Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α1 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states

Searching for the best way to incorporate the proprietary compound GL-II -73 into the nanoemulsion carrier for prospective parenteral application

Searching for the best way to incorporate the proprietary compound GL-II -73 into the nanoemulsion carrier for prospective parenteral application Jelena Đoković1, Bojan Marković2, Dishary Sharmin3, James M Cook3, Miroslav Savić4, Snežana Savić1 1University of pharmacy - Faculty of pharmacy, Department of pharmaceutical technology and cosmetology, 11221 Belgrade, Serbia 2University of pharmacy - Faculty of pharmacy, Department of pharmaceutical chemistry, 11221 Belgrade, Serbia 3Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, Milwaukee, WI, 53201, USA 4University of pharmacy - Faculty of pharmacy, Department of pharmacology, 11221 Belgrade, Serbia The maximum amount of drug that can be incorporated into lipid nanoemulsions (NE) is usually judged by their solubility in the internal phase of the formulation. This can lead to various problems, such as precipitation of the drug after processing the formulation or, depending on the preparation technique used, the use of a large amount of the drug. To this end, it is useful to consider other drug loading methods, especially in the early stages of formulation development. In this study, we aimed to find the best way to achieve the highest loading of GL-II -73 in NEs for future parenteral applications for in vivo animal studies. This ligand acts as a positive allosteric modulator at α-GABAA receptors with combined antidepressant and cognition enhancing effects. NEs were prepared using the high pressure homogenization technique, a standard technique for parenteral NE preparation. The oil phase (medium-chain triglycerides, soy lecithin, and butylated hydroxytoluene) and the aqueous phase (glycerol, polysorbate 80, and 0.01 M phosphate buffer, pH 8) were separately heated to 50 ˚C and mixed until all components were dissolved. The aqueous phase was added to the oil phase and processed first on a rotor-stator homogenizer at 11000 rpm for 1 minute and then on a high-pressure homogenizer at 800 bar for 10 cycles. This resulted in a droplet size of 117.1 ± 1.5 nm, a PDI of 0.060 ± 0.008, a zeta potential of - 43.3 ± 1.3 mV, a pH of 7.89 ± 0.02, and a conductivity of 1061.67 ± 5.51 S/cm, indicating initial suitability for parenteral use. Using the empirical method for drug loading, we were able to dissolve GL-II -73 in the oil phase and achieve a drug concentration of 1.5 mg/ml in NE. For our experimental setup, this required the use of 120 mg of GL-II -73 per experiment. For the passive drug loading procedure, we incubated 1 ml of the placebo NE in the eppendorf tube (in duplicate) with the excess drug (approximately 10 mg per tube) for 72 h. The drug was then added to the eppendorf tube. The excess of the undissolved drug was removed after centrifugation. The drug content in the supernatant was 3.10 ± 0.25 mg/ml, indicating above-average loading of the drug and possibly suggesting localization of the drug in the droplet-stabilising layer, but this needs to be further demonstrated. This approach could contribute to more rational formulation development in the selection of formulation factors. Acknowledgments This research was funded by the Science Fund of the Republic of Serbia, GRANT No 7749108, 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 platform - NanoCellEmоCog

Preformulation and development of preliminary nanoemulsion carrier for patent protected compound GL-II-73

Nanopharmaceuticals offer a good option to avoid some of the difficulties that novel drug candidates confront. They can be tailored to adjust their water solubility, half-life, biodistribution, and govern the release of the integrated medication. Because of the excipients utilized, lipid nanocarriers (liposomes, nanoemulsions (NEs), nanoparticles) have been used to increase brain targeting (Bisso et al., 2020; Ilić et al., 2023). The investigated compound (GL-II-73) - (4R)-8- ethynyl-6-(2-fluorophenyl)-N,N,4-trimethyl-4H- benzo[f]imidazo[1,5-a] [1,4]diazepine-3-carboxamide is imidazobenzodiazepine (IBZD) ligand that acts as positive allosteric modulator on α-GABAA receptors and was shown to possess combined antidepressant and pro- cognitive effects, making it a promising candidate for further research (Prevot et al., 2019). This work aims to investigate the physicochemical features of GL-II-73 to pick the best parenteral nanodelivery system for prospective research to assess its parameters.14th Central European Symposium on Pharmaceutical Technology, 28th - 30th September, Ohrid, N. Macedonia, 2023Poster: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/5049

Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies

INTRODUCTION Recently, the modulation of sigma-2 receptors localized in the brain is proposed to be linked with regulation of mood, anxiety, and cognition [1]. Hence, we hypothesized that novel patent-protected ligand of the pyrazoloquinolinone chemotype (CW-02-79) with a substantial binding affinity for sigma-2 receptors may have a distinct pharmacological profile useful for the treatment of mood, anxiety, and/or cognitive symptoms that usually accompany numerous psychiatric and neurodegenerative disorders. Having in mind that the neuroimmune mechanisms play an important role in pathogenesis of various emotional and cognitive impairments, we aim to test whether modulation of sigma-2 receptors with CW-02-79 results in substantial improvements in neuroimmune and/or behavioral outputs in in vitro cell platforms consisting of human induced pluripotent stem cells and in vivo animal models made to mimic a compromised neuroimmune status. However, very low water solubility of CW-02-79 hinders its administration and reliable efficacy and safety in vitro/in vivo evaluation. In order to avoid usage of non-physiological solvents/vehicles such as dimethyl sulfoxide and consequently, vehicle-related safety issues, nanoemulsions based on biocompatible excipients could be a promising tool for effective preclinical testing of the selected drug candidate. Therefore, firstly, this study aimed to develop biocompatible nanoemulsions (NEs), as carrier for CW-02-79, tailored for the described preclinical studies, using high pressure homogenization (HPH) method. As a first step, preformulation studies were performed to obtain insight into the key properties of CW-02-79 required for further stages of formulation development. Afterward, during NE preparation, the influence of formulation and process parameters on particle size was investigated to obtain NEs with small and uniform particle size suitable for parenteral administration. EXPERIMENTAL METHODS Materials For the preparation of NEs the following ingredients were used: CW-02-79 (synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin—Milwaukee, WI, USA), medium-chain triglycerides (MCT) (Fagron GmbH & KG, Germany), castor oil, polysorbate 80, butylhydroxytoluene, glycerol (Sigma-Aldrich GmbH, Germany), soybean lecithin (Lipoid S75; Lipoid GmbH, Germany) and ultrapure water. Preformulation Studies The solubility of CW-02-79 in different oils and oil mixtures, distilled water, 0.1 M hydrochloride acid, phosphate buffer (pH 7.4), commonly used organic solvents (isopropanol, methanol and dimethyl sulfoxide) at 25 °C was investigated by the shake flask method. CW-02-79 concentration in the obtained supernatants was measured by LC-MS/MS. To gain certain insight into the physical state of CW-02-79, polarization microscopy and differential scanning calorimetry (DSC 1, Mettler–Toledo AG, Switzerland) were used. Preparation and Characterization of NEs Blank and CW-02-79-loaded NEs were prepared by varying the content of the oil phase (20%/30%, w/w) and process parameters (number of homogenization cycles), using hot HPH (EmulsiFlex-C3, Avestin Inc., Canada) at 800 bar and 50°C. The oil to surfactant ratio was kept constant (5:1, w/w) in all tested formulations. Droplet size (Z-ave), polydispersity index (PDI) and zeta potential (ZP) of corresponding NEs, after proper dilution, were determined using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). Conductivity and pH value were measured by the conductometer (CDM230 Radiometer, Denmark) and pH meter (HI 9321, Hanna Instruments Inc, USA), respectively. RESULTS AND DISCUSSION Substance CW-02-79 appeared as a yellow powder, with broad particle size distribution. Results of the solubility study showed that, among the tested oils, the highest solubility of CW-02-79 was achieved in MCT-castor oil mixture (1:1, w/w) which was chosen as the oil phase for NE development. Elevated temperature (50°C) and presence of soybean lecithin as a solubilizer contributed to the loading of the target 2 mg/ml concentration, without precipitation during the storage. After the oil phase selection, blank and CW-02-79-loaded NEs were prepared by varying the content of oil phase, 20% and 30%, w/w (increasing the oil content would reduce the volume to be injected). Polysorbate 80 was added as an additional stabilizer and functional excipient due to its tendency to enhance brain uptake of drugs by acting as P-glycoprotein inhibitor, stealth agent or promoter of receptor-mediated endocytosis [2]. Simultaneously, the impact of the number of homogenization cycles on critical quality attributes of NEs (Z-ave and PDI) was tested. The observed increase in droplet size distribution (Figure 1) with increasing the number of homogenization cycles (> 8 cycles) could be attributed to over-processing (probably caused by increased droplet collision and re-coalescence rates or by insufficient emulsifier concentration in relation to the increasing interfacial area). Interestingly, although larger oil volume fractions generally lead to increased droplet collisions and hence larger droplet size [3], no statistically significant difference regarding droplet size was observed between formulations prepared with 20 and 30% of the oil phase (at 7 HPH cycles, 800 bar, 50°C). Likewise, a relatively narrow particle size distribution (PDI < 0.15) was observed, suggesting that the developed NEs were suitable for parenteral application. Moreover, satisfactory values were observed for all other tested physicochemical parameters (Table 2). Absolute ZP values were above 30 mV, indicating good stability of the system. Furthermore, the incorporation of CW-02-79 did not exert any influence on NE physicochemical properties, irrespective of the oil content. In conclusion, although the formulation prepared with 30% of the oil phase had satisfying physicochemical properties, its relatively high viscosity can restrict syringeability and injectability. On the other hand, owing to satisfying solubilization capacity for CW-02-79 as well as small and uniform droplet size and low viscosity, NE prepared with 20% of the oil phase represents a promising carrier worth exploring further to support the preclinical progress of CW-02-79

Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors

Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors Tijana Stanković1, Tanja Ilić1, Ivana Pantelić1, Anđela Tošić1, Jelena Mitrović1, James M. Cook2, Miroslav Savić3, Snežana Savić1 1 University of Belgrade-Faculty of Pharmacy, Department of Pharmaceutical Technology and Cosmetology, Vojvode Stepe 450, Belgrade, Serbia, 2 University of Wisconsin-Milwaukee, Milwaukee Institute for Drug Discovery, 3210 N. Cramer St. Milwaukee, Wisconsin, United States, 3 University of Belgrade-Faculty of Pharmacy, Department of Pharmacology, Vojvode Stepe 450, Belgrade, Serbia. The poor water solubility of novel patent-protected ligand of the pyrazoloquinolinone chemotype (CW-02-79), with significant binding affinity for sigma-2 receptors in the brain, restricts the development of conventional parenteral formulations and consequently, extensive pharmacological studies during the preclinical investigation. Therefore, we aimed to develop a biocompatible nanocarrier tailored to specific physicochemical properties of CW-02-79, to improve its transport across the blood-brain barrier and achieve the optimal brain disposition. In this context, a detailed analysis of lipophilicity (via log P and log D determination), solubility in various solvents/excipients (using shake-flask method) and crystalline state of CW-02-07 (using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) with melt quenching approach and polarization microsocopy) was performed. After the analysis of key “input” physicochemical descriptors, based on the developed decision tree, nanoemulsions were selected as promising carriers for CW-02-79. The nanoemulsions were prepared using the high pressure homogenization method, varying the process (number of cycles, temperature and pressure) and formulation parameters (the content of the oil phase, the stabilizer mixture composition). Additionally, the influence of the sterilization process (thermal sterilization/aseptic filtration) on the nanoemulsion physicochemical properties was investigated, including droplet size and size distribution, zeta potential, pH, electrical conductivity and osmolality. The obtained results showed that it was possible to formulate CW-02-79-loaded nanoemulsions with 20% oil phase (medium chain triglycerides:castor oil at ratio 1:1), stabilized with the biocompatible emulsifiers (lecithin/polysorbate 80), exhibiting the nano-sized droplets ( ǀ-30ǀ mV), pH (~ 5.7) and osmolality (295 mOsm/kg). The sterilization process did not remarkably affect the physiochemical properties of nanoemulsions, making them suitable for the parenteral administration. Owing to sastifying solubilization capacity for CW-02-79, physicochemical properties and preliminary stability, the nanoemulsions are the promising carriers worth exploring further to support the preclinical evalution of CW-02-79. ACKNOWLEDGEMENT. This research was supported by the Science Fund of the Republic of Serbia, Grant No. 7749108, 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 platform — NanoCellEmoCo

