Introduction

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Cocaine abuse and dependence remain among the nation's most serious drug abuse problems and are associated with many adverse economic, social, and health-related consequences (National Institute on Drug Abuse, 1997; Mendelson and Mello, 1998). Thus far, no effective pharmacotherapy has been devised for cocaine abuse treatment (Rawson et al., 1991; Tutton and Crayton, 1993; Mendelson and Mello, 1996). Although there has been significant progress in clarifying the neurobiological basis of the effects of cocaine (Woolverton and Johnson, 1992; Kuhar, 1993), it is generally acknowledged that a better understanding of the molecular mechanisms of cocaine dependence will be important for the development of more effective treatment medications (Leshner, 1996).

Cocaine binds to the dopamine transporter and blocks the reuptake of dopamine, which results in increased levels of dopamine at the synapse (Kuhar et al., 1991; Kuhar, 1993). In addition, there is abundant evidence that the reinforcing properties of cocaine are well correlated with its actions as an indirect dopamine agonist and are mediated by dopaminergic systems (such as the mesolimbic dopamine system) located in brain (Ritz et al., 1987; Kuhar et al., 1991). One strategy to reduce cocaine abuse has been to develop methods to bind cocaine in blood and prevent it from crossing the blood-brain barrier to reach the central nervous system. Theoretically, such a substance would decrease some of the behavioral and physiologic effects of cocaine. An example of this approach has been the discovery of specific antibodies for both catalyzed degradation (Landry et al., 1993) and immune binding of cocaine (Carrera et al., 1995; Fox et al., 1996; Fox, 1997). The effectiveness of these procedures for reducing the behavioral effects of cocaine is currently under investigation (Fox et al., 1996). New techniques also are being developed to enhance enzyme-catalyzed cocaine hydrolysis in blood (Gorelick, 1997).

We hypothesized that a substance that resembles the structure of the dopamine transporter might also mimic its ability to bind cocaine. The dopamine transporter has been characterized as a glycoprotein that is approximately 20% carbohydrate (Kuhar, 1993). In the course of our studies of cocaine interactions with the endocrine system (Mello and Mendelson, 1997), we noted that portions of the amino acid constituents and N-linked carbohydrate side chains of a glycoprotein hormone, luteinizing hormone (LH), resembled similar molecular structures within portions of the dopamine transporter described by Kuhar and coworkers (Kuhar et al., 1991; Kuhar, 1993). LH is a gonadotropin hormone that is released from the anterior pituitary gonadotropes and does not cross the blood-brain barrier. Accordingly, we hypothesized that high levels of LH might bind to cocaine in blood and decrease levels of free cocaine in plasma. To test this hypothesis, we administered synthetic LH-releasing hormone (LHRH) to male and female rhesus monkeys to stimulate the secretion of LH from the pituitary before i.v. cocaine administration. LHRH is a synthetic decapeptide that acts as endogenous hypothalamic LHRH to stimulate release of LH from the gonadotropes in the anterior pituitary. Synthetic LHRH is used as a provocative test of pituitary function in clinical endocrinology (Rebar, 1991) and effectively stimulates LH release in rhesus monkeys (Mello et al., 1990b, 1995). We now report that LHRH administration to male and female rhesus monkeys was followed by a significant increase in plasma LH levels and a corresponding significant decrease in peak levels of free cocaine measured in plasma.

	Materials and Methods

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Subjects

Four male and three female adult rhesus monkeys (Macaca mulatta) were subjects in studies of the acute effects of LHRH on plasma cocaine levels. Each monkey was studied as his/her own control across experimental conditions. Females were studied during the follicular phase of the menstrual cycle, 6 to 8 days after menstruation onset. Male monkeys weighed 10.4 ± 0.9 kg and females weighed 5.2 ± 0.6 kg. Monkeys were maintained on a diet of jumbo monkey biscuits (PMI Feeds, Inc., St. Louis, MO), multiple vitamins, and fresh fruits and vegetables. Water was continuously available. A 12-h light/dark cycle was in effect throughout the year (lights on from 7:00 AM to 7:00 PM).

