Introduction

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Opioid antagonists are one therapeutic strategy available to clinicians for the treatment of opioid abuse and overdose. However, the identification of selective µ-opioid receptor antagonists has eluded researchers until recently. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP), a peptide antagonist derived from somatostatin analogs, exhibits high affinity and selectivity for µ-opioid receptors in radioligand binding experiments (Pelton et al., 1986; Kazmierski et al., 1988). In vivo, CTAP i.c.v. potently and selectively antagonizes the antinociceptive effects of µ-agonists morphine, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), and [N-Me-Phe³,D-Pro⁴]-morphiceptin (PL017) in various assays, including cold-water tail-flick (Adams et al., 1994) and complete Freund's adjuvant in rats (Hurley and Hammond, 2001) as well as tail-flick (He and Lee, 1998) and hot-plate in mice (Kramer et al., 1989). CTAP lacks µ-agonist activity and fails to antagonize -agonists in a number of assays (Kazmierski et al., 1988; Kramer et al., 1989; Grider and Maklouf, 1991; Adams et al., 1994). Antagonism by CTAP is considered a defining characteristic of µ-opioid receptor mediation.

Recent reports from both in vitro and in vivo studies indicate that CTAP may possess unusual pharmacology under certain conditions. In vitro, some investigators identify CTAP as a neutral antagonist because CTAP blocks both the agonist actions of morphine as well as the inverse agonist actions of naltrexone or -chloronaltrexamine in cells chronically treated with morphine or DAMGO (Wang et al., 1994; Liu and Prather, 2001). Other investigators, however, find that CTAP and traditional opioid antagonist naloxone produce quantitatively similar effects in the guinea pig ileum (Mundey et al., 2000). Similar discrepancies exist in vivo in that some investigators identify CTAP as a neutral antagonist because CTAP fails to produce withdrawal jumping and blocks naloxone-induced withdrawal jumping in morphine-dependent mice (Bilsky et al., 1996). Yet, other investigators report CTAP produces a number of withdrawal signs in morphine-dependent rats (Maldonado et al., 1992). CTAP also seems to interact with the -opioid receptor or DPDPE [D-Pen²,D-Pen⁵]-enkephalin at high doses and under some conditions in vivo (Kramer et al., 1989; He and Lee, 1998; Hurley and Hammond, 2001), further complicating the pharmacological classification of CTAP.

Some of the discrepancies observed for CTAP may result from the limited number of studies evaluating multiple doses of CTAP in combination with more than one opioid agonist. Therefore, the purpose of the present study was to evaluate and compare the potency of CTAP and traditional opioid antagonist naltrexone as competitive antagonists of a range of structurally diverse high- and low-efficacy peptide and alkaloid agonists in the rat tail-withdrawal assay. Lower efficacy alkaloids morphine and buprenorphine and the high-efficacy alkaloid etorphine as well as high-efficacy peptide agonists DAMGO and PL017 (Traynor and Nahorski, 1995; Emmerson et al., 1996; Walker et al., 1998) were examined. If CTAP distinguishes agonists based on their peptide or alkaloid structure, this would suggest that CTAP might bind and interact with peptide and alkaloid agonists differently at the µ-opioid receptor. If CTAP is a competitive antagonist like naloxone or naltrexone, however, CTAP will produce equipotent, parallel, dose-dependent shifts for any agonist that produces antinociceptive effects through the µ-opioid receptor. In the present study, the potency of CTAP as an antagonist of five agonists was compared with the potency of naltrexone, a well characterized alkaloid antagonist. Etorphine, morphine, and naltrexone were injected s.c. and i.c.v. to examine the role of route of administration as a determining factor in the magnitude or pattern of antagonism for either CTAP or naltrexone. The potencies of CTAP and naltrexone were quantified using apparent pA₂ analyses. Specifically, the pA₂ value is defined as the negative logarithm of the dose of antagonist to shift the dose-response curve 2-fold to the right (Arunlakshana and Schild, 1959). Competitive antagonism experiments with multiple doses of antagonists and agonists are essential pharmacological tools to distinguish receptor mechanisms underlying drug actions (Kramer et al., 1989; Walker et al., 1994).

