TY  - JOUR
AU  - Sharmin, Dishary
AU  - Divović, Branka
AU  - Ping, Xingjie
AU  - Cerne, Rok
AU  - Smith, Jodi L.
AU  - Rezvanian, Sepideh
AU  - Mondal, Prithu
AU  - Michelle, Meyer Jean
AU  - Kiley, Molly E.
AU  - Arnold, Leggy A.
AU  - Mian, Md Yeunus
AU  - Pandey, Kamal P.
AU  - Jin, Xiaoming
AU  - Mitrović, Jelena
AU  - Đorović, Đorđe
AU  - Lippa, Arnold
AU  - Cook, James M.
AU  - Golani, Lalit K.
AU  - Scholze, Petra
AU  - Savić, Miroslav
AU  - Witkin, Jeffrey M.
PY  - 2024
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5505
AB  - KRM-II-81 (1) is an imidazodiazepine GABAA receptor (GABAAR) potentiator with broad antiseizure efficacy and a low sedative burden. A brominated analogue, DS-II-73 (5), was synthesized and pharmacologically characterized as a potential backup compound as KRM-II-81 moves forward into development. The synthesis from 2-amino-5-bromophenyl)(pyridin-2yl)methanone (6) was processed in five steps with an overall yield of 38% and without the need for a palladium catalyst. GABAAR binding occurred with a Ki of 150 nM, and only 3 of 41 screened binding sites produced inhibition ≥50% at 10 μM, and the potency to induce cytotoxicity was ≥240 mM. DS-II-73 was selective for α2/3/5- over that of α1-containing GABAARs. Oral exposure of plasma and brain of rats was more than sufficient to functionally impact GABAARs. Tonic convulsions in mice and lethality induced by pentylenetetrazol were suppressed by DS-II-73 after oral administration and latencies to clonic and tonic seizures were prolonged. Cortical slice preparations from a patient with pharmacoresistant epilepsy (mesial temporal lobe) showed decreases in the frequency of local field potentials by DS-II-73. As with KRM-II-81, the motor-impairing effects of DS-II-73 were low compared to diazepam. Molecular docking studies of DS-II-73 with the α1β3γ2L-configured GABAAR showed low interaction with α1His102 that is suggested as a potential molecular mechanism for its low sedative side effects. These findings support the viability of DS-II-73 as a backup molecule for its ethynyl analogue, KRM-II-81, with the human tissue data providing translational credibility.
PB  - American Chemical Society
T2  - ACS Chemical Neuroscience
T1  - New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy
VL  - 15
IS  - 3
SP  - 517
EP  - 526
DO  - 10.1021/acschemneuro.3c00555
ER  - 

@article{
author = "Sharmin, Dishary and Divović, Branka and Ping, Xingjie and Cerne, Rok and Smith, Jodi L. and Rezvanian, Sepideh and Mondal, Prithu and Michelle, Meyer Jean and Kiley, Molly E. and Arnold, Leggy A. and Mian, Md Yeunus and Pandey, Kamal P. and Jin, Xiaoming and Mitrović, Jelena and Đorović, Đorđe and Lippa, Arnold and Cook, James M. and Golani, Lalit K. and Scholze, Petra and Savić, Miroslav and Witkin, Jeffrey M.",
year = "2024",
abstract = "KRM-II-81 (1) is an imidazodiazepine GABAA receptor (GABAAR) potentiator with broad antiseizure efficacy and a low sedative burden. A brominated analogue, DS-II-73 (5), was synthesized and pharmacologically characterized as a potential backup compound as KRM-II-81 moves forward into development. The synthesis from 2-amino-5-bromophenyl)(pyridin-2yl)methanone (6) was processed in five steps with an overall yield of 38% and without the need for a palladium catalyst. GABAAR binding occurred with a Ki of 150 nM, and only 3 of 41 screened binding sites produced inhibition ≥50% at 10 μM, and the potency to induce cytotoxicity was ≥240 mM. DS-II-73 was selective for α2/3/5- over that of α1-containing GABAARs. Oral exposure of plasma and brain of rats was more than sufficient to functionally impact GABAARs. Tonic convulsions in mice and lethality induced by pentylenetetrazol were suppressed by DS-II-73 after oral administration and latencies to clonic and tonic seizures were prolonged. Cortical slice preparations from a patient with pharmacoresistant epilepsy (mesial temporal lobe) showed decreases in the frequency of local field potentials by DS-II-73. As with KRM-II-81, the motor-impairing effects of DS-II-73 were low compared to diazepam. Molecular docking studies of DS-II-73 with the α1β3γ2L-configured GABAAR showed low interaction with α1His102 that is suggested as a potential molecular mechanism for its low sedative side effects. These findings support the viability of DS-II-73 as a backup molecule for its ethynyl analogue, KRM-II-81, with the human tissue data providing translational credibility.",
publisher = "American Chemical Society",
journal = "ACS Chemical Neuroscience",
title = "New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy",
volume = "15",
number = "3",
pages = "517-526",
doi = "10.1021/acschemneuro.3c00555"
}

Sharmin, D., Divović, B., Ping, X., Cerne, R., Smith, J. L., Rezvanian, S., Mondal, P., Michelle, M. J., Kiley, M. E., Arnold, L. A., Mian, M. Y., Pandey, K. P., Jin, X., Mitrović, J., Đorović, Đ., Lippa, A., Cook, J. M., Golani, L. K., Scholze, P., Savić, M.,& Witkin, J. M.. (2024). New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy. in ACS Chemical Neuroscience
American Chemical Society., 15(3), 517-526.
https://doi.org/10.1021/acschemneuro.3c00555

Sharmin D, Divović B, Ping X, Cerne R, Smith JL, Rezvanian S, Mondal P, Michelle MJ, Kiley ME, Arnold LA, Mian MY, Pandey KP, Jin X, Mitrović J, Đorović Đ, Lippa A, Cook JM, Golani LK, Scholze P, Savić M, Witkin JM. New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy. in ACS Chemical Neuroscience. 2024;15(3):517-526.
doi:10.1021/acschemneuro.3c00555 .

Sharmin, Dishary, Divović, Branka, Ping, Xingjie, Cerne, Rok, Smith, Jodi L., Rezvanian, Sepideh, Mondal, Prithu, Michelle, Meyer Jean, Kiley, Molly E., Arnold, Leggy A., Mian, Md Yeunus, Pandey, Kamal P., Jin, Xiaoming, Mitrović, Jelena, Đorović, Đorđe, Lippa, Arnold, Cook, James M., Golani, Lalit K., Scholze, Petra, Savić, Miroslav, Witkin, Jeffrey M., "New Imidazodiazepine Analogue, 5-(8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole, Provides a Simplified Synthetic Scheme, High Oral Plasma and Brain Exposures, and Produces Antiseizure Efficacy in Mice, and Antiepileptogenic Activity in Neural Networks in Brain Slices from a Patient with Mesial Temporal Lobe Epilepsy" in ACS Chemical Neuroscience, 15, no. 3 (2024):517-526,
https://doi.org/10.1021/acschemneuro.3c00555 . .

TY  - JOUR
AU  - Mitrović, Jelena
AU  - Bjelošević Žiberna, Maja
AU  - Vukadinović, Aleksandar
AU  - Knutson, Daniel E.
AU  - Sharmin, Dishary
AU  - Kremenović, Aleksandar
AU  - Ahlin Grabnar, Pegi
AU  - Planinšek, Odon
AU  - Lunter, Dominique
AU  - Cook, James M
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4982
AB  - 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.
PB  - Elsevier B.V.
T2  - European Journal of Pharmaceutical Sciences
T1  - Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study
VL  - 189
DO  - 10.1016/j.ejps.2023.106557
ER  - 

@article{
author = "Mitrović, Jelena and Bjelošević Žiberna, Maja and Vukadinović, Aleksandar and Knutson, Daniel E. and Sharmin, Dishary and Kremenović, Aleksandar and Ahlin Grabnar, Pegi and Planinšek, Odon and Lunter, Dominique and Cook, James M and Savić, Miroslav and Savić, Snežana",
year = "2023",
abstract = "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.",
publisher = "Elsevier B.V.",
journal = "European Journal of Pharmaceutical Sciences",
title = "Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study",
volume = "189",
doi = "10.1016/j.ejps.2023.106557"
}

Mitrović, J., Bjelošević Žiberna, M., Vukadinović, A., Knutson, D. E., Sharmin, D., Kremenović, A., Ahlin Grabnar, P., Planinšek, O., Lunter, D., Cook, J. M., Savić, M.,& Savić, S.. (2023). Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study. in European Journal of Pharmaceutical Sciences
Elsevier B.V.., 189.
https://doi.org/10.1016/j.ejps.2023.106557

Mitrović J, Bjelošević Žiberna M, Vukadinović A, Knutson DE, Sharmin D, Kremenović A, Ahlin Grabnar P, Planinšek O, Lunter D, Cook JM, Savić M, Savić S. Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study. in European Journal of Pharmaceutical Sciences. 2023;189.
doi:10.1016/j.ejps.2023.106557 .

Mitrović, Jelena, Bjelošević Žiberna, Maja, Vukadinović, Aleksandar, Knutson, Daniel E., Sharmin, Dishary, Kremenović, Aleksandar, Ahlin Grabnar, Pegi, Planinšek, Odon, Lunter, Dominique, Cook, James M, Savić, Miroslav, Savić, Snežana, "Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): Process parameters and lyoprotectant selection through the stability study" in European Journal of Pharmaceutical Sciences, 189 (2023),
https://doi.org/10.1016/j.ejps.2023.106557 . .

