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

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

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

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

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

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

Hydrochloride Salt of the GABAkine KRM-II-81

Imidazodiazepine (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a potentiator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. KRM-II-81 is active against many seizure and pain models in rodents, where it exhibits improved pharmacological properties over standard-of-care agents. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption, and distribution, as well as for efficient methods of bulk synthesis, a hydrochloride salt of KRM-II-81 was prepared. KRM-II-81·HCl was produced from the free base with anhydrous hydrochloric acid. The formation of the monohydrochloride salt was confirmed by X-ray crystallography, as well as 1H NMR and 13C NMR analyses. High water solubility and a lower partition coefficient (octanol/water) were exhibited by KRM-II-81·HCl as compared to the free base. Oral administration of either KRM-II-81·HCl or the free base resulted in high concentrations in the brain and plasma of rats. Oral dosing in mice significantly increased the latency to both clonic and tonic convulsions and decreased pentylenetetrazol-induced lethality. The increased water solubility of the HCl salt enables intravenous dosing and the potential for higher concentration formulations compared with the free base without impacting anticonvulsant potency. Thus, KRM-II-81·HCl adds an important new compound to facilitate the development of these imidazodiazepines for clinical evaluation

Effects of α5 GABAA receptor modulation on social interaction, memory, and neuroinflammation in a mouse model of Alzheimer's disease

Aims GABAergic modulation involved in cognitive processing appears to be substantially changed in Alzheimer's disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABAA receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, and neuroinflammation. Methods After 10-day treatment with PAM, NAM, or solvent, 6-month-old transgenic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of IL-1β, IL-6, TNF-α, GFAP, and IBA-1 were determined in hippocampus and prefrontal cortex by qPCR. Results PAM treatment impaired spatial learning in transgenic females compared to solvent-treated transgenic females, and social recognition in transgenic and non-transgenic males. NAM treatment declined social interaction in transgenic and non-transgenic males, while had beneficial effect on cognitive flexibility in non-transgenic males compared to solvent-treated non-transgenic males. Transgenic animals have not fully displayed cognitive symptoms, but neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males compared to pathological controls. Conclusion PAM and NAM failed to exert favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with prominent neuroinflammation

Imidazodiazepine Anticonvulsant, KRM-II-81, Produces Novel, Non-diazepam-like Antiseizure Effects

The need for improved medications for the treatment of epilepsy and chronic pain is essential. Epileptic patients typically take multiple antiseizure drugs without complete seizure freedom, and chronic pain is not fully managed with current medications. A positive allosteric modulator (PAM) of α2/3-containing GABAA receptors (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81 (8) is a lead compound in a series of imidazodiazepines. We previously reported that KRM-II-81 produces broad-based anticonvulsant and antinociceptive efficacy in rodent models and provides a wider margin over motoric side effects than that of other GABAA receptor PAMs. The present series of experiments was designed to fill key missing gaps in prior preclinical studies assessing whether KRM-II-81 could be further differentiated from nonselective GABAA receptor PAMs using the anticonvulsant diazepam (DZP) as a comparator. In multiple chemical seizure provocation models in mice, KRM-II-81 was either equally or more efficacious than DZP. Most strikingly, KRM-II-81 but not DZP blocked the development of seizure sensitivity to the chemoconvulsants cocaine and pentylenetetrazol in seizure kindling models. These and predecessor data have placed KRM-II-81 into consideration for clinical development requiring the manufacture of kilogram amounts of good manufacturing practice material. We describe here a novel synthetic route amenable to kilogram quantity production. The new biological and chemical data provide key steps forward in the development of KRM-II-81 (8) as an improved treatment option for patients suffering from epilepsy

The impact of the aqueous phase on the physicochemical characteristics of nanoemulsions loaded with patent protected compound GL-II-73

INTRODUCTION Temporal lobe epilepsy is characterized by seizures, but can also be associated with mental health problems for which there are no clear treatment regimens. A proprietary compound, GL-II-73 - (4R)-8-ethynyl-6-(2-fluorophenyl)-N,N,4-trimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide, a positive allosteric modulator of α5-containing γ-aminobutyric acid (GABA) receptors, has been shown to be effective in the treatment of these comorbidities [1]. ..

Vasodilatory effects of a variety of positive allosteric modulators of GABAA receptors on rat thoracic aorta

Different subtypes of GABAA (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABAA receptors are expressed on blood vessels, through which positive allosteric modulators of GABAA receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABAA receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABAA receptor modulators: zolpidem (α1-selective), XHe–III–074 (α4-selective), MP–III–022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe–III–074, MP–III–022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10−7 M and 3 × 10−7 M). The present work suggests that the observed vasoactivity is due to modulation of “vascular” GABAA receptors, which after further detailed research may provide a therapeutic target

Postweaning positive modulation of α5GABAA receptors improves autism-like features in prenatal valproate rat model in a sex-specific manner

Autism spectrum disorder (ASD), as a common neurodevelopmental disorder that encompasses impairments in social communication and interaction, as well as repetitive and restrictive behavior, still awaits an effective treatment strategy. The involvement of GABAergic neurotransmission, and especially a deficit of GABA A receptors that contain the α5 subunits, were implicated in pathogenesis of ASD. Therefore, we tested MP-III-022, a positive allosteric modulator (PAM) selective for α5GABAA receptors, in Wistar rats prenatally exposed to valproic acid, as an animal model useful for studying ASD. Postweaning rats of both sexes were treated for 7 days with vehicle or MP-III-022 at two doses pharmacokinetically determined as selective, and thereafter tested in a behavioral battery (social interaction test, elevated plus maze, spontaneous locomotor activ- ity, and standard and reverse Morris water maze). Additional rats were used for establishing a primary neuronal culture and performing calcium imaging, and determination of hippocampal mRNA levels of GABRA5, NKCC1, and KCC2. MP-III-022 prevented impairments in many parameters connected with social, repetitive and restrictive behavioral domains. The lower and higher dose was more effective in males and females, respectively. Intriguingly, MP-III-022 elicited certain changes in control animals similar to those manifested in valproate ani- mals themselves. Behavioral results were mirrored in GABA switch and spontane- ous neuronal activity, assessed with calcium imaging, and also in expression changes of three genes analyzed. Our data support a role of α5GABAA receptors in pathophysiology of ASD, and suggest a potential application of selective PAMs in its treatment, that needs to be researched in a sex-specific manner