Development & Validation of a UPLC-UV Bioanalytical Testing Method
Challenge: A sponsor had developed a promising group of "first in class" potent small molecule compounds for cancer therapy. The research scientists and medicinal chemists used structure-based drug design to derive compounds that target important proteins and therapeutic pathways in cancer. One of these candidates has shown to be a very potent, yet highly selective growth inhibitor of many cancer types, including non-small cell lung cancer, colon cancer and leukemia.
Pre-toxicology studies had been conducted on animal models, however quantitative analytical method for the detection of this candidate in the collected plasma/serum samples had not been suucessfully developed due to the highly challenging stereochemical nature of the active molecule.
Diteba was challenged to develop a quantitative analytical bioassay method capable of detecting the compound in plasma/serum samples and if successful, the sponsor could then proceed with toxicology and clinical studies.
Diteba's scientists, while evaluating the three-dimensional structure and chemical properties of this compound, noted that the selected molecule of the drug candidate is stereochemically very flexible and could easily undergo structural modifications in different solvents or biological fluids. The configuration and conformational isomerism character of the compound resulted in difficulties stabilizing the molecule during bioassay development. Another challenging aspect of the molecule was its nature to act as a strong bidentate chelating ligand and create coordination compounds-complex with metals present in blood. Lastly, very poor solubility in most of the solvents/excipients commonly recommended by FDA made it difficult to formulate a drug product dosage form suitable for administration and further toxicology studies.
Intensive spectral and chemical data analysis (such as UV, IR, NMR, LC/MS, HPLC, XRD) were undertaken in order to assist in the design of optimal and unique conditions whereby the molecule was stabilized during both extraction and chromatographic detection procedures. As a result of Diteba's experimental development work, a sensitive, specific and robust Ultra Performance Liquid Chromatography (UPLC) quantification method in plasma/serum samples was developed and validated. In addition to the method development and validation, a unique solid phase extraction method for extraction of the compound from different biological fluids was also developed and incorporated it into the assay procedures. The developed methods were effectively used throughout the entire toxicology program and qualified for use in further clinical studies. Diteba has also investigated the pre-formulation batches for identifying a suitable dosage form and route of administration.
The R&D data presented demonstrated that an injectable formulation was preferred to an oral formulation. This recommendation was supported also with data obtained from pre-clinical studies.