	Materials and Methods

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Subjects

Male Sprague-Dawley rats (n = 227) (Ace Animals, Inc., Boyertown, PA) were housed individually in cages in a colony room maintained under a 12-h light/dark cycle. Water was freely available in the cages. Rats received 14 to 25 g of Purina rat chow daily to maintain body weights averaging approximately 340 g. Cumulative-dose testing occurred approximately once a week for a group of rats. Generally, one group of rats was tested per antagonist-agonist combination experiment, i.e., 5 to 7 weeks of testing. However, in some of the i.c.v. experiments, two or three groups of rats were required to complete the antagonism experiments. Before testing, the rats were habituated to the restraint tubes for 30 min in the testing room on two separate days.

Apparatus

Eight rodent restraint tubes (Harvard Apparatus, Braintree, MA) were used to restrain the rats during tail-withdrawal studies. A model 280 Series water bath (Precision Scientific, Winchester, VA) with two compartments maintained water temperatures of 40 and 55°C. A hand-held thermos was used to contain the water temperature to be tested. An HI 9060 model microcomputer thermometer (Hanna Instruments, Vila do Conde, Portugal) was used to measure the temperature of the water. Tail-withdrawal latencies were measured by visual observation and recorded manually through a hand-operated digital stopwatch with a time resolution of 1/100 s.

Procedure

Tail-Withdrawal Studies. Dose-response curves were established for all agonists (s.c. and i.c.v.) using a multiple-trial, cumulative-dosing procedure in the warm-water tail-withdrawal procedure as reported previously (Walker et al., 1994, 1998). This procedure allows an entire dose-response curve to be rapidly assessed in a single test session.

Surgery. To deliver drugs centrally, a permanent in-dwelling cannula was placed into the lateral ventricle of each rat (n = 219) using a stereotaxic instrument (Stoelting, Wood Dale, IL). Each rat was anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine i.m. Patency of each cannula was tested by injecting 100 ng of angiotensin II and observing vigorous drinking. These patency tests occurred before, after, and periodically throughout the experiments.

Data Analysis

Latencies for tail withdrawal after drug administration were converted into percentage of maximum possible effect (%MPE) by the formula:
using the control baseline latency for 55°C water measured at the beginning of each experiment. Each rat served as its own control. A value of zero was assigned if the rat withdrew its tail faster than the control latency. If a rat convulsed before the tail-withdrawal measurement, the data were not included in the analyses.

All dose-response curves for individual rats were fitted using the following semilogarithmic form of the logistic dose-response equation:
where E is the %MPE and E_min and E_max are the minimum and maximum of the sigmoid dose-response curve. E_min and E_max were initially set at 0 and 100, respectively. X is the dose of agonist (in milligrams per kilogram) at a particular effect level, ED₅₀ is the agonist dose causing a 50% maximal effect, and h is a slope factor (GraphPad Prism 3.0; GraphPad Software, Inc., San Diego, CA). Intracerebroventricular doses for the alkaloid agonists etorphine and morphine and the antagonist naltrexone were also converted to milligrams per kilogram to facilitate dose comparisons.

Dose-response curves were analyzed for parallelism with the agonist control dose-response curve (Tallarida and Murray, 1987) and these dose-response curves were used in the subsequent apparent pA₂ analyses. Apparent pA₂ values were determined according to the Schild method (Arunlakshana and Schild, 1959) with drug doses (ED₅₀ values) substituted for drug concentrations (Takemori, 1974). These analyses were performed using the computer program from Tallarida and Murray (1987). Schild plot slopes were considered to be significantly different than unity if the 95% CL of the slope did not include 1. Analysis of covariance of multiple regression lines was used to determine whether the slopes of multiple regression lines were significantly different from each other. If the slopes of the regression lines were not different, analysis of covariance was used to detect differences in elevation (apparent pA₂ values) among Schild regressions. When analysis of covariance identified significant differences among slopes or elevations, Tukey's honestly significant difference post hoc tests were performed (Zar, 1996). An apparent pK_B value was determined to estimate antagonist potency using the formula pK_B = log[B/(DR  1)] (Negus et al., 1993) in an experiment in which only one antagonist pretreatment could be performed. Significance was set a P < 0.05.

