Imiquimod Reverses Chronic Toxoplasmosis-Associated Behavioral and Neurocognitive Anomalies in a Rat Model.

Toxoplasma gondii is the etiologic agent of toxoplasmosis, a highly prevalent parasitosis. Toxoplasma gondii (T. gondii) transits in the brain from acute (AT) to chronic toxoplasmosis (CT), under host immune control. In immunocompromised patients, reactivation of CT is potentially life-threatening. Behavioral and neurological complications have been associated with CT. Furthermore, an effective treatment targeting CT is still lacking. We previously reported the efficacy of imiquimod against CT. Here, we demonstrate the molecular effects of imiquimod or imiquimod followed by the clinically used combination of sulfadiazine and pyrimethamine (SDZ + PYR) on CT-associated behavior in a rat model. Imiquimod decreased the number of cysts in the brains of chronically infected rats due to an induced reactivation of bradyzoites into tachyzoites. Importantly, this decrease was more pronounced in rats treated with imiquimod followed by SDZ + PYR. Rats chronically infected with T. gondii exhibited an anxiety-like behavior. Notably, treatment with imiquimod reversed this behavior aberrancy, with even a more pronounced effect with imiquimod followed by SDZ/PYR. Similarly, rats chronically infected with T. gondii exhibited learning deficits, and imiquimod alone or followed by SDZ/PYR reversed this behavior. Our results enhance our knowledge of the implications of CT on behavioral aberrancies and highlight the potency of imiquimod followed by SDZ + PYR on these CT-associated complications.

[1,2,4]triazolo[4,3-a]quinoxaline as Novel Scaffold in the Imiqualines Family: Candidates with Cytotoxic Activities on Melanoma Cell Lines.

Cutaneous melanoma is one of the most aggressive human cancers and is the deadliest form of skin cancer, essentially due to metastases. Novel therapies are always required, since cutaneous melanoma develop resistance to oncogenic pathway inhibition treatment. The Imiqualine family is composed of heterocycles diversely substituted around imidazo[1,2-a]quinoxaline, imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline scaffolds, which display interesting activities on a panel of cancer cell lines, especially melanoma cell lines. We have designed and prepared novel compounds based on the [1,2,4]triazolo[4,3-a]quinoxaline scaffold through a common synthetic route, using 1-chloro-2-hydrazinoquinoxaline and an appropriate aldehyde. Cyclization is ensured by an oxidation-reduction mechanism using chloranil. The substituents on positions 1 and 8 were chosen based on previous structure-activity relationship (SAR) studies conducted within our heterocyclic Imiqualine family. Physicochemical parameters of all compounds have also been predicted. A375 melanoma cell line viability has been evaluated for 16 compounds. Among them, three novel [1,2,4]triazolo[4,3-a]quinoxalines display cytotoxic activities. Compounds 16a and 16b demonstrate relative activities in the micromolar range (respectively, 3158 nM and 3527 nM). Compound 17a shows the best EC50 of the novel series (365 nM), even if EAPB02303 remains the lead of the entire Imiqualine family (3 nM).

Recent Advances on Small-Molecule Antagonists Targeting TLR7.

Toll-like receptor 7 (TLR7) is a class of pattern recognition receptors (PRRs) recognizing the pathogen-associated elements and damage and as such is a major player in the innate immune system. TLR7 triggers the release of pro-inflammatory cytokines or type-I interferons (IFN), which is essential for immunoregulation. Increasing reports also highlight that the abnormal activation of endosomal TLR7 is implicated in various immune-related diseases, carcinogenesis as well as the proliferation of human immunodeficiency virus (HIV). Hence, the design and development of potent and selective TLR7 antagonists based on small molecules or oligonucleotides may offer new tools for the prevention and management of such diseases. In this review, we offer an updated overview of the main structural features and therapeutic potential of small-molecule antagonists of TLR7. Various heterocyclic scaffolds targeting TLR7 binding sites are presented: pyrazoloquinoxaline, quinazoline, purine, imidazopyridine, pyridone, benzanilide, pyrazolopyrimidine/pyridine, benzoxazole, indazole, indole, and quinoline. Additionally, their structure-activity relationships (SAR) studies associated with biological activities and protein binding modes are introduced.

EAPB0503, an Imidazoquinoxaline Derivative Modulates SENP3/ARF Mediated SUMOylation, and Induces NPM1c Degradation in NPM1 Mutant AML.