Animal maintenance and research were conducted in accordance with the guidelines provided by the National Institutes of Health Committee on Laboratory Animal Resources. The facility was licensed by the United States Department of Agriculture, and protocols were approved by the Institutional Animal Care and Use Committee. The health of the monkeys was monitored periodically by consulting veterinarians. Monkeys had visual, auditory, and olfactory contact with other monkeys throughout the study. Environmental enrichment was provided by toys, television, and staff contact.

Sequence of Conditions and Sample Collection Frequency

The acute effects of synthetic LHRH and placebo LHRH on LH and cocaine levels in plasma were examined. LH levels were measured for 30 min before and 30 min after administration of synthetic LHRH (15 or 30 µg/kg i.v.) or placebo LHRH (saline). Cocaine (0.8 mg/kg i.v.) was then administered, and samples were collected for an additional 100 min. LHRH was administered 30 min before cocaine administration, because these doses of LHRH stimulated peak LH release within 30 min in female rhesus monkeys in our previous studies (Mello et al., 1990b, 1995). The 30-min interval between LHRH administration and cocaine infusion was intended to ensure that the maximal effects of cocaine occurred after peak LHRH stimulation of LH. Samples for LH analysis were collected at 10-min intervals until 20 min after cocaine administration and then at 20-min intervals thereafter.

The frequency of sampling for cocaine analyses was dictated by the pharmacokinetics of cocaine in rhesus monkey. Plasma cocaine levels increase rapidly after i.v. administration, and peak plasma cocaine levels were measured at 2 min after i.v. administration in rhesus monkeys (Sarnyai et al., 1996) and in humans (Mendelson et al., 1998; Sholar et al., 1998). A 2-min postcocaine sampling frequency was used in the present study to maximize detectability of changes in plasma cocaine levels during the first 10 min after cocaine administration. During the first 10 min after cocaine administration, five bolus samples for cocaine analysis were collected at 2-min intervals, and, subsequently, samples were collected at 10-min intervals. The duration of sampling was determined, in part, by the half-life of cocaine, which has been estimated to average 80 min after an i.v. bolus injection of 1 mg/kg in rhesus monkey (Misra et al., 1977). The 100-min postcocaine sampling interval used in the present study enabled us to follow cocaine and LH levels during and after the period of maximal cocaine concentrations in plasma.

Venous Catheterization and Blood Collection Procedures

Monkeys were anesthetized with ketamine hydrochloride (5-10 mg/kg, i.m.) for acute venous catheterization. Ketamine does not affect release of pituitary gonadotropins (Ferin et al., 1976; Fuller et al., 1984). Two catheters were implanted in opposite legs, one for injection of LHRH and cocaine solutions and one for withdrawal of blood samples to measure plasma levels of cocaine and LH. A Sur-Flo Intracath containing a 20-gauge needle (i.d. 0.80 × 51 mm; Terumo Medical Corporation, Elkton, MD) was inserted into the saphenous vein using aseptic techniques. After removal of the needle stylet, the catheter was joined to a heparin-impregnated sterile silicon tubing and secured with sutures. The monkey was placed in a standard primate chair for about 60 min before collection of baseline samples began. Monkeys were adapted to chair restraint on several occasions before this study began.

Blood samples for cocaine analysis were collected in tubes containing potassium oxalate and sodium fluoride (2.5 ng/ml) to prevent cocaine hydrolysis by serum esterases (Jatlow and Bailey, 1975). Blood samples for LH analysis were collected in heparinized tubes. Samples were centrifuged, and aliquots of plasma were drawn and stored at 70°C until analysis.

