Abstract
HEPP (D,L-3-hydroxy-3-ethyl-3-phenylpropionamide) is a novel anticonvulsant with promising anticonvulsant profile, which is being actively researched. The potential maternal and embryo/fetal toxicities of HEPP were evaluated in pregnant rats following subcutaneous (s.c.) administration during organogenesis (gestation days 6 through 14, GDs 6–14) and the fetal period (GDs 14–21). Single- and multiple-dose pharmacokinetics were also evaluated at the same periods in order to establish possible correlations with some maternal or embryo/fetal toxicity end points. Embryotoxicity was mainly indicated by a significant dose-concentration dependency in the increase in resorptions, high percentage of fully resorbed litters, and decrease in embryo body weights during the GD6–14 dosing period. No gross external alterations were observed in live fetuses. There was no indication of maternal toxicity; but a marked increase in maternal body weight was evident following dosing from GD14 to GD21. The maternal plasma profile following single subcutaneous dose of 50 mg/kg on both GD14 and GD21 showed a monoexponential elimination pattern. Statistically significant differences between treatments (GD14 versus GD21) were observed in elimination (k el = 0.12 versus 0.15 h−1), absorption (k a = 2.01 versus 3.14 h−1), maximum plasma concentration time points (T max = 1.49 versus 1.01 h); maximum plasma concentration (C max = 40.23 versus 36.31 μg/ml) and areas under the concentration-time curve (AUCs0– ∞ = 421.88 versus 274 μg h/ml. Based on comparisons of C max, T max, and AUCs0– ∞ between the actual data and single intraperitoneal (i.p.) data previously published, the s.c. administration exhibited slower disposition and higher absorbed amount. After multiple-dose administrations of 50 and 100 mg/kg every 12 h (07:00 and 19:00 h), steady-state plasma levels were lower than the computer prediction, and only slight accumulation was observed. In both dosing periods HEPP levels were similar in mothers and offspring at steady-state conditions. The high incidence of embryo death and reduced embryo weight at GD6–14 dosing compared to GD14–21 dosing suggest that embryos are more sensitive to the deleterious effects of HEPP than fetuses; however, the faster elimination observed at late gestation could also contribute to the lower toxicity observed during the fetal period. Because the maternal HEPP plasma levels and the AUC values were positively correlated with embryo/fetal toxicity end points, both pharmacokinetic parameters could be reliable indicators of offspring exposure and consequently of potential toxicity. These data suggest that the length of time that HEPP is present in the maternal plasma at a sufficiently high concentration could be determinant of adverse effects in the offspring.
D,L-3-Hydroxy-3-ethyl-3-phenylpropionamide (HEPP) (Figure 1) is an anticonvulsant drug under investigation, chemically unrelated to all the currently in use antiepileptic drugs (i.e., phenytoin, barbiturates, primidone, carbamazepine, gabapentin, tiagabine, and zozinamide). HEPP exhibits a broad anticonvulsant profile, with particular potency against seizures evoked by pentylenetetrazole and bicuculline (Meza et al. 1990).
An interesting feature of the HEPP anticonvulsant profile is a singular high potency against the γ-aminobutyric acid (GABA)-withdrawal syndrome (GWS), (Brailowsky et al. 1988, 1992). This is a model for focal epilepsy, which has proven to be extremely resistant to all common anticonvulsants (i.e, barbiturates, phenytoin, carbamazepine, valproate, progabide, benzodiazepines, and N-methyl-D-aspartate [NMDA] receptor antagonists). HEPP also has demonstrated a very highly dose-dependent antiepileptic effect against a model of generalized nonconvulsive epilepsy (GNCE), which resembles absence seizures (Brailowsky et al. 1992).
Although the molecular mechanism of action of HEPP is unknown, the molecular structure and the effects on different epilepsy models suggest a possible interaction at the GABA neurotransmission level.
The unique anticonvulsant profile of HEPP against the GWS and GNCE models of epilepsy makes HEPP a promising alternative in the treatment of complex seizure episodes such as generalized absence seizures or status epilepticus. Systematic research has been done on the pharmacological and toxicological profile of HEPP to determine its potential use as an antiepileptic agent. To date phase I clinical studies are being carried out in Mexico.