Nucleophosmin-1 (NPM1) is a pleiotropic protein involved in numerous cellular processes. NPM1 shuttles between the nucleus and the cytoplasm, but exhibits a predominant nucleolar localization, where its fate and functions are exquisitely controlled by dynamic post-translational modifications (PTM). Sentrin/SUMO Specific Peptidase 3 (SENP3) and ARF are two nucleolar proteins involved in NPM1 PTMs. SENP3 antagonizes ARF-mediated NPM1 SUMOylation, to promote ribosomal biogenesis. In Acute Myeloid Leukemia (AML), NPM1 is frequently mutated, and exhibits an aberrant cytoplasmic localization (NPM1c). NPM1c mutations define a separate AML entity with good prognosis in some AML patients, rendering NPM1c as a potential therapeutic target. SENP3-mediated NPM1 de-SUMOylation induces resistance to therapy in NPM1c AML. Here, we demonstrate that the imidazoquinoxaline EAPB0503 prolongs the survival and results in selective reduction in the leukemia burden of NPM1c AML xenograft mice. Indeed, EAPB0503 selectively downregulates HDM2 expression and activates the p53 pathway in NPM1c expressing cells, resulting in apoptosis. Importantly, we unraveled that NPM1c expressing cells exhibit low basal levels of SUMOylation paralleled with high SENP3 and low ARF basal levels. EAPB0503 reverted these molecular players by inducing NPM1c SUMOylation and ubiquitylation, leading to its proteasomal degradation. EAPB0503-induced NPM1c SUMOylation is concurrent with SENP3 downregulation and ARF upregulation in NPM1c expressing cells. Collectively, these results provide a strong rationale for testing therapies modulating NPM1c post-translational modifications in the management of NPM1c AML.

Imiquimod Targets Toxoplasmosis Through Modulating Host Toll-Like Receptor-MyD88 Signaling.

Toxoplasma gondii is a prevalent parasite of medical and veterinary importance. Tachyzoïtes and bradyzoïtes are responsible for acute and chronic toxoplasmosis (AT and CT), respectively. In immunocompetent hosts, AT evolves into a persistent CT, which can reactivate in immunocompromised patients with dire consequences. Imiquimod is an efficient immunomodulatory drug against certain viral and parasitic infections. In vivo, treatment with Imiquimod, throughout AT, reduces the number of brain cysts while rendering the remaining cysts un-infectious. Post-establishment of CT, Imiquimod significantly reduces the number of brain cysts, leading to a delay or abortion of reactivation. At the molecular level, Imiquimod upregulates the expression of Toll-like receptors 7, 11, and 12, following interconversion from bradyzoïtes to tachyzoïtes. Consequently, MyD88 pathway is activated, resulting in the induction of the immune response to control reactivated Toxoplasma foci. This study positions Imiquimod as a potent drug against toxoplasmosis and elucidates its mechanism of action particularly against chronic toxoplasmosis, which is the most prevalent form of the disease.

Imidazo[1,2-a]quinoxalines for melanoma treatment with original mechanism of action.

The malignant transformation of melanocytes causes several thousand deaths each year, making melanoma an important public health concern. Melanoma is the most aggressive skin cancer, which incidence has regularly increased over the past decades. We described here the preparation of new compounds based on the 1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline structure. Different positions of the quinoxaline moiety were screened to introduce novel substituents in order to study their influence on the biological activity. Several alkylamino or alkyloxy groups were also considered to replace the methylamine of our first generation of Imiqualines. Imidazo[1,2-a]pyrazine derivatives were also designed as potential minimal structure. The investigation on A375 melanoma cells displayed interesting in vitro low nanomolar cytotoxic activity. Among them, 9d (EAPB02303) is particularly remarkable since it is 20 times more potent than vemurafenib, the reference clinical therapy used on BRAF mutant melanoma. Contrary to the first generation, EAPB02303 does not inhibit tubulin polymerization, as confirmed by an in vitro assay and a molecular modelisation study. The mechanism of action for EAPB02303 highlighted by a transcriptomic analysis is clearly different from a panel of 12 well-known anticancer drugs. In vivoEAPB02303 treatment reduced tumor size and weight of the A375 human melanoma xenografts in a dose-dependent manner, correlated with a low mitotic index but not with necrosis.

Fused Azolo-Quinoxalines: Candidates for Medicinal Chemistry. A Review of their Biological Applications.

