Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic

Abstract

NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 μgh/mL, a C(max) of 2.16 μg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis.

Figure 1. NOV-002 is a substrate for glutathione reductase

10 μg/ml NOV-002 was treated with 0 or 0.4 μg/ml glutathione reductase in the presence of 200 μM NADPH. Mean concentration profiles for (A) GSSG and (B) GSH were obtained by HPLC-MS analysis. Data are represented as mean ± SEM of n = 3 per group.

Figure 2. Effect of NOV-002 on plasma GSSG and GSH

Mice were treated with 250 mg/kg NOV-002 (i.p.). Orbital blood was collected at 0, 5, 10, 15, 30 and 240 min. Mean plasma concentration-time profiles for (A) GSSG and (B) GSH were obtained by HPLC-MS analysis. Data are represented as mean ± SD of n=7 animals per time-point.

Figure 3. Non-linear pharmacokinetic estimation of NOV-002

The observed (O) and expected (---) plasma concentrations are plotted as a function of time for A) GSSG, the active component of NOV-002 and B) GSH, the primary metabolite. Satisfactory algorithm convergence and acceptance of the modeling results was based on standard methods, including the likelihood ratio test, Akaike’s Information Criterion, goodness of fit plots, and visual inspection of the observed versus predicted concentration-time values. Data were best fit by the initial one-compartment open model with first order absorption and elimination.

Figure 4. Effect of NOV-002 on plasma redox potential and free sulfhydryls

Mice were treated with 250 mg/kg NOV-002 (i.p.). Orbital blood was collected at 0, 5, 10, 15, 30 and 240 min. A) The E_h GSSG/GSH was calculated from the GSSG and GSH concentrations using the Nernst equation. B) Schematic illustrating how the fluorescent probe, ThioGlo^™, binds to free thiols. S-Glutathionylated cysteine residues cannot bind ThioGlo and thereby have lower fluorescence intensities. C) Free protein thiol content of plasma proteins was measured at 0, 5, 10, 15 and 30 min following 250 mg/kg (i.p.). D) Free protein thiol content was measured in control (black) and NOV-002 treated (white) tissues 30 minutes following treatment. The ThioGlo-1 emission (at 513nm) for each treatment group was averaged and plotted as mean ± SD (n = 6); (*=p≤0.001).