Parallel Synthesis of Hexahydro­diimidazo­diazepines Heterocyclic Peptidomimetics and Their in Vitro and in Vivo Activities at μ (MOR), δ (DOR), and κ (KOR) Opioid Receptors

In the development of analgesics with mixed-opioid agonist activity, peripherally selective activity is expected to decrease side effects, minimizing respiratory depression and reinforcing properties generating significantly safer analgesic therapeutics. We synthesized diazaheterocyclics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for μ (MOR), δ (DOR), and κ (KOR) opioid receptors across the series, with the diimidazodiazepine <b>14</b> (2065-14) displaying good affinity for DOR and KOR. Central (icv), intraperitoneal (ip), or oral (po) administration of <b>14</b> produced dose-dependent, opioid-receptor mediated antinociception in the mouse, as determined from a 55 °C warm-water tail-withdrawal assay. Only trace amounts of compound <b>14</b> was found in brain up to 90 min later, suggesting poor BBB penetration and possible peripherally restricted activity. Central administration of <b>14</b> did not produce locomotor effects, acute antinociceptive tolerance, or conditioned-place preference or aversion. The data suggest these diazaheterocyclic mixed activity opioid receptor agonists may hold potential as new analgesics with fewer liabilities of use

Pharmacokinetics and biodistribution of (A) kavain, (B) dihydrokavain, (C) methysticin, (D) dihydromethysticin, and (E) desmethoxyyangonin in the mouse serum, liver, lung, and brain.

<p>Samples were collected 0.5, 1.5, 4, 8, and 24 h after kava treatment. Mice with no kava treatment was used for 0-h timepoint.</p

A stable isotope dilution tandem mass spectrometry method of major kavalactones and its applications

<div><p>Kava is regaining its popularity with detailed characterizations warranted. We developed an ultraperformance liquid chromatography high-resolution tandem mass spectrometry (UPLC-MS/MS) method for major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin and desmethoxyyangonin) with excellent selectivity and specificity. The method has been validated for different matrices following the Food and Drug Administration guidance of analytical procedures and methods validation. The scope of this method has been demonstrated by quantifying these kavalactones in two kava products, characterizing their tissue distribution and pharmacokinetics in mice, and detecting their presence in human urines and plasmas upon kava intake. As expected, the abundances of these kavalactones differed significantly in kava products. All of them exhibited a large volume of distribution with extensive tissue affinity and adequate mean residence time (MRT) in mice. This method also successfully quantified these kavalactones in human body fluids upon kava consumption at the recommended human dose. This UPLC-MS/MS method therefore can be used to characterize kava products and its pharmacokinetics in animals and in humans.</p></div

Reconstructed ion chromatograms at MS/MS scan stage of [²H₂]-DHM, kavain, DHK, methysticin, DHM, and demethoxyyangonin from a control urine, and the urine samples collected from a subject pre- and post-kava.

<p>[²H₂]-DHM was used as the internal standard. The mass extraction window was of ± 5 ppm.</p

The amount of kavain, DHK, methysticin, DHM and desmethoxyyangonin in the urine and plasma samples of two subjects pre- and post-kava administration.

<p>The amount of kavain, DHK, methysticin, DHM and desmethoxyyangonin in the urine and plasma samples of two subjects pre- and post-kava administration.</p

Discovery of Novel Antinociceptive α‑Conotoxin Analogues from the Direct In Vivo Screening of a Synthetic Mixture-Based Combinatorial Library

Marine cone snail venoms consist of large, naturally occurring combinatorial libraries of disulfide-constrained peptide neurotoxins known as conotoxins, which have profound potential in the development of analgesics. In this study, we report a synthetic combinatorial strategy that probes the hypervariable regions of conotoxin frameworks to discover novel analgesic agents by utilizing high diversity mixture-based positional-scanning synthetic combinatorial libraries (PS-SCLs). We hypothesized that the direct in vivo testing of these mixture-based combinatorial library samples during the discovery phase would facilitate the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor activity or liabilities of locomotion and respiration. A PS-SCL was designed based on the α-conotoxin RgIA-ΔR <i>n</i>-loop region and consisted of 10,648 compounds systematically arranged into 66 mixture samples. Mixtures were directly screened in vivo using the mouse 55 °C warm-water tail-withdrawal assay, which allowed deconvolution of amino acid residues at each position that confer antinociceptive activity. A second generation library of 36 individual α-conotoxin analogues was synthesized using systematic combinations of amino acids identified from PS-SCL deconvolution and further screened for antinociceptive activity. Six individual analogues exhibited comparable antinociceptive activity to that of the recognized analgesic α-conotoxin RgIA-ΔR, and were selected for further examination of antinociceptive, respiratory, and locomotor effects. Three lead compounds were identified that produced dose-dependent antinociception without significant respiratory depression or decreased locomotor activity. Our results represent a unique approach for rapidly developing novel lead α-conotoxin analogues as low-liability analgesics with promising therapeutic potential