Heterocyclic compounds hold a huge and recognized place in the field of medicinal chemistry thanks to their multiple biological activities. Their synthetic pathways allow their easy and rapid access due to different bond-forming methodologies and provide a huge amount of multi-functionalized compounds for drug delivery. The syntheses of heterocyclic compounds are today well known for the majority, described and reviewed in an extensive literature. In this review, we choose to gather and classify available information concerning the biological activities of quinoxaline-based compounds annulated at bond a containing one and more nitrogen atoms in the fused azole ring.

Substantial Cellular Penetration of Fluorescent Imidazoquinoxalines.

Fluorescent tools have revolutionized our capability to visualize, probe, study, and understand the biological cellular properties, processes and dynamics, enabling researchers to improve their knowledge for example in cancer field. In this paper, we use the peculiar properties of our Imiqualines derivatives to study their cellular penetration and distribution in a human melanoma cell line A375 using confocal microscopy. Preliminary results on colocalization with the potent protein target c-Kit of our lead are also described.

Agonist and antagonist ligands of toll-like receptors 7 and 8: Ingenious tools for therapeutic purposes.

The discovery of the TLRs family and more precisely its functions opened a variety of gates to modulate immunological host responses. TLRs 7/8 are located in the endosomal compartment and activate a specific signaling pathway in a MyD88-dependant manner. According to their involvement into various autoimmune, inflammatory and malignant diseases, researchers have designed diverse TLRs 7/8 ligands able to boost or block the inherent signal transduction. These modulators are often small synthetic compounds and most act as agonists and to a much lesser extent as antagonists. Some of them have reached preclinical and clinical trials, and only one has been approved by the FDA and EMA, imiquimod. The key to the success of these modulators probably lies in their combination with other therapies as recently demonstrated. We gather in this review more than 360 scientific publications, reviews and patents, relating the extensive work carried out by researchers on the design of TLRs 7/8 modulators, which are classified firstly by their biological activities (agonist or antagonist) and then by their chemical structures, which total syntheses are not discussed here. This review also reports about 90 clinical cases, thereby showing the biological interest of these modulators in multiple pathologies.

An in-line enzymatic microreactor for the middle-up analysis of monoclonal antibodies by capillary electrophoresis.

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.

Novel and Selective TLR7 Antagonists among the Imidazo[1,2-a]pyrazines, Imidazo[1,5-a]quinoxalines, and Pyrazolo[1,5-a]quinoxalines Series.

The Toll-like receptors (TLRs) 7 and 8 play an important role in the immune system activation, and their agonists may therefore serve as promising candidate vaccine adjuvants. However, the chronic immune activation by excessive TLR stimulation is a hallmark of several clinically important infectious and autoimmune diseases, which warrants the search for TLR antagonists. In this study, we have synthesized and characterized a variety of compounds belonging to three heterocyclic chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline. These compounds have been tested for their TLR7 or TLR8 agonistic and antagonistic activities. Several of them are shown to be selective TLR7 antagonists without any TLR7 or TLR8 agonistic activity. The selectivity was confirmed by a comparative ligand-docking study in TLR7 antagonist pocket. Two compounds of the pyrazolo[1,5-a]quinoxaline series (10a and 10b) are potent selective TLR7 antagonists and may be considered as promising starting points for the development of new therapeutic agents.

Imidazo[1,2-a]quinoxalines Derivatives Grafted with Amino Acids: Synthesis and Evaluation on A375 Melanoma Cells.

Imiqualines (imidazoquinoxaline derivatives) are anticancer compounds with high cytotoxic activities on melanoma cell lines. The first generation of imiqualines, with two lead compounds (EAPB0203 and EAPB0503), shows remarkable in vitro (IC50 = 1 570 nM and IC50 = 200 nM, respectively, on the A375 melanoma cell line) and in vivo activity on melanoma xenografts. The second generation derivatives, EAPB02302 and EAPB02303, are more active, with IC50 = 60 nM and IC50 = 10 nM, respectively, on A375 melanoma cell line. The aim of this study was to optimize the bioavailability of imiqualine derivatives, without losing their intrinsic activity. For that, we achieved chemical modulation on the second generation of imiqualines by conjugating amino acids on position 4. A new series of twenty-five compounds was efficiently synthesized by using microwave assistance and tested for its activity on the A375 cell line. In the new series, compounds 11a, 9d and 11b show cytotoxic activities less than second generation compounds, but similar to that of the first generation ones (IC50 = 403 nM, IC50 = 128 nM and IC50 = 584 nM, respectively). The presence of an amino acid leads to significant enhancement of the water solubility for improved drugability.

