Discovery of Spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amines as Potent NOP and Opioid Receptor Agonists.

We report the discovery of spiro[cyclohexane-pyrano[3,4-b]indole]-amines, as functional nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonists with strong efficacy in preclinical models of acute and neuropathic pain. Utilizing 4-(dimethylamino)-4-phenylcyclo-hexanone 1 and tryptophol in an oxa-Pictet-Spengler reaction led to the formation of spiroether 2, representing a novel NOP and opioid peptide receptor agonistic chemotype. This finding initially stems from the systematic derivatization of 1, which resulted in alcohols 3-5, ethers 6 and 7, amines 8-10, 22-24, and 26-28, amides 11 and 25, and urea 12, many with low nanomolar binding affinities at the NOP and mu opioid peptide (MOP) receptors.

Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol.

In a previous communication, our efforts leading from 1 to the identification of spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amine 2a as analgesic NOP and opioid receptor agonist were disclosed and their favorable in vitro and in vivo pharmacological properties revealed. We herein report our efforts to further optimize lead 2a, toward trans-6'-fluoro-4',9'-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1'(3'H)-pyrano[3,4-b]indol]-4-amine (cebranopadol, 3a), which is currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.

Reversibility of opioid receptor occupancy of buprenorphine in vivo.

The slow association and incomplete dissociation of buprenorphine from opioid receptors observed in vitro have been suggested to reduce the accessibility of opioid receptors in vivo. If so, it might be expected that buprenorphine continues to occupy opioid receptors long after the antinociceptive activity has dissipated. To examine this hypothesis, buprenorphine (46.4 microg/kg i.v.) was administered to rats 1, 2, 4 or 8 h before isolation of their forebrain membranes and the maximal binding capacity (Bmax) for [3H]-[D-Ala2, N-methyl-Phe4-Gly5-ol]-enkephalin ([3H]DAMGO) was determined to measure the number of mu-opioid receptor binding sites remaining. Extent and duration of the reduction of Bmax by buprenorphine (ED50 11.2 microg/kg 1 h post-application) correlated with the antinociceptive activity in the rat tail flick (ED50 16.4 microg/kg i.v. 1 h post-application). At 8 h after administration there was still residual antinociception but no further attenuation of Bmax was detectable. Thus receptor occupancy by buprenorphine does not cause impairment of mu-opioid receptor accessibility beyond the duration of its antinociceptive activity. Therefore, no impairment of antinociception in the case of an opioid switch is to be expected.

Biological inactivation and impaired detection of IL-10 by suramin.

Enzyme-linked immunosorbent assay (ELISA) measures the amount of cytokines secreted by cells but "equivalent" ELISAs from different manufacturers may show different results, dependent on the epitopes used to generate the antibodies employed in the ELISAs. This fact was dramatically illustrated in a series of experiments we conducted to analyze the effect of suramin on the lipopolysaccharide-induced interleukin-10 (LPS-induced IL-10) production in human CD14+ monocytes. Use of the human IL-10 DuoSet ELISA from R&D Systems showed no influence of suramin on IL-10 levels, whereas the human IL-10 ELISA from Biosource detected only low amounts of IL-10 in the presence of suramin. The results with the Biosource ELISA reflected not only interference of the recognition of IL-10 in the presence of suramin, but also a block in the biological activity of IL-10. Thus, in the presence of suramin the addition of recombinant biological IL-10 protein could not inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) or interleukin-12p40 (IL-12p40) production. Furthermore, suramin very efficiently neutralizes endogenous IL-10, resulting in a pronounced increase in IL-12 and TNF-alpha synthesis. Impaired recognition of biologically inactive conformations of IL-10 was not unique to suramin, in that heat-treated or chemically reduced recombinant IL-10 was also no longer recognized by the Biosource ELISA. This suggests that at least one of the antibodies employed in the Biosource ELISA is conformation sensitive, while the antibodies employed in the R&D Systems ELISA are insensitive to a number of conformation changes. All in all, our results show that suramin neutralizes the biological activity of IL-10 produced by monocytes. Furthermore, the amount of IL-10 detected by the Biosource ELISA correlates with the biological activity of IL-10, whereas the amount detected by the R&D Systems ELISA includes active as well as several inactive forms of IL-10.