Brain Penetration and Efficacy of Different Mebendazole Polymorphs in a Mouse Brain Tumor Model

Abstract

Purpose: Mebendazole (MBZ), first used as an antiparasitic drug, shows preclinical efficacy in models of glioblastoma and medulloblastoma. Three different mebendazole polymorphs (A, B, and C) exist, and a detailed assessment of the brain penetration, pharmacokinetics, and antitumor properties of each individual mebendazole polymorph is necessary to improve mebendazole-based brain cancer therapy.

Experimental design and results: In this study, various marketed and custom-formulated mebendazole tablets were analyzed for their polymorph content by IR spectroscopy and subsequently tested in an orthotopic GL261 mouse glioma model for efficacy and tolerability. The pharmacokinetics and brain concentration of mebendazole polymorphs and two main metabolites were analyzed by LC/MS. We found that polymorph B and C both increased survival in a GL261 glioma model, as B exhibited greater toxicity. Polymorph A showed no benefit. Polymorph B and C both reached concentrations in the brain that exceeded the IC₅₀ in GL261 cells 29-fold. In addition, polymorph C demonstrated an AUC₀₋₂₄h brain-to-plasma (B/P) ratio of 0.82, whereas B showed higher plasma AUC and lower B/P ratio. In contrast, polymorph A presented markedly lower levels in the plasma and brain. Furthermore, the combination with elacridar was able to significantly improve the efficacy of polymorph C in GL261 glioma and D425 medulloblastoma models in mice.

Conclusions: Among mebendazole polymorphs, C reaches therapeutically effective concentrations in the brain tissue and tumor with fewer side effects, and is the better choice for brain cancer therapy. Its efficacy can be further enhanced by combination with elacridar.

Figure 1. MBZ-C is the most efficacious polymorph with limited toxicity

A and B. Infra-red spectra of MBZ polymorphs and MBZ tablets from different suppliers: S2015 and S2017 (Aurochem), Teva (after 2-year storage at RT), Medley and Janssen. Two peaks represent the -NH and –C=O groups in the molecules. Black arrow heads indicate the peaks of MBZ-C control. C. (Left panal) Kaplan Meier survival curves of mice implanted with GL261-luc glioma and treated with different MBZ polymorphs (A, B and C). A H&E staining of the GL261-luc glioma-bearing mouse brain by coronal cut was shown. 5 days after the tumor implantation, the mice were gavaged with MBZ and control animals were feed with vehicles. One mouse in the MBZ-B group presumably died from drug toxicity as no significant tumor was found in the brain. The p-values of Con vs MBZ-B and Con vs MBZ-C are indicated. The p-value of MBZ-B vs MBZ-C is 0.72. m: median survival in days. Con: n=6; MBZ-A: n=5; MBZ-B: n=6; MBZ-C: n=6. (Right panel) Luciferase counts measured by Xenogen reflected the size of GL261-luc brain tumor in mice treated with MBZ polymorphs for 20 days. D. Survival curves of GL261-luc bearing mice treated with MBZ tablets from different suppliers. Con: n=6; S2015: n=5; S2017: n=6; Medley: n=5; Janssen: n=5.

Figure 2. Plasma and brain distributions of MBZ polymorphs

A. A time course of the MBZ plasma levels in C57BL6 mice after oral gavage of MBZ-A, B or C at 50mg/kg. B. (Left panel) Brain and plasma levels of MBZ-C in a time course after oral gavage at 50mg/kg. Animals were thoroughly perfused with PBS for all brain distribution studies. (Right panel) Brain/plasma (B/P) ratios of MBZ-C. Data were collected from three mice at each time point. C. (Left panel) Brain and plasma levels of MBZ polymorphs at 6 h following oral gavage (50mg/kg). B/P ratio of MBZ polymorphs at 6 h following oral gavage. (Right panel) The mean B/P ratio of MBZ-A is 0.32, MBZ-B is 0.64 and MBZ-C is 0.80. D. MBZ-C distributed equally in the brain and brain tumor. GL261 tumors implanted in the right side of mouse frontal lobe were resected and compared with the contralateral normal brain tissue.

Figure 3. Distribution of MBZ Metabolites

A and B. The plasma and brain levels of MBZ and its metabolites, MBZ-OH (rac dihydro mebendazole, CAS 60254-95-7) and MBZ-NH₂ (2-amino-5-benzoylbenzimidazole, CAS 52329-60-9), were analyzed following oral gavage of 50mg/kg MBZ-C. C. The distributions of MBZ metabolites in the plasma, brain and GL261 brain tumor. At 2.5, 4 and 6 hours after oral gavage of MBZ-C, mice implanted with GL261 for 25 days were sacrificed and the blood, GL261 brain tumor and contra-lateral normal brain tissues were sampled and analyzed. D. IC₅₀ curve of GL261 giloma cells with MBZ and metabolites. GL261 cells were incubated with MBZ or its metabolites for 72 h and the living cells were measured.

Figure 4. Combination of MBZ with elacridar

A. IC₅₀ curve of GL261 glioma cells with elacridar (ELD). IC₅₀=5.8 μM. B. Inhibition of GL261 cells by MBZ (0.25 μM), ELD (1 or 5 μM) or the combination. Cells were incubated with the indicated drugs for 72 h and the living cells were measured by the colorimetric assay. C. ELD elevated the average B/P ratios of MBZ in mice.

Figure 5. Combination of MBZ with elacridar improved the efficacy

A and B. GL261 cells transfected with luciferase were implanted in C57BL6 mice and the treatments were initiated 5 days after the implantation. Elaridar (ELD) was oral gavaged at 50 mg/kg 2 hours before the MBZ administration (50 mg/kg) for the first 7 or 14 days of treatment. Thereafter, MBZ was given five days a week at the same dose for the rest of the therapy. The ELD alone group was gavaged with ELD for 14 daily doses. Animals treated by MBZ and ELD were monitored by Xenogen for tumor luciferase signals starting from 25 days after the tumor implantation (A). C and D. D425 medulloblastoma cells were implanted in the mouse cerebellum and formed a cerebellar tumor (C, H&E staining). Five days after the tumor implantation, mice were treated with vehicle (Con), 7 days of 50 mg/kg elacridar (ELD), 50 mg/kg MBZ-C alone (MBZ) or 7 days of 50mg/kg ELD with 50 mg/kg MBZ-C (MBZ-ELD) following the same dosing regime in B.