LHRH and Cocaine Administration

Synthetic LHRH (gonadorelin hydrochloride; Factrel, 15 or 30 µg/kg i.v.) or placebo LHRH (an equal volume of saline) was injected into the saphenous vein of the leg opposite the exfusion catheter in a single bolus injection and flushed through with sterile saline. Thirty minutes later, cocaine (0.8 mg/kg) was infused into the saphenous vein of the leg opposite the exfusion catheter in a single bolus injection and flushed through with sterile saline. The cocaine dose selected (0.80 mg/kg i.v.) stimulates release of gonadotropins and adrenocorticotropic hormone in rhesus monkey and has been well tolerated by rhesus monkeys in our previous endocrine studies (Mello et al., 1990a,b, 1993; Sarnyai et al., 1996). This dose initially was selected on the basis of reports that in rhesus monkeys, the dose range for potentially lethal convulsions is 3 to 8 mg/kg i.v. cocaine, whereas up to 1 mg/kg cocaine is safe for i.v. administration (Matsuzaki and Misra, 1977; Misra et al., 1977). Moreover, this dose of cocaine is within the behaviorally active range in rhesus monkeys, and lower doses (0.40 mg/kg i.m. injection) have discriminative stimulus effects (Negus et al., 1995, 1996). This cocaine dose is also within the range shown to produce subjective and physiological effects in humans (total dose, 32 to 96 mg divided by 70 kg equals 0.457 to 1.37 mg/kg) (Fischman et al., 1985).

Drugs

Cocaine hydrochloride was obtained from the National Institute on Drug Abuse (Rockville, MD), and solutions were prepared by dissolving cocaine in sterile saline for injection USP. The solution was filter-sterilized using a 0.11-µm Millipore filter.

Synthetic LHRH (gonadorelin hydrochloride; Factrel) was obtained from Wyeth-Ayerst Laboratories (Philadelphia, PA). Ampules containing a diluent of 100 µg of gonadorelin hydrochloride in sterile water were diluted to the appropriate concentration (15 or 30 µg/kg) for individual monkeys.

Plasma LH and Cocaine Analyses

LH Radioimmunoassay. Plasma LH concentrations were determined in duplicate by a double-antibody radioimmunoassay procedure similar to that described by Midgley (1966) using materials prepared by Dr. W. Peckham and following his suggestions. Purified ceropithecus pituitary LH for radioiodination (WP-XV-117-3239), rabbit antiserum (WP-R13, pool D) to human choriogonadotropin, and rhesus pituitary LH reference preparation (NICHD-rhLH, also known as WP-XV-20) were provided by the National Hormone and Pituitary Program, supported by the National Institute of Child Health and Human Development and the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Radioiodination was performed using the chloramine-T reaction (Greenwood et al., 1963) with sodium iodide-125 purchased from New England Nuclear Life Science Products (Billerica, MA). Goat anti-rabbit -globulin was obtained from Calbiochem (La Jolla, CA). Results are expressed in nanograms per milliliter in terms of the reference preparation. The LH assay sensitivity was 9.2 ng/ml and the intra-and interassay c.v.s were 6.6 and 6.9%, respectively.

Plasma Cocaine Analysis. Free plasma cocaine levels were measured in duplicate using a solid-phase extraction method described by Spec Instructions Manual by Ansys with a Hewlett-Packard model 5890 Series II gas chromatograph equipped with a capillary column and a Hewlett-Packard 5971 Series Mass Selective detector (Abusada et al., 1993). The assay sensitivity was 10 ng/ml and the intra-assay c.v. was 2.2%.

Statistical Analysis of LH and Plasma Cocaine Levels

The effects of LHRH and LHRH placebo on LH and plasma cocaine levels were evaluated with a two-way ANOVA for repeated measures with time and LHRH dose as factors. If ANOVA showed a significant main effect, Dunnett's Multiple Comparison Procedure was used to determine which groups were statistically different from each other. ANOVA for repeated measures was also performed for each LHRH dose group, and the significance of group mean values at each sample period were compared with baseline means using Dunnett's test for comparison of multiple experimental groups with a single control group. Probability levels of P < .05-.0001 are reported as statistically significant. The covariance between plasma cocaine levels and LH levels was assessed with correlational techniques (Pearson Product Moment Correlation).

