Elimination of Cocaine by Pregnant Sheep following Single or Multiple Exposures

David J. Burchfield*,1, Ian R. Tebbett{dagger} and Kevin J. Anderson*,{dagger}

* Department of Pediatrics and {dagger} Department of Physiological Sciences, Colleges of Medicine and Veterinary Medicine, University of Florida, Gainesville, Florida

Received April 7, 2001; accepted June 20, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To test the hypothesis that chronic exposure to cocaine would alter drug elimination in pregnant and fetal sheep compared to a single exposure, we administered intravenous cocaine HCl to 8 pregnant sheep daily as a bolus, followed by a 2-h infusion beginning at gestational age 75 days. Eight additional animals received an equivalent volume of saline. Three days after maternal and fetal catheter placement on day 125, ewes in both groups received cocaine HCl, 2 mg/kg, as a bolus. Maternal and fetal plasma samples were serially obtained and analyzed for cocaine and benzoylecognine. Cocaine half-life in the ewes and fetuses exposed to cocaine was no different from that in animals exposed to saline. We conclude that cocaine is rapidly metabolized in pregnant sheep and that chronic administration does not alter drug clearance.

Key Words: cocaine; repetitive; pregnancy; clearance; fetus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cocaine use by pregnant women is a major public health issue. Rates of use vary depending on the population studied; however, it is estimated that 200,000 infants are born each year in the United States who have been prenatally exposed to cocaine.

Through animal research using pregnant sheep, we now know much about the physiological effects of a single dose of cocaine in both the mother and her fetus (Burchfield, 1995Go; Burchfield and Abrams, 1993Go; Burchfield et al., 1990Go, 1991Go; Moore et al., 1986Go; Woods et al., 1987Go). Although the effects of a single dose of cocaine in pregnancy has been well studied, epidemiological studies have revealed that human cocaine use is typically repetitive, not an isolated single exposure.

The rate of clearance of cocaine and its metabolites in the chronically exposed fetus has not been well studied. A recent study of the effect of chronic cocaine infusion in adult rats on urine cocaine and metabolite concentrations showed that cocaine, ecgonine methylester, and benzoylecgonine concentrations varied with the dose administered and the duration of administration (Mets et al., 2000Go). Specifically, urinary concentration of these compounds decreased over the 13 days of infusion. These changes in metabolite concentrations could not be explained by altered cocaine metabolism, but may reflect accumulation in the animal. Given that the fetus has limited metabolic pathways compared to adults, chronic exposure to cocaine in the fetus could result in greater accumulation of the drug and a corresponding change in drug clearance. Alternatively, repetitive exposure to cocaine in the fetus could lead to dispositional tolerance manifested by an increase in the rate of clearance. To test these possibilities, we examined the clearance of cocaine and benzoylecognine in maternal and fetal sheep that had been chronically exposed to cocaine, and compared the results with sheep receiving the drug for the first time.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnant ewes bred in Maryland were delivered to the laboratory 3 days prior to scheduled surgery (72 days gestation). They were fasted for 24 h and anesthetized with halothane. A polyvinyl catheter was surgically placed in the cephalic vein of the ewe and the tip advanced centrally. The proximal end of the catheter was tunneled subcutaneously to the neck, secured with suture, and pulled through a 3 x 3 inch cloth dressing. The dressing was saturated with betadine solution weekly to prevent wound infection.

After placement of the maternal venous catheter, 8 ewes (randomly assigned) received daily cocaine HCl 2.0 mg/kg (free base) intravenously over 30 s, followed by a continuous infusion at 0.2 mg/kg per min. The 2 mg/kg bolus dose to the ewe gave plasma concentrations that approximate that reported in humans (Barnett et al., 1981Go; DeVane et al., 1991Go; London et al., 1986Go) and an infusion of 0.2 mg/ml per min provides a steady-state concentration of approximately 400 ng/ml (Burchfield et al., 1990Go). Through the use of a programmable infusion pump, the infusion was steadily decreased over the next 2 h to simulate cocaine clearance as seen in humans with a presumed t1/2 of 45 min. The cocaine was dissolved in 50 ml of saline immediately prior to infusion, to prevent decomposition. Eight other ewes received an equivalent volume of saline as a bolus, followed by a similarly decreasing infusion rate. Infusions of cocaine or saline continued daily from day 75 through day 128.

Surgical procedures to instrument fetal sheep at 125 days gestation were performed, using methods that have been used in this laboratory for the last 9 years. A hysterotomy was carried out under aseptic conditions and the fetal head and forelegs were delivered. Through a skin incision, catheters (1.07 mm ID) were placed in both brachial arteries and the cephalic vein. Sodium ampicillin, 500 mg, was placed in the amniotic fluid before closing the uterus and an additional 500 mg placed in the peritoneal cavity of the ewe. Catheters (1.65 mm ID) were placed surgically in a femoral artery and vein of the ewe. All catheters were tunneled subcutaneously and exteriorized at the ewe's flank, where they were retained in a pack. Animal care conformed to the policy of the American Physiological Society and was approved by the University of Florida Committee for Care and Use of Animals.

