Given the mounting evidence for involvement of δ opioid receptors in the tolerance and physical dependence of μ opioid receptor agonists, we have investigated the possible physical interaction between μ and δ opioid receptors by using bivalent ligands. Based on reports of suppression of antinociceptive tolerance by the δ antagonist naltrindole (NTI), bivalent ligands [μ-δ agonist-antagonist (MDAN) series] that contain different length spacers, and pharmacophores derived from NTI and the μ agonist oxymorphone, have been synthesized and evaluated by intracerebroventricular (i.c.v.) administration in the tail-flick test in mice. In acute i.c.v. studies, the bivalent ligands functioned as agonists with potencies ranging from 1.6- to 45-fold greater than morphine. In contrast, the monovalent μ agonist analogues were substantially more potent than the MDAN congeners and were essentially equipotent with one another and oxymorphone. Pretreatment with NTI decreased the ED 50 values for MDAN-19 to a greater degree than for MDAN-16 but had no effect on MDAN-21. Chronic i.c.v. studies revealed that MDAN ligands whose spacer was 16 atoms or longer produced less dependence than either morphine or μ monovalent control MA-19. On the other hand, both physical dependence and tolerance were suppressed at MDAN spacer lengths of 19 atoms or greater. These data suggest that physical interaction between the μ and δ opioid receptors modulates μ-mediated tolerance and dependence. Because MDAN-21 was found to be 50-fold more potent than morphine by the i.v. route (i.v.), it offers a previously uncharacterized approach for the development of analgesics devoid of tolerance and dependence.
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Interaction between μ and δ opioid receptors was first suggested by Vaught and Takemori (1), who observed potentiation of morphine-induced antinociception by a δ agonist. Reports that followed also suggested an interaction between μ and δ opioid receptors and its possible significance in morphine tolerance and physical dependence (2–4). Studies with δ antagonists have demonstrated that the chronic effects of morphine can be blocked without significantly diminishing its antinociceptive action (4–7). Subsequent studies by using antisense oligodeoxynucleotides (8, 9) and δ opioid receptor knockout mice (10) have supported these ideas, implicating μ–δ interactions in the development of morphine tolerance and physical dependence. More recently, mixed μ agonist/δ antagonist ligands have been designed as an approach to analgesics devoid of these side effects (11–13).
It has not yet been reported whether the in vivo synergy between μ and δ agonists is a consequence of direct association between receptors or due to functional modulation involving neuronal circuitry. In view of evidence for μ-δ opioid receptor heterodimers in cultured cells (14–16), there is reason to believe that similar interactions may occur in vivo. The development of selective ligands that target associated μ-δ opioid receptors should help to further delineate the pharmacology of these receptors.
We have designed bivalent opioid ligands that contain μ agonist and δ antagonist pharamacophores to address whether physical interaction between μ and δ opioid receptors is required to attenuate μ agonist-induced tolerance and dependence in mice. Several studies have suggested that bivalent ligands may bridge associated opioid receptors. Bivalent ligands of opioid alkaloids (17–24) and peptide agonists derived from enkephalins (25–28) have been characterized as having increased opioid receptor potency and selectivity when compared to corresponding monovalent counterparts. More recently, bivalent ligands have been used as tools to further characterize δ and κ opioid receptor phenotypes. Specifically, unique bivalent probes that target δ 1-κ 2 heterodimers (29) or associated δ 2-κ 1 opioid receptors (30) have been reported.
Here, we report on the design, synthesis, and biological evaluation of the MDAN (MDAN, μ-δ agonist-antagonist) series of bivalent ligands containing a μ opioid agonist and a δ opioid antagonist pharmacophores designed to address the question as to whether μ and δ receptors mediate their actions in a physically associated state. The results of our studies suggest that opioid-induced tolerance and physical dependence are mediated through physical association of μ and δ opioid receptors as heterodimers.
Male ICR mice (Harlan Labs, Indianapolis) that weighed 25–30 g were used throughout these studies. The mice were housed in groups of five at 22–23°C in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal care facility under a 12 h-12 h light/dark cycle. Both food and water were available ad libitum. All procedures were approved by the Louisiana State University Health Sciences Center Animal Care and Use Committee.
Drugs were dissolved in sterile saline (0.9% NaCl). Animals were anesthetized with isoflurane and drugs were administered i.c.v. in a volume of 4 μl into the lateral cerebral ventricle (31). For i.v. administration, animals were restrained by hand, and drugs were injected into a lateral tail vein in a volume of 100 μl.
