Abstract

[Dmt¹]DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂; Dmt = 2′,6′-dimethyltyrosine) binds with high affinity and selectivity to the μ opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt¹]DALDA, we have prepared [³H][Dmt¹]DALDA and compared its binding properties with that of [³H]DAMGO ([d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin). Kinetic studies revealed rapid association of [³H][Dmt¹]DALDA when incubated with mouse brain membranes (K₊₁ = 0.155 nM^–1 min^–1). Dissociation of [³H][Dmt¹]DALDA was also rapid (K_–1 = 0.032 min^–1) and indicated binding to a single site. [³H][Dmt¹]DALDA binds with very high affinity to human μ opioid receptor (hMOR) (K_d = 0.199 nM), and K_d and B_max were reduced by sodium but not Gpp(NH)p [guanosine 5′-(β,γ-imido)triphosphate]. Similar K_d values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human δ opioid receptor) selectivity of [Dmt¹]DALDA (∼10,000) is 8-fold higher than DAMGO. However, [Dmt¹]DALDA is less selective than DAMGO against hKOR (human κ opioid receptor) (26-versus 180-fold). The K_i values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [³H][Dmt¹]DALDA compared with [³H]DAMGO, with the exception of Dmt¹-substituted peptide analogs. All Dmt¹ analogs showed much higher affinity for the μ receptor than corresponding Tyr¹ analogs. [³⁵S]GTPγS (guanosine 5′-O -(3-[³⁵S]thio)triphosphate) binding showed that [Dmt¹]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [³H][Dmt¹]DALDA for the μ receptor, together with its very low nonspecific binding (10–15%) and metabolic stability, make [³H][Dmt¹]DALDA an ideal radioligand for labeling μ receptors.