TY  - JOUR
AU  - Mitrović, Jelena
AU  - Divović-Matović, Branka
AU  - Knutson, Daniel E.
AU  - Petković, Miloš
AU  - Đorović, Đorđe
AU  - Ranđelović, Danijela V.
AU  - Dobričić, Vladimir
AU  - Đoković, Jelena
AU  - Lunter, Dominique J.
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4434
AB  - Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3- 2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nano- particles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (<100 nm), and high encapsulation efficacy (>99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior.
PB  - Elsevier B.V.
T2  - International Journal of Pharmaceutics
T1  - High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances
VL  - 633
DO  - 10.1016/j.ijpharm.2023.122613
ER  - 

@article{
author = "Mitrović, Jelena and Divović-Matović, Branka and Knutson, Daniel E. and Petković, Miloš and Đorović, Đorđe and Ranđelović, Danijela V. and Dobričić, Vladimir and Đoković, Jelena and Lunter, Dominique J. and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2023",
abstract = "Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3- 2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nano- particles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (<100 nm), and high encapsulation efficacy (>99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior.",
publisher = "Elsevier B.V.",
journal = "International Journal of Pharmaceutics",
title = "High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances",
volume = "633",
doi = "10.1016/j.ijpharm.2023.122613"
}

Mitrović, J., Divović-Matović, B., Knutson, D. E., Petković, M., Đorović, Đ., Ranđelović, D. V., Dobričić, V., Đoković, J., Lunter, D. J., Cook, J. M., Savić, M.,& Savić, S.. (2023). High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances. in International Journal of Pharmaceutics
Elsevier B.V.., 633.
https://doi.org/10.1016/j.ijpharm.2023.122613

Mitrović J, Divović-Matović B, Knutson DE, Petković M, Đorović Đ, Ranđelović DV, Dobričić V, Đoković J, Lunter DJ, Cook JM, Savić M, Savić S. High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances. in International Journal of Pharmaceutics. 2023;633.
doi:10.1016/j.ijpharm.2023.122613 .

Mitrović, Jelena, Divović-Matović, Branka, Knutson, Daniel E., Petković, Miloš, Đorović, Đorđe, Ranđelović, Danijela V., Dobričić, Vladimir, Đoković, Jelena, Lunter, Dominique J., Cook, James M., Savić, Miroslav, Savić, Snežana, "High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances" in International Journal of Pharmaceutics, 633 (2023),
https://doi.org/10.1016/j.ijpharm.2023.122613 . .

TY  - CONF
AU  - Stanković, Tijana
AU  - Ilić, Tanja
AU  - Pantelić, Ivana
AU  - Tošić, Anđela
AU  - Mitrović, Jelena
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5000
AB  - 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 (<200 nm) with narrow size distribution (polydispersity index < 0.2), zeta potential (> 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 — NanoCellEmoCog
PB  - International Association of Physical Chemists
C3  - 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6
T1  - Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors
UR  - https://hdl.handle.net/21.15107/rcub_farfar_5000
ER  - 

@conference{
author = "Stanković, Tijana and Ilić, Tanja and Pantelić, Ivana and Tošić, Anđela and Mitrović, Jelena and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2023",
abstract = "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 (<200 nm) with narrow size distribution (polydispersity index < 0.2), zeta potential (> 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 — NanoCellEmoCog",
publisher = "International Association of Physical Chemists",
journal = "10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6",
title = "Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors",
url = "https://hdl.handle.net/21.15107/rcub_farfar_5000"
}

Stanković, T., Ilić, T., Pantelić, I., Tošić, A., Mitrović, J., Cook, J. M., Savić, M.,& Savić, S.. (2023). Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors. in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6
International Association of Physical Chemists..
https://hdl.handle.net/21.15107/rcub_farfar_5000

Stanković T, Ilić T, Pantelić I, Tošić A, Mitrović J, Cook JM, Savić M, Savić S. Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors. in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6. 2023;.
https://hdl.handle.net/21.15107/rcub_farfar_5000 .

Stanković, Tijana, Ilić, Tanja, Pantelić, Ivana, Tošić, Anđela, Mitrović, Jelena, Cook, James M., Savić, Miroslav, Savić, Snežana, "Design of tailor-made biocompatible nanocarrier for novel pyrazoloquinolinone ligand (CW-02-79) based on comprehensive evaluation of critical physicochemical descriptors" in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6 (2023),
https://hdl.handle.net/21.15107/rcub_farfar_5000 .

TY  - CONF
AU  - Ilić, Tanja
AU  - Stanković, Tijana
AU  - Mitrović, Jelena
AU  - Pantelić, Ivana
AU  - Dobričić, Vladimir
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4583
AB  - 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.
C3  - 4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France
T1  - Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies
UR  - https://hdl.handle.net/21.15107/rcub_farfar_4583
ER  - 

@conference{
author = "Ilić, Tanja and Stanković, Tijana and Mitrović, Jelena and Pantelić, Ivana and Dobričić, Vladimir and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2023",
abstract = "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.",
journal = "4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France",
title = "Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies",
url = "https://hdl.handle.net/21.15107/rcub_farfar_4583"
}

Ilić, T., Stanković, T., Mitrović, J., Pantelić, I., Dobričić, V., Cook, J. M., Savić, M.,& Savić, S.. (2023). Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies. in 4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France.
https://hdl.handle.net/21.15107/rcub_farfar_4583

Ilić T, Stanković T, Mitrović J, Pantelić I, Dobričić V, Cook JM, Savić M, Savić S. Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies. in 4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France. 2023;.
https://hdl.handle.net/21.15107/rcub_farfar_4583 .

Ilić, Tanja, Stanković, Tijana, Mitrović, Jelena, Pantelić, Ivana, Dobričić, Vladimir, Cook, James M., Savić, Miroslav, Savić, Snežana, "Biocompatible nanoemulsions as a tool for preclinical testing of CW-02-79, a pyrazoloquinolinone modulator of sigma-2 receptors: preformulation and formulation studies" in 4th European Conference on Pharmaceutics, 20 - 21 March 2023, Marseille, France (2023),
https://hdl.handle.net/21.15107/rcub_farfar_4583 .

TY  - JOUR
AU  - Ilić, Tanja
AU  - Đoković, Jelena
AU  - Nikolić, Ines
AU  - Mitrović, Jelena
AU  - Pantelić, Ivana
AU  - Savić, Snežana
AU  - Savić, Miroslav
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4515
AB  - Contemporary trends in combinatorial chemistry and the design of pharmaceuticals targeting brain disorders have favored the development of drug candidates with increased lipophilicity and poorer water solubility, with the expected improvement in delivery across the blood–brain barrier (BBB). The growing availability of innovative excipients/ligands allowing improved brain targeting and controlled drug release makes the lipid nanocarriers a reasonable choice to overcome the factors impeding drug delivery through the BBB. However, a wide variety of methods, study designs and experimental conditions utilized in the literature hinder their systematic comparison, and thus slows the advances in brain-targeting by lipid-based nanoparticles. This review provides an overview of the methods most commonly utilized during the preclinical testing of liposomes, nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers intended for the treatment of various CNS disorders via the parenteral route. In order to fully elucidate the structure, stability, safety profiles, biodistribution, metabolism, pharmacokinetics and immunological effects of such lipid-based nanoparticles, a transdisciplinary approach to preclinical characterization is mandatory, covering a comprehensive set of physical, chemical, in vitro and in vivo biological testing.
PB  - MDPI
T2  - Pharmaceutics
T1  - Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation
VL  - 15
IS  - 2
DO  - 10.3390/pharmaceutics15020443
ER  - 

@article{
author = "Ilić, Tanja and Đoković, Jelena and Nikolić, Ines and Mitrović, Jelena and Pantelić, Ivana and Savić, Snežana and Savić, Miroslav",
year = "2023",
abstract = "Contemporary trends in combinatorial chemistry and the design of pharmaceuticals targeting brain disorders have favored the development of drug candidates with increased lipophilicity and poorer water solubility, with the expected improvement in delivery across the blood–brain barrier (BBB). The growing availability of innovative excipients/ligands allowing improved brain targeting and controlled drug release makes the lipid nanocarriers a reasonable choice to overcome the factors impeding drug delivery through the BBB. However, a wide variety of methods, study designs and experimental conditions utilized in the literature hinder their systematic comparison, and thus slows the advances in brain-targeting by lipid-based nanoparticles. This review provides an overview of the methods most commonly utilized during the preclinical testing of liposomes, nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers intended for the treatment of various CNS disorders via the parenteral route. In order to fully elucidate the structure, stability, safety profiles, biodistribution, metabolism, pharmacokinetics and immunological effects of such lipid-based nanoparticles, a transdisciplinary approach to preclinical characterization is mandatory, covering a comprehensive set of physical, chemical, in vitro and in vivo biological testing.",
publisher = "MDPI",
journal = "Pharmaceutics",
title = "Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation",
volume = "15",
number = "2",
doi = "10.3390/pharmaceutics15020443"
}

Ilić, T., Đoković, J., Nikolić, I., Mitrović, J., Pantelić, I., Savić, S.,& Savić, M.. (2023). Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation. in Pharmaceutics
MDPI., 15(2).
https://doi.org/10.3390/pharmaceutics15020443

Ilić T, Đoković J, Nikolić I, Mitrović J, Pantelić I, Savić S, Savić M. Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation. in Pharmaceutics. 2023;15(2).
doi:10.3390/pharmaceutics15020443 .

Ilić, Tanja, Đoković, Jelena, Nikolić, Ines, Mitrović, Jelena, Pantelić, Ivana, Savić, Snežana, Savić, Miroslav, "Parenteral Lipid-Based Nanoparticles for CNS Disorders: Integrating Various Facets of Preclinical Evaluation towards More Effective Clinical Translation" in Pharmaceutics, 15, no. 2 (2023),
https://doi.org/10.3390/pharmaceutics15020443 . .

