Lead optimization of purine based orally bioavailable Mps1 (TTK) inhibitors.

Efforts to optimize biological activity, novelty, selectivity and oral bioavailability of Mps1 inhibitors, from a purine based lead MPI-0479605, are described in this Letter. Mps1 biochemical activity and cytotoxicity in HCT-116 cell line were improved. On-target activity confirmation via mechanism based G2/M escape assay was demonstrated. Physico-chemical and ADME properties were optimized to improve oral bioavailability in mouse.

Quantification of recombinant human parathyroid hormone (rhPTH(1-84)) in human plasma by immunoassay: commercial kit evaluation and validation to support pharmacokinetic studies.

Immunoassays utilizing commercial kits designed for diagnostic use can be adapted and validated to meet Good Laboratory Practice (GLP) requirements to support pharmacokinetic (PK) studies. We illustrate in this paper a systematic approach for commercial kit evaluation and GLP-compliant method validation to establish selectivity, sensitivity, linearity, accuracy, precision and stability. Immunoassay kits for human parathyroid hormone (hPTH) quantification from three different vendors were assessed in a side-by-side comparison for their suitability for the PK analysis of recombinant humanPTH (rhPTH) in EDTA plasma. Two immunoradiometric (IRMA) assay kits and one immunoluminometric assay (ILMA) kit were evaluated. Since PTH is present as an endogenous component of human plasma, QC preparation in the biological matrix was handled differently than for a xenobiotic drug compound. The endogenous concentration of PTH was determined in plasma samples from 32 individual lots using the three kits. The lots with the lowest endogenous concentrations of PTH were selected, pooled to form the low QC and spiked with rhPTH to prepare the mid and high QCs. Four evaluation batches were run with each of the three commercial kits to evaluate reference standard linearity, and QC accuracy and precision. Selectivity against PTH peptide fragments PTH(7-84) and PTH(3-84) were assessed by cross-reactivity and accurate spike-recovery to the QC samples at two concentrations. One of the kits was chosen for full method validation because it had the lowest cross-reactivity against hPTH fragments (3-84) and (7-84), a wider dynamic range and the least total error. The accuracy and precision from six validation batches of the QCs were

Characterization of the cellular and antitumor effects of MPI-0479605, a small-molecule inhibitor of the mitotic kinase Mps1.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics.

Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys.

To understand how the carbohydrate moieties of a recombinant glycoprotein affected its pharmacokinetic (PK) properties, the glycan distribution was directly assessed from serial blood samples taken during PK studies in cynomolgus monkeys and humans. The protein studied was an immunoadhesin (lenercept), containing an Fc domain from human immunoglobulin G (IgG-1) and two copies of the extensively glycosylated extra cellular domain of tumor necrosis factor receptor p55. The protein was recovered in pure form using a dual column, immunoaffinity-reversed-phase high-performance liquid chromatography method. The glycans were released and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Alternatively, trypsin was used to obtain glycopeptides, and these were analyzed by MALDI-TOF. The composition versus time profiles show that the distribution of glycans in the Fc domain was not altered over 10 days of circulation, consistent with their sequestration in the interior of the protein. However, the glycan composition in the receptor domain was changed dramatically in the first 24 h and then remained relatively constant. Analysis of the acidic glycans (derived exclusively from the receptor domain) showed that, in the rapid initial phase of clearance, glycans carrying terminal N-acetylglucosamine (tGlcNAc) were selectively cleared from the circulation. This phenomenon occurred similarly in humans and cynomolgus monkeys. Sialic acid content and terminal galactose showed only small changes. These data confirm the correlation of tGlcNAc and half-life of the molecule, and support the hypothesis that the mannose receptor (which can also bind tGlcNAc) causes the variable clearance of this molecule.