The pharmacokinetics of HEPP has been evaluated in several animal species and in male humans. In rabbits, dogs, and rats, HEPP showed a very short elimination half-life following intravenous (i.v.) administration (1.17, 2.10, and 1.81 h, respectively) (Gómez, Cueva-Rolon, and Lehmann 1995; Gómez and Lehmann 1995; Gómez 1993), whereas in humans the half-life of HEPP is relatively long and varies (13 to 45 h) (González-Esquivel et al. 1998). Linearity in kinetic parameters has been observed between 8 and 30 mg/kg following i.v. administration in dogs (Gómez and Lehmann 1995) and between 4 and 8 mg/kg after oral administration in healthy male volunteers (González-Esquivel et al. 1998).
In rats, dogs, and rabbits (Gómez, Cueva-Rolon, and Lehmann 1995; Gómez and Lehmann 1995; Gómez 1993), HEPP distributes extensively in total body water, exhibits low plasma protein binding (19.9% ± 1.1%), and readily crosses the blood-brain barrier. There is also a close relationship between the time courses of HEPP in plasma and brain and the time course of the anticonvulsant effect. Following oral and intraperitoneal administration of HEPP in male rats, the fraction of absorption (F) was 0.6 and 0.8, respectively (Gómez and Lehmann 1995).
In pregnant rats at gestation day (GD)21, HEPP was rapidly transferred to the placenta and fetuses and distributed throughout the total body water when the intraperitoneal administration was used (Luna-Fletes and Gómez 2003). In this same study, the time course of HEPP in fetal and placental tissues paralleled the observed in the maternal plasma and significant changes in the HEPP pharmacokinetics parameters were observed as pregnancy progressed. The maternal plasma half-life (t 1/2) values were 3.83, 3.46, and 2.38 h at GD7, GD12, and GD21, respectively. The volume of distribution (V d) and clearance (Cl) values were also time of pregnancy dependent (Luna-Fletes and Gómez 2003).
Other study showed a significant reduction in the half-life of HEPP in maternal plasma following repeated intraperitoneal administration in rabbits (González-Esquivel, Pérez, and Jung-Cook 2004).
In mice following chronic administration, a gradual decrease in both the HEPP plasmatic concentrations and the anticonvulsant effect were observed (Molina-Jasso 1994). These two latter studies suggest the possibility of enzyme autoinduction in the HEPP metabolic processes.
There are no published studies on the effects of HEPP during gestation. However a relating work on the toxic and teratogenic potential of HEPB (D,L-4-hydroxy-4-ethyl-4-phenylbutyramide, a structural homologue of HEPP; Figure 1) showed a significant decreases in maternal weight and a significant increase in the number of mothers with abnormal, resorbed, or dead fetuses when a intraperitioneal (i.p.) dose of 100 mg/kg was administered in mice (Chamorro et al. 1994).
Given that some studies on the pharmacokinetics of HEPP have already been conducted in humans (González-Esquivel et al. 1998; González-Esquivel, Pérez, and Jung-Cook 2004), and also considering the high incidence of teratogenic effects of all the antiepileptic drugs currently in clinical use (Dansky and Finnell 1991; Koch et al. 1996; Samren, van Duijn, and Christiansens et al. 1999; Holmes et al. 2001; Morrell 2002), studies on the preclinical effects of HEPP during gestation are needed.
Therefore, the purpose of the present study was to evaluate the pharmacokinetics of HEPP following single and repeated subcutaneous administration in pregnant rats.
The possible relationship between some pharmacokinetic parameters, HEPP levels in mothers and embryos/fetuses, and toxicity end points during the organogenesis and the fetal periods was also investigated.
The subcutaneous route of administration was chosen because the half-life of HEPP following oral or i.p. administration in rodents is very short. Consequently, neither of these routes adequately resembles the chronic human epileptic treatment. On the other hand, the subcutaneous route of administration had shown minor fluctuations in steady-state levels, higher area under curve (AUC) and, occasionally, longer half-lives in some chronic treatments (Laslo et al. 2004; Marcucci et al. 2005; Ugwoke et al. 2003).