Drugs

The following compounds were used: morphine sulfate, naltrexone hydrochloride, buprenorphine hydrochloride, etorphine hydrochloride, DAMGO, PL017, and CTAP (supplied by National Institute on Drug Abuse, Rockville, MD).

Morphine, etorphine, and naltrexone were dissolved in physiological saline and injected s.c. into the dorsal flank. Buprenorphine was dissolved in sterile water and injected s.c. into the dorsal flank. CTAP, DAMGO, PL017, naltrexone, morphine, and etorphine were dissolved in filtered, sterile water and injected i.c.v. into the lateral ventricle using a hand-held 50-µl Hamilton syringe. The i.c.v. injections were performed over a period of approximately 1 min. For the first 30 s, 4 to 7 µl of drug was infused into the lateral ventricle. After an additional 30 s, the injector was removed from the guide cannula. A maximum of 25 to 28 µl of total volume was injected over the course of the entire 150-min experiment (i.e., five to six i.c.v. doses).

	Results

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CTAP i.c.v. Antagonism of Alkaloid Agonists Etorphine, Morphine, and Buprenorphine Administered Either s.c. or i.c.v. Etorphine, morphine, and buprenorphine administered s.c. and etorphine and morphine administered i.c.v. produced dose-dependent increases in tail-withdrawal latency until a maximum effect was obtained (Fig. 1). ED₅₀ values were 0.0017 mg/kg and 0.049 µg (0.00015 mg/kg) for etorphine s.c. and i.c.v., respectively; 1.1 mg/kg and 3.1 µg (0.0097 mg/kg) for morphine s.c. and i.c.v., respectively, and 0.01 mg/kg for buprenorphine s.c. Etorphine (left) and morphine (middle) were approximately 10- and 100-fold more potent, respectively, when injected by the i.c.v. route of administration.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   CTAP i.c.v. antagonism of the antinociceptive effects of alkaloid agonists etorphine s.c. and i.c.v., morphine s.c. and i.c.v., and buprenorphine s.c. Ordinate: percentage of maximal antinociceptive response (15 s). Abscissa: cumulative doses of drug, in milligrams per kilogram (top) or micrograms (bottom). Corresponding milligram per kilogram dose range for etorphine i.c.v. was 0.000029 to 0.029 mg/kg and for morphine i.c.v. was 0.0029 to 0.51 mg/kg. Points above C are the antinociceptive effects of CTAP i.c.v. alone. Corresponding milligram per kilogram doses for CTAP were as follows: 0.032 µg (0.000091 mg/kg), 0.1 µg (0.00029 mg/kg), 0.32 µg (0.00091 mg/kg), 1.0 µg (0.0029 mg/kg), 3.2 µg (0.0091 mg/kg), 10 µg (0.029 mg/kg), and 32 µg (0.091 mg/kg). CTAP i.c.v. was administered 30 min before the determination of the agonist dose-response curve. Control dose-response curves are the average of one experiment for etorphine s.c. (n = 13), buprenorphine s.c. (n = 8), etorphine i.c.v. (n = 7), and morphine i.c.v. (n = 7). Control dose-response curves are the average of two experiments for morphine s.c. in a single group of rats (n = 8). Each point in the antagonism experiments represents the mean of one to two observations in five to eight rats for etorphine s.c., morphine s.c., and morphine i.c.v. Each point in the antagonism experiment represents the mean for one observation in eight rats for buprenorphine s.c. and one observation in six to seven rats for etorphine i.c.v. Dose-response curves indicated with  are not parallel to the agonist control dose-response curve. Vertical lines represent S.E.M. During the morphine s.c. experiment, 32 µg of CTAP i.c.v. produced barrel rolling 30 min after administration in two rats. A dose of 3.2 µg of etorphine i.c.v. produced convulsions in one rat. Doses of 32 and 100 µg of morphine i.c.v. produced convulsions in three rats.

