Opioid agonist efficacy predicts the magnitude of tolerance and the regulation of μ-opioid receptors and dynamin-2

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Abstract

It has been proposed that opioid agonist efficacy may play a role in tolerance and the regulation of opioid receptor density. To address this issue, the present studies estimated the in vivo efficacy of three opioid agonists and then examined changes in spinal μ-opioid receptor density following chronic treatment in the mouse. In addition, tolerance and regulation of the trafficking protein dynamin-2 were determined. To evaluate efficacy, the method of irreversible receptor alkylation was employed and the efficacy parameter τ estimated. Mice were injected with the irreversible μ-opioid receptor antagonist clocinnamox (0.32–25.6 mg/kg, i.p), and 24 h later, the analgesic potency of s.c. morphine, oxycodone and etorphine were determined. Clocinnamox dose-dependently antagonized the analgesic effects of morphine, etorphine and oxycodone. The shift to the right of the dose-response curves was greater for morphine and oxycodone compared to etorphine and the highest dose of clocinnamox reduced the maximal effect of morphine and oxycodone, but not etorphine. The order of efficacy calculated from these results was etorphine > morphine > oxycodone. Other mice were infused for 7 days with oxycodone (10–150 mg/kg/day, s.c.) or etorphine (50–250 μg/kg/day, s.c.) and the analgesic potency of s.c. morphine determined. The low efficacy agonist (oxycodone) produced more tolerance than the high efficacy agonist (etorphine) at equi-effective infusion doses. In saturation binding experiments, the low efficacy opioid agonists (morphine, oxycodone) did not regulate the density of spinal μ-opioid receptors, while etorphine produced ≈ 40% reduction in μ-opioid receptor density. Furthermore, etorphine increased spinal dynamin-2 abundance, while oxycodone did not produce any significant change in dynamin-2 abundance. Overall, these data indicate that high efficacy agonists produce less tolerance at equi-effective doses. Furthermore, increased efficacy was associated with μ-opioid receptor downregulation and dynamin-2 upregulation. Conversely, lower efficacy agonists produced more tolerance at equi-effective doses, but did not regulate μ-opioid receptor density or dynamin-2 abundance. Taken together, these studies indicate that agonist efficacy plays an important role in tolerance and regulation of receptors and trafficking proteins.

Introduction

Although tolerance to opioid agonists has been well-documented, studies indicate that the magnitude of tolerance is dependent on the particular opioid agonist that is used (e.g., Duttaroy and Yoburn, 1995, Paronis and Holtzman, 1992, Stevens and Yaksh, 1989, Walker and Young, 2001). For example, if equi-effective doses (e.g., 100 times the ED50) of an opioid agonist are continuously infused in the intact animal, some agonists, such as etorphine, produce substantially less tolerance than other agonists such as morphine (Duttaroy and Yoburn, 1995). In addition, the ability of an opioid agonist to induce analgesic tolerance appears to be inversely related to its effectiveness to internalize and downregulate μ-opioid receptors. Specifically, morphine does not induce μ-opioid receptor internalization and downregulation, either in vitro or in vivo, while etorphine is very effective (Keith et al., 1996, Keith et al., 1998, Patel et al., 2002b, Stafford et al., 2001, Yoburn et al., 2004; however see Heberstock-Debic et al., 2005). Similarly, etorphine, but not morphine, upregulates the trafficking protein dynamin-2; and this may be an important mechanism in accelerating internalization and downregulation (Patel et al., 2002b, Yoburn et al., 2004).

Efficacy is a property of a ligand that may contribute to tolerance and regulation of μ-opioid receptors and trafficking proteins. Efficacy has been defined as “…the property of a molecule that causes the receptor to change its behavior toward the host cell” (Kenakin, 2002a). Efficacy is generally interpreted to be a function of the receptor and its cellular environment (Kenakin, 2002b). Thus, efficacy is not simply a receptor based parameter of drug action; it depends upon activity at the receptor as well as the chain of events that are activated. In contrast, intrinsic efficacy was a term proposed by Furchgott (1966) that was intended to provide “… a method to compare the relative ability of agonists to produce a response for a given receptor occupancy … ” (Kenakin, 2002b). Intrinsic efficacy is a property solely of the drug-receptor complex and, theoretically, is independent of the cellular environment and the assay system.

