BDBM179908 US10752592, Compound nalbuphine US11534436, Compound nalbuphine US10736890, Compound TABLE B.1 US9133125, Table D, nalbuphine
BDBM208977 US9233167, 6-O-mPEG3-Nalbuphine
BDBM208978 US9233167, 6-O-mPEG6-Nalbuphine
BDBM208979 US9233167, 6-O-mPEG9-Nalbuphine
BDBM209007 US9233167, 6-O-mPEG4-Nalbuphine
BDBM209008 US9233167, 6-O-mPEG5-Nalbuphine
BDBM209009 US9233167, 6-O-mPEG8-Nalbuphine
US9233167, 6-O-mPEG7-Nalbuphine BDBM209010
BDBM425933 US10512644, Compound 6-O-mPEG3-Nalbuphine
BDBM425934 US10512644, Compound 6-O-mPEG4-Nalbuphine
BDBM425936 US10512644, Compound 6-O-mPEG6-Nalbuphine
BDBM425938 US10512644, Compound 6-O-mPEG8-Nalbuphine
BDBM425939 US10512644, Compound 6-O-mPEG9-Nalbuphine
US10512644, Compound 6-O-mPEG5-Nalbuphine BDBM425935
US10512644, Compound 6-O-mPEG7-Nalbuphine BDBM425937
US10512644, Compound Nalbuphine US11534436, Compound Table B.1 US10231963, Table B.1 17-cyclobutylmethyl-4,5alpha-epoxymorphinan-3,6alpha,14-triol N-cyclobutylmethyl-4,5alpha-epoxy-3,6alpha,14-morphinantriol NALBUPHINE US9656961, Example 00118 BDBM50105085 CHEMBL895 USRE49340, Rank 9 US9233167, Nalbuphine
- GTPyS binding assay Test compound and/or vehicle was preincubated with the cell membranes and 3 uM GDP in modified HEPES buffer (pH 7.4) for 20 minutes, followed by addition of SPA beads for another 60 minutes at 30° C. The reaction is initiated by 0.3 nM [35S]GTP gamma S for an additional 30 minutes incubation period. Test compound-induced increase of [35S]GTP gamma S binding by 50 percent or more (250%) relative to the receptor subtype-specific agonist response indicates possible opiate receptor agonist activity. 0.1 uM DPDPE, 1 uM U-69593 and 1 uM DAMGO were used as the specific agonists for the delta, kappa and mu opioid receptors respectively. Opioid receptor antagonist activity was measured using inhibition of agonist-induced increase of [35S]GTP gammaS binding response by 50 percent or more (250%). Nalbuphine, 6-O-mPEG3-Nalbuphine, 6-O-mPEG6-Nalbuphine, 6-O-mPEG9-Nalbuphine were screened at concentrations of 10, 1, 0.1, 0.01 and 0.001 uM in both agonist and antagonist mode. EC50 or IC50 values were calculated from the dose-response curves as a measure of the agonist or antagonist activity of the test compounds respectively.
- Receptor Binding of PEG-Nalbuphine Conjugates Briefly, the receptor binding affinity of the nalbuphine and PEG-nalbuphine conjugates was measured using radioligand binding assays in CHO cells that heterologously express the recombinant human mu, delta or the kappa opioid receptor. Cells were plated in 24 well plates at a density of 0.2-0.3*106 cells/well and washed with assay buffer containing 50 mM Tris.HCl and 5 mM MgCl2 (pH 7.4). Competition binding assays were conducted in whole cells incubated with increasing concentrations of test compounds in the presence of appropriate concentration of radioligand. 0.5 nM 3H Naloxone, 0.5 nM 3H Diprenorphine and 0.5 nM 3H DPDPE were used as the competing radioligands for mu, kappa and delta receptors respectively. Incubations were carried out for two hours at room temperature using triplicate wells at each concentration. At the end of the incubation, cells were washed with 50 mM Tris HCl (pH 8.0), solubilized with NaOH and bound radioactivity was measured using a scintillation counter.Specific binding is determined by subtraction of the cpm bound in the presence of 50-100× excess of cold ligand. Binding data assays were analyzed using GraphPad Prism 4.0 and IC50 is generated by non-linear regression from dose-response curves. Ki values were calculated using the Cheng Prusoff equation using the Kd values from saturation isotherms as follows: Ki=IC50/(1+[Ligand]/Kd).
- Binding of PEG-Nalbuphine assays Specific binding is determined by subtraction of the cpm bound in the presence of 50-100× excess of cold ligand. Binding data assays were analyzed using GraphPad Prism 4.0 and IC50 is generated by non-linear regression from dose-response curves. Ki values were calculated using the Cheng Prusoff equation using the Kd values from saturation isotherms as follows: Ki=IC50/(1+[Ligand]/Kd).