Pharmacokinetic Analysis

Estimates of cocaine's primary kinetic parameters (i.e., peak plasma cocaine concentrations, rate constants, apparent volume of distribution, and time to peak plasma concentration) and secondary parameters [i.e., area under the curve (AUC) and initial and terminal phase half-lives] were obtained directly from a nonlinear regression-estimation software program based on the Manual of Pharmacologic Calculations with Computer Programs using PHARM/PCS Version 4.2 (MicroComputer Specialist MCS, Philadelphia, PA). Plasma drug concentrations were fitted to a single dose, one-compartment model with bolus input, first order output, and elimination. Plasma concentrations were weighted by the reciprocal of the predicted concentrations. AUCs (AUC_0-180) were estimated by the linear trapezoidal rule. Estimates of the elimination half-life (T_1/2) were obtained from the computer-fitted model. These pharmacokinetic parameters were analyzed with an ANOVA to determine whether there were any differences between placebo LHRH and 15 and 30 µg/kg LHRH. ANOVAs also were used to compare pharmacokinetic parameters at each dose between males and follicular-phase females.

	Results

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Plasma LH Levels Before and After LHRH Administration

Male LH Levels. The time course of changes in plasma LH levels after placebo LHRH and active LHRH administration in four male rhesus monkeys is shown in Fig. 1 (row 1). All males had normal LH levels before LHRH administration, and there were no significant differences between baseline LH levels across conditions. Baseline LH levels averaged 18 (±1.7), 15.9 (±2.8), and 20.9 (±2.3) ng/ml before placebo LHRH and low- and high-dose LHRH administration, respectively. ANOVA indicated a significant effect of LHRH dose on LH levels (P < .05), and post hoc analysis indicated that relative to placebo LHRH, both 15 µg/kg LHRH (P < .01) and 30 µg/kg LHRH (P < .001) significantly increased plasma LH levels before cocaine administration. At 30 min after placebo LHRH, LH levels did not change appreciably from baseline and averaged 16.3 (±2.3). Ten minutes after administration of cocaine in combination with placebo LHRH, peak LH levels increased slightly above baseline and averaged 20.2 (±2.7). After low- and high-dose LHRH administration, LH increased to average 102 (±13.9) and 122 (±27.5) ng/ml immediately before cocaine administration. Ten minutes after administration of cocaine in combination with 15 or 30 µg/kg LHRH, LH increased to peak levels of 115 (±20.2) and 150.6 (±38.4) ng/ml.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Effects of LHRH on plasma LH (ng/ml) in male and female rhesus monkeys. Data for males are shown in row 1, and data for females are shown in row 2. LH levels before and after administration of placebo LHRH (left), 15 µg/kg i.v. LHRH (center), and 30 µg/kg i.v. LHRH (right) are shown in each row. LH values in ng/ml are shown on the left ordinate. Placebo LHRH or active LHRH was administered at time 0, shown at the first vertical, dotted line. Cocaine was administered 30 min later as shown at the second vertical, dotted line. Time (min) after LHRH administration is shown on the abscissa. Baseline (B) samples are the average (±S.E.M.) of 12 samples in 4 male monkeys or 9 samples in 3 female monkeys. Post-LHRH LH samples are an average (±S.E.M.) of single samples from four male or three female monkeys.

