CASE STUDY : CHALLENGES IN METHOD DEVELOPMENT FOR DRUG INTERACTION STUDIES

 

Current FDA guidance indicates that drug interactions should be "defined during drug development, as part of an adequate assessment of the drug's safety and effectiveness."(1) While in vivo drug interaction studies are typically necessary during late-stage development, in vitro drug interaction studies can sometimes serve as a cost-effective substitute to more costly in vivo drug interaction studies in earlier stages of development. Hence along with pharmacokinetic studies, studies designed to investigate drug-drug interactions represent an important step in the drug development process for new chemical entities.

Pacific BioLabs was recently approached by a client in need of quantitative HPLC test methods for 25 different pharmaceuticals as part of an in vitro drug interaction study. Each drug was to be quantified within three separate buffer formulations designed to simulate in vivo conditions. The list of pharmaceuticals included compounds displaying a broad range of chemical structures, each of which would be expected to display unique retention behavior on an HPLC system. In addition, many of the compounds were to be prepared in only trace quantities representative of bodily concentrations observed in a clinical setting.

Method development began with a chalkboard assessment of the chemical properties of each compound to estimate the most suitable HPLC conditions. A thorough literature search was conducted to examine parameters used in previously published methods. The compounds were subsequently divided into three categories corresponding to molecules with high, intermediate, and low polarity. Basic method conditions were then developed for each of the three categories: a shallow gradient (phosphate buffer/acetonitrile) with a C8 column for high polarity molecules, a moderate gradient (phosphate buffer/acetonitrile) with a C8 column for intermediate polarity molecules, and a steep gradient (H2O + formic acid/acetonitrile + formic acid) with a C18 column for low polarity molecules. These conditions served as a starting point from which more specific conditions for each compound could be adjusted and optimized as necessary.

 

Often common in method development, the occurrence of a number of stumbling blocks necessitated the application of some creative workarounds. Many of the low-polarity molecules displayed poor aqueous solubility even after repeated dilution. In these cases, addition of a small amount of DMSO to the aqueous buffers permitted successful dissolution. On the other end of the polarity scale, some compounds displayed little or no retention on C8/C18 columns.  A pentafluorophenyl (PFP) column, which has a unique bonded-phase that interacts with polar compounds, demonstrated adequate retention for an exceptionally high-polarity compound which failed to retain after repeated adjustments of mobile phase pH and gradient conditions. Adequate detection of compounds present in trace concentrations was accomplished through optimization of UV-wavelength and on-column focusing, although one compound present in extremely low concentrations required MS/MS detection.

Optimal method conditions developed at PBL were qualified to ascertain linearity, accuracy, precision, and benchtop stability. In the end, three of the compounds displayed insufficient benchtop stability and one of the compounds was dropped by the client, leaving a grand total of 21 successfully completed HPLC methods. The successful development of these methods – the majority of which were completed in roughly three months’ time – demonstrates Pacific BioLabs’ capability to respond to client needs with both scientific expertise and timeliness.

For more information about method development services and other contract research services please see our analytical services brochure or inquire by telephone at +1 510 964 9000.

1 "Draft Guidance: Drug Interaction Studies — Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations." FDA, Feb. 2012.