Optimization of dose and route of administration of the P-glycoprotein inhibitor, valspodar (PSC-833) and the P-glycoprotein and breast cancer resistance protein dual-inhibitor, elacridar (GF120918) as dual infusion in rats

Abstract

Transporters can play a key role in the absorption, distribution, metabolism, and excretion of drugs. Understanding these contributions early in drug discovery allows for more accurate projection of the clinical pharmacokinetics. One method to assess the impact of transporters in vivo involves co-dosing specific inhibitors. The objective of the present study was to optimize the dose and route of administration of a P-glycoprotein (P-gp) inhibitor, valspodar (PSC833), and a dual P-gp/breast cancer resistance protein (BCRP) inhibitor, elacridar (GF120918), by assessing the transporters' impact on brain penetration and absorption. A dual-infusion strategy was implemented to allow for flexibility with dose formulation. The chemical inhibitor was dosed intravenously via the femoral artery, and a cassette of known substrates was infused via the jugular vein. Valspodar or elacridar was administered as 4.5-hour constant infusions over a range of doses. To assess the degree of inhibition, the resulting ratios of brain and plasma concentrations, Kp's, of the known substrates were compared to the vehicle control. These data demonstrated that doses greater than 0.9 mg/hr/kg valspodar and 8.9 mg/hr/kg elacridar were sufficient to inhibit P-gp- and BCRP-mediated efflux at the blood-brain barrier in rats without any tolerability issues. Confirmation of BBB restriction by efflux transporters in preclinical species allows for subsequent prediction in humans based upon the proteomic expression at rodent and human BBB. Overall, the approach can also be applied to inhibition of efflux at other tissues (gut absorption, liver clearance) or can be extended to other transporters of interest using alternate inhibitors.

Keywords: BCRP; Breast cancer resistance protein; IVIVC; Kp; P-glycoprotein; P-gp; blood-brain barrier; chemical and genetic knock out; dantrolene; efflux transporter; elacridar; glyburide; loperamide; quinidine; rat; valspodar; verapamil.

FIGURE 1

Inhibition of rat P‐gp at the BBB using valspodar. The P‐gp inhibitor, valspodar (PSC833) dosed from 0.09 to 8 mg/hr/kg as constant infusion over 4.5 hours shows a linear dose dependent increase in free plasma exposure (mean, rat plasma binding =92%) from 32 ng/ml to 2,592 ng/ml. When co‐administered with established P‐gp and BCRP substrates, valspodar, dosed above 0.9 mg/hr/kg does not result in an increase in brain exposure (as measured by the in vivo efflux ratio, brain/plasma ratio with inhibitor: brain/plasma ratio without inhibitor) for the selected P‐gp substrates, loperamide, verapamil and quinidine suggesting complete chemical inhibition at the blood‐brain barrier. The valspodar dose of 0.9 mg/hr/kg yielded a found free plasma concentration of 431.2 +/‐ 95.2 ng/ml which equates to 245 nM, well above the 100 nM invitro IC₅₀. Notably, the formulation and concentrations do not impact the brain concentrations for non‐ or BCRP‐mediated substrates, minoxidil and dantrolene, respectively, suggesting the tight junctions of the BBB were not impacted by the formulation for inhibitor, nor did valspodar inhibitor BCRP at any concentration

FIGURE 2

Inhibition of P‐gp and Bcrp at the rat BBB with elacridar. The multiple transporter inhibitor, elacridar (GF120918) dosed as constant infusion from 0.009 mg/hr/kg to the maximum soluble, 8.9 mg/hr/kg over 4.5 hours showed linear PK with a found total plasma exposure (mean) ranging from 1.6 ng/ml to 3267 ng/ml which equates to 0.025 to 49 ng/ml free and maximum 53 nM free using 1.5% rat plasma free fraction. At the highest concentrations, we observed inhibition of P‐gp and Bcrp, as shown by probe substrates loperamide and verapamil (P‐gp); glyburide and dantrolene (BCRP) in a concentration dependent manner resulting in increased in brain exposure which was quantified by the in vivo efflux ratio, ranging from 4.2 to 46 at 53 nM free elacridar. Although possibly limited by solubility and the dose range from 0.009 to 8.9 mg/hr/kg, no plateau was observed in in vivo efflux ratio as was seen with valspodar. The formulation and range of elacridar did not impact the blood‐brain barrier as seen with the low permeability molecule, atenolol

FIGURE 3

In vivo exposure in relation to in vitro inhibition. The respective free plasma concentration in rat, [I] divided by the in vitro IC₅₀ for valspodar in MDCK‐MDR1 cells (circle), elacridar in Caco‐2 cells (square) and in MDCK‐BCRP cells (triangle). A line of equivalency, where the free plasma concentration is equal to the IC₅₀, is set at 1. The concentration of valspodar in rat plasma is clearly higher than the in vitro IC₅₀ suggesting that significant if not complete inhibition was achieved at the rat BBB. In contrast, due to the limit of solubility with the elacridar formulation, perhaps incomplete inhibition was achieved based upon in vitro IC₅₀ values. Notably, the published IC₅₀ s for elacridar vary significantly across cell lines, labs, and substrates. This range of invitro inhibition makes a direct comparison from invitro to invivo challenging. Future studies using a wider range of substrate and inhibitors to establish an inclusive database, particularly with invivo data (chemical as shown here and genetic) would clarify the interpretation around BCRP

FIGURE 4

(A, B) In vitro to in vivo correlation of P‐glycoprotein. Utilizing the constant infusion approach to inhibiting active P‐gp or Bcrp‐mediated efflux at the rat blood‐brain barrier (BBB) thereby calculating the in vivo efflux ratio (Kp inhibitor / Kp WT), the in vitro to in vivo correlation (IVIVC) has been shown to be linear with the efflux ratios derived from MDCK‐MDR1 (A; NIH/Absorption Systems) and MDCK‐BCRP (B; Solvo Biotechnology) cell lines using commercial and proprietary compounds from in‐house data. These data confirmed previously published MDR1 results from Kalvass, Trapa, et al using different methods. Compounds shown here were screened in vitro against both efflux transporters, P‐gp and BCRP and any dual substrates were not used in this comparison, therefore IVIVC is compared per transporter. Overall the dynamic range of the engineered cell lines appears to be greater than the functional efflux at the rat BBB, as defined by the in vivo efflux ratio. The constant infusion tool inhibiting rat P‐gp and Bcrp allowed for internal calibration of IVIVC to better inform assay cut‐off values, SAR, confirmation of in vitro values, and prediction of brain penetration

FIGURE 5

Inhibition of P‐glycoprotein in the gut. A single oral administration of P‐gp substrates, loperamide or fexofenadine with a constant infusion of the P‐gp‐selective inhibitor, valspodar, for the duration of the study have shown significant impact on their pharmacokinetic profiles, most likely due to improved absorption though alterations to clearance cannot be excluded given the design

FIGURE 6

Inhibition of Bcrp in the gut and biliary compartments. Single oral administration of BCRP substrates, topotecan or sulfasalazine with a constant infusion of the inhibitor, elacridar, over the duration of the study have shown significant impact on their pharmacokinetic profiles. Oral exposure for both molecules increased when dosed with the inhibitor from 2‐to 10‐fold. In addition, the elimination rate of sulfasalazine was also significantly impacted which suggested that the clearance of this molecule is also dependent on active Bcrp‐mediated efflux in the biliary compartment. The constant infusion dose allowed for systemic steady state of the inhibitor, elacridar, over the course of the study informing on rate‐limiting steps in absorption and clearance