Female LH Levels. The time course of changes in plasma LH levels after placebo LHRH and active LHRH in three female rhesus monkeys is shown in Fig. 1 (row 2). All females had normal baseline LH levels that averaged 30 (±2.8), 25 (±4.0), and 40 (±6.4) ng/ml before placebo and low- and high-dose LHRH administration, respectively. There were no significant differences in LH baselines across conditions. ANOVA indicated a significant effect of LHRH dose on LH levels (P < .05), and posthoc analysis indicated that relative to placebo LHRH, both 15 µg/kg LHRH (P < .01) and 30 µg/kg LHRH (P < .05) increased LH levels significantly before cocaine administration. At 30 min after placebo LHRH administration, LH levels averaged 26 (±1.9). Ten minutes after administration of cocaine in combination with placebo LHRH, LH levels averaged 33 (±2) ng/ml, whereas 30 min after administration of 15 or 30 µg/kg LHRH, LH levels increased to average 73 (±25) and 55 (±7.8) ng/ml immediately before cocaine administration. Ten minutes after administration of cocaine in combination with 15 or 30 µg/kg LHRH, LH increased to average 87 (±35) and 58 (±6.5) ng/ml.

Gender Comparisons. Baseline LH levels in females were significantly higher than in males in all three LHRH dose conditions (P < .05). Although both doses of LHRH stimulated significant increases in LH in both males and females, the peak LH increases from pre-LHRH baseline levels were greater in males than in females. After 15 µg/kg LHRH, peak LH levels increased to average 499% above baseline in males and 354% above baseline in females. After 30 µg/kg LHRH, peak LH levels increased to average 529% above baseline in males and 78% above baseline in females.

Plasma Cocaine Levels after Cocaine and LHRH Administration

The time course of changes in plasma cocaine levels after placebo LHRH and 15 and 30 µg/kg LHRH is shown in Fig. 2. After administration of placebo LHRH, plasma cocaine levels rise rapidly, and peak plasma cocaine levels usually occurred within 2 min after cocaine administration in both males and females. Plasma cocaine levels then decreased gradually during the remainder of the 180-min sampling period. Peak plasma cocaine levels were significantly higher after placebo LHRH than after active LHRH administration in both males and females. Comparisons between plasma cocaine levels at corresponding time points indicated that peak plasma cocaine levels after placebo LHRH administration were significantly higher (P < .05) than after administration of 15 and 30 µg/kg LHRH. In males, after administration of 15 and 30 µg/kg LHRH, peak plasma cocaine levels were 47 and 57% lower, respectively, than peak plasma cocaine levels after placebo LHRH administration. In females, the LHRH-associated decrement in peak plasma cocaine levels averaged 36 and 39% below peak plasma cocaine levels after placebo LHRH administration. However, comparisons between plasma cocaine levels in males and females at corresponding time points indicated no significant differences after administration of 15 and 30 µg/kg LHRH. The only significant gender difference in plasma cocaine levels was detected after placebo LHRH administration. Plasma cocaine levels were significantly lower in females than in males at 10 min after cocaine administration (P < .05).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Plasma cocaine levels (ng/ml) after LHRH in male and female rhesus monkeys. The time course of changes in plasma cocaine levels after placebo LHRH or LHRH (15 and 30 µg/kg) administration is shown for males in row 1 and females in row 2. Time (min) after i.v. cocaine administration (0.8 mg/kg) is shown on the abscissa. Plasma cocaine levels (ng/ml) are shown on the left ordinate. Asterisks indicate points after 15 or 30 µg/kg LHRH administration that were statistically significantly different from the corresponding time point after placebo LHRH administration (P < .05).

Pharmacokinetics of Cocaine in Males and Females

Pharmacokinetic analyses of plasma cocaine levels were performed on data from males and females. Table 1 summarizes the pharmacokinetic analyses of plasma cocaine levels after i.v. administration of 0.8 mg/kg cocaine and placebo LHRH, 15 µg/kg LHRH, and 30 µg/kg LHRH. The time to reach peak levels of cocaine in plasma (T_max), the peak levels of cocaine in plasma (C_max), elimination T_1/2, and AUC are shown for males and for females studied during the follicular phase of the menstrual cycle.