CTAP i.c.v. Antagonism of Peptide Agonists PL017 and DAMGO Administered i.c.v. PL017 i.c.v. and DAMGO i.c.v. produced dose-dependent increases in tail-withdrawal latency with ED₅₀ values of 1.9 and 0.11 µg, respectively (Fig. 2). Although CTAP i.c.v. pretreatments 0.0001 µg (0.00000029 mg/kg) to 10 µg (0.029 mg/kg) produced dose-dependent, parallel shifts to the right in the PL017 i.c.v. and DAMGO i.c.v. dose-response curves, the CTAP i.c.v. doses required to antagonize DAMGO i.c.v. were approximately 300-fold lower than those doses required to antagonize PL017 i.c.v. (Fig. 2) and 200- to 300-fold lower than those doses required to antagonize the alkaloid agonists (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   CTAP i.c.v. antagonism of the antinociceptive effects of two peptide agonists, PL017 i.c.v. and DAMGO i.c.v. Abscissa: cumulative doses of drug, in micrograms. Additional corresponding milligram per kilogram doses for CTAP were as follows: 0.0001 µg (0.00000029 mg/kg) and 0.001 µg (0.0000029 mg/kg). Control dose-response curves are the average of one experiment for PL017 i.c.v. (n = 15) and DAMGO i.c.v. (n = 23). Each point in the antagonism experiments represents the mean of one observation in five to eight rats. Doses of 100 µg of PL017 i.c.v. and 1.0 µg of DAMGO i.c.v. each produced convulsions in one rat. In four rats, a dose of 3.2 µg of CTAP i.c.v. caused excessive front paw grooming in the PL017 i.c.v. experiment. Other details as in Fig. 1.

Naltrexone s.c. Antagonism of Peptide Agonists PL017 and DAMGO Administered i.c.v and Alkaloid Agonist Morphine Administered s.c. PL017 i.c.v., DAMGO i.c.v., and morphine s.c. produced dose-dependent increases in tail-withdrawal latency with ED₅₀ values of 0.99 and 0.11 µg, and 1.1 mg/kg, respectively (Fig. 3). Naltrexone s.c. pretreatment of 0.0032 to 1.0 mg/kg produced dose-dependent, parallel shifts to the right in the PL017 i.c.v., DAMGO i.c.v., and morphine s.c. dose-response curves. In these experiments, naltrexone s.c. was approximately equipotent as an antagonist of DAMGO i.c.v., PL017 i.c.v., and morphine s.c.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Naltrexone (NTX) s.c. antagonism of the antinociceptive effects of the peptide agonists PL017 i.c.v. and DAMGO i.c.v. and the alkaloid agonist morphine s.c. Abscissa: cumulative doses of drug, in micrograms (top) or milligrams per kilogram (bottom). Points above N are the antinociceptive effects of NTX alone. Naltrexone s.c. was administered 25 min before the determination of the agonist dose-response curves. Control dose-response curves are the average of one experiment for PL017 (n = 13), DAMGO (n = 23), and morphine s.c. (n = 8). Each point in the antagonism experiment represents the mean of one observation in 7 to 11 rats for PL017 and one observation in five to eight rats for DAMGO i.c.v. and morphine s.c. Doses of 100 and 320 µg of PL017 i.c.v. each produced convulsions in one rat. Doses of 0.1 and 10 µg of DAMGO i.c.v. produced convulsions in two rats and one rat, respectively. Other details as in Fig. 1.

