LPK-26, a novel κ-opioid receptor agonist with potent antinociceptive effects and low dependence potential

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Abstract

Analgesics such as morphine cause many side effects including addiction, but κ-opioid receptor agonist can produce antinociception without morphine-like side effects. With the aim of developing new and potent analgesics with lower abuse potential, we studied the antinociceptive and physical dependent properties of a derivate of ICI-199441, an analogue of (−)U50,488H, named (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (LPK-26). LPK-26 showed a high affinity to κ-opioid receptor with the Ki value of 0.64 nM and the low affinities to μ-opioid receptor and δ-opioid receptor with the Ki values of 1170 nM and > 10,000 nM, respectively. It stimulated [35S]GTPγS binding to G-proteins with an EC50 value of 0.0094 nM. In vivo, LPK-26 was more potent than (−)U50,488H and morphine in analgesia, with the ED50 values of 0.049 mg/kg and 0.0084 mg/kg in hot plat and acetic acid writhing tests, respectively. Moreover, LPK-26 failed to induce physical dependence, but it could suppress naloxone-precipitated jumping in mice when given simultaneously with morphine. Taken together, our results show that LPK-26 is a novel selective κ-opioid receptor agonist with highly potent antinociception effects and low physical dependence potential. It may be valuable for the development of analgesic and drug that can be used to reduce morphine-induced physical dependence.

Introduction

Opioid analgesic drugs such as morphine and fentanyl are widely used for the relief of severe pain. However, clinical use of these drugs is limited by their adverse side effects including respiratory depression and dependence liability. At least three types of opioid receptors (μ, δ, and κ) mediate pharmacological and physiological actions of opioid drugs and endogenous opioid peptides. A considerable number of studies suggest that the μ-opioid receptor mediates most of the opioid actions, including analgesia, tolerance, dependence and reward (for a review, see Pasternak, 1993). Morphine and fentanyl are the selective μ-opioid receptor agonists, and thus the adverse side effects of them may be attributed to selective activation of μ-opioid receptors.

Increasing evidence shows that κ-opioid agonists may provide a strong analgesia free from the abuse potential and the adverse side effects of μ-agonists like morphine (Lahti et al., 1982, Lahti et al., 1985, Clark and Pasternak, 1988, Hunter et al., 1990). Moreover, activation of the κ-opioid receptor exerts an opposite effect on morphine-induced adverse actions, such as tolerance, reward, and impairment of learning and memory (for a review, see Pan, 1998). These studies suggest that κ-opioid receptor agonists would have potential advantages over μ-opioid receptor agonists as analgesics. Therefore, in the search for alternative analgesics to μ-opioid receptor agonists such as morphine, considerable attention has been focused on the development of κ-opioid receptor agonists.

With the aim of developing new and potent κ-opioid receptor agonists with lower abuse potential than available opiates, we modified the arylacetamide moiety of ICI-199441, an analogue of (−)U50,488H, by incorporating 3-pyrroline into the basic amino functionality, producing a new series of 3-pyrroline containing arylacetamides, which could be eventually modified to peripherally acting agonists (Mo et al., 2002). Among these compounds, LPK-26 (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (Fig. 1) is one of the compounds with high affinity for κ-opioid receptor (Mo et al., 2002). In this study, we determined LPK-26 binding affinity and its ability to stimulate guanosine 5′-O-(3-[35S]thio) triphosphate ([35S]GTPγS) binding to G-proteins and examined the antinociceptive activities of LPK-26 by acetic acid writhing and hot plate tests. Meanwhile, we also detected its potential to develop physical dependence and effect on morphine physical dependence.

Section snippets

Cell culture

CHO cells were transfected with human κ-, rat μ- or rat δ-opioid receptors by using Lipofectamine (Invitrogen) as per manufacturer's protocol. CHO cells stably expressing human κ-, rat μ- or rat δ-opioid receptors were maintained in F12 medium (Gibco) with 10% fetal calf serum and 0.25 mg/ml G418 (Roche). Cells were incubated in a humidified atmosphere consisting of 5% CO2, 95% air at 37 °C. For receptor binding and [35S]GTPγS binding experiments, cells were seeded into 175-cm3 flasks. When

Expression levels of μ-, δ- and κ-opioid receptors in CHO cells

The rat μ-, δ-opioid receptor and human κ-opioid receptor were stably expressed in CHO cells. Saturation binding of [3H]diprenorphine to μ-, δ- and κ-opioid receptors was performed on membrane, and Kd and Bmax values were determined. [3H]Diprenorphine bound to the μ-, δ- and κ-opioid receptors with Kd values of 0.29 ± 0.02 nM, 0.25 ± 0.03 nM and 0.20 ± 0.03 nM, respectively (n = 4), and the Bmax values of 576 ± 95 fmol/mg protein, 1597 ± 366 fmol/mg protein and 765 ± 35 fmol/mg protein, respectively (n = 4).

Binding affinity of LPK-26 and (−)U50,488H for human κ-opioid receptor

Discussion

The present study demonstrated that LPK-26 was a full κ-opioid receptor agonist with high potency. In vitro analyses showed that LPK-26 exhibited high affinity to κ-opioid receptor with Ki of 0.64 nM, which is ~ 10-fold less than the prototypic selective κ-opioid receptor agonist (−)U50,488H. Agonist-stimulated binding of [35S]GTPγS to G-proteins has been used as a sensitive assay of activation of many G protein-coupled receptors. It has been reported that enhancement of [35S]GTPγS binding by

Acknowledgements

This work was supported by the National Basic Research Program grant from the Ministry of Science and Technology of China G2003CB515401 and National Science Fund for Distinguished Young Scholar from the National Natural Science Foundation of China 30425002 and fund provided by Chinese Academy of Sciences (J.-G. Liu).

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    These authors contributed equally to this work.

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