Studies on ligands of the kappa opioid receptor.

详细信息   

• 作者：DiMattio ; Kelly M.
• 学历：Ph.D.
• 年：2016
• 毕业院校：Temple University.bPharmacology.
• Department：Behavioralsciences.
• ISBN：9781321996760
• CBH：3719347
• FileSize：2815215
• Pages：134

文摘

This thesis is comprised of three parts. In the first part, we investigated zyklophin, a novel selective short-acting kappa opioid receptor KOPR) antagonist, and its effects on scratching behaviors in Swiss-Webster mice. We investigated whether zyklophin was able to induce scratching in a dose-dependent fashion, and whether this scratching behavior could be blocked by pretreatment with nor-binaltorphimine norBNI). We also used KOPR -/- mice to further clarify the role of the KOPR in this behavior. In the second part, we examined the role of the divergent amino acid at position 6.58 in the mu opioid receptor MOPR) and the KOPR on the binding of betafunaltrexamine beta-FNA). In the third part, we investigated the concept of functional selectivity, or ligand bias, at the KOPR. Our first set of studies revealed that zyklophin 0.1, 0.3 and 1 mg/kg, s.c., behind the neck), induced vigorous scratching in a dose-dependent manner. 0.3 mg/kg zyklophin induced 150 scratches over a 30 minute period. The scratching was not blocked by pretreatment with 20 mg/kg norBNI i.p.) 18-20 hours before injection of 0.3 mg/kg zyklophin s.c. in the nape of the neck. The scratching also persisted in KOPR -/- mice, in which the absence of the KOPR was confirmed by [3H]U69,593 binding 2 nM). In our second set of studies, we mutated the lysine at position 303 in the MOPR to glutamic acid K303E), and the glutamic acid at the equivalent position in the KOPR to lysine E297K). We transfected these mutant receptors into mouse neuroblastoma N2A) cells. We found that the mutations had no effect on [3H]diprenorphine binding affinity or competition binding with [3H]diprenorphine and beta-FNA indicating a functional intact opioid receptor. The mutations also did not affect [35S]GTPgammaS binding EC50 or Emax values. The mutation K303E in the MOPR reduced irreversible binding by 2/3 compared to the wildtype MOPR. Finally, we found that there were several ligands that displayed bias at the hKOPR and the mKOPR. At the hKOPR, using dynorphin A as the reference ligand to calculate bias, ICI-199441 was the only G biased ligand, while enadoline, nalbuphine, v pentazocine, salvinorin A, tifluadom and butorphanol were arrestin-biased. At the mKOPR, only salvinorin B methoxymethyl ether MOM-SalB) was G-biased, and salvinorin B ethoxymethyl ether EOM-SalB), ICI-199441, U50,488H, nalfurafine and 12-epi-salvinorin A 12epiSalA) were beta-arrestin-biased. Enadoline and salvinorin A were slightly arrestin biased with respect to dynorphin A. From the in vitro data at the mKOPR, we selected MOM-SalB as our G biased ligand, U50,488H as our arrestin biased ligand and additionally chose to investigate nalfurafine due to its use in clinical studies. We hypothesized that U50,488H and nalfurafine would produce aversion at lower doses than analgesia or anti-pruritic effects. We found that nalfurafine was the only ligand studied to have a separation between doses that produced analgesia and anti-scratching effects, with A50 values of 5.8 and 8 ig/kg, respectively, and only produced significant dysphoria at a dose of 20 ig/kg. U50,488H and MOM-SalB produced dysphoria at all doses tested 0.25-10 mg/kg and 0.01-0.3 mg/kg, respectively). U50,488H produced a dose-dependent analgesia and anti-scratching with A50 values of 0.58 mg/kg and 2.07 mg/kg, respectively. MOM-SalB was more potent than U50,488H in producing dose-dependent analgesia and anti-scratching, with A50 values of 0.017 mg/kg and 0.070 mg/kg, respectively. Therefore, we concluded that the in vitro bias is not able to accurately predict in vivo behaviors, and nalfurafine is the first selective full agonist at the KOPR to show ligand bias in vivo. Abstract shortened by UMI.).