(M1430-10-66) Assessment of In Vitro and In Vivo Pre-clinical Pharmacokinetics of EPZ015666; a Novel Small Molecule for Medulloblastoma Treatment

Purpose: Medulloblastoma (MB) is the second most common malignant pediatric brain tumor, constituting about 20% of all pediatric brain malignancies.¹ Current MB therapies involve chemotherapy and radiation therapy but fail to cure approximately one-third of patients.² Group 3 MB has the worst prognosis of the MB subgroups with a 5-year survival rate of less than 50%.³ It has been reported that Protein arginine methyltransferase 5 (PRMT5), is overexpressed in MYC amplified tumors, therefore, EPZ015666 (EPZ) an inhibitor of PRMT5 can be a potential treatment for MYC-driven group 3 medulloblastoma.⁴ However, the ability of EPZ to cross the blood-brain barrier and achieve therapeutic concentrations in the brain is currently unknown. Due to its potent activity against MB cells in vitro, we evaluated the protein binding, metabolic stability, pharmacokinetic (PK) profile, tissue distribution pattern and brain penetration of EPZ.

Methods: A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify EPZ in mouse plasma and tissues. In vitro protein binding was determined by Rapid equilibrium dialysis (RED) method. A parallel artificial membrane permeability assay (PAMPA) was performed to determine the apparent permeability of the substance. Metabolic stability was evaluated in mouse (MLM) and human liver microsomes (HLM) followed by in vivo PK and biodistribution studies post single oral (PO) dose of 100 mg/kg EPZ in BALB/c mice(n=3). Plasma and tissue samples were analyzed for EPZ concentrations and pharmacokinetic data was processed with Phoenix 8.3 software simulating through non-compartmental analysis (NCA).

Results: The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using the precursor ion → product ion combinations of 384.0 >146.1 m/z for EPZ and 250 >175.9 for internal standard (IS) Oxibendazole. The Chromatographic separation was achieved using Synergi 4µm Polar-RP 80 A LC Column (150×2mm) with a C18 guard column (Phenomenex,Torrance CA). The mobile phase consisted of 0.1% formic acid in water and methanol using a gradient flow at a total flow rate of 0.25 mL/min. The MS/MS response was linear over the concentration range from 0.2–500 ng/mL for EPZ, correlation coefficient (r²) of 0.98. Precision (% RSD) and accuracy (% bias) were within acceptable limits as per FDA guidelines. Plasma and tissues sample prepared using solid phase extraction (SPE). EPZ was stable under storage and laboratory handling conditions. The validated LC-MS/MS method was successfully applied to in-vitro and in-vivo pharmacokinetic (PK) and bio-distribution studies of EPZ in mouse. In vitro plasma and brain protein binding of EPZ was approximately 32%. In both mouse liver microsomes and human liver microsomes studies EPZ showed complete stability after 1 hour of incubation (Figure 1). This represents that EPZ is stable in Mouse liver microsomes and Human liver microsomes suggesting no non-CYP and CYP mediated metabolism. EPZ showed high apparent permeability (P_app) in PAMPA study with a value of 18.6 ± 4.43 x 10^-6 cm/s. Following oral administration of EPZ (100 mg/kg) in BALB/c mice, the C_max (ng/mL) was achieved at 4496.95 ± 811.88 which was higher than IC₅₀ value of EPZ in HD-MB03 (2032.23 ng/mL) and D-341 (1840.51 ng/mL) cell lines (Figure 2). Plasma apparent clearance (CL/F; L/hr/kg) was 14.6 ± 4.0. The concentration of EPZ in the brain at 24 hours was 68.99 ± 2.74 ng/mL. The brain-to-plasma concentration ratio (K_p, _brain) for 2 and 24 hr was found to be 0.31 and 6.05 (mean ± SD, n=3) (Table 1).

Conclusion: In summary, these studies demonstrate metabolic stability and CNS penetration for a novel PRMT5 inhibitor (EPZ015666) and form the basis for future efficacy studies in mouse models of MB.

References: (1) Sidharth Mahapatra, M. J. A. Medulloblastoma. 2023. https://www.ncbi.nlm.nih.gov/books/NBK431069/ (accessed.
(2) Liu, Y.-P.; Zheng, C.-C.; Huang, Y.-N.; He, M.-L.; Xu, W. W.; Li, B. Molecular mechanisms of chemo- and radiotherapy resistance and the potential implications for cancer treatment. MedComm 2021, 2 (3), 315-340. DOI: https://doi.org/10.1002/mco2.55.
(3) Khatua, S.; Song, A.; Citla Sridhar, D.; Mack, S. C. Childhood Medulloblastoma: Current Therapies, Emerging Molecular Landscape and Newer Therapeutic Insights. Curr Neuropharmacol 2018, 16 (7), 1045-1058. DOI: 10.2174/1570159X15666171129111324 From NLM Medline.
(4) Chaturvedi, N. K.; Mahapatra, S.; Kesherwani, V.; Kling, M. J.; Shukla, M.; Ray, S.; Kanchan, R.; Perumal, N.; McGuire, T. R.; Sharp, J. G.; et al. Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma. BMC Cancer 2019, 19 (1), 1056. DOI: 10.1186/s12885-019-6291-z.

Acknowledgements: This work was partially funded by a grant from the Child Health Research Institute and the Pediatric Cancer Research Group to NKC.

Figure 1: Metabolic Stability of EPZ in mouse (MLM) and human liver microsomes (HLM) (n=3)

Figure 2: Plasma concentration vs time profile following a single oral (PO) dose of 100 mg/kg EPZ in BALB/c mice (n=3)

Table 1: Plasma and tissue distribution study of EPZ after 100 mg/kg oral dose in mice (Mean ± SD, n=3)