Time to Reach Peak Plasma Cocaine Levels. In males, the T_max after placebo LHRH averaged 3 min and did not change significantly as a function of LHRH administration condition. In females, the T_max averaged 2 min after placebo LHRH and increased to 4.7 (±2.7) min after active LHRH. There were no statistically significant differences between males and females on this pharmacokinetic parameter.

Peak Levels of Plasma Cocaine. In males, peak levels of free plasma cocaine were significantly higher after placebo LHRH administration than after 15 or 30 µg/kg LHRH administration (P < .02-.04). Although peak plasma cocaine levels were lower after 30 µg/kg LHRH than after 15 µg/kg LHRH, this difference was not statistically significant. In females, peak plasma cocaine levels were significantly higher after placebo LHRH than after 30 µg/kg LHRH (P < .04). Peak plasma cocaine levels were also higher after administration of placebo LHRH than after 15 µg/kg LHRH, but this difference was not significant (P = .06), in part because the pharmacokinetics program does not compare plasma concentrations at corresponding time points. Moreover, there were no gender differences in peak levels of cocaine in plasma after placebo LHRH administration or 15 or 30 µg/kg LHRH administration.

Elimination T_1/2 of Plasma Cocaine. The elimination T_1/2 of plasma cocaine did not differ significantly as a function of the dose of LHRH. In males, the T_1/2 averaged between 43 and 61 min. In females, the T_1/2 averaged between 56 and 61 min. The elimination T_1/2 of plasma cocaine after placebo LHRH or 15 or 30 µg/kg LHRH administration did not differ significantly in males and females.

AUC. In males, the AUC was greatest after placebo LHRH, but this was not significantly different from the AUC after 15 and 30 µg/kg LHRH administration. In females, the AUC was significantly greater after placebo LHRH than after 15 µg/kg LHRH (P < .03) but not after 30 µg/kg. Comparisons between males and females showed no differences in AUC measured during the placebo LHRH condition or the 15 or 30 µg/kg condition.

Covariance Between Peak Levels of LH and Cocaine in Plasma

Figure 3 shows the relationship between peak levels of plasma cocaine 2 min after i.v. cocaine injection and LH levels measured immediately before i.v. cocaine administration. In males there was a significant negative correlation between LH levels and peak plasma cocaine levels (P < .01). LHRH-induced stimulation of LH secretion was accompanied by an LHRH dose-dependent decrease in peak plasma cocaine levels. In females, although LHRH administration was associated with significant attenuation of peak plasma cocaine levels, there was no significant correlation between LH and plasma cocaine levels. In males, LH levels above 70 ng/ml were sufficient to decrease plasma cocaine levels compared with the placebo LHRH baseline. In females, LH levels above 45 ng/ml were sufficient to decrease plasma cocaine levels compared with the placebo LHRH baseline.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Correlation between peak plasma cocaine levels and LH levels. The correlation between plasma cocaine levels (ng/ml) 2 min after i.v. cocaine injection and LH levels (ng/ml) measured immediately before i.v. cocaine administration is shown for male (row 1) and female rhesus monkeys (row 2).

	Discussion

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LHRH-induced increases in LH release were associated with significantly lower peak levels of cocaine in plasma than after placebo LHRH administration when LH levels remained at normal baseline levels (Figs. 1 and 2). Moreover, peak plasma cocaine levels were inversely correlated with peak LH levels in rhesus males but not in females (Fig. 3). The mechanisms by which LHRH administration resulted in a significant decrease in peak plasma cocaine levels are unknown. Pharmacokinetic analyses indicated that the significant reduction in peak plasma cocaine levels was not accompanied by changes in the rate of cocaine absorption or metabolism as inferred from the rate of elimination. The cocaine assay used in this study measures free cocaine in plasma, and there is a possibility that cocaine might also be sequestered in body lipid and not detected by this analytic procedure. In this discussion we will consider these issues, as well as the possibility that LH, a glycoprotein hormone, bound cocaine in plasma. Alternatively, LHRH may have stimulated release of another hormone such as follicle-stimulating hormone (FSH), which is similar in structure to LH, or the combination of LHRH and LH may have stimulated release of an unknown entity that bound to cocaine in blood.

