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Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study.

ABSTRACT: Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1→217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2→232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5-500ng/mL with a mean r(2)=0.9987. The mean inter-day accuracy and precision for all calibration standards were 93-118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5ng/mL using a sample volume of 20μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice.

SUBMITTER: Khaliq T

PROVIDER: S-EPMC4956519 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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Project description:The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTP?S functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTP?S assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the first opioid pan antagonist with high affinity at all four opioid receptor subtypes. Our SAR studies show that the 3,4-dimethyl groups of the well-known trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist scaffold may be removed without significant loss in binding affinity or antagonist potency to obtain an opioid pan antagonist such as AT-076.

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Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study.

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