, North Chicago, IL). Reported assay sensitivity is 0.5 Izg/mL (distinguishable from 0 Ixg/mL with 95% confidence), with a concentration range of 0 to 40 ixg/mL without sample dilution. Assay accuracy (average recovery) is 99.1 --- 1.60% in the range 2.5 to 40 Ixg/mL. Reproduceability of the assay shows a standard deviation of < 2% for intraassay variability and < 3% for interassay variability when tested in the range of 7.5 to 30 Ixg/mL.

controlled conditions of lighting (12 h light/12 h dark), temperature (22 _+ 1 °C), and relative humidity (4555%). Food (Purina Rat Chow, Ralston Purina Co.) and water were provided ad libitum. Virgin females weighing 255 --- 30 g were mated with fertile males ovemight. The presence of a copulatory plug or sperm in vaginal smears the following moming was indicative of a positive mating, and this day was considered gestational day (GD) 0. Dams were housed individually upon successful mating and randomly assigned to one of the oral or i.v. treatment groups listed in Tables 1 and 2, respectively. Phenytoin (2,2-diphenylhydantoin) was administered orally by gavage at levels of 150, 375, 750, 1125, and 1500 mg/kg daily on GDs 8 to 17. The oral phenytoin suspension was prepared by homogenizing 2,2-diphenylhydantoin sodium salt (Sigma Chemical Co., St. Louis, MO) in distilled water. Vehicle controls received distilled water (pH 11.0) by the same method. For each pregnant animal, dosage was based on the sodium salt concentration and calculated according to the daily maternal weight and a constant dosing volume of 10 mL/kg. On GD 6, animals were anesthetized with ketamine hydrochloride (Keteset, Bristol Laboratories,

Table 2. Reproductive outcome in rats treated with phenytoin during organogenesis: iv dosing Dose (mg/kg/day) 0 25 50 75

Pregnant animals (n)

Maternal deaths (n)

8 (4) c 8(4) 9(4) 8(4)

0 0 0 0

Maternal wt g a i n (g)a (X --- SD)b 22 21 19 9

± ±± ±

27 10 8 14

a(GD 20 - GD 8) weight minus gravid uterine weight. bValues are means of litter values ± SD. CNumber of dams (n) used in pharmacokinetic sampling.

R__esorptions (X % --- SD)b 10 7 7 33

± + ± ---

12 6 7 42

Fetal death (X % ± SD) b 0 2 --- 3 0 15 ~ 32

Phenytoin embryotoxicity and kinetics • J. R. ROWLANOEr AL,

193

Table 3. Pharmacokinetic parameters (X --- SD) of total plasma phenytoin in the rat on GD 8 and 17: oral dosing Dose (mg/kg/day)

N

tl/2 (h)

150 150 1125 1125

5 5 6 4

5 . 4 9 _+ 2.19 15.71 ± 9.53 a * *

(GD (GD (GD (GD

8) 17) 8) 17)

AUC (IJ,g h/mL) 205 906 1550 2113

± ± + -+

100 336 a 452 445

VOID (mL/kg)

CI (mL/h/kg)

6733 ± 3401 3542 -+ 1038 * *

995 185 772 550

__. 744 ___ 66 a ± 196 --- 115

CMAX (p,g/mL) 13.4 30.2 39.4 53,7

___ ± --±

3.6 4,7 a 16.6 11.9

TMAX (h) 8.8 9.6 6,4 6,4

___ 1.8 ± 3.6 --- 6.1 ± 6.5

aSignificant increase (P -< 0.05) over corresponding G D 8 parameter. *Indeterminable due to absent elimination phase,

Cross reactivity with p-hydroxydiphenylhydantoin (pHPPH) is reported as 9.67%. All animals were observed daily during treatment for signs of toxicity. Necropsies were performed on dams that died during the study. Dams were euthanized on GD 20 by CO 2 anesthesia and cervical dislocation. The gravid uterine weight, position and number of fetuses, and resorption sites were recorded. All viable fetuses were sexed, weighed, and measured (crown-rump length), and examined for external abnormalities. Fresh dissections were carried out on all fetuses according to the methods of Stuckhardt and Poppe (23). Fetal heads from one-half of the litter were examined by the razor-blade method of Wilson (24) following fixation in Bouin's solution. All skeletons were double-stained with Alcian Blue and Alizarin Red S (25) and evaluated for degree and normality of ossification (26,27). In treated and control fetuses, reduced ossification was noted if the following criteria were noted in one or more of the seven skeletal areas; 1) skull, -> 2 of 8 bones (frontal, nasal, parietal, interparietal, occipital, maxilla, mandible, and zygomatic arch) less than 75% control size; 2) steruebrae, -< 3 of 6 ossification centers; 3) thoracic vertebrae, -> 1 of 13 bilobed, unilateral, split, or nonossified centra; 4) lumbar vertebrae, -> 1 of 6 bilobed, unilateral, split, or nonossified centra; 5) pelvis, -> 1 of 5 bones not ossified; 6) metacarpals, < 3 of 4 ossification centers; and 7) metatarsals, < 4 of 5 ossification centers.