Three days following fetal surgery, ewes were brought into the laboratory and maternal and fetal blood pressure and heart rate were continuously recorded for 2 h. Arterial blood (0.3 ml) was removed from the fetus for measurement of blood gases and glucose. Following a 2-h observation period, cocaine, 2.0 mg/kg as free base dissolved in 30 ml saline, was administered intravenously over 30 s to both cocaine-exposed and control ewes. Maternal and fetal blood samples (1.0 ml) were obtained in heparinized syringes before cocaine administration and at 0.5, 2, 4, 8, 12, 16, 20, and 30 min later. Samples were immediately transferred to conical tubes containing 10 µl of 6 N HCl and centrifuged for 5 min. Plasma was separated and stored at –70°C until analyzed. Animals were sacrificed following the last sample, using an overdose of barbiturate followed by saturated KCl.

Cocaine (C17H21NO4) and its major metabolite in sheep, benzolecognine (C16H19NO4), were measured by a gas chromatographic-mass spectroscopy technique as previously described in detail (Phillips et al., 1996Go). This technique, with a level of detection of 1 ng/ml for benzolecognine and 2 ng/ml for cocaine, is capable of detecting other metabolites as well, if present in quantities above 5 ng/ml. Concentrations were plotted and half-life was determined, assuming linearity. Plasma half-life was compared by 2-sided Student's t-test. Differences were considered significant if p <= 0.05. Data are presented as means ± SD.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All animals underwent the protocol as outlined. Of the 8 ewes that received cocaine for 53 days, none had measurable cocaine concentrations on the day of the experiment, and only one had a measurable concentration of benzoylecognine (41 ng/ml). Likewise, no fetus had measurable cocaine concentrations, but 3 had low benzoylecognine concentrations (28, 130, and 317 ng/ml).

Half-life for cocaine was not different between the saline and the experimental groups in either the ewe or the fetus (Fig. 1Go). Cocaine-exposed ewes eliminated cocaine with a t1/2 2.00 ± 0.50 min compared to 1.92 ± 0.33 min in saline-treated controls (not significant). Likewise, cocaine-exposed fetuses eliminated cocaine with t1/2 2.38 ± 0.96 min vs. 2.33 ± 0.97 min in controls (not significant).



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FIG. 1. Maternal (top) and fetal (bottom) cocaine concentrations over time, following a 2 mg/kg bolus of cocaine hydrochloride to ewes exposed to saline (filled circle) or cocaine (filled square) for 53 days.

 
Similar to the findings for cocaine, benzoylecognine concentrations followed a similar pattern in both groups of ewes and fetuses. Thirty min after cocaine administration, control ewes had a mean benzoylecognine concentration of 211 ± 100 ng/ml vs 161 ± 93 ng/ml in chronically cocaine-exposed ewes (not significant). In control fetuses, benzoylecognine concentration at 30 min was 215 ± 134 ng/ml, while in cocaine-exposed fetuses it measured 146 ± 53 ng/ml (not significant).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cocaine use in pregnancy is prevalent and multiple exposures are more common than a single use (Frank et al., 1988Go; Graham and Koren, 1991Go). Therefore, the question of whether cocaine clearance is altered in the mother or fetus by prior exposure is pertinent. Clearly, studies of the effects of cocaine do not accurately reflect the human experience if only a single dose is administered.

Following a single intravenous dose of cocaine in pregnant ewes, placental transfer occurs quickly, with cocaine appearing in the fetus by 30 s (DeVane et al., 1991Go). Fetal-placental clearance of cocaine is a rapid, first-order pharmacokinetic process, with plasma benzoylecgonine concentrations accumulating significantly with prolonged cocaine exposure (Downs et al., 1996Go). In this study we have demonstrated that, following chronic cocaine administration in pregnant sheep, there are no differences in clearance or metabolism of a single dose in the mother or fetus. These findings are similar to previous studies which have shown no effect of cocaine pretreatment on the pharmacokinetics of a single dose of cocaine in rats (Mets et al., 2000Go; Pan and Hedaya 1999Go). The significance of these findings is that the bioavailability of a single dose of cocaine is likely unaltered, whether the subject had previously experienced multiple exposures or not. Thus, tolerance or sensitization to cocaine is likely to be unaffected by changes in metabolism or clearance following chronic use.

Limited metabolism of cocaine to benzoylecgonine by the fetus and human newborns has been reported and is considered to be associated with an immature esterase system (Browne et al., 1992Go; Dusick et al., 1993Go). Chronic maternal use of cocaine could therefore result in an accumulation of cocaine in the fetus, resulting in elevated drug levels and a greater potential for neurotoxicity. Evidence of inhibited metabolism and elimination of cocaine or benzoylecgonine was, however, not apparent in this sheep study.

Several possible mechanisms for neurotoxicity induced by cocaine or its active metabolites have been suggested: alterations of sodium channel and monoamine transporter development, release of epinephrine from the adrenal medulla with subsequent hyperglycemia, vasoconstriction with subsequent hypoxia, and decrease of nutrient supply, calcium ion chelation, superoxide formation, or infarction following repeated ischemia and reperfusion, enzyme inhibition, reduced neurotrophic activity, altered gene expression, and plasma membrane changes (Olsen, 1995Go). We hypothesized that cocaine administered to sheep during the last half of pregnancy would up regulate drug clearance mechanisms, increasing the rate of elimination. The data from these experiments clearly do not support this hypothesis. Therefore, it is likely that following a single dose to the ewe, the concentration of cocaine in the central nervous system is consistent between animals chronically exposed versus animals receiving their first exposure. This suggests that each dose of cocaine administered to the mother would have a similar toxicological effect on the fetal nervous system.


    NOTES
 
1 To whom correspondence should be addressed at Department of Pediatrics, Box 100296, University of Florida, Gainesville, FL 32610. Fax: (352) 846-3937. E-mail: burchdj{at}peds.ufl.edu. Back


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