A Repurposed Drug Selection Pipeline to Identify CNS-Penetrant Drug Candidates for Glioblastoma

Abstract

Background: Glioblastoma is an aggressive and incurable type of brain cancer. Little progress has been made in the development of effective new therapies in the past decades. The blood-brain barrier (BBB) and drug efflux pumps, which together hamper drug delivery to these tumors, play a pivotal role in the gap between promising preclinical findings and failure in clinical trials. Therefore, selecting drugs that can reach the tumor region in pharmacologically effective concentrations is of major importance.

Methods: In the current study, we utilized a drug selection platform to identify candidate drugs by combining in vitro oncological drug screening data and pharmacokinetic (PK) profiles for central nervous system (CNS) penetration using the multiparameter optimization (MPO) score. Furthermore, we developed intracranial patient-derived xenograft (PDX) models that recapitulated the in situ characteristics of glioblastoma and characterized them in terms of vascular integrity, BBB permeability and expression of ATP-binding cassette (ABC) transporters. Omacetaxine mepesuccinate (OMA) was selected as a proof-of-concept drug candidate to validate our drug selection pipeline.

Results: We assessed OMA's PK profile in three different orthotopic mouse PDX models and found that OMA reaches the brain tumor tissue at concentrations ranging from 2- to 11-fold higher than in vitro IC₅₀ values on patient-derived glioblastoma cell cultures.

Conclusions: This study demonstrates that OMA, a drug selected for its in vitro anti-glioma activity and CNS- MPO score, achieves brain tumor tissue concentrations exceeding its in vitro IC₅₀ values in patient-derived glioblastoma cell cultures, as shown in three orthotopic mouse PDX models. We emphasize the importance of such approaches at the preclinical level, highlighting both their significance and limitations in identifying compounds with potential clinical implementation in glioblastoma.

Keywords: ABC transporters; BBB; CNS-MPO; PDX models; glioblastoma; pharmacokinetics.

Figure 1

Pharmacokinetic analysis of OMA in vivo indicates accumulation in the brain tumor tissue. LC/MS/MS analysis determined the drug concentration after a single dose of 1.5 mg/kg of OMA in tumor-bearing mice. (A) Assessment of omacetaxine’s concentration in plasma (ng/mL), (B) liver (ng/g), (C) brain tumor tissue (ng/g) at 1, 2 and 4 h and (D) B/P ratio at 1, 2 and 4 h in GS832 PDX model (n = 5–10). (E) Assessment of drug concentration in plasma (ng/mL), (F) liver (ng/g) and (G) brain tumor tissue (ng/g) at 1 h and (H) B/P ratio at 1 h in GS607 PDX model (n = 4). Assessment of drug concentration in (I) plasma (ng/mL), (J) liver (ng/g) and (K) brain tumor tissue (ng/g) at 1 h and (L) B/P ratio at 1 h in GBM8 PDX model (n = 7).

Figure 2

Assessment of the BBB permeability in three glioblastoma PDX models. The permeability of the BBB was evaluated by T2W, T1W pre- and post-gadolinium MRI imaging. Histological visualization of the tumor by H/E staining of mouse brain coronal slices. MRI images were obtained 70 days post-tumor implantation for GS607 and GS832-PDX, and 30 days post-implantation for GBM8. Black dashed lines indicate the tumor areas in the mouse brain slices, while red arrows denote the tumor regions in the MRI images. The number of mice used in this experiment was 3 per model.

Figure 3

Characterization of the vascular integrity of the glioblastoma PDX models, GS607, GS832 and GBM8. The integrity of the vasculature was assessed by immunofluorescence staining for ZO-1 and GLUT-1, and GSC infiltration by Nestin staining. The number of mice used in this experiment varied between 3 and 6 per model. Scale bar 100 μm.

Figure 4

(A) Assessment of P-gp and BCRP transporter’s expression in the glioblastoma PDX models by IHC staining, magnification ×20 and scale bar 100 μm. Red arrows indicate the expression of the P-gp and BCRP transporters in the mouse brain tumor areas. The number of mice used in this experiment varied between 3 to 8 per model. (B) Assessment of omacetaxine’s in vitro affinity for P-gp and BCRP transporters by LC/MS-MS. The graphs depict the concentration of OMA as a percentage (y-axis) in the basal and apical compartments of LLC cells expressing murine Abcb1a and human ABCB1 and MDCK cells expressing murine Abcg2 and human ABCG2, over time in hours (x-axis). Zosuquidar was added to MDCK cells to inhibit endogenous (canine) P-gp. All experiments were performed in 3 biological replicates.

Figure 4

(A) Assessment of P-gp and BCRP transporter’s expression in the glioblastoma PDX models by IHC staining, magnification ×20 and scale bar 100 μm. Red arrows indicate the expression of the P-gp and BCRP transporters in the mouse brain tumor areas. The number of mice used in this experiment varied between 3 to 8 per model. (B) Assessment of omacetaxine’s in vitro affinity for P-gp and BCRP transporters by LC/MS-MS. The graphs depict the concentration of OMA as a percentage (y-axis) in the basal and apical compartments of LLC cells expressing murine Abcb1a and human ABCB1 and MDCK cells expressing murine Abcg2 and human ABCG2, over time in hours (x-axis). Zosuquidar was added to MDCK cells to inhibit endogenous (canine) P-gp. All experiments were performed in 3 biological replicates.