Pharmacokinetics The number of animals assigned to pharmacokinetics (PK) in the oral and i.v. dose groups is presented in Tables 3 and 4, respectively. Due to the long half-life ( t l / 2 ) of orally administered phenytoin, serial blood sampling in the oral dose groups was performed on two schedules as follows: Group A -- Pretreatment, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h following a single dose on GD 8 and 16 to obtain drug elimination data over a 48-h period; Group B -- Pretreatment, 0.5, 1, 2, 4, 8, 12, and 24 h following the first dose, and 1, 4, 8, 12, and 24 h following a second dose in order to examine drug accumulation data. Following i.v. dosing, samples were collected pretreatment, and 5, 15, and 30 min, 1, 2, 4, 6, 8, and 24 h posttreatment. Blood (0.2 mL) was collected into nonheparinized syringes and immediately transferred into Eppendorf tubes with 5.0 p,L of heparin, and centrifuged in an Eppendorf microcentrifuge at 7,000 × G for 2 min. The plasma was recovered and stored at - 8 0 °C until analysis. Total plasma phenytoin concentration was determined using the Abbott TDx drug analyzer. TM

Data analyses The mean fetal weights and measurements, percent resorbed, dead, and malformed fetuses, and maternal weight data were calculated for each litter within each treatment group, and the litter mean values were corn-

Table 4. Pharmacokinetic parameters (X -+ SD) of total plasma phenytoin in the rat: iv dosing Dose (mg/kg/day)

N

tl/2 (h)

AUC (p,g h/mL)

Vol D (mL/kg)

C1 (mL/h/kg)

CMAX (p,g/mL)

TMAX (h)

25 50 75 G D 17

4 4 4

0.81 ± 0 . 0 8 1.42 --- 0 . 1 3 3.48 ± 1.35

33 --+ 6 104 ___ 14 258 +-- 87

906 ___ 131 998 --+ 134 1433 ± 213

784 ± 146 489 ± 62 319 --- 118

2 3 . 9 --- 5,5 4 7 , 7 ± 8.4 7 4 , 0 ___ 30.3

0.08 + 0.0 0 . 0 8 __. 0 . 0 0 . 0 8 --+ 0 . 0

25 50 75

3 4 4

1.71 --- 0 . 4 3 a 3 . 2 4 --- 1.10 a 6.48 ± 2 . 9 0

113 ± 68 248 ± 20 a 638 --- 244 a

627 --- 234 944 ___ 333 1092 ± 262

279 ± 154 a 203 ± 15 a 130 --- 46 a

34.7 ± 8,5 55.5 ± 10.6 72.8 ± 13.3

0.08 ± 0.0 0.08 ± 0.0 0 . 1 9 ± 0.21

GD 8

aSignificant c h a n g e (P --- 0.05) c o m p a r e d to corresponding G D 8 parameter.

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T a b l e 5. E f f e c t s o f p h e n y t o i n t r e a t m e n t o f p r e n a t a l d e v e l o p m e n t in rats: oral d o s i n g Fetal weight (g) (X 4- SD) a

Dose (mg/kg/day) 0 150 375 750 1125

Male 3.81 3.36 2.78 2.27 1.93

± ± 4± ---

0.61 0.60 0.58 b 0.53 b 0.16 b

Fetal c r o w n - r u m p (cm) (X 4- SD)"

Female 3.36 3.26 2.61 2.19 1.80

----± ± 4-

Male

0.36 0.42 0.50 b 0.57 b 0.21 b

3.56 3.41 3.22 3.04 2.81

Female

_ 0.33 _ 0.28 _ 0.29 ___ 0.31 b 4- 0.15 b

3.42 3.36 3.14 2.98 2.82

± ± 4--4-

0.20 0.26 0.27 0.34 b 0.17 b

Reduced ossification

Malformed fetuses

(X % 4- SD) a

(X % --- SD) a

27.0 17.8 37.4 50.3 84.4

2.2 6.1 5.3 39.6 62.2

--4_+ 44-

34.5 20.6 37.1 44.1 31.2 b

_+ 4.1 4- 8.0 4- 8.1 -- 38.4 b 4- 19.4 b

aValues are means of litter values ± SD. bp <: 0.05.

