Staurosporine analysis and its pharmacokinetics in the blood of rats

Abstract

Staurosporine (Stsp), a protein kinase inhibitor, has been found to have a differential effect on the proliferation of normal and transformed cells in vitro (Crissman et al., Pruc. Nut. Acud. Sci. USA 88 [1991]). Hence, Stsp might be used in cell-cycle-specific cancer therapy to arrest normal proliferating cells in G1, while permitting tumor cells to continue proliferation. The patient could then be treated with a therapeutic agent of maximum toxicity for actively proliferating tumor cells. Essentially nothing is known, however, about the distribution and pharmacokinetics of Stsp in the body.To facilitate such in vivo investigations of Stsp, we have developed a High-Performance Liquid Chromatography (HPLC) method for measuring the levels of Stsp in blood. Using a rat model, plasma and red blood cells (RBC) containing Stsp are treated with acetone, which precipitates proteins and permits extraction of the Stsp. The acetone extract containing Stsp is diluted 2: 1 with aqueous 0.2% trifluoroacetic acid (TFA) and then subjected to reversed-phase HPLC on a Pondapak C18 column. A linear elution gradient of acetonitrile running from water/O.2% TFA to acetonitrile/O.2% TFA in 60 min at 1 mumin elutes Stsp as a sharp peak at -35 min which can be detected by UV absorption at 292 nm. HPLC of plasma spiked with Stsp standards produced a linear calibration curve that can be used to quantify Stsp in blood samples.Using this analytical method, Stsp was measured in both plasma and RBC of rat blood. In vitro studies in which whole blood was incubated with Stsp indicated that RBC took up Stsp at a rapid rate, thereby diminishing its concentration in plasma. However, the binding of Stsp to RBC was found to be weak in vitro, resulting in a steady state equilibrium in which an RBC:plasma ratio of 2: 1 was maintained over a wide range of Stsp concentrations.In vivo, Stsp was rapidly sequestered in some other tissue compartment, which rapidly decreased the concentration of Stsp in plasma to nondetectable levels. Using a postchromatography computerized analysis program that amplified the Stsp UV absorption peak from the HPLC, nanogram levels of Stsp were detected in vivo. Using this detection system for pharmacokinetic studies it was found that, in vivo, Stsp had a half-life of 51.6 min in plasma and 75.3 min in RBC. Tissue adsorption studies demonstrated that up to 99% of the Stsp was adsorbed by the heart and lung tissue in one pass through these organs. Extrapolation of the data from these studies suggest that 1-pg Stsp injections should produce a 2to 7-ndml plasma Stsp level in vivo which is in the effective range to produce G1 arrest in normal cells. The short half-life of Stsp in plasma indicates that it will be necessary to infuse Stsp at some low level following the initial bolus injection in order to maintain Stsp levels in plasma at the 1to lO-ng/ml level for the 2to 3-day period necessary to achieve G1 arrest in vivo.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

“…8,34 Previous gradient loading methods have only supported liposomal retention of weakly basic anthracyclines such as doxorubicin and to some extent the camptothecin analogs. 54,55 The loading methodology and liposomal composition we describe resulted in high ($96%) STS retention at 2 hours in human serum, with 82% remaining at 6 hours ( Figure 2).…”

“…6,7 However, translational development has not proved feasible because of poor pharmacokinetics and toxicity. 6,8 A number of less potent but more stable STS analogs are in clinical trials, but these too are constrained by toxicity, which includes hyperglycemia with acidosis, pulmonary dysfunction, hypotension, creatinine elevation, diarrhea, nausea, and vomiting. 9,10 Our aim was to assess whether liposomal encapsulation of STS would reduce toxicity and allow a comparatively low systemic dose to exert a significant antitumor effect in vivo.…”

“…Similarly, our results for the large tumor group (180-200 mm 3 ) indicate that free STS at the doses we used had no detectable effect on tumor growth. 8 We did not administer free STS to mice bearing the smaller …”

In vivo and in vitro effects of staurosporine on the DNA distributions in and proliferation of various rodent cell populations

“…Study Series V Our findings from HPLC analysis of STSP in the plasma and erythrocyte compartments (Gurley et al, 1994) following bolus i.v. injection of STSP provided a more analytical basis for STSP dosing in rats.…”