EAPB0503: An Imiquimod analog with potent in vitro activity against cutaneous leishmaniasis caused by Leishmania major and Leishmania tropica.

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL.

Liquid chromatography-electrospray ionization-tandem mass spectrometry method for quantitative estimation of new imiqualine leads with potent anticancer activities in rat and mouse plasma. Application to a pharmacokinetic study in mice.

Imidazoquinoxaline derivatives (imiqualines) are a new series of anticancer compounds. Two lead compounds (EAPB0203 and EAPB0503) with remarkable in vitro and in vivo activity on melanoma and T-cell lymphomas have been previously identified. The modulation of the chemical structure of the most active compound, EAPB0503, has led to the synthesis of two compounds, EAPB02302 and EAPB02303, 7 and 40 times more active than EAPB0503 against A375 human melanoma cancer cell line, respectively. The aim of this study was to develop and validate a sensitive and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry method to simultaneously quantify EAPB02303 and its potential active metabolite, EAPB02302, in rat and mouse plasma. Analytes were detected in multiple reaction monitoring acquisition mode using an electrospray ionization detector in positive ion mode. Following a liquid-liquid extraction with ethyl acetate, analytes and internal standard were separated by HPLC reversed-phase on a C18 RP18 Nucleoshell column (2.7µm, 4.6×100mm). The method was validated according to FDA and EMA Bioanalytical Method Validation guidelines. The robustness of the method was assessed by introducing small variations in nine nominal analytical parameters. Statistical interpretation was performed by mean of the Student's t-test. Standard curves were generated via unweighted quadratic regression of calibrators (EAPB02303: 1.95-1000ng/mL, EAPB02302: 7.81-1000ng/mL in rat plasma; EAPB02303: 0.98-1000ng/mL, EAPB02302: 1.95-1000ng/mL in mouse plasma). From the analysis of QC samples, intra- and inter-assay precision and accuracy studies demonstrated %R.S.Ds. <12.5% and percent deviation from nominal concentration <7%. Matrix effects (mean matrix factors from 91.8-108.5% in rat plasma; and from 90.4-102.4% in mouse plasma) and stability assays (recoveries >87%) were acceptable and in accordance with the guidelines. No quantifiable carryover effect was observed. The LLOQs were 1.95ng/mL for EAPB02303 and 7.81ng/mL for EAPB02302 in rat plasma, and 0.98ng/mL and 1.95ng/mL for the two compounds in mouse plasma, respectively. This method was successfully implemented to support a mouse pharmacokinetic study following a single intraperitoneal administration of EAPB02303 in male C57Bl/6 mice. The obtained pharmacokinetic parameters of EAPB02303 would be useful to optimize the dosing and the rhythm of administration for subsequent preclinical in vivo activity studies.

Raman chemical imaging for spectroscopic screening and direct quantification of falsified drugs.

Falsified drugs are a threat to the health of patients. The analytical control of such products contributes to the fight against this global issue. Raman chemical imaging is a method that relies on consecutive measurements at the surface of a sample, combining spectroscopy, microscopy and chemometrics. This article explores the capabilities of this analytical technique proposing an innovative methodology with spectroscopic screening for the identification of chemical compounds and the direct quantification of the active substance (without prior calibration). Two chemometric methods were used: Multivariate Curve Analysis - Alternate Least Squares for the qualitative analysis and Direct Classical Least Squares for the quantitative analysis. The methodology was optimized with samples prepared in the laboratory and validation parameters were studied. The methodology was then applied to real (authentic and falsified) samples of Viagra® and Plavix®. Despite the presence of fluorescence emission in some samples, the methodology succeeded in the detection of active pharmaceutical ingredients, and in the discrimination of three salts of clopidogrel (in generic formulations of Plavix®). The quantitative deviation from the reference method ranged from -15% to +24% of the active substance content. This deviation may be considered to be acceptable since it is sufficient for assessing the risk to the health of patients and for quickly alerting the health authorities.

Imidazo[1,2-a]pyrazine, Imidazo[1,5-a]quinoxaline and Pyrazolo[1,5-a]quinoxaline derivatives as IKK1 and IKK2 inhibitors.

The transcription nuclear factor NF-κB plays a pivotal role in chronic and acute inflammatory diseases. Among the several and diverse strategies for inhibiting NF-κB, one of the most effective approach considered by the pharmaceutical industry seems to be offered by the development of IKK inhibitors. In a former study, two potential IKK2 inhibitors have been highlighted among a series of imidazo[1,2-a]quinoxaline derivatives. In order to enhance this activity, we present herein the synthesis of twenty-one new compounds based on the imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline or pyrazolo[1,5-a]quinoxaline structures. Their potential to inhibit IKK1 and IKK2 activities is also tested.