Antinociception was evaluated by the radiant heat tail-flick assay (32). Time required to flick the tail in response to a focused beam of light was recorded. Each animal served as its own control and was used only once. Mice were tested once before injection (control time) and again once after injection (drug time) at the time of peak drug response as determined by pilot time course studies. The light intensity was adjusted so that control times were between 1.5–2.5 sec. A 10-sec cut-off drug time was set to minimize the risk of tissue damage. Percent maximum possible effect (%MPE) was calculated as: [(drug time (sec) - control time (sec))/(10 sec - control time (sec))] × 100 = %MPE.
Graded dose–response curves of at least four doses with at least eight mice per dose were generated from the %MPE data. ED 50 values with 95% confidence intervals (C.I.) were computed with GraphPad prism by using nonlinear regression methods.
Osmotic minipumps (model 1003D, Alzet, Durect Corp. Cupertino, CA) were filled with saline or the drug solution to be tested. The dose of each drug was 12 times its ED 50/h. These doses were based on methods previously developed in our laboratory for chronic i.c.v. infusion of morphine in mice (33). The minipumps were connected by a 1.6–1.8 cm length of PE-60 tubing to a 3-mm-long cannula (osmotic pump connector cannula, Plastics One, Roanoke, VA) and primed in sterile saline at 37°C overnight.
For pump implantation, mice were anesthetized with Avertin [2,2,2-tribromoethanol (370 mg/kg, i.p.)/t-amyl alcohol (0.16 mg/kg, i.p.)]. The scalp was shaved, an incision was made along the midline, and the skull was scraped clean of periosteum. Hemostats were used to make a pocket under the skin between the shoulder blades. A micro drill was used to drill a hole ≈1.6 mm lateral and 0.6 mm caudal to bregma. The minipump was placed in the pocket between the shoulder blades, the cannula was inserted through the drilled hole into the lateral ventricle, and the cannula pedestal was affixed to the skull with cyanoacrylate glue. The animals were allowed to recover on a heating pad and were returned to their cages in the animal facility for 3 days.
On the fourth day after implantation of the minipump, mice were injected with naloxone (1 mg/kg, s.c.) and placed into Plexiglas cylinders for 10 min (34). The number of vertical jumps were counted as withdrawal signs, indicating development of physical dependence. Wet shakes were also observed in some animals but were not recorded.
The minipumps were then removed, and mice were returned to their home cage for 4 h. To test for development of tolerance, animals were then injected i.c.v. with the test drug into the contralateral cerebroventricle and antinociception was measured with the tail-flick test at the predetermined time of peak drug activity.
ED 50 values were considered significantly different when the 95% C.I. intervals did not overlap.
Preparation and characterization data for all of the compounds are published as supporting information, which is published on the PNAS web site.
The pharmacophores (1 and 2 in Fig. 1) chosen for the MDAN series incorporate the μ opioid agonist oxymorphone 1 (35) and the δ opioid antagonist naltrindole (NTI) 2 (36), linked through a variable length spacer. The NTI pharmacophore was selected because of the report that δ opioid antagonists suppress tolerance and dependence without a substantial diminution of efficacy (5). The spacer features a central diamine flanked by adjacent diglycolic acid moieties. Spacer length was varied by changing the number of methylenes in the central diamine portion. Because prior studies have provided evidence for bridging of associated opioid receptors by bivalent ligands whose spacers are in the range of 22 Å, we bracketed this length with shorter and longer length spacers (22). The spacers varied from 16 atoms (MDAN-16, 3) to 21 atoms (MDAN-21, 8). The constitution of the spacers was motivated by our desire to maintain a favorable hydrophilic-hydrophobic balance of the bivalent ligands. Matched monovalent control compounds were synthesized for the μ monovalent series (MA-16–MA-21, 9–13)¶ (MA, μ agonist) along with a δ monovalent control (DN-20, 14) (DN, δ antagonist) in an effort to factor out possible effects of the spacer on activity in the bivalent ligands. Final compounds are illustrated in Fig. 2.
Key pharmacophores: oxymorphone (1, μ agonist) and NTI (2, δ antagonist).
Final compounds.
To determine whether spacer length affects the antinociceptive potency of our bivalent ligand series, compounds 3–8 (MDAN series) were acutely evaluated after i.c.v. administration to mice. These bivalent ligands were all less potent than the μ monovalent ligands 9–13 (MA series) (Table 1). Increasing the distance between the μ and δ pharmacophores of the MDAN ligands was associated with increased antinociceptive potency. In contrast, acute administration of the μ monovalent compounds (MA series) revealed that the spacer l