MATERIALS AND METHODS
Drugs and Chemicals
HEPP and HEPA (D,L-2-hydroxy-2-ethyl-2-phenylacetamide) (Figure 1), which was used as internal standard in the analytical method, were kindly donated by Dr. Guillermo Carvajal of the Department of Biochemistry of the National School of Biological Sciences, National Polytechnic Institute, Mexico DF. The vehicle was commercial corn oil (Mazola). Water for high-performance liquid chromatography (HPLC) was purified using a Milli-Q water purification system (Millipore, Eschborn, Germany). All solvents used in the HEPP analysis were HPLC-grade, and all other chemicals were of the highest available commercial grade (Merck, Darmstad, Germany).
Experimental Animals
Virgin, female Wistar rats (240 to 260 g) from our facilities (CINVESTAV-IPN, Mexico) were housed in a controlled environment (14-h light/dark cycle, light period 06:00 to 20:00 h; temperature of 22°C ± 2°C), with free access to food (Purina Rat Chow; Ralston Rations, Kansas, USA) and purified commercial water. When proestrus occurred, two females were placed overnight with a male of proven fertility. The following day was considered day 0 of gestation (GD0) if a copulatory plug or spermatozoa were detected in the vaginal smear. Mexican laboratory animal care regulations (NOM-062-ZOO-1999) and institutional ethics committee requirements were observed in all experimental protocols.
Single Subcutaneous Administration Design
Pregnant rats on GD14 or GD21 were administered with a single 50 mg/kg subcutaneous (s.c.) dose of HEPP. This dose was chosen to enable comparison of results with those of previous pharmacokinetic studies. HEPP was dissolved in 0.1 ml ethyl alcohol, suspended in corn oil, and then injected into a s.c, deposit under a fold of the animal’s neck in a final volume of 2 ml/kg. After administration, 1 ml of blood samples were taken from the tail vein at 0.25, 0.33, 0.5, 0.6, 1, 4, 8, 12, and 24 h. Five blood samples were withdrawn from each rat at different sampling times so that taken together, seven samples for each time point were available for HEPP analyses. Water via oral gavages was periodically administered. All blood samples were centrifuged at 1875 × g, the plasma separated, and stored in polypropylene tubes at −30°C until analysis.
Multiple Subcutaneous Administration Design
Groups of 10 healthy pregnant rats were randomly selected and assigned to a control group or to two HEPP dosage schedules (50 or 100 mg/kg subcutaneously every 12 h [07:00 and 19:00 h] per treatment period). Vehicle (control) and HEPP dose groups were administered during the rat organogenesis period (GDs 6–14) and the fetal period (GDs 14–21). The HEPP was suspended in the oil vehicle and administered subcutaneously as previously described. The animals in the control treatment received only the corresponding amount of vehicle. The 50 mg/kg dose was selected because this dose is the equivalent median effective dose (DE50) reported in previous pharmacodynamic studies (Meza et al. 1990), and this dose has been used in all the previous pharmacokinetic studies (Gómez and Lehman 1995; Gómez, Cueva-Rolon, and Lehmann 1995; Luna-Fletes and Gómez 2003). The 100 mg/kg dose was chosen in order to evaluate any possible dose dependency.
The dams were housed individually in propylene cages and their behavior and health observed daily. Body weight, food and water consumption, and gross external clinical signs were monitored daily. HEPP concentrations at steady state were verified by determination of two blood plasma samples taken from the tail vein of each rat before the morning dose (07:00 h) and at 6 h post administration (13:00 h) on GD9 and GD11 for the GD6–14 groups, and on GD17 and GD19 for the GD14–21 groups. Two final blood samples were taken on GD14 and GD21 prior to the morning dose, and again at 13:00 h before sacrifice (a handling time of 10 min was considered for each sampling time point). The animals were euthanized by excess carbon dioxide, the gravid uterus was removed by cesarean surgery, weighed, and examined to determine the number and status of implants. Placental weight, number of live embryos or fetuses, weight of embryos or fetuses, and resorption sites were recorded. Five embryos per dam were pooled and homogenized for further HEPP determination. Live fetuses at GD21 were examined for external alterations.
