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JM-20 is a 1,5-benzodiazepine compound fused to a dihydropyridine fraction with different pharmacological properties. However, its potential toxic effects on blood cells have not yet been reported. Thus, the present study aimed to investigate, for the first time, the possible cytotoxicity of JM-20 through cell viability, cell cycle, morphology changes, reactive species (RS) to DCFH-DA, and lipid peroxidation in human leukocytes, its hemolytic effect on human erythrocytes, and its potential DNA genotoxicity using plasmid DNA in vitro. Furthermore, the compound's ability to reduce the DPPH radical was also measured. Human blood was obtained from healthy volunteers (30 ± 10 years old), and the leukocytes or erythrocytes were immediately isolated and treated with different concentrations of JM-20. A cytoprotective effect was exhibited by 10 μM JM-20 against 1 mM tert-butyl hydroperoxide (t-but-OOH) in the leukocytes. However, the highest tested concentrations of the compound (20 and 50 μM) changed the morphology and caused a significant decrease in the cell viability of leukocytes (p < 0.05, in comparison with Control). All tested concentrations of JM-20 also resulted in a significant increase in intracellular RS as measured by DCFH-DA in these cells (p < 0.05, in comparison with Control). On the other hand, the results point out a potent antioxidant effect of JM-20, which was similar to the classical antioxidant α-tocopherol. The IC 50 value of JM-20 against the lipid peroxidation induced by (FeII) was 1.051 μM ± 0.21, while the IC 50 value of α-tocopherol in this parameter was 1.065 μM ± 0.34. Additionally, 50 and 100 μM JM-20 reduced the DPPH radical in a statistically similar way to the 100 μM α-tocopherol (p < 0.05, in comparison with the control). No significant hemolysis in erythrocytes, no cell cycle changes in leukocytes, and no genotoxic effects in plasmid DNA were induced by JM-20 at any tested concentration. The in silico pharmacokinetic and toxicological properties of JM-20, derivatives, and nifedipine were also studied. Here, our findings demonstrate that JM-20 and its putative metabolites exhibit similar characteristics to nifedipine, and the in vitro and in silico data support the low toxicity of JM-20 to mammals., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cancer remains a worldwide cause of morbidity and mortality. Investigational research efforts have included the administration of tumor-derived extracts to healthy animals. Having previously demonstrated that the administration of non-transmissible, human cancer-derived homogenates induced malignant tumors in mice, here, we examined the consequences of administering 50 or 100 µg of protein of crude homogenates from mammary carcinoma, pancreatic adenocarcinoma, and melanoma samples in 6 inoculations per week during 2 months. The concurrent control mice received homogenates of healthy donor-skin cosmetic surgery fragments. Mammary carcinoma homogenate administration did not provoke the deterioration or mortality of the animals. Multiple foci of lung adenocarcinomas with a broad expression of malignity histomarkers coexisting with small cell-like carcinomas were found. Disseminated cells, positive to classic epithelial markers, were detected in lymphoid nodes. The administration of pancreatic tumor and melanoma homogenates progressively deteriorated animal health. Pancreatic tumor induced poorly differentiated lung adenocarcinomas and pancreatic islet hyperplasia. Melanoma affected lungs with solid pseudopapillary adenocarcinomas. Giant atypical hepatocytes were also observed. The kidney exhibited dispersed foci of neoplastic cells within a desmoplastic matrix. Nuclear overlapping with hyperchromatic nuclei, mitotic figures, and prominent nuclear atypia was identified in epidermal cells. None of these changes were ever detected in the control mice. Furthermore, the incubation of zebrafish embryos with breast tumor homogenates induced the expression of c-M yc and HER-2 as tumor markers, contrasting to embryos exposed to healthy tissue-derived material. This study confirms and extends our hypothesis that tumor homogenates contain and may act as vectors for "malignancy drivers," which ultimately implement a carcinogenesis process in otherwise healthy mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Berlanga-Acosta, Arteaga-Hernandez, Garcia-Ojalvo, Duvergel-Calderin, Rodriguez-Touseiro, Lopez-Marin, Suarez-Alba, Fuentes-Morales, Mendoza-Fuentes, Fernández-Puentes, Nuñez-Figueredo and Guillen-Nieto.)

