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, 29 (5), 901-11

Pharmacokinetics and Derivation of an Anticancer Dosing Regimen for PAC-1, a Preferential Small Molecule Activator of procaspase-3, in Healthy Dogs

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Pharmacokinetics and Derivation of an Anticancer Dosing Regimen for PAC-1, a Preferential Small Molecule Activator of procaspase-3, in Healthy Dogs

Pamela W Lucas et al. Invest New Drugs.

Abstract

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Figures

Figure 1
Figure 1
Demonstration of procaspase-3 levels in immortalized cell lines. The relative expression levels of procaspase-3 in 17-71 (left), GL-1 (middle), and CA46 (right) B-cell lymphoma lines were determined. Staining for procaspase-3 is represented by shaded grey line and non-specific staining by an isotype control antibody is marked with open black line. Mean fluorescent intensity (MFI) ratios expressed as the fraction (procaspase-3 MFI)/(isotype control MFI) was used to compare relative differences or similarities in basal procaspase-3 levels among cell lines.
Figure 2
Figure 2
PAC-1 induces apoptosis. The selective ability of PAC-1 to induce cell death through apoptosis, not necrosis, is demonstrated in the 17-71 cell line. Maximal apoptosis is induced with 48 hour exposure durations and 20 µM concentrations, with approximately 70% of cells staining only for Annexin-V-FITC (right lower quadrant), an early apoptotic marker.
Figure 3
Figure 3
PAC-1 exerts time- and dose-dependent apoptotic effects. The ability of PAC-1 to induce apoptosis is influenced by exposure durations and concentrations. Treatment conditions which resulted in significant increases in the percentage of apoptotic cells compared to control cells incubated with media (M) alone represented by double asterisks with p < 0.01. 17-71 (a, b) and CA46 (e, f) cell lines are more sensitive to the apoptotic effects of PAC-1 in comparison with the GL-1 (c, d) cell line. VC = Vehicle control; all PAC-1 concentrations are in µM. Error bars represent standard deviation, n = 5.
Figure 4
Figure 4
Observed apoptosis correlates with caspase-3 activity in cell lysate. Time- and dose-dependent apoptotic death induced by PAC-1 is correlated with increases in caspase-3 activity. A 10 µM concentration of PAC-1 significantly increases caspase-3 activity (as measured by mean optical densities) in 17-71 (a), GL-1 (b), and CA46 (c) cell lines, but requires greater than 4 hours of drug exposure. At 10 µM PAC-1 concentrations, treatment conditions which induce significant increases in caspase-3 activities above vehicle control treated cells (Hour 0) is represented by double asterisk, p < 0.01. Error bars represent standard deviation, n = 4. Similar results were observed for PAC-1 concentrations of 20 µM (data not shown).
Figure 5
Figure 5
Pharmacokinetics of PAC-1 in healthy dogs. The pharmacokinetic profiles of (a) intravenous and (b) oral PAC-1 were characterized in 6 healthy dogs using a crossover design. Intravenous PAC-1 produces higher and more consistent peak plasma concentrations in comparison with orally administered PAC-1. Error bars represent standard deviation, n = 6.
Figure 6
Figure 6
Continuous rate infusion of PAC-1. A pharmacokinetically-derived dosing regimen, designed to achieve and maintain steady state plasma concentrations of PAC-1 predicted to exert time- and dose-dependent cytotoxic effects, was evaluated in healthy dogs. The achievement of predicted PAC-1 cytotoxic concentrations ≥ 10 µM, as well as durations of exposure ≥ 24 hours was technically feasible and biologically well-tolerated by all dogs. Error bars represent standard deviation, n = 4.

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