Optimization binding studies of opioid receptors, saturation and competition, using [³H]-DAMGO

Opioid analgesics are prescribed for the moderate to severe pain in the clinic. New analogs of µ-opioid receptors are introduced because they may have less adverse effects and better efficacy. However, these new analogs have to be screened for their receptor affinity before entering clinical trial phases. A common method to do such screening is using radioligand-binding-assay, which is a fast and precise screening technique if the assays are done at an optimum condition. One of the main challenges in this type of screening is to separate free/unbound radioligands from bound radioligands. In this study, we applied a centrifugation method instead of a filtration method to separate free radioligands from bound radioligands, and also optimized the conditions for radioligand receptor binding studies of µ-opioid receptors, saturation, and the competition.

We used the midbrain and brainstem of naltrexone-treated rats as a source of µ-opioid receptors, and [³H]-DAMGO as the radioligand. Naloxone was also used to determine non-specific binding. A given amount of membrane protein was incubated with an increasing amount of radioligand at 37 °C to saturate the receptors at equilibrium and the amount of radioligand saturated in the receptors were used in competition studies.

160 µg membrane protein saturated with 20 nM [³H]-DAMGO at 37 °C for 35 min with K_d (15.06 nM, 95% CI 8.117-22.00) and B_max (0.4750 pmol/mg, 95% CI 0.3839-0.5660).

Applying the centrifugation method instead of the filtration to separate free from bound radioligand produced repeatable and reliable results. The optimum conditions for radioligand binding were used in competition studies which resulted in the expected outcomes.