pared with other treatment groups using a computerized Generalized Linear Model (GLM) for Analysis of Variance (ANOVA) and weighted Tukey's Standardized Range Test (SAS Institute, Inc., Cary, NC) with litter as the unit of analysis. Due to the possible influence of PK sampling on developmental toxicity, fetal parameters within the same dose groups from both PK and non-PK groups were compared (ANOVA and weighted Tukey's standardized range test). Total phenytoin (bound + free) plasma half-life (tl/2), area under the concentration curve (AUC), maximal plasma concentration (Cmax) and time of peak concentration ( T m a x ) , apparent volume of distribution (VOID), and drug clearance (C1) were examined in both oral and i.v. dosing regimens. PK parameters were calculated using the BASIC exponential stripping program, ESTRIP (28). Half-life values were calculated using the best fit (r 2) value of the elimination phase. AUC values were calculated using the trapezoidal method with extrapolation after the terminal sample. As an exception, AUC values in the 1125 mg/kg oral dose group were presented as AUC per 48 h sampling period with no extrapolation due to the absent elimination phase. Dunnett's t test was used to analyze PK parameters between identical dose groups on the first and last day of treatment. "Significance" in the text denotes P -< 0.05.

RESULTS

Oral dosing Oral administration of 150 to 1500 mg/kg/day phenytoin was associated with mild to severe maternal toxicity. Mild lethargy and ataxia were observed only 1 day during treatment in the 150 mg/kg (1/9 dams) and 375 mg/kg (1/8 dams) dose groups, while moderate to severe lethargy, ataxia, and imbalance were observed 2 to 10 days in the 750 mg/kg (3/8 dams), 1125 mg/kg (8/8 dams), and 1500 mg/kg (4/4 dams) dose groups. Maternal deaths occurred between GD 10 and 17 at the 1125 mg/kg (2/8 dams) and 1500 mg/kg (3/4 dams) dose groups. Postmortem examination of the 750 to 1500 mg/kg dams revealed mild to moderate periacinar hepatic necrosis; acute, multifocal, necrohemorrhagic gastritis; and decreased food content in the intestinal tract. Increasing amounts of precipitated drug were found lining the gastric mucosa in the 375 to 1500 mg/kg dose groups at GD 20 laparotomy. Maternal weight gain (GD 20 minus GD 8 weight, minus gravid uterus) was reduced significantly in a dose-related manner in the 375 to 1125 mg/kg dose groups (Table 1). The marked increase in embryonic loss in the 1125 mg/kg group resulted from 2/6 litters with a 100% resorption. Fetal deaths occurred in 1/6 litters (11/14 pups) and approximated GD 14 to 16 (stages 25-28)

Table 6. Major organ systems affected by prenatal phenytoin treatment: oral dosing

Dose (mg/kg/day)

0 150 375 750 1125

Fetuses/ Litters 86/6 103/9 102/8 102/8 42/4

Cardiovascular X % 4-- SD a

Uro__ genital X % ± SD ~

Skeletal CranioVertebrae/ __ facial __ ribs X % ± SD" X % ± SD"

2.2 2.8 0 9.8 16.0

0

0

± ±

4.1 8.3

± 14.1 +--- 25.7

aMean percent affected fetuses per litter. bp _< 0.05.

2.5 ± 3.8 0 14.6 -- 20.3 29.2 -- 11.7 b

0 0 20.8 ----- 34.6 62.5 -- 47.9 b

0 0 1.0 ± 2.9 2.5 ----- 7.1 6.3 Z 12.5

Phenytoin embryotoxicityand kinetics • J. R. Row~ayDET~.

195

Fig. 1. Abnormal craniofacial development in rat fetuses following 1125 mg/kg oral and 75 mg/kg i.v. administration of phenytoin. Compared to the control (a), note the overall growth retardation in both fetuses (b,c), the shortened snout and highly arched palate (b) and thin, tapered snout (c). The two types of head malformations were seen in both oral and i.v. dose groups.

