(M1030-10-63) In Vitro and In Vivo Pharmacokinetics of a Novel B7-H3 Inhibitor (Ni1) for the Treatment of Group 3 Medulloblastoma

Purpose: Amplification of MYC, poor prognosis, and early metastasis are commonly associated with pediatric patients diagnosed with group 3 medulloblastoma (MB), with a 5-year survival rate of less than 50%.¹ B7-H3 (CD276), a member of the B7 superfamily of immune checkpoints, has limited expression in healthy tissues but is overexpressed in various cancers, including group 3 MB.^{2, 3} Ni1 is a novel small molecule B7-H3 inhibitor with in vitro cytotoxicity against MB cell. As a novel compound for treating medulloblastoma, little is known about the in vivo disposition and the brain partitioning of Ni1. The current study objective was to establish a preclinical pharmacokinetic (PK) profile of Ni1, a potential therapeutic for MB.

Methods: A rapid and robust liquid-chromatography tandem mass spectrometry (LC-MS/MS) analytical method was developed for the quantification of Ni1 in biological matrices using protein precipitation and passing it through phospholipid removing Phree plate as an extraction method of choice. The developed analytical method was utilized to determine the in vitro preclinical characteristics of Ni1, including stability in physiological media, thermodynamic solubility, permeability, metabolic stability, and protein binding. In vivo PK profile was established by performing the non-compartmental analysis (NCA) using Phoenix WinNonlin followed by biodistribution studies in healthy male Balb/c mice after a single intraperitoneal (IP) injection of Ni1 at a dose of 10 mg/kg.

Results: The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using the precursor ion > product ion combinations of 501.05 >258.10 m/z for Ni1 and 497.15 >181.10 m/z for internal standard (IS, a structure analog) in positive electrospray ionization (ESI) mode. The chromatographic separation of Ni1 from the biological matrix was achieved using an Acquity BEH C-18 column (2.1*100 mm, 1.7 𝜇m) with an Acquity C-18 guard column. The mobile phase consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile:methanol (1:1, mobile phase B) using a gradient flow at a flow rate of 0.25 mL/min with injection volume held constant at 2 µL. The MS/MS response was linear over a concentration range of 0.20–1000 ng/mL for Ni1 with a correlation coefficient (r²) of more than 0.98. Precision (% RSD) and accuracy (% bias) were within the acceptable limits set forth by FDA. Ni1 was stable under storage and laboratory handling conditions. In vitro stability studies in physiological fluids like simulated gastric fluid, simulated intestinal fluid, and mouse plasma suggested that Ni1 was stable, remaining more than 85% and 75% at the end of 2 h and 97% at the end of 4 h, respectively. Ni1 was also stable in the presence of mouse and human liver microsomes with slow to moderate metabolism and an intrinsic clearance of 5.53 ± 0.40 and 12.67 ± 0.40 µL/(min*mg protein), respectively. Low aqueous solubility (0.14 ± 0.04 mg/mL at pH 7.4) and low apparent permeability (3.64 x 10^-6 cm/s) across an artificial membrane (Parallel Artificial Membrane Permeability Assay, PAMPA) were also associated with the molecule. Extensive in vitro plasma and brain protein binding proposed an unbound fraction (F_u) of less than 2% (Table 1). The in vivo preclinical PK studies suggested rapid absorption ( < 1 h) to achieve a maximum concentration (C_max) of 178.93 ± 32.79 ng/mL after a single intraperitoneal injection. The elimination half-life was approximately 8 h, with a high volume of distribution of 87.24 ± 53.46 L/kg, and a systemic clearance of 7.17 ± 2.43 L/hr/kg. The biodistribution study Ni1 displayed maximum brain concentrations of 1091.09 ± 174.46 ng/g in 8 h resulting in a high brain-to-plasma ratio with brain concentrations approximately 14-fold higher than the corresponding plasma concentrations (Figure 1). Owing to the high volume of distribution, a result of NCA, Ni1 was found to be well distributed in other tissues as well, like the spleen, kidney, liver, and lungs, with concentrations staying well above the IC₅₀ value up to 8 h with no physical signs of toxicity (Figure 2). Finally, physiological-based pharmacokinetic modeling is ongoing to simulate the plasma concentrations for different doses and dose intervals of Ni1.

Conclusion: The PK study demonstrated high brain penetration of a Ni1 with slow metabolism and formed the basis for future efficacy studies in a mouse model of MB. In conclusion, Ni1, a small molecule inhibitor of B7-H3, might be a viable drug candidate for enhancing immune response, alleviating the tumor's aggressiveness, and thereby improving the survival rate in patients suffering from group 3 MB.

References: (1) Li, S.; Poolen, G. C.; van Vliet, L. C.; Schipper, J. G.; Broekhuizen, R.; Monnikhof, M.; Van Hecke, W.; Vermeulen, J. F.; Bovenschen, N. Pediatric medulloblastoma express immune checkpoint B7-H3. Clin Transl Oncol 2022, 24 (6), 1204-1208. DOI: 10.1007/s12094-021-02762-y From NLM Medline.
(2) Getu, A. A.; Tigabu, A.; Zhou, M.; Lu, J.; Fodstad, O.; Tan, M. New frontiers in immune checkpoint B7-H3 (CD276) research and drug development. Mol Cancer 2023, 22 (1), 43. DOI: 10.1186/s12943-023-01751-9 From NLM Medline.
(3) Chapoval, A. I.; Ni, J.; Lau, J. S.; Wilcox, R. A.; Flies, D. B.; Liu, D.; Dong, H.; Sica, G. L.; Zhu, G.; Tamada, K.; et al. B7-H3: A costimulatory molecule for T cell activation and IFN-γ production. Nature Immunology 2001, 2 (3), 269-274. DOI: 10.1038/85339.

Acknowledgements: This project was partially funded by Edna Ittner Pediatric Research Support Fund.

Table 1: In vitro preclinical pharmacokinetic studies (Mean ± SD, n=3)

Figure 1: Plasma and brain concentration-time profile of Ni1 following an intra-peritoneal (IP) dose of 10 mg/kg (Mean ± SD, n=5)

Figure 2: Biodistribution of Ni1 at 2, 8, and 24 h after a single IP dose of 10 mg/kg (Mean ± SD, n=5)