Equipment and Chromatographic Conditions
All HPLC analyses were done with a Hewlett-Packard HP 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with an isocratic HP 1100 pump, an HP 1100 UV diode-array detector operated at 219 nm, and a Rheodyne 7125 injection valve with a 100-μl loop. Separation was performed in a C18 reverse-phase column (4.6 mm × 15 cm), with 5-μm particle size (Beckman Coulter, Fullerton, CA, USA), protected by a guard column packed with the same material. Elution was performed under isocratic conditions, using a mobile phase of acetonitrile:water (25:75 v/v) at a flow rate of 1.5 ml/min.
Drug Assay
Plasma, embryo, and fetus samples were analyzed for HEPP levels using the high-pressure liquid chromatography method of Gómez and Lehmann (1991, 1992). Briefly, HEPP and the HEPA internal standard were extracted from 0.5 ml of the alkalinized samples (plasma or tissue) using dichloromethane. The organic layer was collected in glass tubes and evaporated with a nitrogen stream. Following evaporation, the drug residue was dissolved in 100 μl of the mobile phase and injected into the chromatograph. Assay detection limit was 0.01 μg/ml, and calibration curves were prepared for each blank matrix.
Single Subcutaneous Administration Pharmacokinetics Analysis
Pharmacokinetic parameters were calculated from plasma concentration–time data using the best-fit equation as calculated based on residuals analysis, the coefficient of determination, and F test. The elimination rate constant (k el) was estimated from the linear least-square regression of the terminal phase of the ln concentration-time profile. Elimination half-life (t 1/2) was calculated as 0.693/k el. The area under the drug concentration-time curve (AUC0–∞) was determined using the linear trapezoidal rule to the last measured concentration (C last), with extrapolation to infinity by adding C last /k el. Time to peak (T max) was estimated as:
Peak concentration was estimated as:
Mean residence time: MRT = AUMC0–∞/(AUC0–∞). AUMC0–∞ is the area under the first-moment curve and were calculated using the statistical moment theory (Yamaoka, Nakagawa, and Uno 1972).
The apparent volume of distribution: V d = Cl MRT and total body clearance: Cl = dose·F/AUC0–∞. Based on previous research (Gómez et al., 1995), a bioavailability factor of F = 0.8 was used.
Statistical comparisons between rate constants were carried out using a parallelism test (Tallarida and Murray 1986), with differences considered significant at p < .05.
Best fit was calculated by using the nonlinear interactive PkCalc program (Shumaker 1986).
Multiple Subcutaneous Administration Analysis
Experimental minimum plasma concentration (C min) was represented by the plasma concentration of the sample collected just before the 07:00 h dose on GD9, GD11, GD14 (GD6–14 group) and at GD17, GD19, and GD21 (GD14–21 group). Average experimental plasma concentration (C ave) was obtained from the sample collected at 13:00 h (6 h after the morning dose) on the above gestation days.
Predicted drug concentrations at steady state and the accumulation ratio were calculated from the best-fit equations obtained for the single subcutaneous dose, using the following equations (Rowland and Tozer 1980; PK solutions 2005):
where τ is the constant dose interval (12 h) and F the fraction of absorption.
The C min at steady state,
where f ss is the fraction of the steady state.
Data Analysis
Dams or litters and implants were used as statistical analysis units, and values were expressed as mean ± SD. For comparisons of groups, a one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls multiple comparison test was performed, if statistical differences were found. Kruskal-Wallis test followed by Dunn’s test was used for the analysis of data expressed in percent. The minimum significance level in all analyses was p < .05. Calculations were made using the GraphPad InStat program (version 2.03; GraphPad Software, San Diego, CA, USA).
