Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood-brain barrier in mice and rats

Abstract

1. TXD258, a new taxoid antitumor agent, is a poor substrate for the P-glycoprotein (P-gp) in Caco-2 cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice under a variety of conditions (dose and infusion time, species and plasma concentration) using conventional in vivo pharmacokinetic techniques and in situ brain perfusion. 2. Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m(-2) for 45 s or 1 h and rats were infused with 15 and 60 mg m(-2) over 2.3 min. The radioactivity in the plasma and brains was measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of TXD258 increased linearly in mice with the infused dose, the brain content increased more than proportionally with the dose between 15 and 90 mg m(-2). This nonlinear uptake of TXD258 also occurred in the plasma and brain of the rat. 3. These findings suggest that the protein-mediated efflux across the blood-brain barrier (BBB) becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P-gp substrate at the BBB of mice and rats. The P-gp of both species was saturated at the half-inhibitory concentration ( approximately 13 micro M) produced by i.v. infusion. 4 Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation of the P-gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P-gp-mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity.

Figure 1

Chemical structure of [¹⁴C]-TXD258.

Figure 2

Average changes in the plasma and brain concentrations of TXD258 over time. [¹⁴C]-TXD258 (15 mg m⁻²) was infused into mice for 45 s and into rats for 2.3 min. Samples were taken from four mice and two rats at each time and pooled to obtain an average analytical determination.

Figure 3

(a) Relations between the average areas under the curves (AUC_{0 – 168 h}) or maximal concentration (C_max; inset) in the brains and plasma of mice infused intravenously with [¹⁴C]-TXD258 at 15, 30, 45 and 90 mg m⁻² for 45 s. Values in parentheses represent the increases in the AUC obtained at 30, 45 or 90 mg m⁻² compared to the value measured at 15 mg m⁻². (b) Rat brain and plasma AUC_{0 – 168 h} or C_max (inset) values. The rats were given an intra-venous infusion of [¹⁴C]-TXD258 at 15 and 60 mg m⁻² lasting 2.3 min.

Figure 4

Concentration-dependent brain transport of [¹⁴C]-TXD258 (expressed as a brain transport coefficient K_in) measured by in situ brain perfusion in rats and mice. Animals were perfused with TXD258 via the common carotid artery for 60 s. Dotted (rats) and solid (mice) curves represent data fitted with the Hill equation and an apparent TXD258 IC₅₀ value of ∼13 μM. Data are presented as means±s.d. of n=4 – 8 animals per point.

Figure 5

The brain transport coefficient (K_in; μl s⁻¹ g⁻¹) was measured by in situ brain perfusion in P-gp-proficient mice (a) and rats (b) with or without the chemical P-gp modulator (±)-verapamil (150 μM) and in P-gp-deficient mdr1a(−/−) mice. Each group of animals was perfused with [¹⁴C]-TXD258 at a noninhibiting concentration (5.4 μM). P-gp chemical modulation or disruption produced an approximately three-fold increase in the brain TXD258 transport in mice and a 4.7-fold increase in rats. Data are presented as means ±s.d. of n=5 – 8 animals per group. ^***P<0.001 compared to the control group.

Figure 6

The apparent distribution volume (V_d; μl g⁻¹) of [¹⁴C]-TXD258 was measured in controls after [¹⁴C]-TXD258 had been accumulated for 60 s. The animals in the two other groups, underwent a 30 s tracer-free ‘wash-out' with or without (±)-verapamil (150 μM) after the 60 s of brain accumulation of [¹⁴C]-TXD258. Both mice and rats were perfused with [¹⁴C]-TXD258 at 5.4 μM. Data are presented as means ±s.d. of n=5 – 6 animals per group. ^*P<0.05, ^***P<0.001, compared to their respective controls.