(M1230-10-63) Characterization of Preclinical In Vitro and In Vivo Pharmacokinetics Properties for JNJ-64619178, a New Therapeutic for the Treatment of Medulloblastoma

Purpose: Medulloblastoma is the most common malignant pediatric brain tumor and accounts for around 12 to 25% of all central nervous systems tumors in children [1-3]. Among four distinct molecular subgroups of medulloblastoma, namely - wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4, group 3 tumors typically predominate in the pediatric age spectrum and represent approximately 45% of medulloblastoma tumors in the infant population [4, 5]. Amplification or overexpression of the MYC oncogene is a hallmark of group 3 tumors [6]. Previous studies observed overexpression of protein arginine methyltransferase 5 (PRMT5), a key epigenetic enzyme in MYC-driven primary medulloblastoma tumors, suggesting a potential therapeutic target for the treatment of medulloblastoma [7]. Although several PRMT5 inhibitors are currently in clinical trials for a wide variety of cancers, their ability to cross the blood-brain barrier and potential in the therapy of medulloblastoma are largely unexplored. In this context, we aimed to assess in vitro and in vivo pharmacokinetic (PK) properties and brain-penetrance properties of a selective small molecule inhibitor of PRMT5, JNJ-64619178.

Methods: A LC-MS/MS method was developed and validated for the analysis of JNJ-64619178 in in vitro incubation systems and mouse plasma and tissues. Microsomal stability was evaluated through incubation in mouse (MLM) and human liver microsomes (HLM) for 1 hour. A rapid equilibrium dialysis (RED) method was applied to determine the plasma and brain protein binding of JNJ-64619178. A parallel artificial membrane permeability assay (PAMPA) was performed to determine the apparent permeability of the substance. Finally, to characterize the pharmacokinetics (PK) and biodistribution, we have performed a pilot PK analysis of JNJ-64619178 in healthy BALB/c mice. Following a single oral (PO) dose of JNJ-64619178 (10 mg/kg) in mice (n=4), blood samples were collected 0.25, 0.5, 1, 2, 4, 8 and 24 hours. Plasma and tissue samples were analyzed for JNJ-64619178 concentrations. Non-compartmental analysis (NCA) was performed using Pheonix software version 8.3 to estimate PK parameters from the observed plasma concentration-time profile data.

Results: The mass spectrometric conditions were optimized in positive electrospray ionization (ESI) mode and the multiple reaction monitoring (MRM) transitions were obtained as 484.95 > 135.1 and 286.1 > 202.05 for JNJ-64619178 and probenecid (internal standard) respectively. Column separation parameters were optimized using Synergi 4 µm Polar-RP (150 × 2 mm) column protected with a C18 guard column (Phenomenex, Torrance CA). 0.1% formic acid in water (A) and methanol (B) were selected as mobile phase using a gradient flow at a total flow rate of 0.25 mL/min. The assay was linear over the concentration range from 0.2–500 ng/mL for JNJ-64619178 with a correlation coefficient (r²) of 0.988. Inter and intra-day accuracy and precision were within acceptable limit for the analyte. In vitro metabolic stability studies showed that the percent remaining concentration of JNJ-64619178 after 1 hour incubation was 60.3±12.4 and 72.4±2.6 % respectively in MLM and HLM (figure 1). The calculated mean intrinsic clearance (CL_int) values in MLM and HLM were 41.88±15.77 and 7.49±0.46 mL/min/kg respectively, which signifies the rate of disappearance was significantly lower in HLM compared to MLM. JNJ-64619178 exhibited very high protein binding to mouse plasma and brain tissue, with a value of > 94%. Stability (%) in mouse plasma and brain matrices for approximately 4 hours was almost 100 %. JNJ-64619178 showed high apparent permeability (P_app) in PAMPA study with a value of 7.12 ± 4.66 (× 10^-6) cm/s. Following the oral administration of JNJ-64619178 (10 mg/kg), the maximum plasma concentration (C_max) achieved was 2781.61 ± 1033.62 ng/mL which was above the IC₅₀ of JNJ in HD-MB cell line (1.7 µM or 821.71 ng/mL) (figure 2). Plasma clearance was 1.5 L/hr/kg and volume of distribution was 5.6 L/kg. Although, brain to plasma concentration ratio at 2 hour was 0.04, the compound was well-retained in the brain environment even after 24 hours with a ratio of 14.89 (figure 3).

Conclusion: JNJ-64619178 was identified as a potential therapeutic for medulloblastoma with good PK properties in terms of in vitro and in vivo studies. Using the PK data derived from the pilot PK study, dosing strategies should be evaluated to assess efficacy.

References: 1. Dhall, G., Medulloblastoma. Journal of Child Neurology, 2009. 24(11): p. 1418-1430.
2. Millard, N.E. and K.C. De Braganca, Medulloblastoma. J Child Neurol, 2016. 31(12): p. 1341-53.
3. Bartlett, F., R. Kortmann, and F. Saran, Medulloblastoma. Clinical Oncology, 2013. 25(1): p. 36-45.
4. Orr, B.A., Pathology, diagnostics, and classification of medulloblastoma. Brain Pathol, 2020. 30(3): p. 664-678.
5. Northcott, P.A., et al., Medulloblastoma comprises four distinct molecular variants. J Clin Oncol, 2011. 29(11): p. 1408-14.
6. Tao, R., et al., MYC drives group 3 medulloblastoma through transformation of Sox2+ astrocyte progenitor cells. Cancer research, 2019. 79(8): p. 1967-1980.
7. Chaturvedi, N.K., et al., Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma. BMC Cancer, 2019. 19(1): p. 1056.

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 studies of JNJ-64619178 in mouse (MLM) and human liver microsomes (HLM)

Figure 2. Plasma concentration-time profile following the oral administration of JNJ-64619178 (10 mg/kg) in mice (n=4)

Figure 3. Plasma and brain tissue distribution study of JNJ-64619178 in mice (n=4)