Naltrexone i.c.v. Antagonism of Alkaloid Agonists Etorphine and Morphine Administered Either s.c. or i.c.v. Etorphine s.c. and i.c.v. as well as morphine s.c. and i.c.v. produced dose-dependent increases in tail-withdrawal latency with ED₅₀ values of 0.0011 mg/kg, 0.20 µg (0.00063 mg/kg), 1.2 mg/kg, and 12 µg (0.038 mg/kg), respectively (Fig. 4). In these experiments, etorphine was 1.7-fold and morphine was 32-fold more potent administered i.c.v. than s.c. Naltrexone i.c.v. pretreatments of 1 µg (0.0029 mg/kg) to 32 µg (0.091 mg/kg) produced dose-dependent, parallel shifts to the right in the etorphine and morphine s.c. and i.c.v. dose-response curves. Naltrexone i.c.v. pretreatment of 10 µg (0.029 mg/kg) produced approximately a 10-fold shift in the morphine i.c.v. dose-response curve similar to the morphine s.c. dose-response curve. Additional morphine i.c.v. experiments could not be completed because naltrexone i.c.v. did not seem to block the convulsions produced by morphine i.c.v. These data indicate that the alkaloid antagonist naltrexone was approximately equipotent by either central or systemic routes of administration as an antagonist of etorphine s.c., morphine i.c.v. and morphine s.c.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Naltrexone i.c.v. antagonism of the antinociceptive effects of the alkaloid agonists etorphine and morphine administered s.c. or i.c.v. Abscissa: cumulative doses of drug, in milligrams per kilogram (top) or micrograms (bottom). Corresponding milligram per kilogram dose range for etorphine i.c.v. was 0.000029 to 0.029 mg/kg and for morphine i.c.v. was 0.0029 to 0.51 mg/kg. Corresponding milligram per kilogram doses for naltrexone were as follows: 0.032 µg (0.000091 mg/kg), 1.0 µg (0.0029 mg/kg), 3.2 µg (0.0091 mg/kg), 10 µg (0.029 mg/kg), and 32 µg (0.091 mg/kg). Naltrexone i.c.v. was administered 30 min before the determination of the agonist dose-response curves. Control dose-response curves are the average of one experiment for etorphine s.c. (n = 8), morphine s.c. (n = 6), morphine i.c.v. (n = 8), and etorphine i.c.v. (n = 8). Each dose-response curve in the antagonism experiments represents the mean of one observation in five to eight rats per curve. A low dose of 0.1 µg of etorphine i.c.v. produced convulsions in one rat and a dose of 1 µg of etorphine i.c.v. produced convulsions in another rat. High doses of either 100 and 320 µg of morphine i.c.v. produced convulsions in four rats. Other details as in Fig. 1.

Naltrexone i.c.v. Antagonism of Peptide Agonists PL017 and DAMGO Administered i.c.v. PL017 i.c.v. and DAMGO i.c.v. produced dose-dependent increases in tail-withdrawal latency with ED₅₀ values of 0.63 and 0.17 µg, respectively (Fig. 5). Naltrexone i.c.v. pretreatments of 1 µg (0.0029 mg/kg) to 32 µg (0.091 mg/kg) produced dose-dependent, parallel shifts to the right in the PL017 i.c.v. and DAMGO i.c.v. dose-response curves. Although CTAP i.c.v. was more potent as an antagonist of DAMGO i.c.v. (Fig. 1), naltrexone i.c.v. was equipotent as an antagonist of DAMGO i.c.v. and PL017 i.c.v. (Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Naltrexone i.c.v. antagonism of the antinociceptive effects of the peptide agonists PL017 i.c.v. and DAMGO i.c.v. Control dose-response curves are the average of one experiment for PL017 i.c.v. (n = 8) and DAMGO i.c.v. (n = 15). Each point in the antagonism experiment represents the mean of one observation in seven to eight rats for PL017 and one observation in five to eight rats for DAMGO i.c.v. PL017 i.c.v., 180 µg produced convulsions in one rat. Other details as in Figs. 2 and 4.

Schild Analyses for CTAP and Naltrexone Antagonism of Peptide and Alkaloid Opioid Agonists. ED₅₀ values from all antagonism dose-response curves that were parallel to initial control agonist dose-response curves were used to compare the potency of CTAP i.c.v. and naltrexone i.c.v. or s.c. as antagonists of DAMGO, PL017, morphine, etorphine, and buprenorphine using apparent pA₂ analyses (Fig. 6; Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Schild plots for CTAP, naltrexone i.c.v., and naltrexone s.c. as antagonists of the antinociceptive effects of morphine, etorphine, DAMGO, PL017, and buprenorphine. Ordinate: logarithm of the quantity [A' (ED50 of the agonist in the presence of antagonist)/A (ED50 of the agonist alone)  1]. The data for the ED50 values are determined from Figs. 1 to 5 (see text for further details). Abscissa: negative logarithm of molar doses of antagonist.