RESULTS
Pharmacokinetics of Single Subcutaneous Administration
The concentration-time profiles for HEPP in pregnant rats at GD14 and GD21 following a single subcutaneous dose of 50 mg/kg are shown in Figure 2. Derivative pharmacokinetic parameters from analysis of mean plasma levels in seven samples from each collection time are shown in Table 1. A mono-exponential equation could correctly describe HEPP’s elimination phase in the two treatment periods. Significant differences in rate constants (k a and k el) were observed when comparing the two treatment periods using a parallelism test (Tallarida and Murray 1986). Consequently, considerably faster absorption and elimination processes were observed at the later gestation stage (GD21) than in the earlier stage (GD14). Peak plasma concentration (C max) displayed similar values, but time to peak concentration (T max) was shorter at GD21 (1.01 h) than at GD14 (1.49 h), suggesting that absorption was significantly faster later in gestation. The elimination phase was also shorter at GD21 (k el = 0.12 h−1) versus GD14 (k el = 0.15 h−1), meaning elimination half-life at this stage was also shorter (t 1/2 el = 0.22 h for GD21 versus 0.34 h for GD14); this is consistent with the observed decrease in the MRT value at GD21 (MRT = 8.10 h at GD14 versus 6.47 h at GD21). A significant decrease in AUC0–∞ at GD21 was also observed (AUC0–∞ (GD21/GD14) = 0.650). The values of V d (0.82 and 1.00 for GD14 and GD21, respectively) were similar to those observed in a previous study reported by Luna-Fletes and Gómez (2003). These V d values suggest an extensive distribution of HEPP in body fluids during pregnancy. The slight increase in V d observed at late gestation (GD21) is possibly associated with the expansion in the plasmatic volume and with the gradual increase in both the extracellular fluid space and the total body water occurred as pregnancy progresses (Fredericksen 2001).
The elimination half life significantly decreased at the end of gestation (GD21 versus GD14), in concordance with the notable increase in clearance (Cl) values and the decrease in MRT values (Table 1).
These results suggest age-gestational dependency in HEPP pharmacokinetics during pregnancy in rats.
Prediction of Blood Levels Following Multiple-Dose Administration
The mean experimental and predicted steady-state HEPP concentrations observed in dams after repeated s.c. dosing in the GD6–14 and GD14–21 groups are shown in Table 2. Observed HEPP levels at steady state after repeated dose administration during the GD6–14 period were lower than the predicted from the kinetic parameters obtained for the single administration. However during the GD14–21 period, a more close relationship between observed and predicted values was observed. In agreement with the relatively short half-life observed after the single-dose administration, no important accumulation was reached following multiple-dose treatment.
Effects on Mothers and Offspring
The effects of HEPP on dams, embryos, and fetuses after treatment in the GD6–14 and GD14–21 groups are summarized in Table 3. Subcutaneous administration of HEPP caused no maternal deaths in any of the administration regimens. Nevertheless, high proportions of the dams administered the highest dose (100 mg/kg) in both gestation periods exhibited piloerection and decreased motor activity within minutes after administration. Toxicity in offspring was particularly marked in the GD6–14 period and was manifest in a high incidence of resorptions and embryonic death. High percentages of dams with all fetuses resorbed, and a high incidence of resorption rates in the remaining animals, were observed, which were dose and concentration dependent. Although the incidence of resorptions or dead fetuses was lower in the dams treated during the GD14–21 period than in those treated during the GD6–14 period, a significant increase in the number of resorptions per litter and a significant decrease in live fetuses versus the control group were also observed at the high dose (100 mg/kg). Percentage of resorptions compared to embryo/fetal HEPP concentrations is shown in Table 4.
Another important indicator of prenatal toxicity was a dose-related decrease in embryo weight in both the 50 and 100 mg/kg groups in the GD6–14 period. Although the effects were less marked in the GD14–21 period, a significant decrease in mean fetal body weight was observed at the 100 mg/kg dose. No external gross clinical abnormalities were observed in live fetuses.
Mean implantation site and uterine and placental weights showed no statistically significant differences in any treatment compared to the control.
Increases in maternal body weight and water intake were not affected in the GD6–14 group, but a very significant, dose-dependent increase in mean dam body weight gain was observed in the GD14–21 group. This increase was progressive and very notable at near term, and was accompanied by a significant increase in water intake and in food consumption. This effect has also been reported after chronic treatment of pregnant mice with HEPB, the superior homologue of HEPP (Chamorro et al. 1994). This effect could be related with the GABAergic properties of HEPP, because similar results have been also reported after chronic human therapy with valproic acid (VPA) (Luef et al. 2002, 2003) and gabapentin (DeToledo et al. 1997). Similar findings were also observed during the preclinical anticonvulsant evaluation of avermectin analogs, which are antiparasitic agents with GABA-like properties (Wise et al. 1997). It has been postulated that GABAergic mechanisms are involved in pancreatic beta-cell modulation and insulin secretion (Luef et al. 2002).