(29). Laparotomy was performed on only one dam in the 1500 mg/kg group, revealing 100% resorption. Interlitter variability precluded statistically significant differences in prenatal mortality (Table 1). A significant dose-dependent reduction in fetal weights was evident in the 375 to 1125 mg/kg dose groups in addition to a significant decrease in crownrump lengths in the 750 and 1125 mg/kg groups (Table 5). Dose-dependent intrauterine growth retardation (IUGR) was also evidenced by reduced appendicular and axial skeleton ossification with significance at the 1125 mg/kg dose. The percentage of malformed fetuses per litter was significantly increased at 750 and 1125 mg/kg phenytoin (Table 5). The incidence of specific defects among treatment groups is summarized in Table 6. Major cardiovascular malformations, occurring mainly in the 1125 mg/kg group, included absent aortic arch between the left carotid and subclavian (n = 4) and transposition of the great vessels (n = 1), all with a ventricular septal defect (VSD). Other observations included variations in the brachiocephalic branching pattern (n = 6), shortened and rounded cardiac apices (n = 10), and enlarged right ventricles (n = 2) and auricle (n = 1). Two control cases displayed situs inversus of the heart and great vessels with ventricular septal defects. An increase in urogenital anomalies occurred at the 750 mg/kg dose, although significance was only present at the 1125

mg/kg level. Defects included hydronephrosis (n = 8), ectopic kidneys (n = 5), undescended testes (n = 2), and uterine horn hypoplasia (n = 2). Necrotic (n = 1) and hemorrhagic (n = 1) liver lobes also were observed. Craniofacial malformations were observed mainly in the 750 and 1125 dose groups and were characterized by highly arched palates (n = 18), often in association with a thin, tapered snout (n = 10) or shortened snout (n = 2) (Figure 1); agnathia (n = 2) with aglossia (n = 1); cleft lip and palate (n = 1); anophthalmia (n = 1); and macrocephaly (n = 1). Skeletal malformations consisted of hemivertebrae and fused vertebrae (n = 5) in the cervical and thoracic regions. In addition, three fetuses from one litter in the 1125 mg/kg dose group displayed ectromelic humeri with abnormal lateral curvature in the distal region of the shaft. The number of rudimentary ribs observed in the oral and i.v. dose groups were neither dose-related nor considered malformations, but rather developmental variations (30,31). No significant differences in fetal parameters were evident in PK compared w~th non-PK groups. Pharmacokinetics Total phenytoin plasma levels following oral administration on GD 8 and 17 are summarized in Table 4 and Figures 2 and 3. On GD 8, phenytoin levels in the 150 mg/kg group peaked at 8.8 --- 1.8 h and declined to minimal levels within 24 h. In contrast, phenytoin

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plasma levels on GD 8 in the 1125 mg/kg group approached maximal levels soon after dosing ( - 3 0 min) and remained relatively constant throughout the 48 h sampling period. Due to these continuously high pheny-

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Phenytoinembryotoxicityand kinetics • J. R. ROWLANDEr AL.

197

Table 7. Effects of phenytoin treatment on prenatal development in rats: iv dosing

Dose (mg/kg/day) 0 25 50 75

Fetal weight (g) Male Female (X-- ± SD)a (X- --- SD)~ 3.80 3.71 3.46 2.63

± 0.41 ± 0.24 --- 0.32 ± 0.59b

3.31 3.46 3.29 2.56

± ±

0.68 0.22 0.34 0.46b

Fetal crown-rump (cm) Male Female (X- ± SD)~ (X-- ± SD)~ 3.64 3.63 3.56 3.19

- 0.17 ± 0.08 +-- 0.14 --- 0.24b

3.45 3.51 3.48 3.15

± 0.32 --- 0.13 ± 0.16 --- 0.16b

Reduced ossification (X-% - SD)a

Malformed fetuses (X % -+ SD)a

13.9 5.1 12.0 63.1

3.6 4.1 10.4 43.5

± 21.0 - 7.1 ± 12.1 --- 38.4b

± ±

6.6 6.2 9.8 45.0b

aValues are mean of litter values --.SD. bp < 0.05.

also prevented calculation of tl/2 and Vol D. Comparison of GD 8 and 17 plasma profiles in the 1125 mg/kg group revealed a notable, although not statistically significant, increase in the maximal plasma concentration (39.4 ___ 16.6 ixg/mL on GD 8; 53.7 ___ 11.9 ~g/mL on GD 17). In order to examine drug accumulation data, total plasma phenytoin was monitored over 2 consecutive days of dosing on GD 8 and 9, then again on GD 16 and 17 (Group B, Figure 3). A slight increase in the 150 mg/kg Group B was evident in the Cmax values from GD 8 to 9 (GD 8, 15.6 --- 0.6 t~g/mL; GD 9, 17.1 _ 9.2 I~g/mL, respectively), with a more notable increase in Cm~x evident in the 1125 mg/kg Group B from GD 8 to GD 9 (27.1 ___ 5.6 to 37.7 --- 14.2 Ixg/mL, respectively). Negligible change in the profiles from GD 16 to 17 at 150 or 1125 mg/kg were evident.