LHRH Effects on Plasma LH Levels. The time course and magnitude of LHRH stimulation of LH was comparable to that reported in our previous studies (Mello et al., 1990b, 1995). Although LHRH stimulated a significant increase in LH above basal levels within 30 min in all monkeys, there were gender-specific differences in the magnitude of the LH response (Fig. 1). LHRH stimulated greater LH responses in male than in female rhesus monkeys. This is consistent with normative data on LHRH effects on LH in men and women reported in the Physicians' Desk Reference (1997). Men reached peak LH levels more rapidly and had a higher percentage increase in LH than follicular-phase women after 100 µg i.v. LHRH (1997). Although estradiol levels are relatively low during the early follicular phase of the menstrual cycle in rhesus monkeys (Knobil and Hotchkiss, 1988), the well established negative-feedback relationship between estradiol and LH may have contributed to the differences between males and females in the LH response to LHRH observed in this study (Yen, 1991). Estradiol inhibition of follicular-phase LH may also have contributed to our finding that LHRH resulted in a dose-dependent LH increase in rhesus males but not in females (Fig. 1). We administered LHRH on a microgram per kilogram basis in an effort to minimize intersubject variability. However, in many previous clinical studies, a single dose of LHRH was administered and not adjusted for body weight. Under those conditions, LH responses also were not LHRH dose-dependent (1-450 µg) in human males (Rebar et al., 1973). In previous studies in rhesus males, i.v. administration of 1, 5, and 25 µg of LHRH also resulted in relatively small differences in peak LH levels (Toivola et al., 1978).

Cocaine Interactions with LH. LH is secreted from anterior pituitary gonadotrophs in response to pulsatile release of endogenous LHRH from the hypothalamus or to synthetic LHRH stimulation. Some gonadotropes produce only LH or only FSH, whereas other gonadotropes produce both LH and FSH (Chin, 1984). LH has a molecular weight of 28,000 (Catt and Dugau, 1991) and does not cross the blood-brain barrier. Like all glycoprotein hormones, LH is composed of an  and  subunit (Chin, 1984), and the potential contribution of these subunits to the effects observed is unclear. Because high LH levels appear to reduce the peak level of cocaine in plasma, it is tempting to postulate that stimulation of LH results in binding cocaine to LH in blood before it reaches the brain. However, the degree to which LHRH-stimulated LH interacts with cocaine to reduce peak levels of cocaine in plasma is unknown. Although LH is a glycoprotein hormone with carbohydrate side chains that are similar to those on the dopamine transporter (Kuhar, 1993), these carbohydrate side chains are probably cocaine-recognition sites rather than cocaine-binding sites per se. Kuhar reported that enzymatic removal of sialic acid residues on the carbohydrate side chains of the dopamine transporter did not alter binding of a cocaine analog, but dopamine transport was altered in synaptasomes (Kuhar, 1993). At present, the basic molecular mechanisms underlying cocaine binding to the dopamine transporter are unknown. For example, recent studies have shown that an amine nitrogen similar to the amine of dopamine is not essential for cocaine binding to the dopamine transporter (Madras et al., 1998).

Cocaine Pharmacokinetics in Rhesus Monkeys. Although peak levels of plasma cocaine (C_max) after LHRH administration were reduced significantly compared with placebo LHRH treatment, there were no LHRH-related differences in other pharmacokinetic parameters. Specifically, the T_max and the elimination T_1/2 did not change significantly after LHRH administration. By analogy, administration of an anticocaine monoclonal antibody to mice did not significantly change the rate of cocaine metabolism or the ratio of cocaine to its metabolites (benzoylecgonine and ecgonine methylester) (Fox et al., 1996). Rather, the cocaine vaccine is thought to bind cocaine in the peripheral circulation and inhibit its entry into brain (Fox, 1997). Consistent with this hypothesis, higher levels of [³H]cocaine were measured in plasma of immunized mice than in controls, but lower levels were measured in brain tissue (Fox, 1997). In the present study, lower levels of cocaine were measured in plasma after LHRH administration. Cocaine bound to LH would not be detectable with the analytic procedures for plasma cocaine described in this report. However, LH degraded in liver and kidney would release bound cocaine, and it would be metabolized rapidly by esterases in these tissues and in blood. Coincidentally, the elimination T_1/2 of circulating LH is 53 (±5.4) min (Veldhuis and Johnson, 1988; Veldhuis et al., 1989), and this is very similar to the T_1/2 of cocaine shown in Table 1.