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1 H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodiazepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a promising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the development of worms after 48 h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM-20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, computational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl 3 ) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl 3 -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cerebral ischemia constitutes the most frequent type of cerebrovascular disease. The reduction of blood supply to the brain initiates the ischemic cascade starting from ionic imbalance to subsequent glutamate excitotoxicity, neuroinflammation and oxidative stress, eventually causing neuronal death. Previously, the authors have demonstrated the in vitro cytoprotective and antioxidant effects of a new arylidene malonate derivative, KM-34, against oxidizing agents like hydrogen peroxide, glutamate or Fe 3+ /ascorbate. Here, we examined for the first time the neuroprotective effect of KM-34 on ischemia/reperfusion models. In vitro, treatment with 10 and 50 μM KM-34 reduced the cellular death (propidium iodide incorporation) induced by oxygen glucose deprivation (OGD) in rat organotypic hippocampal slices cultures. In vivo, stroke was induced in male Wistar rats through middle cerebral artery occlusion (MCAO), followed by 23 h of reperfusion. KM-34 was orally administered 105 min after MCAO onset. We noticed that 1 mg/kg KM-34 reduced infarct volume and neurological score, and increased the latency to fall in the Hanging Wire test compared to vehicle-treated ischemic animals. While ischemic and sham-operated groups showed similar horizontal locomotor activity, vertical counts decreased after MCAO, suggesting that vertical movements are more sensitive to the ischemic injury. Treatment with KM-34 also alleviated the mitochondrial impairment (ROS generation, swelling and membrane potential dissipation) induced by transient MCAO but not significant alterations were found in oxidative stress parameters. Overall, the study provides preclinical evidences confirming the neuroprotective effects of a novel synthetic molecule and paved the way for future investigations regarding its therapeutic potential against brain ischemia/reperfusion injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The main function of AChE is the hydrolysis of the neurotransmitter acetylcholine (ACh) at the neuromuscular and in cholinergic brain synapses. In some pathologies, loss of cholinergic neurons may be associated with a deficiency of ACh in specific brain areas. Consequently, the study of new safe drugs that inhibit AChE is important, because they can increase ACh levels in the synaptic cleft without adverse effects. Here, we evaluated the effects of JM-20 (a benzodiazepine-dihydropyridine hybrid molecule) on cholinesterase (ChE) activities from distinct sources (AChE from Electrophorus electricus (EeAChE), human erythrocyte membranes (HsAChE (ghost)), total erythrocyte (HsAChE (erythrocyte)) and BChE from plasma (HsBChE) and purified enzyme from the horse (EcBChE)). Kinetic parameters were determined in the presence of 0.05-1.6 mM of substrate concentration. The interactions ChEs with JM-20 were performed using molecular docking simulations. JM-20 inhibited all tested AChE but not BChE. The IC 50 values were 123 nM ± 0.2 (EeAChE), 158 nM ± 0.1 (ghost HsAChE), and 172 nM ± 0.2 (erythrocytic HsAChE). JM-20 caused a mixed type of inhibition (it altered K m and V max of AChE). The molecular docking indicated the binding poses and the most plausible active isomer of JM-20. Besides giving important data for future drug design, our results help us understand the mode of action of JM-20 as a specific inhibitor of AChE enzymes., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The etiology of Parkinson's disease is not completely understood and is believed to be multifactorial. Neuronal disorders associated to oxidative stress and mitochondrial dysfunction are widely considered major consequences. The aim of this study was to investigate the effect of the synthetic arylidenmalonate derivative 5-(3,4-dihydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (KM-34), in oxidative stress and mitochondrial dysfunction induced by 6-hydroxydopamine (6-OHDA). Pretreatment (2 h) with KM-34 (1 and 10 μM) markedly attenuated 6-OHDA-induced PC12 cell death in a concentration-dependent manner. KM-34 also inhibited H 2 O 2 generation, mitochondrial swelling, and membrane potential dissipation after 6-OHDA-induced mitochondrial damage. In vivo, KM-34 treatment (1 and 2 mg/Kg) reduced percentage of asymmetry (cylinder test) and increased the vertical exploration (open field) with respect to untreated injured animals; KM-34 also reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in substantia nigra pars compacta. These results demonstrate that KM-34 present biological effects associated to mitoprotection and neuroprotection in vitro, moreover, glial response and neuroprotection in SNpc in vivo. We suggest that KM-34 could be a putative neuroprotective agent for inhibiting the progressive neurodegenerative disease associated to oxidative stress and mitochondrial dysfunction.

