Binding affinity to and dependence on some opioids in Sf9 insect cells expressing human mu-opioid receptor.

AIM

To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine, fentanyl, heroin, and pethidine in Sf9 insect cells expressing human mu-opioid receptor (Sf9-mu cells).

METHODS

Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for mu-opioid receptor in Sf9-mu cells. [3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using degree 55 mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice.

RESULTS

All drugs displayed antinociceptive activity and produced physical dependence in mice. The K(i) values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85+/-0.20) nmol, (59.1+/-11.7) nmol, (0.36+/-0.13) micromol, and (12.2+/-3.8) micromol respectively. The binding affinities of these drugs for mu-opioid receptor in Sf9-mu cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-mu cells. The dependence index in Sf9-mu cells was calculated as K(i) value in competitive binding assay over EC(50) value in naloxone-precipitated cAMP assay. The physical dependence index in mice was calculated as antinociceptive ED(50)/withdrawal jumping cumulative ED(50). There was a good linear correlation between dependence index in Sf9-mu cells and physical dependence index in mice.

CONCLUSION

The Sf9-mu cells could be used as a cell model to evaluate the receptor binding affinity and physical dependent liability of analgesic agents.