Intravenous dosing I.v. administration of 25, 50, and 75 mg/kg/day phenytoin was associated with mild to severe maternal toxicity. Slight imbalance was observed 1 to 2 days in the 25 mg/kg group (3/8 dams), while mild to severe imbalance and ataxia were observed 7 to 10 days in the 50 mg/kg group (7/8 dams) and 75 mg/kg group (8/8 dams). One maternal death occurred on GD 12 at the 100 mg/kg dose. Due to the excessive ataxia, this dose was discontinued. No gross anatomic evidence of maternal toxicity was evident in this 100 mg/kg dam or remain-

ing dose groups at laparotomy on GD 20. No significant decreases in maternal weight gain were evident (Table 2). In the 75 mg/kg group, the increase in embryonic loss resulted from 2/6 litters with 100% resorption (Table 2). The fetal deaths occurred in 2/6 litters (4/5 pups, 1/15 pups). The 4 deaths in the first litter were estimated at GD 17.5 to 18.5 (stages 31-32) (29). Palate development in these same pups, however, appeared retarded, and corresponded to GD 16-17 (stages 28-30) (29). The one death in the second litter approximated GD 14 (stage 25) (29). Interlitter variability precluded statistically significant increases in both resorptions and fetal deaths. A significant reduction in fetal weights and c r o w n rump lengths occurred only at the 75 mg/kg dose (Table 7). A significant decrease in male PK compared with non-PK fetal weight, crown-rump length measurements (males only), and reduced ossification was evident at the 75 mg/kg dose, although both groups were significantly below control values. All other PK and non-PK i.v. parameters at identical doses were very similar. A significant increase in malformed fetuses was evident at the 75 mg/kg level (Table 7). Craniofacial malformations comprised a large part of this increase, and consisted of thin, tapered snouts (n = 23) accompanied by arched palates (n = 8) (Figure 1). Increases in both cardiovascular and urogenital anomalies were minor, and were distributed evenly throughout treatment

Table 8. Major organ systems affected by prenatal phenytoin treatment: iv dosing

Dose (mg/kg/day) 0 25 50 75

Fetuses/ Litters

Cardio_._vascular X % --- SDa

95/8 10o/8 102/9 58/6

0 1.9 --- 3.5 1.6 - 3.2 2.7 --- 4.1

aMean percent affected fetuses per litter. bp
Uro__ genital X % ± SDa 1.8 2.3 7.4 1.4

-.+ 5.1 --- 4.3 - 9.8 _ 3.4

Skeletal CranioVertebrae/ _ facial ribs X % --- SDa X % ± SD a 1.8 - 5.1 0 1.4 - 4.2 39.5 ± 48.0b

1.81 --. 5.1 0 0 0

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Reproductive Toxicology

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Volume 4, Number 3, 1990

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Fig. 4. Maternal plasma concentration of total phenytoin following a single i.v. dose of 25 (n = 3, GD 8; n = 4, GD 17), 50 (n = 4), or 75 mg/kg (n = 4) on a) GD 8 and b) GD 17. Plotted values represent mean ± standard deviation.

groups (Table 8). Isolated cardiovascular findings included enlarged right (n = 5) and left (n = 1) ventricles, while urogenital anomalies consisted of hydronephrosis (n = 8), and hypoplastic and/or ectopic kidneys (n = 3). Skeletal malformations consisted of only one control fetus with 2 fused ribs.

were apparent. Ct values in corresponding dose groups decreased by approximately 2.5-fold from GD 8 to 17. A slight increase in the Cmax values at the 25 mg/kg and 50 mg/kg dose was evident while the 75 mg/kg dose remained constant. DISCUSSION