Objective: JM-20, a novel hybrid synthetic molecule, has been reported to have antioxidant, mitoprotective, anti-excitotoxic, anti-apoptotic and anti-inflammatory properties. However, the neuroprotective effect of JM-20 against memory impairment in preclinical AD-like models has not been analyzed. The aim of this study was to evaluate the potential neuroprotection of JM-20 that preserves essential memory process from cholinergic dysfunction and other molecular damages., Methods: The effects of JM-20 on scopolamine (1 mg/kg)-induced cognitive disorders were studied. Male Wistar rats (220-230 g) were treated with JM-20 and/or scopolamine, and behavioral tasks were performed. The AChE activity, superoxide dismutase activity, catalase activity, MDA and T-SH level on brain tissue were determined by spectrophotometric methods. Mitochondrial functionality parameters were measured after behavioral tests. Histological analyses on hippocampus and prefrontal cortex were processed with hematoxylin and eosin, and neuronal and axonal damage were determined., Results: The behavioral, biochemical and histopathological studies revealed that oral pre-treatment with JM-20 (8 mg/kg) significantly attenuated the scopolamine-induced memory deficits, mitochondrial malfunction, oxidative stress, and prevented AChE hyperactivity probably due to specific inhibition of AChE enzyme. It was also observed marked histological protection on hippocampal and prefrontal-cortex regions., Conclusions: The multimodal action of this molecule could mediate the memory protection here observed and suggest that it may modulate different pathological aspects of memory deficits associated with AD in humans.

Stroke is frequently associated with severe neurological decline and mortality, and its incidence is expected to increase due to aging population. The only available pharmacological treatment for cerebral ischemia is thrombolysis, with narrow therapeutic windows. Efforts aimed to identify new therapeutics are crucial. In this study, we look into plausible molecular and cellular targets for JM-20, a new hybrid molecule, against ischemic stroke in vivo. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) following 23 h of reperfusion. Animals treated with 8 mg/kg JM-20 (p.o., 1 h after reperfusion) showed minimal neurological impairment and lower GABA and IL-1β levels in CSF when compared to damaged rats that received vehicle. Immunocontent of pro-survival, phosphorylated Akt protein decreased in the cortex after 24 h as result of the ischemic insult, accompanied by decreased number of NeuN + cells in the peri-infarct cortex, cornu ammonis 1 (CA1) and dentate gyrus (DG) areas. Widespread reactive astrogliosis in both cortex and hippocampus (CA1, CA3, and DG areas) was observed 24 h post-ischemia. JM-20 prevented the activated Akt reduction, neuronal death, and astrocytes reactivity throughout the brain. Overall, the results reinforce the pharmacological potential of JM-20 as neuroprotective agent and provide important evidences about its molecular and cellular targets in this model of cerebral ischemia.