	Discussion

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In this study, the in vivo antagonist properties of the peptide CTAP were characterized and compared with a traditional opioid antagonist naltrexone in the rat tail-withdrawal assay. CTAP was a more potent antagonist of the antinociceptive effects of five high- and low-efficacy peptide and alkaloid µ-agonists than naltrexone. In general, CTAP was 10- to 100-fold more potent than naltrexone as an antagonist of µ-agonists. For example, the apparent pA₂ value for CTAP with morphine s.c. was 9.5, whereas the apparent pA₂ values for naltrexone i.c.v and s.c. with morphine s.c. were 8.1 and 8.5, respectively. The apparent pA₂ values for naltrexone s.c. obtained in the present study are similar to those values obtained in other antinociception assays (Takemori and Portoghese, 1984; Garner et al., 1997). Similarly, CTAP was approximately equipotent as an antagonist of µ-agonists morphine and PL017 in the present study as in other antinociception procedures in rats and mice (Kramer et al., 1989; Adams et al., 1994; Takasuna et al., 1994). Taken together with radioligand binding and guinea pig ileum studies (Pelton et al., 1986; Kazmierski et al., 1988; Kramer et al., 1989), the data from the present experiment indicate CTAP is a potent µ-antagonist.

However, higher pretreatment doses of 10 and 32 µg (0.029 and 0.091 mg/kg) CTAP produced nonparallel shifts in the morphine s.c. and etorphine s.c. dose-response curves. CTAP (10 µg; 0.029 mg/kg;) produced dose-dependent, parallel shifts in the dose-response curves for highly µ-opioid, receptor-selective peptide agonists PL017 or DAMGO (Chang et al., 1983; Schiller et al., 1989). The difference between the competitive antagonism of high-efficacy agonists DAMGO and PL017 and the noncompetitive antagonism of etorphine s.c. and morphine s.c. may implicate CTAP is an insurmountable antagonist. Indeed, the finding that CTAP pretreatments produced a progressive flattening of the morphine s.c. dose-response curve at a dose lower (3.2 µg; 0.0091 mg/kg) than that for the etorphine s.c. dose-response curve suggests that CTAP may distinguish these agonists according to relative efficacy as seen in previous studies with insurmountable antagonists -funaltrexamine and clocinnamox (Tiano et al., 1998; Walker et al., 1998). Further experiments with high doses of CTAP and other lower efficacy agonists are required to test this hypothesis.

Alternatively, this difference between the competitive antagonism of DAMGO and PL017 and the noncompetitive antagonism of etorphine s.c. and morphine s.c. suggests that etorphine and morphine may interact with additional opioid receptors when producing antinociceptive effects. In vitro, etorphine binds with similar affinities to µ-, -, and -opioid receptor sites and morphine has at best 100-fold selectivity for µ- compared with - and -receptors (Kosterlitz and Paterson, 1980; Neil, 1984; Goldstein and Naidu, 1989). However, in vivo data with the antagonist naltrexone indicate morphine s.c. and etorphine s.c. produce their antinociceptive effects predominantly through the µ-opioid receptor in this antinociception assay in rats, even at high doses (Walker et al., 1994, 1996).

The noncompetitive antagonism of morphine s.c. and etorphine s.c. produced by high doses of CTAP may therefore indicate that CTAP, as opposed to etorphine or morphine, interacts with -opioid receptors in some manner. For example, in previous experiments CTAP noncompetitively blocks -agonist DPDPE antinociception in the mouse (Kramer et al., 1989), although this may be a µ-mediated effect in the spinal cord (He and Lee, 1998). In addition, the agonist effects of high doses of CTAP in the mouse vas deferens were blocked by -antagonist ICI 174,864, suggesting that CTAP may possess weak -agonist activity in mice (Kramer et al., 1989). Generally, because CTAP is so potent, most investigators test CTAP doses of 1.0 µg and lower (Kramer et al., 1989; Adams et al., 1994). However, the data in the present study include a wide range of CTAP doses with a series of alkaloid and peptide agonists, revealing a complex in vivo characterization of CTAP.

Another unique pharmacological interaction observed in the present study was the extremely potent antagonism of the µ-agonist DAMGO by CTAP. The apparent pA₂ value for CTAP with DAMGO was 2 to 3 log units higher than the apparent pA₂ values for CTAP with the other peptide and alkaloid agonists. Previous investigators determined apparent pA₂ values of approximately 11 for CTAP i.t. as an antagonist of DAMGO i.t. in a mouse radiant-heat tail-flick assay (He and Lee, 1998; Riba et al., 2002). One explanation for the unique CTAP and DAMGO interaction in the present study may be related to the manner in which these peptides bind to the µ-opioid receptor. DAMGO binds into the first extracellular loop of the µ-opioid receptor, whereas other agonists bind into the middle of the third intracellular loop to the C terminus (Onogi et al., 1995). CTAP binding selectivity was found to reside in a more extended receptor region (Xue et al., 1995). Conceivably, because DAMGO binds differently than other agonists within the µ-opioid receptor, CTAP may interact with DAMGO at the binding site in a manner that either increases the potency for CTAP or decreases the potency of DAMGO.