Recently, it has been proposed that the Operational Model of Drug Agonism (Black and Leff, 1983, Black et al., 1985, Leff et al., 1990) is a preferred approach for characterizing agonist efficacy (e.g., Kenakin, 2004). A key feature of the operational model is the term τ. τ is a dimensionless proportionality factor that can relate receptor occupancy and receptor stimulus. τ is a transducer constant and characterizes the propensity of a system and an agonist to produce a response (Kenakin, 2004). τ is analogous to the efficacy term, since it is a property of both the tissue, assay and the receptor system (Motulsky and Christopoulos, 2004). Estimation of τ using the operational model requires depletion of receptors, typically using an antagonist that will irreversibly bind to the receptor. In the present study, we employed the irreversible μ-opioid receptor antagonist clocinnamox and the data analysis method of Zernig et al., 1995, Zernig et al., 1996 to calculate τ values for 3 opioid agonists following clocinnamox treatment. Clocinnamox has been shown to dose-dependently decrease μ-opioid receptor BMAX in the intact animal (Burke et al., 1994, Chan et al., 1995, Paronis and Woods, 1997). Furthermore, clocinnamox produces a dose-dependent shift to the right of opioid agonist dose-response functions (Barrett et al., 2003, Burke et al., 1994, Chan et al., 1995, Comer et al., 1992, Zernig et al., 1995).

We have proposed that ligand efficacy is a determinant of the regulation of μ-opioid receptors and trafficking proteins, as well as changes in opioid agonist potency following treatment (Duttaroy and Yoburn, 1995, Patel et al., 2002b, Yoburn et al., 1993). This suggestion is based on results from experiments using chronic treatment with opioid agonists and antagonists in the intact animal. High efficacy opioid agonists (e.g., etorphine) can reliably decrease μ-opioid receptor density, regulate μ-opioid receptor mRNA levels and upregulate dynamin-2 (e.g. Yoburn et al., 1993, Sehba et al., 1997, Duttaroy and Yoburn, 2000, Patel et al., 2002b). Furthermore, at equi-effective doses, high efficacy opioid agonists produce less tolerance. Conversely, opioid antagonists reliably increase μ-opioid receptor density, decrease dynamin-2 abundance, but do not alter μ-opioid receptor mRNA levels in vivo (e.g.Rajashekara et al., 2003, Duttaroy et al., 1999, Unterwald et al., 1995). In addition, chronic opioid antagonist treatment produces a significant shift to the left of the agonist dose-response function for several outcome measures including analgesia and lethality (i.e., supersensitivity; Tempel et al., 1982, Paronis and Holtzman, 1991, Patel et al., 2002a, Yoburn, 1988). Therefore, from a broad perspective, the direction of receptor regulation (upregulation, downregulation) covaries with efficacy for agonists and antagonists. Similarly, following chronic agonist or antagonist treatment, the change in potency of agonists (tolerance, supersensitivity) also covaries with efficacy. Given these data, we hypothesized that, following chronic opioid treatment, the efficacy of a ligand can be used to predict changes in potency of agonists, as well as regulation of μ-opioid receptor density and dynamin-2. In this study, we determined the efficacy of oxycodone, morphine and etorphine and then evaluated tolerance and regulation of μ-opioid receptor density and dynamin-2 in the intact mouse. Overall, we report that efficacy does predict tolerance and regulation of μ-opioid receptors and dynamin-2.

Section snippets

Subjects

Male, Swiss–Webster mice (20–25 g) were obtained from Taconic Farms (Germantown, NY, USA). Mice were housed (5–10/cage) under standard colony conditions with food and water available ad libitum. All animal treatment protocols were approved by the St. John's University Institutional Animal Care and Use Committee.

General procedure

Initially, analgesic (tailflick, see below) ED50's were determined for the opioid agonists oxycodone and etorphine. Other groups of mice (N = 8–10/group) were injected i.p. with saline or

Results

The quantal ED50's (percent analgesic) were determined for oxycodone and etorphine using both the standard and cumulative dose-response protocols (Fig. 1, Fig. 2). The ED50 for oxycodone using the standard dosing protocol was 0.81 ± 0.10 mg/kg and using the cumulative dosing protocol was 1.74 ± 0.46 mg/kg. The ED50 for etorphine using the standard dosing protocol was 0.85 ± 0.17 μg/kg and using the cumulative dosing protocol was 1.12 ± 0.07 μg/kg. Similar results were observed when graded data (mean

Discussion

Opioid agonists vary in the magnitude of tolerance produced following chronic treatment. Several studies suggest that opioid agonists with lower efficacy will induce more tolerance than higher efficacy agonists (Duttaroy and Yoburn, 1995, Paronis and Holtzman, 1992, Stevens and Yaksh, 1989, Walker and Young, 2001). Previously, we have used an equi-effective dosing approach to directly determine the magnitude of tolerance produced by different opioid agonists (Duttaroy and Yoburn, 1995). This

Acknowledgements

These data represent a portion of a thesis presented by the first author (MP) to the faculty of the College of Pharmacy and Allied Health Professions, St. John's University, in partial fulfillment of the requirements for the M.S. degree in Pharmaceutical Sciences. We thank Dr. M.T. Turnock, Qiuyu Zhang, Pinky Sharma for advice and technical assistance. We also thank Dr. Terry Kenakin for several discussions regarding the nature of efficacy. This work was supported in part by DA 19959 and a seed

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