Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study

Recently, nanocrystal dispersions have been considered as a promising formulation strategy to improve the bioavailability of the deuterated pyrazoloquinolinone ligand DK-I-56-1 (7‑methoxy-2-(4‑methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one). In the current study, the freeze-drying process (formulation and process parameters) was investigated to improve the storage stability of the previously developed formulation. Different combinations of lyoprotectant (sucrose or trehalose) and bulking agent (mannitol) were varied while formulations were freeze-dried under two conditions (primary drying at -10 or -45 °C). The obtained lyophilizates were characterized in terms of particle size, solid state properties and morphology, while the interactions within the samples were analyzed by Fourier transform infrared spectroscopy. In the preliminary study, three formulations were selected based on the high redispersibility index values (around 95%). The temperature of primary drying had no significant effect on particle size, but stability during storage was impaired for samples dried at -10 °C. Samples dried at lower temperature were more homogeneous and remained stable for three months. It was found that the optimal ratio of sucrose or trehalose to mannitol was 3:2 at a total concentration of 10% to achieve the best stability (particle size < 1.0 μm, polydispersity index < 0.250). The amorphous state of lyoprotectants probably provided a high degree of interaction with nanocrystals, while the crystalline mannitol provided an elegant cake structure. Sucrose was superior to trehalose in maintaining particle size during freeze-drying, while trehalose was more effective in keeping particle size within limits during storage. In conclusion, results demonstrated that the appropriate combination of sucrose/trehalose and mannitol together with the appropriate selection of lyophilization process parameters could yield nanocrystals with satisfactory stability

Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1); process parameters and cryoprotectant selection through stability study

1. INTRODUCTION Nanocrystal dispersions are considered as the universal formulation strategy for brick dust substances. However, the stability of these systems to aggregation represents a big issue. To overcome this, nanocrystal dispersions are usually solidified by freeze-drying (lyophilization). During this process the risk of aggregation is considered to be high, due to ice formation and/or water loss. To prevent the aggregation, For the particle size preservation, therefore, it is necessary to add cryoprotectants/lyoprotectants, among which sugars are most commonly used. To ensure good structure of the cake, bulking agents are often included in formulations, as well [1,2], although in nanocrystalline dispersions the combination of cryoprotectants and bulking agents is not frequent nor much investigated. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), patent protected pyrazoloquinolinone ligand, have been developed recently, and characterized in terms of physicochemical properties and pharmacokinetics after intraperitoneal administration in mice. These formulations were stable for three weeks [3]. Our aim in this study was to improve the stability by freeze-drying, and investigate the influence of different concentrations and physical form of cryoprotectants (sucrose, trehalose) and bulking agent (mannitol) as well as different primary drying conditions on the aggregation prevention. 2. MATERIALS AND METHODS 2.1. Materials DK-I-56-1 was synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin—Milwaukee, USA. The following other materials were used: polysorbate 80, poloxamer 407, sucrose, mannitol (Sigma-Aldrich Laborchemikalien GmbH, Germany) and trehalose (Carl Roth GmbH, Germany). 2.2. Lyophilization Nanocrystal dispersions stabilized by polysorbate 80 and poloxamer 407 were prepared by wet ball milling [3]. After addition of mannitol (M), sucrose (S), or trehalose (T) alone or in combination samples were freeze- dried. Two processes were applied: (1) freezing at -80 °C (3 h), primary drying at -10 °C, 0.340 mbar, secondary drying at 25 °C (24 h) or (2) freezing at -50 °C (3 h), primary drying at -45 °C, 0.2 mbar (21 h), secondary drying at 20 °C (30 h). Samples were stored in crimped vials at 25 °C (lyophilization 1) or 2-8 ºC (lyophilization 2) for three months. 2.3. Physicochemical characterization Particle size (z-ave) was measured by Zetasizer Nano ZS (Malvern Instruments, UK) and Mastersizer (Malvern Mastersizer 2000 Malvern, UK). Redispersibility index (RDI) was calculated as z-ave (before)/z-ave (after) and expressed in percentages. Physical state of samples was determined by differential scanning calorimetry (DSC1; Mettler Toledo, Switzerland),powder X-ray diffraction (Rigaku Smartlab X-ray Diffractometer) and polarized light microscopy (PLM) (Carl Zeiss ApoTome Imager Z1 microscope Zeiss, Germany). 3. RESULTS AND DISCUSSION Right after preparation, nanocrystal dispersions were with submicron particle size around 160 nm, and PDI below 0.2, suggesting narrow size distribution. In the cryoprotectant screening phase, sucrose and/or mannitol were added in different concentrations. It was shown that 10% of the total stabilizer concentration was needed for the particle size preservation: the achieved RDI was above 95%, while cakes with sucrose alone or in combination with mannitol in ratio 1:1 or 3:2 were also with satisfied appearance (Figure 1). Lyophilization was conducted above or below the glass transition temperature of the maximally freeze-concentrated solution (Tg’) (around -39 ºC). When primary drying was performed at -10 °C, no aggregation was noticed right after lyophilization, but particle size increased significantly, lowering down the RDI to < 50%, after one month storage at 25 °C. This was confirmed by laser diffraction. In lyophilization 2, with primary drying at temperature below Tg’, trehalose was also used in the same concentration as sucrose and in combination with mannitol. Interestingly, in this process parameters setup, sucrose or trehalose alone did not prevent aggregation during freeze-drying. Particle size remained almost unchanged in formulation S+M 3+2 (RDI 95%) or slightly higher in T+M 3+2 (RDI 90%), after three months storage, suggesting it was most probably the optimal combination for the stabilization. Physical state analysis revealed that sucrose and mannitol in samples lyophilized by process 1 were in crystalline state, as well as sucrose when used alone in lyophilization 2. Trehalose, on the other hand was amorphous in all samples containing it. Amorphous state of lyoprotectants allows maximal hydrogen bonding due to higher molecule flexibility and availability of hydroxyl groups [3]. Surprisingly, mannitol as a substance with high crystallization tendency was with low crystallinity in lyophilizates. These observations were confirmed by PLM. It is possible that it formed interactions with sucrose or nanocrystal stabilizers [4]. 4. CONCLUSION Results from this study demonstrated freeze- drying as an important technique for the improvement of nanocrystals stability. However, the selection of cryoprotectant and bulking agent ratio beside process parameters (primary drying at -45 ºC) was crucial to get freeze-dried samples with good stability. Sucrose or trehalose in combination with mannitol (ratio 3+2) in total concentration 10% successfully hindered aggregation, thus prolonging the stability to 3 months at 2-8 ºC. 5. REFERENCES 1. Van Eerdenbrugh, B., et al. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. International journal of pharmaceutics, 2008. 364(1): 64-75. 2. Trenkenschuh, E., and Friess, W. Freeze-drying of nanoparticles: How to overcome colloidal instability by formulation and process optimization. European Journal of Pharmaceutics and Biopharmaceutics, 2021.165: 345-360. 3. Mitrović, J.R., et al. Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand DK-I-60-3 by nanonization: A knowledge-based approach. Pharmaceutics, 2021. 13(8): 1188. 4. Kumar, S., et al. Sugars as bulking agents to prevent nano-crystal aggregation during spray or freeze-drying. International journal of pharmaceutics, 2014. 471(1-2): 303-311. ACKNOWLEDGMENT This research was supported by the Science Fund of the Republic of Serbia, grant No. 7749108, project Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma-2 receptors: In vitro/in vivo delineation by nano- and hiPSC-based platforms-NanoCellEmoCog

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