Evaluation of the cassette dosing approach for assessing the pharmacokinetics of geldanamycin analogues in mice

Purpose: There is currently much interest in developing analogues of the benzoquinone ansamycin geldanamycin that may overcome the limitations of 17( allylamino)-17-demethoxygeldanamycin (17AAG), which is the first known inhibitor of heat shock protein 90 (Hsp90) to enter clinical trials. Studies were performed to assess whether cassette dosing, the coadministration of several compounds to a single animal, is a suitable approach to evaluate the preclinical pharmacokinetics of geldanamycin analogues in high throughput. Methods: Five geldanamycin analogues ( 17AAG, NSC 255110, NSC 682300, NSC 683661, NSC 683663) were administered intravenously to mice in combination at 5 mg/kg each and as single agents at 5 mg/kg and 50 mg/kg, or 12.5 mg/kg for NSC 682300. The compounds were also incubated with mouse liver microsomes individually and in combination at 15 muM each. Quantitative analysis was performed by LC/MS/MS. Plasma and tissue pharmacokinetic parameters were evaluated by non-compartmental analysis. In vitro metabolic stability was assessed by monitoring disappearance of the parent compound. Results: Of the compounds that were detectable following individual administration at 5 mg/kg, 17AAG and NSC 683661 exhibited nonlinear pharmacokinetics. In addition, the plasma area under the curve (AUC) and the half-life of these compounds was greater following cassette dosing at 5 mg/kg compared to single administration at the same dose. When pharmacokinetic parameters were calculated up to the same time point following cassette and individual administration at the higher dose, three of the compounds displayed non-linear increases in AUC and slower clearances following cassette compared to single compound dosing. When all measurable concentrations at the higher dose were included, the half-life of NSC 683663 was nine-fold longer following individual compared to cassette administration. 17AAG displayed the highest AUC following cassette dosing, whereas NSC 683663 displayed the highest AUC following single-compound dosing. Excluding NSC 683663, the rank order from the highest to the lowest AUC was the same; however, NSC 682300, which ranked fifth, was administered at a four-fold lower individual dose than the other compounds. Exposure of the liver and kidneys to the compounds was greater than that of plasma. Despite being administered at a lower dose, NSC 682300 displayed the highest kidney AUC of the five compounds. The same ranking was maintained between cassette and single compound dosing in the kidney. With the exception of NSC 682300, in vitro metabolic stability was predictive of in vivo pharmacokinetics in the plasma and liver. The extent of metabolism of four of the five compounds was lower following microsomal incubation in combination compared to incubation alone, suggestive of likely drug-drug interaction in the cassette. However, for 17AAG this may be partly due to metabolism of NSC 683661 and NSC 683663 to this compound. Conclusions: Whilst cassette dosing has advantages for use in drug discovery, it is probably unsuitable to evaluate the pharmacokinetics of geldanamycin analogues due to non-linear pharmacokinetics and drug-drug interaction. The issues identified for this compound series should also be considered in assessing the suitability of cassette dosing for other chemotypes.