Pharmacokinetics Total phenytoin plasma levels following i.v. administration on GD 8 and 17 are summarized in Table 8 and Figure 4. On GD 8, a significant dose-dependent 2-fold increase in tl/2 occurred from the 25 to 50 mg/kg dose, while a disproportionately greater 4-fold increase occurred from 25 to 75 mg/kg. A significant 3-fold increase in AUC values occurred from 25 mg/kg to 50 mg/kg, with an 8-fold increase from the 25 to 75 mg/kg dose. C1 values revealed a significant dose-dependent decrease, while a slight dose-dependent increase in Vol D was apparent. On GD 17, significant changes in kinetic parameters with increasing dose were similar to those observed on GD 8, with approximate 2- and 4-fold increases in t~/z and 2- and 6-fold increases in AUC from 25 to 50 mg/kg and 25 to 75 mg/kg, respectively. A slight dose-dependent increase in Vol D was evident on GD 17, while C t values significantly decreased. A significant dose-dependent increase in Cmax was evident on both GD 8 and 17. Comparing GD 8 with GD 17, significant 2- to 3-fold increases in tl/2 and AUC occurred for corresponding doses, while no significant changes in VolD

In both the oral and i.v. dose ranges examined, phenytoin caused severe maternal toxicity and variable developmental toxicity in the Sprague-Dawley rat. The manifestations of developmental toxicity observed in this investigation -- embryolethality, IUGR, and defects encompassing the cardiovascular, urogenital, craniofacial, and skeletal systems -- have been variably demonstrated in rats (15-17), mice (8-14), and rabbits (18). Three of the manifestations associated with the "hydantoin syndrome" -- IUGR, craniofacial defects, and appendicular defects -- were evident in this study. The ectromelia observed in this investigation has been reported in other rodent studies, in addition to adactyly and syndactyly (9,13,15-17). In contrast to the relatively minor phalangeal/nail hypoplasia often associated with the human hydantoin syndrome, recent case reports reveal prenatal phenytoin exposure in association with more dramatic limb reduction including adactyly (32,33) and forearm deficiency (34). In addition, evidence in humans concerning an increased incidence of major cardiac, urogenital, and CNS anomalies has incited debate over the specificity of the syndrome (2,35-39). Developmental toxicity following oral phenytoin

Phenytoinembryotoxicityand kinetics • J. R. ROWLANDETAL. administration was more marked in terms of an increase in IUGR and number/variability of malformations when compared to i.v. dosing. Cardiovascular, urogenital, craniofacial, and skeletal defects all contributed to the significant increase in malformed fetuses at the oral 750 and 1125 mg/kg dose levels. With i.v. administration, the only significant increase in malformations occurred in the 75 mg/kg dose group and consisted almost exclusively of craniofacial defects similar to those seen in the oral dose group. At oral doses of 700 to 1000 mg/kg during 3 consecutive or alternate days from GD 9 to 15 in the albino rat (average serum phenytoin levels from GD 9 to 14 at 1000 mg/kg were ~17 ~g/mL), Lorente et al. (16) describes craniofacial defects (arched palate, narrow palate, shortened snout) similar to the abnormal orofacial and craniofacial features observed in the current study. Although manifestations of abnormal or delayed palate development were evident in both the i.v. and oral dosing regimens, incidence of cleft palate or cleft palate/lip in this investigation, as well as in Lorente et al. (16), was extremely low. Maternal toxicity has not been consistently addressed in developmental toxicity studies, including those testing phenytoin (40-42). Although the correlations between maternal and developmental toxicity remain to be elucidated (42-46), consideration of matemal influence on the conceptus remains an important factor. In this investigation, oral phenytoin administration resulted in noticeable dose-dependent matemal toxicity evidenced by maternal deaths and a significant decrease in maternal weight gain. These signs of maternal toxicity appeared to parallel the increase in digestive complications, and may have occurred due to the insoluble nature of phenytoin in the acidic contents ( p H i 2 ) of the stomach (19). The substantial accumulation of precipitated phenytoin along the gastric mucosa may have contributed to the necrohemorrhagic gastritis and decrease in maternal weight gain. Similar manifestations of toxicity, including decreased food intake and body weight gain, and intestinal hemorrhage have been reported in poultry following oral administration of 100 to 2500 mg/kg phenytoin (47). Without pair-fed comparisons, it is difficult to conclude whether the adverse maternal influences contributed to the observed increases i n developmental toxicity. Other pair-fed studies in experimental animals have demonstrated a positive correlation between decreased maternal weight gain and IUGR (48-50), however, associations between teratogenicity and maternal toxicity remain inconclusive (45). In contrast to the oral dosing regimen, i.v. dosing resulted in only slight maternal toxicity as evidenced by reduced maternal weight gain. The i.v. compared with oral dosing regimen provides a valuable comparison in this regard, since with i.v. dosing 1) direct systemic phenytoin administration appears to minimize maternal

