Pharmacokinetics,protein binding and metabolism of a quinoxaline urea analog as an NF‐κB inhibitor in mice and rats by LC‐MS/MS

13–197 is a novel NF‐κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13–197 in mice and rats. A valid, sensitive and selective LC‐MS/MS method was developed. This method was validated for the quantification of 13–197, in the range of 0.1 or 0.2‐500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13–197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12‐fold shorter T_max than in rats. Tissue concentrations were 1.3–69.2‐fold higher in mice than in rats at 72 h after intravenous administration. 13–197 is well distributed to the peripheral tissues and has relatively high tissue–plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13–197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13–197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography‐triple quadrupole mass spectrometric method for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma and its application to microdose clinical trial

A liquid chromatography–triple quadrupole mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma to support a microdose clinical trial. The method consisted of a liquid–liquid extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₃‐AGM‐130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10–2000 pg/mL for AGM‐130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between‐run accuracy ranged from 98.1 to 101.0%. AGM‐130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM‐130 was also stable in human plasma at room temperature for 6 h and through three freeze–thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC‐MS/MS method for determination of AGM‐130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

Tumor-Cell-Specific Targeting of Ibrutinib: Introducing Electrostatic Antibody-Inhibitor Conjugates (AiCs)

Ibrutinib is an inhibitor of Bruton's tyrosine kinase that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities. To minimize its toxicities, we linked ibrutinib to a cell-targeted, internalizing antibody. To this end, we synthesized a poly-anionic derivate, ibrutinib-Cy3.5, that retains full functionality. This anionic inhibitor is complexed by our anti-CD20-protamine targeting conjugate and free protamine, and thereby spontaneously assembles into an electrostatically stabilized vesicular nanocarrier. The complexation led to an accumulation of the drug driven by the CD20 antigen internalization to the intended cells and an amplification of its pharmacological effectivity. In vivo, we observed a significant enrichment of the drug in xenograft lymphoma tumors in immune-compromised mice and a significantly better response to lower doses compared to the original drug.     

Determination of GDC‐0980 (apitolisib), a small molecule dual phosphatidylinositide 3‐kinase/mammalian target of rapamycin inhibitor in dog plasma by LC‐MS/MS to support a GLP toxicology study

An LC‐MS/MS method for the determination of GDC‐0980 (apitolisib) concentrations in dog plasma has been developed and validated for the first time to support pre‐clinical drug development. Following protein precipitation with acetonitrile, the resulting samples were analyzed using reverse‐phase chromatography on a Metasil AQ column. The mass analysis was performed on a triple quadruple mass spectrometer coupled with an electrospray interface in positive ionization mode. The selected reaction monitoring transitions monitored were m/z 499.3 → 341.1 for GDC‐0980 and m/z 507.3 → 341.1 for IS. The method was validated over the calibration curve range 0.250–250 ng/mL with linear regression and 1/x² weighting. Relative standard deviation (RSD) ranged from 0.0 to 10.9% and accuracy ranged from 93.4 to 113.6% of nominal. Stable‐labeled internal standard GDC‐0980‐d₈ was used to minimize matrix effects. This assay was used for the measurement of GDC‐0980 dog plasma concentrations to determine toxicokinetic parameters after oral administration of GDC‐0980 (0.03, 0.1 and 0.3 mg/kg) to beagle dogs in a GLP toxicology study. Peak concentration ranged from 3.23 to 84.9 ng/mL. GDC‐0980 was rapidly absorbed with a mean time to peak concentration ranging from 1.3 to 2.4 h. Mean area under the concentration–time curve from 0 to 24 hours ranged from 54.4 to 542 ng h/mL. Copyright © 2015 John Wiley & Sons, Ltd.     

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

Conformationally Constrained Peptidomimetics as Inhibitors of the Protein Arginine Methyl Transferases

Protein arginine N‐methyl transferases (PRMTs) belong to a family of enzymes that modulate the epigenetic code through modifications of histones. In the present study, peptides emerging from a phage display screening were modified in the search for PRMT inhibitors through substitution with non‐proteinogenic amino acids, N‐alkylation of the peptide backbone, and incorporation of constrained dipeptide mimics. One of the modified peptides ( 23 ) showed an increased inhibitory activity towards several PRMTs in the low μm range and the conformational preference of this peptide was investigated and compared with the original hit using circular dichroism and NMR spectroscopy. Introducing two constrained tryptophan residue mimics (l ‐Aia) spaced by a single amino acid was found to induce a unique turn structure stabilized by a hydrogen bond and aromatic π‐stacking interaction between the two l ‐Aia residues.     

Hollow fiber based affinity selection combined with high performance liquid chromatography–mass spectroscopy for rapid screening lipase inhibitors from lotus leaf

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography–mass spectrometry. Three flavonoids including quercetin-3-O-β-d-arabinopyranosyl-(1 → 2)-β-d-galactopyranoside, quercetin-3-O-β-d-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources.     

An Efficient Method for Synthesis of Tofacitinib Citrate

An efficient and mild synthetic method was developed for tofacitinib citrate from 3‐amino‐4‐methylpyridine and 4‐chloro‐7H‐pyrrolo[2,3‐d]pyrimidine. The related reactions were systematically optimized. Sodium hydride instead of potassium tert‐butoxide employed in the methoxycarbonylation reaction of compound 9 made the reaction proceed effectively to present compound 8 in a better yield. The replacement of benzaldehyde with benzyl bromide simplified the protection process of amino group. Red‐Al provided a cost‐effective method for the reduction of amides. The introduction of tosyl group into compound 10 enhanced the nucleophilic substitution of 10 with compound 4 dramatically. Thus, under the optimized conditions, tofacitinib citrate was obtained in 13.3% yield (based on compound 9 ) with a purity of 99.9%, much better than the reported yield 8.6%. This cost‐effective and environmental friendly process is more suitable for scale‐up production.     

Design and synthesis of novel diamide derivatives of glycine as antihyperglycemic agents

DPP-4 inhibition is one of the most extensively explored approaches for the management of type 2 diabetes (T2D). Most DPP-4 inhibitors in the market contain a proline mimetic active pharmacophore. Herein, we report the design, synthesis, and preliminary evaluation of a series of novel diamide derivatives of glycine, devoid of the proline mimic, for the treatment of T2D. As predicted from in silico studies, the diamide derivatives of glycine exhibited comparable DPP-4 inhibition with the standard as confirmed by the preliminary in vitro studies. Compound 6b was found to be the most potent (IC₅₀ 94.82 nM) DPP-4 inhibitor among all the molecules synthesized in the series.