TY  - CONF
AU  - Nikolić, Ines
AU  - Petrović, Marija
AU  - Mitrović, Jelena
AU  - Sublet, Emmanuelle
AU  - Jordan, Oliver
AU  - Savić, Snežana
AU  - Borchard, Gerrit
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5007
AB  - It is well known that the characterization of nanomedicines can pursue different levels of complexity, both in the development stage and in the quality control process [1]. In line with physicochemical aspects, even more obstacles are encountered in biological safety assessment, while anticipation of their immunogenic potential represents an additional challenge. Moreover, interactions between the test reagents and the nanomaterial have been identified as one of the most important issues in toxicity testing that influence market authorization of nanomedicines, which ought to be resolved [1]. The European Nanomedicine Characterization Laboratory – the reference laboratory for nanomedicines, provides protocols for 2 colorimetric cytotoxicity assays employing LLC-PK1 (porcine kidney epithelial cells) and Hep-G2 (human hepatocarcinoma cells) cell lines. However, the latest recommendations in the field underline the demand for enhancing the testing procedures, while proposing incorporation of immune cells as target cell lines for toxicity evaluation, aiming to provide more reliable conclusions on nanomedicine safety in preclinical level.
In this study 2 inherently different types of pharmaceutical nanosystemes were selected: nanoemulsion (NE) and solid lipid nanoparticles (LNP) and subjected to a set of orthogonal toxicity evaluation assays. Adjusted WST-1 (assessing mitochondrial activity as an indicator of cellular well-being) and LDH (lactate dehydrogenase release evaluation as an indicator of cell membrane damage) assays have been performed as the colorimetric tests, while propidium-iodide (PI)-based assay was developed as a fluore-scent counterpart (able to directly distinguish live from dead cells), using RAW 246.7 cell line (murine macrophages – immune system cell line). Starting concentration of the tested nanoformulations was 50 % v/v, and they were subsequently diluted with the factor of 2, to create a total of 8 concentrations. Incubation time was 4 h.
Presented assays rely on completely different biological bases. Therefore, their careful combination can address some shortcomings in the in vitro evaluations established so far. Although similar toxicity trends were observed regardless the assay used, it was evident that the LDH assay required specific consideration. Since the supernatant is the subject of the analysis (not the cells directly), containing not only the enzyme of interest, but also the nanoformulations, in the wells corresponding to the 3 highest concentration of the NE/LNP pronounced scattering effects were observed. Such an event could be easily overlooked, potentially affecting the conclusions. However, it was overcome by careful design of control and blank wells (each test concentration was coupled with its own blank well containing no cells, but the same concentration of the NE/LNP in the culture medium). In contrast, absorbance measurements in WST-1 assay were performed in the absence of the NE/LNP, avoiding any interactions or scattering effects. Finally, developed PI-based assay proved to be the most relevant method. Based on the penetration of PI into the dead cell only, attaching to their DNA, the concentration of the dead vs. live cells could be directly estimated. What is more, after the incubation time, the measurements can be performed in the nanoformulation-free environment, surpas-sing the potential interactions. Notably, cell viability obtained in the PI-based assay followed the same trend as in the WST-1 assay but with significant difference in the obtained values for the first 3 concentrations (Figure 1).
PB  - International Association of Physical Chemists
C3  - 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6
T1  - Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays
SP  - 21
EP  - 21
UR  - https://hdl.handle.net/21.15107/rcub_farfar_5007
ER  - 

@conference{
author = "Nikolić, Ines and Petrović, Marija and Mitrović, Jelena and Sublet, Emmanuelle and Jordan, Oliver and Savić, Snežana and Borchard, Gerrit",
year = "2023",
abstract = "It is well known that the characterization of nanomedicines can pursue different levels of complexity, both in the development stage and in the quality control process [1]. In line with physicochemical aspects, even more obstacles are encountered in biological safety assessment, while anticipation of their immunogenic potential represents an additional challenge. Moreover, interactions between the test reagents and the nanomaterial have been identified as one of the most important issues in toxicity testing that influence market authorization of nanomedicines, which ought to be resolved [1]. The European Nanomedicine Characterization Laboratory – the reference laboratory for nanomedicines, provides protocols for 2 colorimetric cytotoxicity assays employing LLC-PK1 (porcine kidney epithelial cells) and Hep-G2 (human hepatocarcinoma cells) cell lines. However, the latest recommendations in the field underline the demand for enhancing the testing procedures, while proposing incorporation of immune cells as target cell lines for toxicity evaluation, aiming to provide more reliable conclusions on nanomedicine safety in preclinical level.
In this study 2 inherently different types of pharmaceutical nanosystemes were selected: nanoemulsion (NE) and solid lipid nanoparticles (LNP) and subjected to a set of orthogonal toxicity evaluation assays. Adjusted WST-1 (assessing mitochondrial activity as an indicator of cellular well-being) and LDH (lactate dehydrogenase release evaluation as an indicator of cell membrane damage) assays have been performed as the colorimetric tests, while propidium-iodide (PI)-based assay was developed as a fluore-scent counterpart (able to directly distinguish live from dead cells), using RAW 246.7 cell line (murine macrophages – immune system cell line). Starting concentration of the tested nanoformulations was 50 % v/v, and they were subsequently diluted with the factor of 2, to create a total of 8 concentrations. Incubation time was 4 h.
Presented assays rely on completely different biological bases. Therefore, their careful combination can address some shortcomings in the in vitro evaluations established so far. Although similar toxicity trends were observed regardless the assay used, it was evident that the LDH assay required specific consideration. Since the supernatant is the subject of the analysis (not the cells directly), containing not only the enzyme of interest, but also the nanoformulations, in the wells corresponding to the 3 highest concentration of the NE/LNP pronounced scattering effects were observed. Such an event could be easily overlooked, potentially affecting the conclusions. However, it was overcome by careful design of control and blank wells (each test concentration was coupled with its own blank well containing no cells, but the same concentration of the NE/LNP in the culture medium). In contrast, absorbance measurements in WST-1 assay were performed in the absence of the NE/LNP, avoiding any interactions or scattering effects. Finally, developed PI-based assay proved to be the most relevant method. Based on the penetration of PI into the dead cell only, attaching to their DNA, the concentration of the dead vs. live cells could be directly estimated. What is more, after the incubation time, the measurements can be performed in the nanoformulation-free environment, surpas-sing the potential interactions. Notably, cell viability obtained in the PI-based assay followed the same trend as in the WST-1 assay but with significant difference in the obtained values for the first 3 concentrations (Figure 1).",
publisher = "International Association of Physical Chemists",
journal = "10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6",
title = "Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays",
pages = "21-21",
url = "https://hdl.handle.net/21.15107/rcub_farfar_5007"
}

Nikolić, I., Petrović, M., Mitrović, J., Sublet, E., Jordan, O., Savić, S.,& Borchard, G.. (2023). Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays. in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6
International Association of Physical Chemists., 21-21.
https://hdl.handle.net/21.15107/rcub_farfar_5007

Nikolić I, Petrović M, Mitrović J, Sublet E, Jordan O, Savić S, Borchard G. Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays. in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6. 2023;:21-21.
https://hdl.handle.net/21.15107/rcub_farfar_5007 .

Nikolić, Ines, Petrović, Marija, Mitrović, Jelena, Sublet, Emmanuelle, Jordan, Oliver, Savić, Snežana, Borchard, Gerrit, "Navigating towards improved cytotoxicity assessment in nanomedicine development: Shifting from colorimetric to fluorescence-based assays" in 10th IAPC Meeting Tenth World Conference on Physico-Chemical Methods in Drug Discovery & Sixth World Conference on ADMET and DMPK Belgrade, Serbia, September 4-6 (2023):21-21,
https://hdl.handle.net/21.15107/rcub_farfar_5007 .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Ilić, Tanja
AU  - Jančić, Ivan
AU  - Bufan, Biljana
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2023
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5090
AB  - Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration
Jelena Mitrović1, Tanja Ilić1, Ivan Jančić2, Biljana Bufan2, Miroslav Savić3, Snežana Savić1
1 Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
2 Department of Microbiology and Immunology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
3 Department of Pharmacology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
Estimation of endotoxin level in parenteral formulations is a prerequisite for numerous in vitro tests in preclinical studies and for future clinical development. However, the Limulus amoebocyte lysate (LAL) test in formulations containing nanoparticles could often lead to misinterpretation of results. Therefore, we tested if endotoxins could be detected in nanocrystal dispersions by the commercial gel clot assay kit. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one) were prepared by wet-ball milling, lyophilized and reconstituted with water for injection prior experiment. Different dilutions of nanocrystal dispersion in LAL reagent water were prepared as well as positive and negative control. Despite difficulties to detect gel clots, they were visible in the sample at dilutions 1:75 and below. According to the protocol, the endotoxin limit was estimated to be 25.00 EU/ml, which corresponds to <12.50 EU/mg of DK-I-56-1. This value relates to the endotoxin limit for diazepam, with the similar dosing regimen as proposed for DK-I-56-1.
C3  - NANOGVA Symposium, October 5th - 6th, 2023, Geneva, Switzerland
T1  - Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration
UR  - https://hdl.handle.net/21.15107/rcub_farfar_5090
ER  - 

@conference{
author = "Mitrović, Jelena and Ilić, Tanja and Jančić, Ivan and Bufan, Biljana and Savić, Miroslav and Savić, Snežana",
year = "2023",
abstract = "Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration
Jelena Mitrović1, Tanja Ilić1, Ivan Jančić2, Biljana Bufan2, Miroslav Savić3, Snežana Savić1
1 Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
2 Department of Microbiology and Immunology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
3 Department of Pharmacology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia
Estimation of endotoxin level in parenteral formulations is a prerequisite for numerous in vitro tests in preclinical studies and for future clinical development. However, the Limulus amoebocyte lysate (LAL) test in formulations containing nanoparticles could often lead to misinterpretation of results. Therefore, we tested if endotoxins could be detected in nanocrystal dispersions by the commercial gel clot assay kit. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one) were prepared by wet-ball milling, lyophilized and reconstituted with water for injection prior experiment. Different dilutions of nanocrystal dispersion in LAL reagent water were prepared as well as positive and negative control. Despite difficulties to detect gel clots, they were visible in the sample at dilutions 1:75 and below. According to the protocol, the endotoxin limit was estimated to be 25.00 EU/ml, which corresponds to <12.50 EU/mg of DK-I-56-1. This value relates to the endotoxin limit for diazepam, with the similar dosing regimen as proposed for DK-I-56-1.",
journal = "NANOGVA Symposium, October 5th - 6th, 2023, Geneva, Switzerland",
title = "Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration",
url = "https://hdl.handle.net/21.15107/rcub_farfar_5090"
}

Mitrović, J., Ilić, T., Jančić, I., Bufan, B., Savić, M.,& Savić, S.. (2023). Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration. in NANOGVA Symposium, October 5th - 6th, 2023, Geneva, Switzerland.
https://hdl.handle.net/21.15107/rcub_farfar_5090

Mitrović J, Ilić T, Jančić I, Bufan B, Savić M, Savić S. Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration. in NANOGVA Symposium, October 5th - 6th, 2023, Geneva, Switzerland. 2023;.
https://hdl.handle.net/21.15107/rcub_farfar_5090 .