Ischemic stroke is a major cause of death and disability worldwide. Thrombolysis by tissue plasminogen activator is the only pharmacological treatment approved for clinical practice, but has a narrow therapeutic window and poor efficacy when the cell death cascade is activated. Numerous drugs that are thought to protect neurons against injury have previously failed in human trials despite showing efficacy in experimental models of stroke. Herein, we reviewed the main pre-clinical results of the neuroprotective effects of JM-20, a new hybrid molecule, against brain ischemia. JM-20 appears to protect the brain from ischemic damage by interfering with several elements of the ischemic cascade: antiexcitotoxic, anticalcic, antioxidant, antiapoptotic, and anti-inflammatory. Its ability to protect not only neurons but also glial cells together with its ability to target and preserve mitochondrial function makes JM-20 a promising molecule that may be able to shield the whole neurovascular unit. The multimodal and multi-cell action of JM-20 may explain the high degree of protection observed in a rat model of brain ischemia, as assayed through histological (hematoxylin-eosin, and luxol fast blue staining), neurochemical (glutamate and aspartate levels in cerebrospinal fluid), mitochondrial functionality and behavioural (neurological scale) analysis at doses of 4 and 8mg/kg. Furthermore, the wide therapeutic window of JM-20 of 8h also suggests that this molecule could be of potential interest in situations where brain perfusion is compromised., (Copyright © 2018 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Free radicals are important mediators in a number of neurodegenerative diseases and molecules capable of scavenging reactive oxygen species (ROS) may be a feasible strategy for protecting neuronal cells. In this sense, polyphenols have been studied for their antioxidant effects, KM-34 (5-(3, 4-dydroxyl-benzylidene)-2, 2-dimethyl-1, 3-dioxane-4, 6-Dione) is a novel synthetic catechol with potential neuroprotective and antioxidant properties. We have assessed the antioxidant (as scavenging and iron-chelating compound) and neuroprotectant in vitro (in PC12 cell injury induced by H 2 O 2 , glutamate or FeSO 4 /AA) of KM-34. KM-34 was found to be a potent antioxidant, as shown by (i) inhibition of iron induced-brain lipid peroxidation, (ii) inhibition of 2-deoxyribose degradation, (iii) inhibition of superoxide radicals generation (IC 50 =11.04 μM) and (iv) inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reduction (IC 50 =16.26 μM). The overall anti-oxidant action of KM-34 appears to be a combination of a direct reaction with free radicals and chelating the metal ions responsible for the production of ROS. Our work suggests that the antioxidant properties of KM-34 may provide future therapeutic approaches for neurodegenerative disorders., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© Georg Thieme Verlag KG Stuttgart · New York.)

Ethnopharmacological Relevance: Amburana cearensis (Allemao) A.C.Sm. is a medicinal plant of the Brazilian Caatinga reported to present antioxidant and anti-inflammatory activity. This study aimed to evaluate the neuroprotective effect of the extracts obtained from the seeds of A. cearensis in primary cultures of cerebellar cells subjected to excitotoxicity induced by glutamate and brain mitochondria submitted to oxidative stress., Materials: and methods: Primary cultures of cerebellar cells were treated with the ethanol (ETAC), hexane (EHAC), dichloromethane (EDAC) and ethyl acetate (EAAC) extracts of the seeds of A.cearensis and subjected to excitotoxicity induced by glutamate (10µM). Mitochondria isolated from rat brains were submitted to oxidative stress and treated with ETAC., Results: Only the EHAC extract reduced cell viability by 30% after 72h of treatment. Morphological analyses by Immunofluorescence showed positive staining for glutamine synthetase, β-III tubulin, GFAP and IBA1 similar to control cultures, indicating a better preservation of astrocytes, neurons and microglia, after excitotoxic damage induced by glutamate in cerebellar cultures treated with the extracts. The ETAC extract also protected mitochondria isolated from rat brains from oxidative stress, reducing the swelling, dissipation of the membrane potential, ROS production and calcium influx., Conclusion: Thus, this study suggests that the seed extracts from A. Cearensis exhibit neuroprotective potential against oxidative stress and excitotoxicity induced by glutamate and can be considered a potential therapeutic agent in the treatment of neurodegenerative