Alternatively, the pharmacodynamics of CTAP and DAMGO in vivo may account for the dramatic increase in CTAP potency. The Schild regression for CTAP antagonism of DAMGO is shallow, indicating the metabolism of DAMGO may be more rapid than CTAP or perhaps CTAP is accumulating in the brain. CTAP was found to be stable in the blood and serum of rats (t_1/2 > 500 min) with diffusional blood-central nervous system penetration quantitatively similar to morphine (Abbruscato et al., 1997; Egleton et al., 1998). Elimination half-life for DAMGO has been measured as only 15 min in sheep (Szeto et al., 2001). Conceivably, DAMGO may be metabolized by peptidases at a rate faster or through a different pathway than CTAP, a cyclic peptide, resulting in an observed increase in potency for CTAP. The observation that the potency for CTAP as an antagonist of PL017 was similar to the alkaloid agonists in the present study is supported by data indicating that PL017 i.c.v. can produce antinociception for as long as 4 h in rats (Chang et al., 1983). PL017 may last as long as CTAP in the brain and represent a more compatible peptide agonist for in vivo studies with CTAP in rats.

The apparent pA₂ values for CTAP i.c.v and naltrexone i.c.v. were significantly higher with etorphine i.c.v. compared with etorphine s.c. This observation may result from some unique properties of etorphine. The apparent pA₂ values were similar for naltrexone i.c.v. and naltrexone s.c. as antagonists of the other µ-agonists with the exception of the values obtained with the agonist PL017. Etorphine is highly lipophilic (Medzihradsky et al., 1992) and would be expected to diffuse more rapidly out of the central nervous system than other agonists such as PL017, which has a limited ability to cross the blood-brain barrier (Shook et al., 1987). Conceivably, if etorphine i.c.v. diffuses more rapidly out of the brain after central administration than either CTAP i.c.v. or naltrexone i.c.v., the antagonists may seem more potent as reflected in the higher apparent pA₂ values. Previous studies have compared the relative potencies of etorphine, morphine, and PL017 administered by central and systemic routes of administration (Lange et al., 1980; Shook et al., 1989) but little comparative data exists for the potency of naltrexone or CTAP to block these agonists via multiple routes of administration. Additional studies with other highly lipophilic agonists or antagonists would be required to test the hypothesis that the higher apparent pA₂ values for CTAP i.c.v. and naltrexone i.c.v. result from the rapid diffusion of etorphine i.c.v. out of the brain.

In summary, CTAP is a more potent antagonist than naltrexone injected by either s.c. or i.c.v. routes of administration. CTAP antagonism, however, is not universally equipotent or competitive, may be insurmountable, and at high doses CTAP may interact with -opioid receptors in vivo. Furthermore, CTAP may distinguish some peptide agonists such as DAMGO from other agonists based on binding interactions within the µ-opioid receptor or pharmacodynamic properties of these peptides. The results of this study with CTAP and naltrexone are important for a variety of reasons. Often single doses of CTAP in combination with single doses of single opioid agonists are used to characterize an observed in vivo or in vitro effect as specifically µ-opioid receptor-mediated. Additionally, recent in vitro and in vivo studies propose that CTAP may be differentiated from naltrexone or naloxone based on neutral antagonist versus inverse agonist activity at the µ-opioid receptor (Wang et al., 1994; Bilsky et al., 1996). Although this is an exciting possibility, the present study reveals that some of the unusual pharmacological properties of CTAP may be related to either noncompetitive interactions with the µ-opioid receptor or perhaps other receptors in vivo. Nevertheless, these data indicate that the peptide antagonist CTAP possesses a unique pharmacology that warrants additional in vitro and in vivo characterization.