Mitrović, Jelena, Ilić, Tanja, Jančić, Ivan, Bufan, Biljana, Savić, Miroslav, Savić, Snežana, "Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration" in NANOGVA Symposium, October 5th - 6th, 2023, Geneva, Switzerland (2023),
https://hdl.handle.net/21.15107/rcub_farfar_5090 .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Bjelošević, Maja
AU  - Knutson, Daniel E.
AU  - Kremenović, Aleksandar
AU  - Lunter, Dominique
AU  - Ahlin Grabnar, Pegi
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2022
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4270
AB  - 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.
C3  - 9th BBBB International conference on pharmaceutical sciences; 15th - 17th September, Ljubljana, Slovenia
T1  - Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): process parameters and cryoprotectant selection through stability study
UR  - https://hdl.handle.net/21.15107/rcub_farfar_4270
ER  - 

@conference{
author = "Mitrović, Jelena and Bjelošević, Maja and Knutson, Daniel E. and Kremenović, Aleksandar and Lunter, Dominique and Ahlin Grabnar, Pegi and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2022",
abstract = "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.",
journal = "9th BBBB International conference on pharmaceutical sciences; 15th - 17th September, Ljubljana, Slovenia",
title = "Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): process parameters and cryoprotectant selection through stability study",
url = "https://hdl.handle.net/21.15107/rcub_farfar_4270"
}

Mitrović, J., Bjelošević, M., Knutson, D. E., Kremenović, A., Lunter, D., Ahlin Grabnar, P., Cook, J. M., Savić, M.,& Savić, S.. (2022). Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): process parameters and cryoprotectant selection through stability study. in 9th BBBB International conference on pharmaceutical sciences; 15th - 17th September, Ljubljana, Slovenia.
https://hdl.handle.net/21.15107/rcub_farfar_4270

Mitrović J, Bjelošević M, Knutson DE, Kremenović A, Lunter D, Ahlin Grabnar P, Cook JM, Savić M, Savić S. Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): process parameters and cryoprotectant selection through stability study. in 9th BBBB International conference on pharmaceutical sciences; 15th - 17th September, Ljubljana, Slovenia. 2022;.
https://hdl.handle.net/21.15107/rcub_farfar_4270 .

Mitrović, Jelena, Bjelošević, Maja, Knutson, Daniel E., Kremenović, Aleksandar, Lunter, Dominique, Ahlin Grabnar, Pegi, Cook, James M., Savić, Miroslav, Savić, Snežana, "Freeze-dried nanocrystal dispersion of novel deuterated pyrazoloquinolinone ligand (DK-I-56-1): process parameters and cryoprotectant selection through stability study" in 9th BBBB International conference on pharmaceutical sciences; 15th - 17th September, Ljubljana, Slovenia (2022),
https://hdl.handle.net/21.15107/rcub_farfar_4270 .

TY  - JOUR
AU  - Divović-Matović, Branka
AU  - Knutson, Dan
AU  - Mitrović, Jelena
AU  - Stevanović, Vladimir
AU  - Stanojević, Boban
AU  - Savić, Snežana
AU  - Cook, James
AU  - Savić, Miroslav
PY  - 2022
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4289
AB  - Several pyrazoloquinolinone (PQ) ligands were recently discovered as func-tionally selective positive modulators at the PQ site ofα6-containing GABAAreceptors. PQs are also neutral modulators at the benzodiazepine site. Weassessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of maleSprague–Dawley rats. An excellent behavioural safety profile of all tested PQswas demonstrated in the spontaneous locomotor activity, rotarod, loss of right-ing reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029and its analogues prevented the ataxic effects of the benzodiazepine, asassessed in the rotarod test and during monitoring of rat locomotor activityafter awakening from the loss of righting reflex. Published electrophysiologicalprofiles of PQ ligands imply that positive modulation elicited atα6-GABAAreceptors that contain theγ2 andδsubunit, rather than their neutral modula-tory action at the benzodiazepine site, may prevent the ataxic action of diaze-pam. Thus, PZ-II-029 and its deuterated analogues are not prone to untowardinteractions with benzodiazepines and may indeed completely abolish theirataxic action, seen at therapeutic, and especially toxic concentrations.
PB  - John Wiley and Sons Inc
T2  - Basic and Clinical Pharmacology and Toxicology
T1  - Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature
VL  - 131
IS  - 6
SP  - 514
EP  - 524
DO  - 10.1111/bcpt.13801
ER  - 

@article{
author = "Divović-Matović, Branka and Knutson, Dan and Mitrović, Jelena and Stevanović, Vladimir and Stanojević, Boban and Savić, Snežana and Cook, James and Savić, Miroslav",
year = "2022",
abstract = "Several pyrazoloquinolinone (PQ) ligands were recently discovered as func-tionally selective positive modulators at the PQ site ofα6-containing GABAAreceptors. PQs are also neutral modulators at the benzodiazepine site. Weassessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of maleSprague–Dawley rats. An excellent behavioural safety profile of all tested PQswas demonstrated in the spontaneous locomotor activity, rotarod, loss of right-ing reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029and its analogues prevented the ataxic effects of the benzodiazepine, asassessed in the rotarod test and during monitoring of rat locomotor activityafter awakening from the loss of righting reflex. Published electrophysiologicalprofiles of PQ ligands imply that positive modulation elicited atα6-GABAAreceptors that contain theγ2 andδsubunit, rather than their neutral modula-tory action at the benzodiazepine site, may prevent the ataxic action of diaze-pam. Thus, PZ-II-029 and its deuterated analogues are not prone to untowardinteractions with benzodiazepines and may indeed completely abolish theirataxic action, seen at therapeutic, and especially toxic concentrations.",
publisher = "John Wiley and Sons Inc",
journal = "Basic and Clinical Pharmacology and Toxicology",
title = "Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature",
volume = "131",
number = "6",
pages = "514-524",
doi = "10.1111/bcpt.13801"
}

Divović-Matović, B., Knutson, D., Mitrović, J., Stevanović, V., Stanojević, B., Savić, S., Cook, J.,& Savić, M.. (2022). Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature. in Basic and Clinical Pharmacology and Toxicology
John Wiley and Sons Inc., 131(6), 514-524.
https://doi.org/10.1111/bcpt.13801

Divović-Matović B, Knutson D, Mitrović J, Stevanović V, Stanojević B, Savić S, Cook J, Savić M. Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature. in Basic and Clinical Pharmacology and Toxicology. 2022;131(6):514-524.
doi:10.1111/bcpt.13801 .

Divović-Matović, Branka, Knutson, Dan, Mitrović, Jelena, Stevanović, Vladimir, Stanojević, Boban, Savić, Snežana, Cook, James, Savić, Miroslav, "Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature" in Basic and Clinical Pharmacology and Toxicology, 131, no. 6 (2022):514-524,
https://doi.org/10.1111/bcpt.13801 . .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Knutson, Daniel
AU  - Nikolić, Ines
AU  - Cook, James
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2022
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4285
AB  - After parenteral administration, nanoparticles interact with different proteins,
forming a shell called corona, which further influence nanoparticles’ biodistribution. Protein
adsorption is affected by particle size and shape, but also by molecular interactions of
chemical groups from the particle surface and amino-acid residues of the proteins. In human
plasma, albumin is the most abundant protein so it is frequently used for the investigation of
protein-nanoparticle interactions (1). In this study we investigated the attachment of bovine
serum albumin (BSA) to recently developed nanocrystals (2) of DK-I-56-1 (7-methoxy-2-
(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), stabilized by
polysorbate 80 (NS2) or the combination of polysorbate 80 and poloxamer 407 (NS4).
Nanocrystal dispersion was incubated in medium containing 0.1% or 1% BSA in phosphate
buffer saline (pH 7,4) at 37 °C for 1 h. Particle size analysis was conducted by dynamic light
scattering in 10 min interval, at 37 °C on Zetasizer ZS90 (Malvern Instruments Ltd.,
Worcestershire, UK). It was shown that albumin adsorption was influenced by the
nanocrystal formulation and albumin concentration, but not incubation time. In a medium
with 0.1% BSA, no particle size difference was noticed in either formulation. However, in
case of NS2, after the addition of 1% albumin, particle size and particle size distribution
increased, which indicated albumin binding. On the other hand, in formulation NS4, with
higher albumin concentration two peaks were visible, one from the free albumin, and one
from nanocrystal particles. Therefore, it could be concluded that the affinity of albumin was
influenced mainly by the interaction with the nanocrystal stabilizers.
C3  - 8. Kongres farmaceuta Srbije; 12.-15. oktobar, Beograd, Srbija
T1  - Investigation of albumin adsorption on DK-I-56-1 nanocrystals by dynamic light scattering
UR  - https://hdl.handle.net/21.15107/rcub_farfar_4285
ER  - 

@conference{
author = "Mitrović, Jelena and Knutson, Daniel and Nikolić, Ines and Cook, James and Savić, Miroslav and Savić, Snežana",
year = "2022",
abstract = "After parenteral administration, nanoparticles interact with different proteins,
forming a shell called corona, which further influence nanoparticles’ biodistribution. Protein
adsorption is affected by particle size and shape, but also by molecular interactions of
chemical groups from the particle surface and amino-acid residues of the proteins. In human
plasma, albumin is the most abundant protein so it is frequently used for the investigation of
protein-nanoparticle interactions (1). In this study we investigated the attachment of bovine
serum albumin (BSA) to recently developed nanocrystals (2) of DK-I-56-1 (7-methoxy-2-
(4-methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), stabilized by
polysorbate 80 (NS2) or the combination of polysorbate 80 and poloxamer 407 (NS4).
Nanocrystal dispersion was incubated in medium containing 0.1% or 1% BSA in phosphate
buffer saline (pH 7,4) at 37 °C for 1 h. Particle size analysis was conducted by dynamic light
scattering in 10 min interval, at 37 °C on Zetasizer ZS90 (Malvern Instruments Ltd.,
Worcestershire, UK). It was shown that albumin adsorption was influenced by the
nanocrystal formulation and albumin concentration, but not incubation time. In a medium
with 0.1% BSA, no particle size difference was noticed in either formulation. However, in
case of NS2, after the addition of 1% albumin, particle size and particle size distribution
increased, which indicated albumin binding. On the other hand, in formulation NS4, with
higher albumin concentration two peaks were visible, one from the free albumin, and one
from nanocrystal particles. Therefore, it could be concluded that the affinity of albumin was
influenced mainly by the interaction with the nanocrystal stabilizers.",
journal = "8. Kongres farmaceuta Srbije; 12.-15. oktobar, Beograd, Srbija",
title = "Investigation of albumin adsorption on DK-I-56-1 nanocrystals by dynamic light scattering",
url = "https://hdl.handle.net/21.15107/rcub_farfar_4285"
}

Mitrović, J., Knutson, D., Nikolić, I., Cook, J., Savić, M.,& Savić, S.. (2022). Investigation of albumin adsorption on DK-I-56-1 nanocrystals by dynamic light scattering. in 8. Kongres farmaceuta Srbije; 12.-15. oktobar, Beograd, Srbija.
https://hdl.handle.net/21.15107/rcub_farfar_4285

Mitrović J, Knutson D, Nikolić I, Cook J, Savić M, Savić S. Investigation of albumin adsorption on DK-I-56-1 nanocrystals by dynamic light scattering. in 8. Kongres farmaceuta Srbije; 12.-15. oktobar, Beograd, Srbija. 2022;.
https://hdl.handle.net/21.15107/rcub_farfar_4285 .

