Phase II attrition of clinical candidates in the
drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of
drug levels at the site of action.
PD 0332991, a highly specific inhibitor of
cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid
tumor. A clinical presurgical study will be required to better understand how
PD 0332991 affects signaling pathways and how the intratumoral concentration of
PD 0332991 correlates with plasma PK parameters and molecular alterations in
breast cancer tissues after
PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate
PD 0332991 levels in
tumor tissue samples from a xenograft mouse model for the determination of
drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify
PD 0332991 in mouse
tumor tissues from MDA-MB-231-Luc human
breast tumor xenografts in SCID-beige mice; (2) to quantify
PD 0332991 levels in mouse
tumor tissues after
oral administration of
PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse
tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure
PD 0332991 levels in
tumor tissues in MDA-MB-231-Luc human
breast tumor xenografts in SCID beige mice. The mean
tumor concentrations at 6h post-oral
PD 0332991 administration
at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.