DISCUSSION
Several studies have demonstrated HEPP effectiveness in different epilepsy models (Meza et al. 1990; Brailowsky et al. 1992). However, the short plasma time course, which is linearly correlated with short brain time course, and brief anticonvulsant effect (Gómez, Cueva-Rolon, and Lehmann 1995; Brailowsky et al. 1992) limit the use of rodent models in chronic HEPP studies. Because HEPP’s half-life in humans is much longer than in rats (González-Esquivel et al. 1998), chronic studies in rats using conventional administration routes (oral, i.p., or i.v.) would reproduce neither the potential clinical situation of the epileptic patient nor an accurate toxicological risk assessment of chronic treatments.
Many studies have demonstrated the close relationship between drug effect and administration route in some drugs (Yuan et al. 2002; Allison and Pratt 2006; Marks, Morris, and Weeks 1987; Nau 1986), and subcutaneous administration has been reported to maintain more consistent drug plasma levels, with minor fluctuations in steady state (Allison and Pratt 2006; Laslo et al. 2004; Lespine et al. 2002; Nau 1985). Given this, the present study was designed to evaluate HEPP pharmacokinetics after single subcutaneous administration on GD14 (late organogenesis) and on GD21 (late gestation). The relationship between administration route and accumulation patterns, and the correlation between pharmacokinetics and HEPP-induced effects in dams and offspring, following multiple s.c. administration twice per day were also evaluated. Data from a previous study following i.p. administration in similar conditions (Luna-Fletes and Gómez 2003) were utilized for comparative purposes. Subcutaneous administration resulted in slower absorption and elimination patterns than did i.p. administration (Luna-Fletes and Gómez 2003), as shown by a significant decrease in the rate constants (k a and k el) and a significant increase in C max, T max, and AUC values following s.c. administration. The slower disposition kinetic of HEPP after s.c. administration may be associated with a slower and more sustained absorption from the subcutaneous deposit, as demonstrated by the comparisons of AUC values (AUCs.c./AUCi.p. = 2.15 at GD14 and 1.28 at GD21) (Table 1). The significant decrease in AUC values, the significant increase in the clearance (Cl) values, and the significant decrease in the MRT values observed at GD21 with respect to GD14 in this study are indicative of faster elimination, probably associated with the marked physiological changes of the late pregnancy (increase in cardiac output with regional blood flow changes, in particular the progressive increase in uterine blood flow; increase in renal blood flow associated with increased glomerular filtration; increase in the activity of hepatic drug-metabolizing enzymes in the mother and progressive activities of some metabolic enzymes in the fetus; and decrease in the concentration of serum albumin) (Morgan 1997).
Although there is no published research on HEPP metabolism, indirect evidence suggest that metabolism may be the most important elimination process for this drug. A study in human volunteers (González-Esquivel et al. 1998) reported only 6% unmetabolized HEPP in urine 72 h after oral administration. Additionally, significant tolerance to HEPP’s anticonvulsant effect was observed after chronic administration in mice (Molina-Jasso 1994). This tolerance was progressive from days 4 to 14 of treatment and was associated with significant decreases in HEPP plasma levels. Medina et al. (1998) also reported significant decreases in t 1/2, MRT, and V d, as well as a significant increase in body clearance of HEPP after chronic phenytoin administration in rabbits. These authors suggest that HEPP may share metabolic pathways with phenytoin.
In the present study, steady-state conditions after repeated s.c. administration of HEPP were reached 2 days after treatment was begun (3.19 doses for GD14 and 2.59 doses for GD21), confirming the predictions obtained after single s.c. administration (Table 2).
The experimental plasma concentrations at steady state during GD6–14 period were lower than the predicted; however, a very close relationship was obtained for the GD14–21 period. These findings are consistent with the faster elimination pattern observed at GD21 in the single-dose pharmacokinetic studies. The changes in the volume of fluids as pregnancy progresses also can explain these data.
The lower accumulation index observed here (1.31 for GDs 6–14 and 1.20 for GDs 14–21) indicated no relevant accumulation.
The HEPP kinetic in fetuses was not analyzed here; but our previous research (Luna-Fletes and Gómez 2003) indicates that HEPP readily transfers from dams to fetuses and exhibits parallel elimination kinetics in mothers and fetuses. The present finding agree well because the HEPP concentrations in embryonic (GD14) and in fetal tissues (GD21) were practically the same as maternal plasma HEPP concentrations under both dosing regimens (embryo or fetus/maternal ratio ≈1.0, Table 4). This means that the levels of HEPP in maternal plasma may reflect HEPP exposure in embryos and fetuses.