Mitrović, Jelena, Knutson, Daniel, Nikolić, Ines, Cook, James, Savić, Miroslav, Savić, Snežana, "Investigation of albumin adsorption on DK-I-56-1 nanocrystals by dynamic light scattering" in 8. Kongres farmaceuta Srbije; 12.-15. oktobar, Beograd, Srbija (2022),
https://hdl.handle.net/21.15107/rcub_farfar_4285 .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Petković, Miloš
AU  - Ranđelović, Danijela
AU  - Đoković, Jelena
AU  - Knutson, Daniel
AU  - Cook, James
AU  - Savić, Vladimir
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2022
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4097
AB  - Lipid nanoparticles are being intensively investigated for
the formulation of the drugs with poor solubility substances
(1). They represent colloid dispersions of the particles with
lipid matrix that is solid at room and body temperature.
Because of the low capacity of triglycerides for the drug
substances incorporation, alternatively, high amounts of
lecithin could be added to increase the solubilization (2).
This was used for the incorporation of DK-I-60-3 (7-
methoxy-d3-2-(4-methoxyd3-phenyl)-2,5-dihydro-
3Hpyrazolo[4,3-c]quinolin-3-one), novel deuterated
pyrazoloquinolinone ligand, with very low solubility in
water as well as in oils (3,4). However, because of
amphiphilic nature of lecithin, its localization within
nanoparticles should be analyzed, especially with respect
to stability, drug loading capacity and drug localization,
because it may additionally influence the drug release
mechanism (2).
C3  - 13th World meeting on pharmaceutics, biopharmaceutics and pharmaceutical technology, 28-31 March 2022, Rotterdam, Netherlands
T1  - Physicochemical/structural investigation of lipid nanoparticles with high lecithin amounts loaded with patent protected pyrazoloquinolinone ligand DK-I-60-3
UR  - https://hdl.handle.net/21.15107/rcub_farfar_4097
ER  - 

@conference{
author = "Mitrović, Jelena and Petković, Miloš and Ranđelović, Danijela and Đoković, Jelena and Knutson, Daniel and Cook, James and Savić, Vladimir and Savić, Miroslav and Savić, Snežana",
year = "2022",
abstract = "Lipid nanoparticles are being intensively investigated for
the formulation of the drugs with poor solubility substances
(1). They represent colloid dispersions of the particles with
lipid matrix that is solid at room and body temperature.
Because of the low capacity of triglycerides for the drug
substances incorporation, alternatively, high amounts of
lecithin could be added to increase the solubilization (2).
This was used for the incorporation of DK-I-60-3 (7-
methoxy-d3-2-(4-methoxyd3-phenyl)-2,5-dihydro-
3Hpyrazolo[4,3-c]quinolin-3-one), novel deuterated
pyrazoloquinolinone ligand, with very low solubility in
water as well as in oils (3,4). However, because of
amphiphilic nature of lecithin, its localization within
nanoparticles should be analyzed, especially with respect
to stability, drug loading capacity and drug localization,
because it may additionally influence the drug release
mechanism (2).",
journal = "13th World meeting on pharmaceutics, biopharmaceutics and pharmaceutical technology, 28-31 March 2022, Rotterdam, Netherlands",
title = "Physicochemical/structural investigation of lipid nanoparticles with high lecithin amounts loaded with patent protected pyrazoloquinolinone ligand DK-I-60-3",
url = "https://hdl.handle.net/21.15107/rcub_farfar_4097"
}

Mitrović, J., Petković, M., Ranđelović, D., Đoković, J., Knutson, D., Cook, J., Savić, V., Savić, M.,& Savić, S.. (2022). Physicochemical/structural investigation of lipid nanoparticles with high lecithin amounts loaded with patent protected pyrazoloquinolinone ligand DK-I-60-3. in 13th World meeting on pharmaceutics, biopharmaceutics and pharmaceutical technology, 28-31 March 2022, Rotterdam, Netherlands.
https://hdl.handle.net/21.15107/rcub_farfar_4097

Mitrović J, Petković M, Ranđelović D, Đoković J, Knutson D, Cook J, Savić V, Savić M, Savić S. Physicochemical/structural investigation of lipid nanoparticles with high lecithin amounts loaded with patent protected pyrazoloquinolinone ligand DK-I-60-3. in 13th World meeting on pharmaceutics, biopharmaceutics and pharmaceutical technology, 28-31 March 2022, Rotterdam, Netherlands. 2022;.
https://hdl.handle.net/21.15107/rcub_farfar_4097 .

Mitrović, Jelena, Petković, Miloš, Ranđelović, Danijela, Đoković, Jelena, Knutson, Daniel, Cook, James, Savić, Vladimir, Savić, Miroslav, Savić, Snežana, "Physicochemical/structural investigation of lipid nanoparticles with high lecithin amounts loaded with patent protected pyrazoloquinolinone ligand DK-I-60-3" in 13th World meeting on pharmaceutics, biopharmaceutics and pharmaceutical technology, 28-31 March 2022, Rotterdam, Netherlands (2022),
https://hdl.handle.net/21.15107/rcub_farfar_4097 .

TY  - JOUR
AU  - Đoković, Jelena
AU  - Demisli, Sotiria
AU  - Savić, Sanela
AU  - Marković, Bojan
AU  - Cekić, Nebojša D.
AU  - Ranđelović, Danijela V.
AU  - Mitrović, Jelena
AU  - Lunter, Dominique Jasmin
AU  - Papadimitriou, Vassiliki
AU  - Xenakis, Aristotelis
AU  - Savić, Snežana
PY  - 2022
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/4265
AB  - A nanotechnology-based approach to drug delivery presents one of the biggest trends in biomedical science that can provide increased active concentration, bioavailability, and safety compared to conventional drug-delivery systems. Nanoemulsions stand out amongst other nanocarriers for being biodegradable, biocompatible, and relatively easy to manufacture. For improved drug-delivery properties, longer circulation for the nanoemulsion droplets should be provided, to allow the active to reach the target site. One of the strategies used for this purpose is PEGylation. The aim of this research was assessing the impact of the oil phase selection, soybean or fish oil mixtures with medium chain triglycerides, on the physicochemical characteristics and injectability of curcumin-loaded PEGylated nanoemulsions. Electron paramagnetic resonance spectroscopy demonstrated the structural impact of the oil phase on the stabilizing layer of nanoemulsions, with a more pronounced stabilizing effect of curcumin observed in the fish oil nanoemulsion compared to the soybean oil one. The design of the experiment study, employed to simultaneously assess the impact of the oil phase, different PEGylated phospholipids and their concentrations, as well as the presence of curcumin, showed that not only the investigated factors alone, but also their interactions, had a significant influence on the critical quality attributes of the PEGylated nanoemulsions. Detailed physicochemical characterization of the NEs found all formulations were appropriate for parenteral administration and remained stable during two years of storage, with the preserved antioxidant activity demonstrated by DPPH and FRAP assays. In vitro release studies showed a more pronounced release of curcumin from the fish oil NEs compared to that from the soybean oil ones. The innovative in vitro injectability assessment, designed to mimic intravenous application, proved that all formulations tested in selected experimental setting could be employed in prospective in vivo studies. Overall, the current study shows the importance of oil phase selection when formulating PEGylated nanoemulsions
PB  - MDPI
T2  - Pharmaceutics
T1  - The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions
VL  - 14
IS  - 8
DO  - 10.3390/pharmaceutics14081666
ER  - 

@article{
author = "Đoković, Jelena and Demisli, Sotiria and Savić, Sanela and Marković, Bojan and Cekić, Nebojša D. and Ranđelović, Danijela V. and Mitrović, Jelena and Lunter, Dominique Jasmin and Papadimitriou, Vassiliki and Xenakis, Aristotelis and Savić, Snežana",
year = "2022",
abstract = "A nanotechnology-based approach to drug delivery presents one of the biggest trends in biomedical science that can provide increased active concentration, bioavailability, and safety compared to conventional drug-delivery systems. Nanoemulsions stand out amongst other nanocarriers for being biodegradable, biocompatible, and relatively easy to manufacture. For improved drug-delivery properties, longer circulation for the nanoemulsion droplets should be provided, to allow the active to reach the target site. One of the strategies used for this purpose is PEGylation. The aim of this research was assessing the impact of the oil phase selection, soybean or fish oil mixtures with medium chain triglycerides, on the physicochemical characteristics and injectability of curcumin-loaded PEGylated nanoemulsions. Electron paramagnetic resonance spectroscopy demonstrated the structural impact of the oil phase on the stabilizing layer of nanoemulsions, with a more pronounced stabilizing effect of curcumin observed in the fish oil nanoemulsion compared to the soybean oil one. The design of the experiment study, employed to simultaneously assess the impact of the oil phase, different PEGylated phospholipids and their concentrations, as well as the presence of curcumin, showed that not only the investigated factors alone, but also their interactions, had a significant influence on the critical quality attributes of the PEGylated nanoemulsions. Detailed physicochemical characterization of the NEs found all formulations were appropriate for parenteral administration and remained stable during two years of storage, with the preserved antioxidant activity demonstrated by DPPH and FRAP assays. In vitro release studies showed a more pronounced release of curcumin from the fish oil NEs compared to that from the soybean oil ones. The innovative in vitro injectability assessment, designed to mimic intravenous application, proved that all formulations tested in selected experimental setting could be employed in prospective in vivo studies. Overall, the current study shows the importance of oil phase selection when formulating PEGylated nanoemulsions",
publisher = "MDPI",
journal = "Pharmaceutics",
title = "The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions",
volume = "14",
number = "8",
doi = "10.3390/pharmaceutics14081666"
}

Đoković, J., Demisli, S., Savić, S., Marković, B., Cekić, N. D., Ranđelović, D. V., Mitrović, J., Lunter, D. J., Papadimitriou, V., Xenakis, A.,& Savić, S.. (2022). The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions. in Pharmaceutics
MDPI., 14(8).
https://doi.org/10.3390/pharmaceutics14081666

Đoković J, Demisli S, Savić S, Marković B, Cekić ND, Ranđelović DV, Mitrović J, Lunter DJ, Papadimitriou V, Xenakis A, Savić S. The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions. in Pharmaceutics. 2022;14(8).
doi:10.3390/pharmaceutics14081666 .