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weight loss, and 2) more effective isolation of possible primary developmental toxicity is possible (neurotoxic influences still remain). Concerning this latter aspect, it is interesting that the high plasma levels and severe neurotoxicity evident in the 8/8 dams at the 75 mg/kg i.v. dose, while associated with only a minor decrease in maternal weight gain, was associated with a significant increase in IUGR and teratogenicity. The advent of neurotoxic manifestations (severe lethargy, ataxia, and imbalance) was consistently associated with phenytoin plasma levels -> 30 ixg/mL. Consistent correlations between severe neurotoxic manifestations and plasma levels > 25-30 Izg/mL have been demonstrated in the mouse, rat, rabbit, rhesus monkey, and human (51-54). The extremely wide range of oral doses (4 to 4,000 mg/kg oral) necessary to achieve the more limited range of plasma phenytoin in these studies emphasizes the relative predictive capability of plasma levels, as opposed to dose, as far as neurotoxic manifestations are concerned. Both oral and i.v. dosing regimens resulted in a dramatic increase in phenytoin exposure between the beginning and end of the treatment period. Two- to four-fold increases in tl/2 and AUC were observed in all i.v. dose groups and in the low-dose oral group from GD 8 to 17. With experimental studies in the mouse demonstrating a positive correlation between increased plasma phenytoin levels and frequency of congenital malformations (14,55), these substantial increases in phenytoin exposure in the Sprague-Dawley rat following chronic administration must also be considered when evaluating developmental toxicity. Comparison of the oral with i.v. exposure profiles reveals that the sustained absorption following oral phenytoin administration results in greater maternal drug exposure, as evidenced by AUC values (e.g., 2113 _+ 445 p,g.h/mL in the 1125 mg/kg oral dose compared with 638 _ 244 ixg.h/mL in the 75 mg/kg i.v. high dose on GD 17). This increased phenytoin exposure in the high oral compared with i.v. dose groups was associated with greater decreases in fetal weights (48% compared with 27%) and crown-rump lengths (19% compared with 11%) as well as greater increases in reduced ossification (84% compared with 63%) and malformations (62% compared with 45%). However, larger AUC values were not consistently associated with an increased incidence in developmentally toxic manifestations. For example, although the AUC value in the 150 mg&g oral dose (906 __ 336 p,g.h/mL) is larger than that of the 75 mg/kg i.v. dose (638 + 244 ixg.h/mL), the exposure to high plasma levels (e.g., > 30 p,g/mL) at the 75 mg/kg i.v. dose (--1.5 h on GD 8; ~ > 6 h on GD 17) is associated with a more significant increase in prenatal mortality, IUGR, and teratogenicity. This suggests that the attainment of a certain plasma threshold level may be

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more essential than a large AUC value in the induction of developmental toxicity. It could be further speculated that the prolonged exposure to phenytoin levels > 30 p,g/mL at the 1125 mg/kg oral dose as opposed to the more limited exposure at the 75 mg/kg i.v. dose may be responsible for the increase in observed developmental toxicity. Due to the increased accumulation of precipitated phenytoin in the stomach at the 375 to 1500 mg/kg dose levels, it is possible that the insoluble excess drug was not contributing to increased plasma levels. In isolated intestinal preparations in the Wistar rat, Woodbury and Swinyard (19) demonstrated that phenytoin plasma levels reached a plateau at an oral dose of 66 mg/kg; no further increases in plasma phenytoin were observed above this dose. In contrast to these findings, a substantial increase in plasma levels from the 150 to 1125 mg/kg dose was evident using the Sprague-Dawley rat in the current study. In addition, the rise in plasma levels following the second 1125 mg/kg dose in Group B from GD 8 to 9, and less substantial, but noticeable increase immediately following the GD 17 dosing suggests that absorption capabilities were not completely saturated, even at the relatively large 1125 mg/kg dose. With the Sprague-Dawley rat, in order to achieve steady-state phenytoin levels throughout gestation and thus more closely replicate the human therapeutic situation, an oral dose in the 150 mg/kg range prior to and throughout gestation might minimize maternal toxicity while maintaining phenytoin plasma levels in the potentially teratogenic range of - 3 0 ~g/mL. Roberts et al. (56) implemented a similar study design by exposing A/J mice, which are highly susceptible to cleft palate induction following acute phenytoin exposure, and C57BL/6J mice, which are less susceptible, to chronic oral doses of phenytoin at teratogenic steady-state plasma levels (> 20 Ixg/mL in the mouse) prior to and throughout gestation. Interestingly, this dosing regimen produced a relatively low proportion of clefting and other defects in both A/J and C57BL/6J strains. Pharmacokinetics Phenytoin exhibits dose-dependent pharmacokinetics in rats due to a combination of capacity-limited kinetics and p-hydroxyphenytoin (p-HPPH) inhibition (57). This occurs due to saturation of the hepatic enzyme system necessary for the conversion of phenytoin to its major metabolite, conjugated p-HPPH. In addition, the p-HPPH metabolite accumulates and competes with phenytoin in the initial hydroxylation process. In the nonpregnant rat, Chou and Levy (58) have suggested the onset of this capacity-limited "saturation" and p-HPPH inhibition occurs with i.v. doses >-- 10 mg/kg; at 30 mg/kg a subsequent decreased C1 and 2-fold increase in tl/2 was observed. This kinetic behavior results in termi-