Fluorescence Study of Imidazoquinoxalines.

The fluorescence properties of eleven novel derivatives based on the imidazo[1,2-a]quinoxaline structures have been studied. The absorption and emission spectra of these compounds have been recorded in dimethylsulfoxide solution. The phenyl substituting group on position 1 gives them particular properties thanks to the diverse hydroxy or methoxy decorating moieties, especially when they are multiplied or mixed. The investigated fluorescence auto-quenching revealed that the decreasing fluorescence intensity correlated only with the chemical structures of the aromatic compounds.

Imidazoquinoxaline derivative EAPB0503: A promising drug targeting mutant nucleophosmin 1 in acute myeloid leukemia.

BACKGROUND: Nucleophosmin 1 (NPM1) is a nucleocytoplasmic shuttling protein mainly localized in the nucleolus. NPM1 is frequently mutated in acute myeloid leukemia (AML). NPM1c oligomerizes with wild-type nucleophosmin 1 (wt-NPM1), and this leads to its continuous cytoplasmic delocalization and contributes to leukemogenesis. Recent studies have shown that Cytoplasmic NPM1 (NPM1c) degradation leads to growth arrest and apoptosis of NPM1c AML cells and corrects wt-NPM1 normal nucleolar localization. METHODS: AML cells expressing wt-NPM1 or NPM1c or transfected with wt-NPM1 or NPM1c as well as wt-NPM1 and NPM1c AML xenograft mice were used. Cell growth was assessed with trypan blue or a CellTiter 96 proliferation kit. The cell cycle was studied with a propidium iodide (PI) assay. Caspase-mediated intrinsic apoptosis was assessed with annexin V/PI, the mitochondrial membrane potential, and poly(adenosine diphosphate ribose) polymerase cleavage. The expression of NPM1, p53, phosphorylated p53, and p21 was analyzed via immunoblotting. Localization was performed with confocal microscopy. The leukemia burden was evaluated by flow cytometry with an anti-human CD45 antibody. RESULTS: The imidazoquinoxaline 1-(3-methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (EAPB0503) induced selective proteasome-mediated degradation of NPM1c, restored wt-NPM1 nucleolar localization in NPM1c AML cells, and thus yielded selective growth arrest and apoptosis. Introducing NPM1c to cells normally harboring wt-NPM1 sensitized them to EAPB0503 and led to their growth arrest. Moreover, EAPB0503 selectively reduced the leukemia burden in NPM1c AML xenograft mice. CONCLUSIONS: These findings further reinforce the idea of targeting the NPM1c oncoprotein to eradicate leukemic cells and warrant a broader preclinical evaluation and then a clinical evaluation of this promising drug. Cancer 2017;123:1662-1673. © 2017 American Cancer Society.

Lipid nanocapsules formulation and cellular activities evaluation of a promising anticancer agent: EAPB0503.

OBJECTIVE: EAPB0503, lead compound of imiqualines, presented high antitumor activities but also a very low water solubility which was critical for further preclinical studies. To apply to EAPB0503, a robust and safe lipid formulation already used for poor soluble anticancer agents for injectable administration at a concentration higher than 1 mg/mL. MATERIALS AND METHODS: Physicochemical properties of EAPB0503 were determined to consider an adapted formulation. In a second time, lipid nanocapsules (LNC) formulations based on the phase-inversion process were developed for EAPB0503 encapsulation. Then, EAPB0503 loaded-LNC were tested in vitro on different cell lines and compared to standard EAPB0503 solutions. RESULTS: Optimized EAPB0503 LNC displayed an average size of 111.7 ± 0.9 nm and a low polydispersity index of 0.059 ± 0.002. The obtained loading efficiency was higher than 96% with a drug loading of 1.7 mg/mL. A stability study showed stability during 4 weeks stored at 25°C. In vitro results highlighted similar efficiencies between LNC and standard EAPB0503 solutions prepared in dimethyl sulfoxide. CONCLUSION: In view of results obtained for loading efficiency and drug loading, the use of a LNC formulation is very interesting to permit the solubilization of a lipophilic drug and to improve its bioavailability. Preliminary tested pharmaceutical formulation applied to EAPB0503 significantly improved its water solubility and will be soon considered for future preclinical in vivo studies.

New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122µM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.