Đoković, Jelena, Demisli, Sotiria, Savić, Sanela, Marković, Bojan, Cekić, Nebojša D., Ranđelović, Danijela V., Mitrović, Jelena, Lunter, Dominique Jasmin, Papadimitriou, Vassiliki, Xenakis, Aristotelis, Savić, Snežana, "The Impact of the Oil Phase Selection on Physicochemical Properties, Long-Term Stability, In Vitro Performance and Injectability of Curcumin-Loaded PEGylated Nanoemulsions" in Pharmaceutics, 14, no. 8 (2022),
https://doi.org/10.3390/pharmaceutics14081666 . .

TY  - JOUR
AU  - Mitrović, Jelena
AU  - Divović-Matović, Branka
AU  - Knutson, Daniel
AU  - Đoković, Jelena
AU  - Kremenović, Aleksandar
AU  - Dobričić, Vladimir
AU  - Ranđelović, Danijela
AU  - Pantelić, Ivana
AU  - Cook, James
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2021
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/3934
AB  - Poor water solubility of new chemical entities is considered as one of the main obstacles in drug development, as it usually leads to low bioavailability after administration. To overcome these problems, the selection of the appropriate formulation technology needs to be based on the physicochemical properties of the drug and introduced in the early stages of drug research. One example of the new potential drug substance with poor solubility is DK-I-60-3, deuterated pyrazoloquinolinone, designed for the treatment of various neuropsychiatric disorders. In this research, based on preformulation studies, nanocrystal technology was chosen to improve the oral bioavailability of DK-I-60-3. Nanocrystal dispersions stabilized by sodium lauryl sulfate and polyvinylpyrrolidone were prepared by modified wet media milling technique, with the selection of appropriate process and formulation parameters. The nanoparticles characterization included particle size and zeta potential measurements, differential scanning calorimetry, X-ray powder diffraction, dissolution and solubility study, and in vivo pharmacokinetic experiments. Developed formulations had small uniform particle sizes and were stable for three months. Nanonization caused decreased crystallite size and induced crystal defects formation, as well as a DK-I-60-3 solubility increase. Furthermore, after oral administration of the developed formulations in rats, two to three-fold bioavailability enhancement was observed in plasma and investigated organs, including the brain.
PB  - MDPI AG
T2  - Pharmaceutics
T1  - Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach
VL  - 13
IS  - 8
DO  - 10.3390/pharmaceutics13081188
ER  - 

@article{
author = "Mitrović, Jelena and Divović-Matović, Branka and Knutson, Daniel and Đoković, Jelena and Kremenović, Aleksandar and Dobričić, Vladimir and Ranđelović, Danijela and Pantelić, Ivana and Cook, James and Savić, Miroslav and Savić, Snežana",
year = "2021",
abstract = "Poor water solubility of new chemical entities is considered as one of the main obstacles in drug development, as it usually leads to low bioavailability after administration. To overcome these problems, the selection of the appropriate formulation technology needs to be based on the physicochemical properties of the drug and introduced in the early stages of drug research. One example of the new potential drug substance with poor solubility is DK-I-60-3, deuterated pyrazoloquinolinone, designed for the treatment of various neuropsychiatric disorders. In this research, based on preformulation studies, nanocrystal technology was chosen to improve the oral bioavailability of DK-I-60-3. Nanocrystal dispersions stabilized by sodium lauryl sulfate and polyvinylpyrrolidone were prepared by modified wet media milling technique, with the selection of appropriate process and formulation parameters. The nanoparticles characterization included particle size and zeta potential measurements, differential scanning calorimetry, X-ray powder diffraction, dissolution and solubility study, and in vivo pharmacokinetic experiments. Developed formulations had small uniform particle sizes and were stable for three months. Nanonization caused decreased crystallite size and induced crystal defects formation, as well as a DK-I-60-3 solubility increase. Furthermore, after oral administration of the developed formulations in rats, two to three-fold bioavailability enhancement was observed in plasma and investigated organs, including the brain.",
publisher = "MDPI AG",
journal = "Pharmaceutics",
title = "Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach",
volume = "13",
number = "8",
doi = "10.3390/pharmaceutics13081188"
}

Mitrović, J., Divović-Matović, B., Knutson, D., Đoković, J., Kremenović, A., Dobričić, V., Ranđelović, D., Pantelić, I., Cook, J., Savić, M.,& Savić, S.. (2021). Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach. in Pharmaceutics
MDPI AG., 13(8).
https://doi.org/10.3390/pharmaceutics13081188

Mitrović J, Divović-Matović B, Knutson D, Đoković J, Kremenović A, Dobričić V, Ranđelović D, Pantelić I, Cook J, Savić M, Savić S. Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach. in Pharmaceutics. 2021;13(8).
doi:10.3390/pharmaceutics13081188 .

Mitrović, Jelena, Divović-Matović, Branka, Knutson, Daniel, Đoković, Jelena, Kremenović, Aleksandar, Dobričić, Vladimir, Ranđelović, Danijela, Pantelić, Ivana, Cook, James, Savić, Miroslav, Savić, Snežana, "Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach" in Pharmaceutics, 13, no. 8 (2021),
https://doi.org/10.3390/pharmaceutics13081188 . .

TY  - JOUR
AU  - Đoković, Jelena
AU  - Savić, Sanela
AU  - Mitrović, Jelena
AU  - Nikolić, Ines
AU  - Marković, Bojan
AU  - Ranđelović, Danijela
AU  - Antić-Stanković, Jelena
AU  - Božić, Dragana
AU  - Cekić, Nebojša
AU  - Stevanović, Vladimir
AU  - Batinić, Bojan
AU  - Aranđelović, Jovana
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2021
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5541
AB  - The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index <0.15, zeta potential of −40 mV, and acceptable osmolality of about 550 mOsm/kg. After two years of storage at room temperature, all formulations remained stable. Moreover, antioxidant activity remained intact, as demonstrated by DPPH (IC50 values 0.078–0.075 mg/mL after two years) and FRAPS assays. In vitro release testing proved that PEGylated phospholipids slowed down the curcumin release from nanoemulsions. The nanoemulsion carrier has been proven safe by the MTT test conducted with MRC-5 cell line, and effective on LS cell line. Results from the pharmacokinetic pilot study implied the PEGylated nanoemulsions improved plasma residence of curcumin 20 min after intravenous administration, compared to the non-PEGylated nanoemulsion (two-fold higher) or curcumin solution (three-fold higher). Overall, conclusion suggests that developed PEGylated nanoemulsions present an acceptable delivery system for parenteral administration of curcumin, being effective in preserving its stability and antioxidant capacity at the level highly comparable to the initial findings.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats
VL  - 22
IS  - 15
DO  - 10.3390/ijms22157991
ER  - 

@article{
author = "Đoković, Jelena and Savić, Sanela and Mitrović, Jelena and Nikolić, Ines and Marković, Bojan and Ranđelović, Danijela and Antić-Stanković, Jelena and Božić, Dragana and Cekić, Nebojša and Stevanović, Vladimir and Batinić, Bojan and Aranđelović, Jovana and Savić, Miroslav and Savić, Snežana",
year = "2021",
abstract = "The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index <0.15, zeta potential of −40 mV, and acceptable osmolality of about 550 mOsm/kg. After two years of storage at room temperature, all formulations remained stable. Moreover, antioxidant activity remained intact, as demonstrated by DPPH (IC50 values 0.078–0.075 mg/mL after two years) and FRAPS assays. In vitro release testing proved that PEGylated phospholipids slowed down the curcumin release from nanoemulsions. The nanoemulsion carrier has been proven safe by the MTT test conducted with MRC-5 cell line, and effective on LS cell line. Results from the pharmacokinetic pilot study implied the PEGylated nanoemulsions improved plasma residence of curcumin 20 min after intravenous administration, compared to the non-PEGylated nanoemulsion (two-fold higher) or curcumin solution (three-fold higher). Overall, conclusion suggests that developed PEGylated nanoemulsions present an acceptable delivery system for parenteral administration of curcumin, being effective in preserving its stability and antioxidant capacity at the level highly comparable to the initial findings.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats",
volume = "22",
number = "15",
doi = "10.3390/ijms22157991"
}

Đoković, J., Savić, S., Mitrović, J., Nikolić, I., Marković, B., Ranđelović, D., Antić-Stanković, J., Božić, D., Cekić, N., Stevanović, V., Batinić, B., Aranđelović, J., Savić, M.,& Savić, S.. (2021). Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats. in International Journal of Molecular Sciences
MDPI., 22(15).
https://doi.org/10.3390/ijms22157991

Đoković J, Savić S, Mitrović J, Nikolić I, Marković B, Ranđelović D, Antić-Stanković J, Božić D, Cekić N, Stevanović V, Batinić B, Aranđelović J, Savić M, Savić S. Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats. in International Journal of Molecular Sciences. 2021;22(15).
doi:10.3390/ijms22157991 .

