Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein-Protein Interaction.

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 µM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Design and characterization of a new miniaturized rotating disk equipment for in vitro dissolution rate studies.

A miniaturized apparatus for the determination of the apparent in vitro dissolution rate has been designed, constructed and characterized. The miniaturized apparatus was based on a low volume dissolution cell and a disk in a rotating magnetic bar. The disk tablet is pressed directly into the bar with a press designed and constructed for this purpose. It requires approximately 5 mg of substance. The disk was positioned eccentrically on the bar with an external flow of medium to increase the rate of solvent flow over the disk surface. Six different drug substances were used. The dissolution media were sodium phosphate buffer, pH 7.0, and ammonium acetate buffer, pH 6.8. All quantifications were made by integrating the dissolution cell with high-performance liquid chromatography (HPLC) using diode-array detection (DAD). The obtained results were compared with data from a conventional rotating disk equipment, where the disk was centrically mounted. The dissolution rates at 100 rpm seemed to be on an average of 2-3 times higher for the miniaturized apparatus (RSD 0.2-56%). The preliminary studies of this prototype indicate that the miniaturized rotating disk is a promising design for the qualitative estimation of dissolution rates of substances, for example during screening in early drug discovery.

Single run measurements of drug-protein binding by high-performance frontal analysis capillary electrophoresis and mass spectrometry.

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry.

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.

Rapid screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis combined with short-end injection.

A method applying pressure-assisted capillary electrophoresis combined with short-end injection has been developed for the rapid screening of the pKa values of pharmaceuticals. The electrophoretic separation is performed on a short capillary length with short-end injection under an applied pressure, and the effective mobility is measured in a series of 10 different buffers with constant ionic strength (I = 0.05). The application of pressure not only reduces migration times, particularly in lower pH buffers, but also improves the repeatability of effective mobility measurements. The influence of pressure on the effective mobility was investigated at various pH values. It was observed for the first time that an increase in pressure resulted in a slight decrease in the effective mobility when the pH was above the pKa for acidic analytes, whereas an increased effective mobility with increasing pressures was observed when the pH was below the pKa. However, the observed effective mobility shift by the applied pressure did not significantly affect the determined pKa values. The determined pKa values were in good agreement with published data. Furthermore, a stacking condition was applied to increase the sensitivity, and a concentration down to 2 microM could readily be detected with UV detection using a 50 microm I.D. capillary. This technique is particularly suitable for measurement of pKa values for compounds with poor aqueous solubility. The method also omits the commonly used preconditioning steps with sodium hydroxide and water. The exclusion of excessive preconditioning steps and the use of pressure reduces the total cycling analysis time, and makes it possible to determine the pKa in less than 40 min per compound without loss of accuracy.