(M0930-01-05) Lipid-Stabilized Nanocrystal Vesicles for Cheat-Free Testing of Novel Drugs: The Case of Zuranolone

Purpose: Drug transfer from pre-clinical development to first-in human (FIH) clinical testing often represents a bottle-neck. In vitro bioactivity screening and preclinical in vivo tests, employ solubilization strategies based on surfactant micelles or dimethyl sulfoxide solutions, unviable for later application to humans where solid dosage forms are preferred. It would be therefore massively beneficial to unify the formulation approach at all stages of drug development and use the same form of the API during early-stage bioactivity screening studies as in FIH trials. Neurosteroids are endogenous steroid molecules which are synthesized de novo in the nervous system from cholesterol [1]. The synthetic analogues of neurosteroids are called neuroactive steroids. These compounds are rationally designed and synthesized with modified physicochemical or biological properties (e.g. solubility, permeability, affinity), some of them (such as low aqueous solubility) so far gatekeeping their pharmaceutical use [2, 3]. This contribution discusses the utilization of phospholipidic stabilization of nanocrystals (NCs) as a means of bridging this issue. Our previous results show that these can be repeatedly prepared, and their surface stabilization can be adjusted in order to alter their cell uptake and interactions [4, 5].

Methods: Small-scale milling experiments were performed in doses of zuranolone ranging from 20 to 100 mg in a 25 ml amber vial using 5 g 0.4-0.5 mm ZrO₂ milling pearls. Phospholipids (dipalmitoylphosphatidylcholine, sodium dipalmitoylphosphoglycerole), (N-(carbonyl-methoxypolyethylen-glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanol-amine sodium salt) were used as stabilizers in their molar ratio of 85:10:5, totaling two thirds of zuranolone added in mass (e. g. 40 mg lipids per 60 mg zuranolone). After 24 hours of milling at 600 rpm, draining and dilution, the suspensions were stored at 6 °C for up to 24 months. Particle size analysis and X-ray powder diffraction was performed on raw zuranolone and shock-frozen zuranolone NCs both fresh and 24 months old. In vivo experiments were done in male Wistar albino rats (n = 98). All formulations and vehicles were administered i.p. (0.25, 0.5 or 1 mg/kg). Pentylenetetrazol (PTZ, Sigma-Aldrich, 50 mg/mL in water, dose 100 mg/kg s.c.) was administered 20 min after zuranolone formulations. Seizure severity and incidence of generalized seizures (GS) were registered and statistically evaluated [6].

Results: PSD of zuranolone NCs measured by DLS confirmed the qualitative TEM observation (Figures 1, 2) and provided a number-mean particle size d_1,0 = 114 ± 39 nm for the fresh NCs, with a polydispersity index (PDI) of 0.311. The NCs revealed remarkably good temporal stability time, having number-mean particle size d_1,0 = 122 ± 41 nm (PDI  0.388) after 24 months of storage. This prefaced the creation of a solubility-independent concentration range of NCs. NCs retained their crystallinity throughout storage. The described procedure has repeatedly yielded stable nanosuspensions of concentrations up to 100 mg/ml, exceeding even solubility of zuranolone in pure dimethyl sulfoxide (78 mg/ml). In P12 rats, seizure severity expressed as a score was significantly suppressed by nanocrystal formulation of zuranolone in doses of 0.5 and 1 mg/kg, administered 20 min before injection of PTZ, whereas zuranolone dissolved in CDX was ineffective. The protective effect was driven mostly by decreased incidence of generalized seizures. In P25 rats zuranolone in a dose of 0.5 mg/kg significantly decreased seizure severity in both formulations. Both formulations equally suppressed the incidence of GS in doses 0.5 and 1 mg/kg. The significant effect on the incidence of GS in P12 animals after the administration of a lower dose using the NCs than CDX solution might be caused by higher concentration in the brain, possibly arising from differences of brain maturity corresponding to age of rats used.

Conclusion: Suspensions of phospholipid-stabilized zuranolone NCs have been repeatedly prepared by wet stirred media milling. These retained their PSD, colloidal stability, and crystallinity over 24 months of storage. A broad range of easy-to-handle solutions up to 100 mg/mL of zuranolone NCs was obtained, exceeding the solubility of in pure DMSO (82 mg/mL). The severity of PTZ-induced seizures was greatly diminished in P12 rats, while administration of zuranolone in cyclodextrin solution showed no such effect. Moreover, the GS incidence was decreased only as a consequence of administration of NCs. Conversely, in P25 rats, the seizure severity and incidence were decreased after the administration of either tested formulation. Owing to their non-xenobiotic composition, the same NCs can be used in all development stages without much adjustment, reducing the time of transfer and nearly eliminating drug reformulation.

References: 1. Baulieu, E. E. Neurosteroids: A novel function of the brain. Psychoneuroendocrinology (1998). 23, 963–987.
2. Kudova, E. Rapid effects of neurosteroids on neuronal plasticity and their physiological and pathological implications. Neurosci. Lett. (2021), 750, 135771.
3. Scott, L. J. Brexanolone: First Global Approval. Drugs (2019), 779–783.
4. Hládek, F., Chvíla, S., et al., Systematic Investigation of Wet-Milling Kinetics and Colloidal Stability of Pharmaceutical Nanocrystals, Crystal Growth & Design (2022), 22(12), 6928-6940.
5. Lizoňová, D. et al., Surface stabilization determines macrophage uptake, cytotoxicity, and bioactivity of curcumin nanocrystals, International Journal of Pharmaceutics (2022), 626, 122133.
6. Pohl, M., Mares, P. Effects of flunarizine on Metrazol-induced seizures in developing rats. Epilepsy Res. (1987), 1, 302–305.

Acknowledgements: We would like to thank Erik Sonntag for help with HPLC. F. Š. would like to acknowledge the financial support from the Czech Science Foundation (project no. 19-26127X). S. Ch. would like to acknowledge the support from The Pharmaceutical Applied Research Centre (The PARC). E. K. was supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104) – Funded by the European Union – Next Generation EU, by the Czech Academy of Sciences institutional support RVO:61388963, and by the European Regional Development Fund–ERDF/ESF Project “PharmaBrain”, No. CZ.02.1.01/0.0/0.0/16_025/0007444. H. K. would like to acknowledge the Czech Academy of Sciences institutional support RVO:67985823.


Graphic 1. Schematic depiction of the workflow: Design, preparation and analysis of zuranolone nanocrystals, subsequent in vivo test and its evaluation.