The most relevant data regarding HEPP effects in offspring following repeated s.c. administration to pregnant rats were the significant dose- and concentration-dependent decreases in embryo/fetus weight in both periods of treatment and the significant increase in resorption rates at the GD6–14 period (Tables 3 and 4). As shown in Table 3, the actual exposure to HEPP on embryos and fetuses on a μg/g basis is similar in both periods, as indicated by the average embryo/fetal concentration. Consequently, the higher incidence of deaths and the larger reduction in body weight observed at the GD6–14 period suggest that embryos are more sensitive than fetus to the deleterious effects of HEPP. Earlier in the pregnancy, the developing embryo is more fragile and embryonic death is more easily induced by toxic insults. However, the significant association between embryo/fetal end points such as the incidence in resorptions and the decrease in embryo/fetal weight with the levels of the drug in maternal plasma, in addition to the close relationship between maternal and fetal concentrations, suggest that the toxicity may be a function of HEPP exposure level. The pharmacokinetic parameters following single administration corroborate the aforementioned hypothesis. The higher AUC values at GD14 with respect to GD21 may be associated with higher embryo than fetuses exposure because the AUC is an estimate of the whole exposure during the treatment. The faster HEPP elimination in mothers and the possible contribution of fetal metabolism in late gestation (Anderson 2005) may also contribute to the lower fetal HEPP exposure during the GD14–21 period.
The significant decrease in embryos’ and fetuses’ body weights in both HEPP treatment periods deserves special concern. The United States Environmental Protection Agency (EPA) developmental toxicity guidelines (EPA/600/FR-91/001) established that lower embryo/fetus weight could be a sensitive indicator of toxicity in offspring.
The significant concentration-dependent increase in resorption rate is also an important finding because similar data have been reported for known teratogens such as valproic acid (Nau 1985, 1986), and other anticonvulsants in clinical use (e.g., phenytoin, phenobarbital, carbamazepine, thrimetadione) (Vorhess 1983; Finnell, Nau, and Yerby 1995).
The mechanism of action of these effects is still not well known. Recent studies have associated the common membrane-stabilizing properties of these drugs with both the GABAergic activity and the toxic effects during the prenatal development (Danielsson et al. 2000). In this sense, the magnitude of the deleterious effects was higher during the embryogenesis and was joined with hipoxic/ischemic episodes in the embryo, related to the drug levels (Azarbayjani and Danielsson 1998).
Overall, our present data indicate probable toxicity of HEPP during early pregnancy in rats. Because no significant toxicological findings were observed in the dams, the toxicological end points in the offspring suggest that HEPP is embryotoxic in rats at doses below those that cause significant maternal toxicity.
Although the effects observed here are preliminar, they merit special attention given the potential for HEPP use in pregnant women and the fact that HEPP is already in the clinical phase of investigation in Mexico (González-Esquivel et al. 1998; González-Esquivel, Pérez, and Jung-Cook 2004).
In summary, the present data following subcutaneous administration, together with previous findings using intraperitoneal administration, demonstrated that the pharmacokinetic parameters of HEPP in pregnant rats are dependent on both the route of administration and the gestational age. The adverse effects observed here in the offspring demonstrated that the organogenesis is the period of maximum susceptibility to deleterious effect of HEPP in the developing rat.
There was a direct relationship among the concentration of HEPP in maternal plasma, the elimination half-life, and the area under the maternal concentration-time curve (AUC) with the embryo/fetal toxic effects. Therefore these pharmacokinetic parameters could be reliable predictors of HEPP’s toxic effects on offspring during pregnancy.
Further conventional developmental studies of HEPP in animals are needed, but an adequate experimental protocol taken into consideration pharmacokinetics factors is advisable. Given the close relationship between drug concentration and adverse effects, the determination of drug levels in mothers and offspring during treatments is also desirable.
Footnotes
Figures and Tables
The author thanks Dr. Guillermo Carvajal of the Biochemistry Department, School of Biological Sciences, IPN, Mexico City, for his generous donation of HEPP and HEPA.