A major objective of this proposal is to synthesize and biologically evaluate irreversible affinity ligands that will bind selectively to mu, delta, and kappa receptors both in vivo and in vitro with the goal of finding a new modality for treating heroin addiction and preliminary studies have shown that the thiol-containing morphine derivative act as long-term mu-selective antagonists in a mouse tail flick test when administered by intracerebroventricular (i.c.v) injection. 14Beta- (Dithioglycolamido)-bis-7,8-dihydromorphinone (TAMO) acted as a short- term agonist and a long-term high affinity antagonist in supraspinal antinociceptive tests when given by either i.c.v. or intraperitoneal (i.p.) injection. The corresponding N-cyclopropylmethyl derivative, N- CPM-TAMO, acted as a short-term kappa agonist in the mouse writhing test and as a long-term mu-selective high affinity antagonist in the mouse tail flick test after i.c.v. injection. These ligands inhibited mu opioid binding in bovine striatal membranes. The synthesis of thiol- containing mu, kappa, and delta affinity ligands is described in this proposal. Selected ligands will be tested for their effects on supraspinal and spinal antinociception, development of physical dependence, respiratory depression and gastrointestinal motility. Different routes of administration including oral will be used. Biochemical studies will be directed to determine specificity and irreversibility of the ligands in membrane bioassays. Affinity ligands that are active in vitro and in vivo, particularly by the oral route, will be radiolabelled to characterize the proteins that are specifically labelled by the radiolabelled ligands. Another objective of this proposal is to synthesize and evaluate biologically photoaffinity ligands that bind covalently to the mu receptor. The desirable properties of such a ligand are: (1) It must photolyze at wavelengths which do not destroy the receptor (>295 nm); (2) must wash out before irradiation; (3) should be a high affinity mu- selective agent. A ligand has been synthesized which meets these criteria but its potency is not as great as one might wish. This ligand will be tritium labelled and the radiolabelled ligand photolyzed in membranes. More potent ligands will be synthesized. Preliminary studies have shown that it is possible to prepare fluorescent affinity ligands which bind irreversible to mu receptors. Other ligands have been prepared which have fluorescence emission spectra at wavelengths at which tissue autofluorescence does not interfere. These wash out easily. Based on out experience with non-equilibrium ligands, the design and synthesis of non-equilibrium fluorescence ligands are planned which will have emission spectra at wavelengths where tissue fluorescence is not a problem.
Showing the most recent 10 out of 14 publications