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

PWZ-029, a compound with moderate inverse agonist functional selectivity at GABA(A) receptors containing alpha 5 subunits, improves passive, but not active, avoidance learning in rats

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABA(A) receptors containing alpha 1, alpha 2, alpha 3 or alpha 5 subunits, and may have numerous experimental and clinical applications. The ability of non-selective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the alpha 1 and/or alpha 5 subunit-containing GABA(A) receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesized and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at alpha 5-containing GABA(A) receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil. and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2'F) selective for GABA(A) receptors containing the alpha 5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist beta-CCt exhibiting a preferential affinity for alpha 1-subunit-containing receptors. These data suggest that moderate negative modulation at GABA(A) receptors containing the alpha 5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined

Odloženi bihejvioralni efekti SH-I-048A, novog neselektivnog pozitivnog modulatora GABAA receptora, nakon povrede perifernog nerva pacova

The complex clinical picture of mono-neuropathy following injury of peripheral nerve is often accompanied by changes in the patients' affective states. It has been shown that positive allosteric modulators of GABAA receptors can decrease nociceptive transmission in animal pain models. However, recent findings suggest a possibility that at least some of the antinociceptive effects of benzodiazepines, related to neuropathic painful stimuli, may to a significant degree be a consequence of their anxiolytic action. In this study we evaluated the possible delayed effects of SH-I-048A, a newly- synthesized high-efficacy nonselective positive modulator of GABAA receptors, on anxiety-like behavior and locomotor activity in Wistar rats with a previously induced peripheral nerve injury. Assessment of behavioral parameters, using spontaneous locomotor activity and elevated plus maze tests, was performed 48h after completion of single-day three i.p. injections treatment consisting of zero, one, two or three doses of SH-I-048A (10 mg/kg). In general, rats' behavior observed 72 h after a moderate peripheral nerve injury did not indicate the persistence of sequelae of mono-neuropathic pain. The rats treated with three doses of SH-I-048A displayed an enhanced immobility time in the locomotor activity test, without concomitant decrease of the total distance traveled. On the other hand, in the group treated with two doses of SH-I-048A, a decrease in the emotional reactivity in the elevated plus maze test was observed. Subtle changes in the regimen of dosing of SH-I-048A affect locomotor activity and anxiety-related behavior in animals with peripheral nerve injury.Kompleksna klinička slika neuropatije nakon povrede perifernog nerva često je praćena i promenama afektivnog stanja pacijenata. U animalnim modelima bola pokazano je da pozitivni alosterni modulatori GABAA receptora mogu da smanje nociceptivnu transmisiju. Međutim, skorašnji nalazi ukazuju i na mogućnost da je prividni antinociceptivni efekat benzodiazepina nakon delovanja akutnog bolnog stimulusa u značajnoj meri posledica anksiolitičnog efekta. U ovoj studiji ispitani su mogući odloženi efekti novosintetisanog, neselektivnog liganda GABAA receptora (SH-I-048A) na lokomotornu aktivnost i ponašanje u vezi sa anksioznošću kod pacova soja Wistar prethodno podvrgnutih povredi perifernog nerva. Procena bihejvioralnih parametara sprovedena je testovima spontane lokomotorne aktivnosti i uzdignutog plus lavirinta 48 sati nakon što su životinje u periodu od 24 sata primile rastvarač, jednu, dve ili tri doze SH-I-048A (10 mg/kg). Generalno, ponašanje pacova praćeno 72 sata nakon umerene povrede perifernog nerva nije ukazivalo na prisustvo perzistentnih posledica neuropatskog bola. Pacovi prethodno tretirani sa tri doze SH-I-048A ispoljili su povećano vreme imobilnosti u testu lokomotorne aktivnosti, bez značajnog smanjenja ukupnog pređenog puta. S druge strane, u grupi koja je tretirana sa dve doze SH-I-048A zapažena je smanjena emocionalna reaktivnost u uzdignutom plus lavirintu. Kod životinja sa povredom perifernog nerva, suptilne razlike u režimu doziranja pozitivnog modulatora GABAA, mogu značajno da utiču na ponašanje vezano za lomotornu aktivnost i nivo anksioznosti

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