Đoković, Jelena, Savić, Sanela, Mitrović, Jelena, Nikolić, Ines, Marković, Bojan, Ranđelović, Danijela, Antić-Stanković, Jelena, Božić, Dragana, Cekić, Nebojša, Stevanović, Vladimir, Batinić, Bojan, Aranđelović, Jovana, Savić, Miroslav, Savić, Snežana, "Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats" in International Journal of Molecular Sciences, 22, no. 15 (2021),
https://doi.org/10.3390/ijms22157991 . .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Bjelošević, Maja
AU  - Đoković, Jelena
AU  - Ahlin Grabnar, Pegi
AU  - Planinšek, Odon
AU  - Knutson, Daniel
AU  - Cook, James
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2020
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/3744
AB  - Despite of good pharmacodynamics of DK-I-56-1, novel deuterated pyrazoloquinolinones ligand, low solubility limits its administration. Nanosuspensions can help to overcome this problem, but its small particle size usually leads to particle agglomeration in short period of time. This phenomenon can be prevented by performing lyophilization. In this study cryoprotectants selection as well as characterization (particle size measurements after redispersion, scanning electron microscopy, differential scanning calorimetry and thermogravimetric measurements) of obtained freeze dried preparations was carried out. It was observed that sucrose/mannitol ratio 1:1 and 3:2 in total concentration of 10% can preserve particle size during lyophilization. However, after stability study conducted during one month storage at 25 °C and 40 °C, particle size remained in submicron range only in one sample. Changes in particle size were also followed by changes in polymorphic form of mannitol. It can be concluded that changes of crystal forms in freeze dried preparations during storage could jeopardize their stability, and therefore should be carefully examined.
T1  - Nanosuspensions of novel deuterated pyrazoloquinolinones ligand (DK-I-56-1): lyophilization procedure development through cryoprotectant selection and stability study
UR  - https://hdl.handle.net/21.15107/rcub_farfar_3744
ER  - 

@conference{
author = "Mitrović, Jelena and Bjelošević, Maja and Đoković, Jelena and Ahlin Grabnar, Pegi and Planinšek, Odon and Knutson, Daniel and Cook, James and Savić, Miroslav and Savić, Snežana",
year = "2020",
abstract = "Despite of good pharmacodynamics of DK-I-56-1, novel deuterated pyrazoloquinolinones ligand, low solubility limits its administration. Nanosuspensions can help to overcome this problem, but its small particle size usually leads to particle agglomeration in short period of time. This phenomenon can be prevented by performing lyophilization. In this study cryoprotectants selection as well as characterization (particle size measurements after redispersion, scanning electron microscopy, differential scanning calorimetry and thermogravimetric measurements) of obtained freeze dried preparations was carried out. It was observed that sucrose/mannitol ratio 1:1 and 3:2 in total concentration of 10% can preserve particle size during lyophilization. However, after stability study conducted during one month storage at 25 °C and 40 °C, particle size remained in submicron range only in one sample. Changes in particle size were also followed by changes in polymorphic form of mannitol. It can be concluded that changes of crystal forms in freeze dried preparations during storage could jeopardize their stability, and therefore should be carefully examined.",
title = "Nanosuspensions of novel deuterated pyrazoloquinolinones ligand (DK-I-56-1): lyophilization procedure development through cryoprotectant selection and stability study",
url = "https://hdl.handle.net/21.15107/rcub_farfar_3744"
}

Mitrović, J., Bjelošević, M., Đoković, J., Ahlin Grabnar, P., Planinšek, O., Knutson, D., Cook, J., Savić, M.,& Savić, S.. (2020). Nanosuspensions of novel deuterated pyrazoloquinolinones ligand (DK-I-56-1): lyophilization procedure development through cryoprotectant selection and stability study. .
https://hdl.handle.net/21.15107/rcub_farfar_3744

Mitrović J, Bjelošević M, Đoković J, Ahlin Grabnar P, Planinšek O, Knutson D, Cook J, Savić M, Savić S. Nanosuspensions of novel deuterated pyrazoloquinolinones ligand (DK-I-56-1): lyophilization procedure development through cryoprotectant selection and stability study. 2020;.
https://hdl.handle.net/21.15107/rcub_farfar_3744 .

Mitrović, Jelena, Bjelošević, Maja, Đoković, Jelena, Ahlin Grabnar, Pegi, Planinšek, Odon, Knutson, Daniel, Cook, James, Savić, Miroslav, Savić, Snežana, "Nanosuspensions of novel deuterated pyrazoloquinolinones ligand (DK-I-56-1): lyophilization procedure development through cryoprotectant selection and stability study" (2020),
https://hdl.handle.net/21.15107/rcub_farfar_3744 .

TY  - JOUR
AU  - Mitrović, Jelena
AU  - Divović, Branka
AU  - Knutson, Daniel E.
AU  - Đoković, Jelena
AU  - Vulić, Predrag
AU  - Ranđelović, Danijela
AU  - Dobričić, Vladimir
AU  - Čalija, Bojan
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2020
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/3640
AB  - DK-I-56–1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), a recently developed deuterated pyrazoloquinolinone, has been recognized as a lead candidate for treatment of various neuropsychiatric disorders. During preclinical investigation of poorly water-soluble compounds such as DK-I-56–1, the application of nanotechnology could be advantageous due to improved safety and possibly increased bioavailability of nanosized formulation. DK-I-56–1 nanosuspensions stabilized by polysorbate 80, alone or in combination with poloxamers 188 i.e. 407 or d-α-tocopheryl polyethylene glycol 1000 succinate, were prepared using a small-scale media milling device. With particle size 208.7–250.6 nm and polydispersity index <0.250, selected nanodiseprsions were stable for three weeks. Pharmacokinetic and biodistribution studies following intraperitoneal administration of three types of formulation in mice indicated high plasma DK-I-56–1 levels after solution (10,228.6 ± 1037.2 ngh/ml) and nanosuspension (6770.4 ± 770.7 ngh/ml) but not suspension administration (966.0 ± 58.1 ngh/ml). However, distribution of DK-I-56–1 after solution was heavily influenced by its composition, and brain availability of nanosuspension was superior to that of solution formulation. In spontaneous locomotor activity test, the expected hyperlocomotor effect was observed after nanosuspension administration, without compromising impact of the vehicle/excipients used. Therefore, nanonization of drug compound assembled with proper selection of stabilizers may seemingly contribute further thorough testing of DK-I-56–1 preclinical efficacy.
PB  - Elsevier B.V.
T2  - European Journal of Pharmaceutical Sciences
T1  - Nanocrystal dispersion of DK-I-56–1, a poorly soluble pyrazoloquinolinone positive modulator of α6 GABAA receptors: Formulation approach toward improved in vivo performance
VL  - 152
DO  - 10.1016/j.ejps.2020.105432
ER  - 

@article{
author = "Mitrović, Jelena and Divović, Branka and Knutson, Daniel E. and Đoković, Jelena and Vulić, Predrag and Ranđelović, Danijela and Dobričić, Vladimir and Čalija, Bojan and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2020",
abstract = "DK-I-56–1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one), a recently developed deuterated pyrazoloquinolinone, has been recognized as a lead candidate for treatment of various neuropsychiatric disorders. During preclinical investigation of poorly water-soluble compounds such as DK-I-56–1, the application of nanotechnology could be advantageous due to improved safety and possibly increased bioavailability of nanosized formulation. DK-I-56–1 nanosuspensions stabilized by polysorbate 80, alone or in combination with poloxamers 188 i.e. 407 or d-α-tocopheryl polyethylene glycol 1000 succinate, were prepared using a small-scale media milling device. With particle size 208.7–250.6 nm and polydispersity index <0.250, selected nanodiseprsions were stable for three weeks. Pharmacokinetic and biodistribution studies following intraperitoneal administration of three types of formulation in mice indicated high plasma DK-I-56–1 levels after solution (10,228.6 ± 1037.2 ngh/ml) and nanosuspension (6770.4 ± 770.7 ngh/ml) but not suspension administration (966.0 ± 58.1 ngh/ml). However, distribution of DK-I-56–1 after solution was heavily influenced by its composition, and brain availability of nanosuspension was superior to that of solution formulation. In spontaneous locomotor activity test, the expected hyperlocomotor effect was observed after nanosuspension administration, without compromising impact of the vehicle/excipients used. Therefore, nanonization of drug compound assembled with proper selection of stabilizers may seemingly contribute further thorough testing of DK-I-56–1 preclinical efficacy.",
publisher = "Elsevier B.V.",
journal = "European Journal of Pharmaceutical Sciences",
title = "Nanocrystal dispersion of DK-I-56–1, a poorly soluble pyrazoloquinolinone positive modulator of α6 GABAA receptors: Formulation approach toward improved in vivo performance",
volume = "152",
doi = "10.1016/j.ejps.2020.105432"
}

Mitrović, J., Divović, B., Knutson, D. E., Đoković, J., Vulić, P., Ranđelović, D., Dobričić, V., Čalija, B., Cook, J. M., Savić, M.,& Savić, S.. (2020). Nanocrystal dispersion of DK-I-56–1, a poorly soluble pyrazoloquinolinone positive modulator of α6 GABAA receptors: Formulation approach toward improved in vivo performance. in European Journal of Pharmaceutical Sciences
Elsevier B.V.., 152.
https://doi.org/10.1016/j.ejps.2020.105432

Mitrović J, Divović B, Knutson DE, Đoković J, Vulić P, Ranđelović D, Dobričić V, Čalija B, Cook JM, Savić M, Savić S. Nanocrystal dispersion of DK-I-56–1, a poorly soluble pyrazoloquinolinone positive modulator of α6 GABAA receptors: Formulation approach toward improved in vivo performance. in European Journal of Pharmaceutical Sciences. 2020;152.
doi:10.1016/j.ejps.2020.105432 .

Mitrović, Jelena, Divović, Branka, Knutson, Daniel E., Đoković, Jelena, Vulić, Predrag, Ranđelović, Danijela, Dobričić, Vladimir, Čalija, Bojan, Cook, James M., Savić, Miroslav, Savić, Snežana, "Nanocrystal dispersion of DK-I-56–1, a poorly soluble pyrazoloquinolinone positive modulator of α6 GABAA receptors: Formulation approach toward improved in vivo performance" in European Journal of Pharmaceutical Sciences, 152 (2020),
https://doi.org/10.1016/j.ejps.2020.105432 . .