Pharmacokinetic Analyses of Plasma Cocaine: Gender Comparisons. We are unaware of any previous comparisons of the pharmacokinetics of plasma cocaine levels in both male and female rhesus monkeys. Although both males and females were studied by Misra and et al. (1997), data were not reported separately for each gender. In the present study, the quantitative pharmacokinetic profiles indicated that after placebo LHRH administration, there were no gender differences in peak levels of cocaine in plasma after administration of 0.8 mg/kg i.v. cocaine, T_max, the elimination T_1/2, or the AUC. These findings suggest that after i.v. administration, the pharmacokinetic parameters of cocaine are similar in male and female rhesus monkeys.

Summary and Conclusions. Synthetic LHRH was administered to stimulate the secretion of LH from pituitary gonadotropes in rhesus monkeys before i.v. cocaine administration. Synthetic LHRH administration resulted in a significant increase in plasma LH levels in both male and female rhesus monkeys. C_max levels of cocaine were decreased after LHRH stimulation of LH secretion compared with peak plasma cocaine levels after placebo LHRH administration in both male and female rhesus monkeys. The reduction in C_max levels was related to the magnitude of LHRH-induced increases in plasma LH levels. These observations are consistent with the hypothesis that cocaine may bind rapidly to an LH molecular entity in plasma that resembles a portion of the molecular structure of the dopamine transporter in brain. Binding of cocaine to a segment of the LH molecule in plasma should reduce significantly the amount of cocaine that crosses the blood-brain barrier. Cocaine binding occurred rapidly (in less than 2 min), and T_max levels did not differ significantly in monkeys that received active LHRH and placebo LHRH. Clinical studies have shown that maximal arterial cocaine concentrations occurred within 15 s after i.v. administration (Evans et al., 1996). It is possible that cocaine binding occurred in arterial pools in rhesus monkeys because of the rapid time course of the initial event. However, only venous samples were collected in this study. The clinical relevance of these findings in rhesus monkey remain to be determined, and studies of the effects of human chorionic gonadotropin and synthetic LHRH on the effects of cocaine in humans are ongoing. The feasibility of long-term synthetic LHRH administration has been demonstrated in clinical studies of infertility (Filicori et al., 1994; Crowley et al., 1985). Synthetic LHRH (2.5-5.0 µg i.v.) was administered every 60 to 90 min over 505 menstrual cycles to women with various types of infertility disorders, and no adverse side effects were reported (Filicori et al., 1994). We recognize that sustained high levels of LH in gonadally intact men and women could disrupt regulation of the hypothalamic-pituitary gonadal axis, and the ratio of risks to benefits compared with the adverse effects of cocaine itself is unknown. However, the maximal LH levels achieved in rhesus monkeys after LHRH stimulation ranged between 87 and 150 ng/ml, and these are less than those after ovariectomy in rhesus monkeys (N.K.M, unpublished data) or after natural menopause in women (Jaffe, 1991). If it were possible to identify and isolate the molecular elements of LH that bind to cocaine, then a new medication, with less direct biological activity than LHRH, could be developed to attenuate the effects of cocaine. Data obtained in these studies suggest a new approach to the development of medications designed to rapidly bind cocaine in blood and prevent it from crossing the blood-brain barrier.