Helming Tan

An Automated Screening Assay for Determination of Aqueous Equilibrium Solubility Enabling SPR Study During Drug Lead Optimization

Aqueous solubility is one of the most critical physicochemical properties to be determined in the process of drug lead optimization. Particularly, an equilibrium solubility method is highly valuable to the study of structure property relationship (SPR), while meeting the needs of analytical sensitivity, reproducibility, and throughput. In this report, an automated solubility assay in a 96-well library format was designed and developed by means of robotic liquid handling, centrifugal separation, and HPLC-UV quantification. Requiring 1 mg of solid compound, this assay was used to determine the equilibrium solubility in three user-selected media, that is, 0.01 N HCl, phosphate buffer saline (PBS), and fasted state simulated intestinal fluid (SIF), with a throughput of up to 192 compounds a week. The assay parameters, including the equilibration time and the separation technique, were optimized to ensure that the thermodynamic solubility was measured at the presence of excess solid compound. A fast gradient HPLC method was developed with single-point on-plate calibration for each compound, followed by a customized 96-well chromatographic data analysis. The reporting solubility range was 1–200 μg/mL, appropriate for oral drug candidate selection at the stage of discovery lead optimization. Based on the test results obtained on the commercially available drugs and Amgen research compounds, this assay was considered to be equivalent to the conventional shake-flask methods. Examples were given to demonstrate that the thermodynamic solubility determined by this assay enabled the SPR study to support drug lead optimization.

Antibody Solubility Behavior in Monovalent Salt Solutions Reveals Specific Anion Effects at Low Ionic Strength

Protein solubility was measured using the crystalline precipitate of a recombinant therapeutic antibody, in monovalent salt solutions containing KF, KCl, and KSCN (up to ∼ 0.7 M) at different pH conditions. For all three anions, the antibody solubility demonstrated complex behavior, both monotonic and nonmonotonic, with dependence on pH and salt concentration. At pH 7.1, close to the isoelectric point (pI) of 7.2, a typical salting-in behavior was observed with the salting-in constants of 12.7, 8.0, and 2.8 M for KSCN, KCl, and KF, respectively, suggesting that the anions follow the order of SCN(-) > Cl(-) > F(-) for increasing antibody solubility. Nonmonotonic behavior, as described by an initial solubility decrease followed by a solubility increase with ionic strength, was observed at pH 5.3, far below its pI. The effectiveness of the anion for reducing the solubility followed the order of SCN(-) > Cl(-) > F(-) . After the solubility reached the minimum, the anions effectiveness for raising the antibody solubility was in agreement with that at pH 7.1. The mechanisms for the above phenomena are discussed based upon specific binding of the anions to the antibody surface. The mechanistic view of anion binding and its charge neutralization effect at pH 5.3 was supported by the results from the effective charge and zeta-potential measurements.

High-throughput 96-well solvent mediated sonic blending synthesis and on-plate solid/solution stability characterization of pharmaceutical cocrystals.

A 96-well high-throughput cocrystal screening workflow has been developed consisting of solvent-mediated sonic blending synthesis and on-plate solid/solution stability characterization by XRPD. A strategy of cocrystallization screening in selected blend solvents including water mixtures is proposed to not only manipulate solubility of the cocrystal components but also differentiate physical stability of the cocrystal products. Caffeine-oxalic acid and theophylline-oxalic acid cocrystals were prepared and evaluated in relation to saturation levels of the cocrystal components and stability of the cocrystal products in anhydrous and hydrous solvents. AMG 517 was screened with a number of coformers, and solid/solution stability of the resulting cocrystals on the 96-well plate was investigated. A stability trend was observed and confirmed that cocrystals comprised of lower aqueous solubility coformers tended to be more stable in water. Furthermore, cocrystals which could be isolated under hydrous solvent blending condition exhibited superior physical stability to those which could only be obtained under anhydrous condition. This integrated HTS workflow provides an efficient route in an API-sparing approach to screen and identify cocrystal candidates with proper solubility and solid/solution stability properties.

Abstract 711: Small molecule compounds that target cell division cycle 7 (Cdc7) kinase inhibit cell proliferation and tumor growth.

Cdc7 is an essential, serine/threonine protein kinase that activates the initiation of DNA synthesis at replication origins. Cdc7 also promotes cell cycle checkpoint activation in response to replication stress. As a key regulator of S phase entry and progression, Cdc7 kinase is a potential target for cancer therapy, with a distinct mechanism of action from known drugs that inhibit DNA replication. Following a high throughput screen for inhibitors of Cdc7 kinase activity, we investigated structure-activity relationships of azole-based compounds and optimized the compounds for potency and pharmacokinetic properties. Here we present the characterization of one of these compounds as a potent, selective, bioavailable Cdc7 kinase inhibitor. In cells, Cdc7 inhibition decreases MCM2 phosphorylation and DNA synthesis, causes DNA damage, and slows S phase progression. Cdc7 inhibition also induces chromosome missegregation leading to cell lethality in vitro and tumor growth inhibition in vivo. Cdc7 inhibition provides a new approach to target cancers, either as a single agent or in combination with chemotherapy. Citation Format: Julie Bailis, Li Fang, Jessica Orf, Scott Heller, Tammy Bush, Matthew Bourbeau, Sonia Escobar, Michael Frohn, Paul Harrington, Faye Hsieh, Alexander Pickrell, Kelvin Sham, Aaron Siegmund, Helming Tan, Leeanne Zalameda, John Allen, Dineli Wickramasinghe. Small molecule compounds that target cell division cycle 7 (Cdc7) kinase inhibit cell proliferation and tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 711. doi:10.1158/1538-7445.AM2013-711