TY  - CONF
AU  - Mitrović, Jelena
AU  - Divović, Branka
AU  - Dobričić, Vladimir
AU  - Čalija, Bojan
AU  - Vulić, Jelena P.
AU  - Knutson, Daniel E.
AU  - Cook, James M.
AU  - Savić, Miroslav
AU  - Savić, Snežana
PY  - 2019
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/5542
AB  - Deuterated pyrazoloquinolinones, GABAn receptor ligands, are being investigated for neuropsychiatric disorders treatment. Among others, DK-l-56-1 (7-Methoxy-2-(4-methoxy-d3- p h e n yl ) - 2, 5 -d i h yd ro - 3 H - py r azolo -14,3 -clq u i n o I i n - 3-one) was chosen as the lead component [1]. Because of its low solubility in water (6.27 t0.74 pg/ml) nanosuspension formulation for prospective parenteral administration was carried out [2]. ...
C3  - 10th International Congress Nanotechnology in Medicine & Biology, 15 - 17. April 2019, Graz / Austria
T1  - Nanonization of new patent protected substances - carrier selection for preclinical research: physicochemical and in vivo behavioral characterization
UR  - https://hdl.handle.net/21.15107/rcub_farfar_5542
ER  - 

@conference{
author = "Mitrović, Jelena and Divović, Branka and Dobričić, Vladimir and Čalija, Bojan and Vulić, Jelena P. and Knutson, Daniel E. and Cook, James M. and Savić, Miroslav and Savić, Snežana",
year = "2019",
abstract = "Deuterated pyrazoloquinolinones, GABAn receptor ligands, are being investigated for neuropsychiatric disorders treatment. Among others, DK-l-56-1 (7-Methoxy-2-(4-methoxy-d3- p h e n yl ) - 2, 5 -d i h yd ro - 3 H - py r azolo -14,3 -clq u i n o I i n - 3-one) was chosen as the lead component [1]. Because of its low solubility in water (6.27 t0.74 pg/ml) nanosuspension formulation for prospective parenteral administration was carried out [2]. ...",
journal = "10th International Congress Nanotechnology in Medicine & Biology, 15 - 17. April 2019, Graz / Austria",
title = "Nanonization of new patent protected substances - carrier selection for preclinical research: physicochemical and in vivo behavioral characterization",
url = "https://hdl.handle.net/21.15107/rcub_farfar_5542"
}

Mitrović, J., Divović, B., Dobričić, V., Čalija, B., Vulić, J. P., Knutson, D. E., Cook, J. M., Savić, M.,& Savić, S.. (2019). Nanonization of new patent protected substances - carrier selection for preclinical research: physicochemical and in vivo behavioral characterization. in 10th International Congress Nanotechnology in Medicine & Biology, 15 - 17. April 2019, Graz / Austria.
https://hdl.handle.net/21.15107/rcub_farfar_5542

Mitrović J, Divović B, Dobričić V, Čalija B, Vulić JP, Knutson DE, Cook JM, Savić M, Savić S. Nanonization of new patent protected substances - carrier selection for preclinical research: physicochemical and in vivo behavioral characterization. in 10th International Congress Nanotechnology in Medicine & Biology, 15 - 17. April 2019, Graz / Austria. 2019;.
https://hdl.handle.net/21.15107/rcub_farfar_5542 .

Mitrović, Jelena, Divović, Branka, Dobričić, Vladimir, Čalija, Bojan, Vulić, Jelena P., Knutson, Daniel E., Cook, James M., Savić, Miroslav, Savić, Snežana, "Nanonization of new patent protected substances - carrier selection for preclinical research: physicochemical and in vivo behavioral characterization" in 10th International Congress Nanotechnology in Medicine & Biology, 15 - 17. April 2019, Graz / Austria (2019),
https://hdl.handle.net/21.15107/rcub_farfar_5542 .

TY  - JOUR
AU  - Milenković, Anđela
AU  - Miljić, Dragana
AU  - Mitrović, Jelena
AU  - Protić, Ana
AU  - Otašević, Biljana
PY  - 2016
UR  - https://farfar.pharmacy.bg.ac.rs/handle/123456789/2729
AB  - In recent years, there is aim to reduce toxic solvents use in chromatographic techniques. Beta-cyclodextrins (β-CD) are commonly used for this purpose, as substance retention modulators. By increasing their concentration in the mobile phase, generally there is a decrease in analyte retention time, and consequently, a decrease in the organic solvent ratio in the mobile phase. The aim of this study was to investigate β-CD and selected sartans inclusion complexes formation. The emphasis was on the influence of the aqueous phase pH value, the drug-β-CD molar ratio and the organic solvent type on complex formation. The mass spectrometry investigation included characterization of the formed complex drug: β-CD at different molar ratios (1:1, 1:2, 1:5 and 1:10), and at different pH values of the aqueous phase (5, 7 and 9) using triple quadrupole mass analyzer. The mobile phase and solvent in which the complexes were formed consisted of acetonitrile and water mixture (50:50, v/v) and methanol and water mixture (50:50, v/v). Measurements of these complexes signal intensity after electrospray ionization in negative mode. Observed m/z values indicated formation of drug-β-CD in 1:1 ratio at all pH values of water phase and independently of organic solvent type and β-CD ratio. It was noted that formed complex signal intensity increased only by increasing β-CD ratio.
AB  - Poslednjih godina sve više se teži smanjenju upotrebe toksičnih rastvarača u hromatografskim metodama. U te svrhe često se upotrebljavaju beta-ciklodekstrini (β-CD) kao modulatori retencije supstanci. Povećavanjem njihove koncentracije u mobilnoj fazi uglavnom dolazi do smanjenja retencije analita i, posledično, do smanjenja udela organskog rastvarača u mobilnoj fazi. Cilj rada bio je ispitivanje načina formiranja inkluzionih kompleksa β-CD i odabranih lekova iz grupe sartana, sa posebnim osvrtom na uticaj pH vrednosti vodene faze, molarnog odnosa leka i β-CD i vrste organskog rastvarača na formiranje kompleksa. Primenom metode masene spektrometrije, na triplkvadrupolskom masenom analizatoru, vršena je karakterizacija formiranog kompleksa β-CD i leka pri različitom molarnom odnosu lek:β-CD (1:1, 1:2, 1:5 i 1:10) i pri pH vrednostima vodene faze 5, 7 i 9. Mobilna faza i rastvarač u kome su formirani kompleksi bila je smeša acetonitrila i vode, kao i smeša metanola i vode (50:50, V/V). Merenja intenziteta signala nagrađenih kompleksa nakon elektrosprej jonizacije vršena su u negativnom modu. Uočene su m/z vrednosti koje potiču od masa kompleksa leka i β-CD u odnosu 1:1 pri svim pH vrednostima vodene faze i nezavisno od vrste organskog rastvarača i udela β-CD. Uočeno je da intenzitet signala nastalog kompleksa raste jedino povećanjem udela β-CD.
PB  - Savez farmaceutskih udruženja Srbije, Beograd
T2  - Arhiv za farmaciju
T1  - Monitoring of complex forming of the active pharmaceutical substance and β-cyclodextrin as an additive of the mobile phase using mass spectrometry
T1  - Praćenje građenja kompleksa aktivne farmaceutske supstance i β-ciklodekstrina kao aditiva mobilne faze primenom masene spektrometrije
VL  - 66
IS  - 4
SP  - 147
EP  - 160
DO  - 10.5937/arhfarm1604147M
ER  - 

@article{
author = "Milenković, Anđela and Miljić, Dragana and Mitrović, Jelena and Protić, Ana and Otašević, Biljana",
year = "2016",
abstract = "In recent years, there is aim to reduce toxic solvents use in chromatographic techniques. Beta-cyclodextrins (β-CD) are commonly used for this purpose, as substance retention modulators. By increasing their concentration in the mobile phase, generally there is a decrease in analyte retention time, and consequently, a decrease in the organic solvent ratio in the mobile phase. The aim of this study was to investigate β-CD and selected sartans inclusion complexes formation. The emphasis was on the influence of the aqueous phase pH value, the drug-β-CD molar ratio and the organic solvent type on complex formation. The mass spectrometry investigation included characterization of the formed complex drug: β-CD at different molar ratios (1:1, 1:2, 1:5 and 1:10), and at different pH values of the aqueous phase (5, 7 and 9) using triple quadrupole mass analyzer. The mobile phase and solvent in which the complexes were formed consisted of acetonitrile and water mixture (50:50, v/v) and methanol and water mixture (50:50, v/v). Measurements of these complexes signal intensity after electrospray ionization in negative mode. Observed m/z values indicated formation of drug-β-CD in 1:1 ratio at all pH values of water phase and independently of organic solvent type and β-CD ratio. It was noted that formed complex signal intensity increased only by increasing β-CD ratio., Poslednjih godina sve više se teži smanjenju upotrebe toksičnih rastvarača u hromatografskim metodama. U te svrhe često se upotrebljavaju beta-ciklodekstrini (β-CD) kao modulatori retencije supstanci. Povećavanjem njihove koncentracije u mobilnoj fazi uglavnom dolazi do smanjenja retencije analita i, posledično, do smanjenja udela organskog rastvarača u mobilnoj fazi. Cilj rada bio je ispitivanje načina formiranja inkluzionih kompleksa β-CD i odabranih lekova iz grupe sartana, sa posebnim osvrtom na uticaj pH vrednosti vodene faze, molarnog odnosa leka i β-CD i vrste organskog rastvarača na formiranje kompleksa. Primenom metode masene spektrometrije, na triplkvadrupolskom masenom analizatoru, vršena je karakterizacija formiranog kompleksa β-CD i leka pri različitom molarnom odnosu lek:β-CD (1:1, 1:2, 1:5 i 1:10) i pri pH vrednostima vodene faze 5, 7 i 9. Mobilna faza i rastvarač u kome su formirani kompleksi bila je smeša acetonitrila i vode, kao i smeša metanola i vode (50:50, V/V). Merenja intenziteta signala nagrađenih kompleksa nakon elektrosprej jonizacije vršena su u negativnom modu. Uočene su m/z vrednosti koje potiču od masa kompleksa leka i β-CD u odnosu 1:1 pri svim pH vrednostima vodene faze i nezavisno od vrste organskog rastvarača i udela β-CD. Uočeno je da intenzitet signala nastalog kompleksa raste jedino povećanjem udela β-CD.",
publisher = "Savez farmaceutskih udruženja Srbije, Beograd",
journal = "Arhiv za farmaciju",
title = "Monitoring of complex forming of the active pharmaceutical substance and β-cyclodextrin as an additive of the mobile phase using mass spectrometry, Praćenje građenja kompleksa aktivne farmaceutske supstance i β-ciklodekstrina kao aditiva mobilne faze primenom masene spektrometrije",
volume = "66",
number = "4",
pages = "147-160",
doi = "10.5937/arhfarm1604147M"
}

Milenković, A., Miljić, D., Mitrović, J., Protić, A.,& Otašević, B.. (2016). Monitoring of complex forming of the active pharmaceutical substance and β-cyclodextrin as an additive of the mobile phase using mass spectrometry. in Arhiv za farmaciju
Savez farmaceutskih udruženja Srbije, Beograd., 66(4), 147-160.
https://doi.org/10.5937/arhfarm1604147M

Milenković A, Miljić D, Mitrović J, Protić A, Otašević B. Monitoring of complex forming of the active pharmaceutical substance and β-cyclodextrin as an additive of the mobile phase using mass spectrometry. in Arhiv za farmaciju. 2016;66(4):147-160.
doi:10.5937/arhfarm1604147M .

Milenković, Anđela, Miljić, Dragana, Mitrović, Jelena, Protić, Ana, Otašević, Biljana, "Monitoring of complex forming of the active pharmaceutical substance and β-cyclodextrin as an additive of the mobile phase using mass spectrometry" in Arhiv za farmaciju, 66, no. 4 (2016):147-160,
https://doi.org/10.5937/arhfarm1604147M . .