Volume4, Number3, 1990 nal plasma concentrations that appear to decline exponentially with time, but also in an apparent half-life that increases with increasing dose. Particularly applicable to i.v. dosing, this also necessitates approximation of k e and subsequent half-life values from a convex elimination profile that is not indicative of first order kinetics. In addition, pregnancy appears to accentuate increased phenytoin levels and subsequent exposure in the rat, particularly at the high doses used in this study. At equivalant 30 mg/kg doses, Chou and Levy (58) demonstrated decreased C1 values in pregnant rats compared with nonpregnant rats, and suggested that p-HPPH inhibition may be more significant during pregnancy. In addition, Westmoreland and Bass (59) found that plasma phenytoin levels were approximately 1.5- to 2-fold greater after a single dose and 2- to 3-fold greater after one week of chronic 100 mg/kg i.p. phenytoin treatment in pregnant compared with nonpregnant rats. It is thus apparent that the "saturation" of capacitylimited elimination in combination with p-HPPH inhibition results in steady-state phenytoin accumulation (exhibited by increased tl/2 and decreased C1) over the 10-day treatment period in the current investigation. Following oral administration, sustained phenytoin absorption further contributes to the steady-state accumulation and subsequent increase in maternal exposure by the end of the treatment period. Do these increases in maternal phenytoin exposure translate into increased exposure to the conceptus? It has been shown that phenytoin can readily pass the placenta in humans (60-64), rhesus monkeys (65), and mice (66,67). In the rat, fetal/maternal phenytoin ratios vary from one-half (68) to unity or greater (17,59,60). Although only total plasma phenytoin (bound + free) was measured in the present study, Chou and Levy (59) present a literature summary revealing that the free fraction of total phenytoin (portion unbound to plasma proteins that can readily cross the placental barrier) in pregnant rats is relatively constant (--21 to 33%). This free fraction also appears to be concentration-independent over a wide plasma range ( - 1 . 8 to 30 p,g/mL) in both pregnant and nonpregnant rats (69). Thus, maternal phenytoin plasma levels may be predictive of embryonic/ fetal exposure; however, supplemental studies are necessary to compare the concentration of phenytoin in the embryonic and maternal compartments during peak embryotoxic sensitivity. In summary, the dose-dependent increases in matemal plasma phenytoin in both the oral and i.v. dose groups were associated with increased matemal and developmental toxicity in the Sprague-Dawley rat. Comparison of oral and i.v. kinetic profiles suggested that a "threshold" plasma phenytoin level appeared more significant than large drug exposures (as measured by AUC) in the induction of developmental toxicity. The

Phenytoin embryotoxicity and kinetics • J. R. ROWLANDET AL.

dose-dependent kinetic nature of phenytoin also appeared responsible for the significant increase in maternal phenytoin exposure following chronic treatment from GD 8 to 17. These findings emphasize the importance of monitoring maternal phenytoin plasma levels in the evaluation of phenytoin-induced developmental toxicity. - - We thank Carol A. Bracco-Dominguez and Dr. Matthew J. Cukierski for their technical assistance and Drs. Alan R. Buckpitt, Allen C. Enders, and Jon M. Rowland for critical review and discussion.

Acknowledgments

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