diseases., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Stroke represents one of the first causes of mortality and morbidity worldwide. We evaluated the therapeutic potential of a novel semi-synthetic spirosteroid sapogenin derivative "S15" in a transient middle cerebral artery occlusion (tMCAO) focal ischemia model in rat. S15-treated rats had significantly reduced infarct volumes and improved neurological functions at 24h post-reperfusion, compared with ischemia. Corresponding gene expression changes in brain were characterized by mRNA sequencing and qPCR approaches. Next, we applied geneset, pathway and transcription factor motif enrichment analysis to identify relevant signaling networks responsible for neuronal damage upon ischemia-reperfusion or neuroprotection upon pretreatment with S15. As expected, ischemia-reperfusion brain damage strongly modulates transcriptional programs associated with immune responses, increased differentiation of immune cells as well as reduced (cat)ion transport and synaptic activity. Interestingly, S15-dependent neuroprotection regulates inflammation-associated genes involved in phagosome specific resolution of tissue damage, chemotaxis and anti-inflammatory alternative activation of microglia. Altogether our transcriptome wide RNA sequencing and integrated pathway analysis provides new clues in the neuroprotective properties of a novel spirosteroid S15 or neuronal damage in rat brains subjected to ischemia, which opens new perspectives for successful treatment of stroke., (Copyright © 2017. Published by Elsevier Inc.)

Objective: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action., Methods: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function., Results: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H 2 O 2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly., Conclusion: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

Two spirosteroid analogues were synthesized and evaluated for their in vitro neuroprotective activities in PC12 cells, against glutamate-induced excitotoxicity and mitochondrial damage in glucose deprivation conditions, as well as their anti-inflammatory potential in LPS/IFNγ-stimulated microglia primary cultures. We also evaluated the in vitro anti-excitotoxic and anti-inflammatory activities of natural and endogenous steroids. Our results show that the plant-derived steroid solasodine decreased PC12 glutamate-induced excitotoxicity, but not the cell death induced by mitochondrial damage and glucose deprivation. Among the two synthetic spirosteroid analogues, only the (25R)-5α-spirostan-3,6-one (S15) protected PC12 against ischemia-related in vitro models and inhibited NO production, as well as the release of IL-1β by stimulated primary microglia. These findings provide further insights into the role of specific modifications of the A and B rings of sapogenins for their neuroprotective potential.

Cerebral ischemia is the third most common cause of death and a major cause of disability worldwide. Beyond a shortage of essential metabolites, ischemia triggers many interconnected pathophysiological events, including excitotoxicity, oxidative stress, inflammation and apoptosis. Here, we investigated the neuroprotective mechanisms of JM-20, a novel synthetic molecule, focusing on the phosphoinositide-3-kinase (PI3K)/Akt survival pathway and glial cell response as potential targets of JM-20. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD) to achieve ischemic/reperfusion damage in vitro. Treatment with JM-20 at 0.1 and 10 μM reduced PI incorporation (indicative of cell death) after OGD. OGD decreased the phosphorylation of Akt (pro-survival) and GSK 3β (pro-apoptotic), resulting in respective inhibition and activation of these proteins. Treatment with JM20 prevented the reduced phosphorylation of these proteins after OGD, representing a shift from pro-apoptotic to pro-survival signaling. The OGD-induced activation of caspase-3 was also attenuated by JM-20 treatment at 10 μM. Moreover, in cultures treated with JM-20 and exposed to OGD conditioning, we observed a decrease in activated microglia, as well as a decrease in interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α release into the culture medium, while the level of the anti-inflammatory IL-10 increased. GFAP immunostaining and IB4 labeling showed that JM-20 treatment significantly augmented GFAP immunoreactivity after OGD, when compared with cultures exposed to OGD only, suggesting the activation of astroglial cells. Our results confirm that JM-20 has a strong neuroprotective effect against ischemic injury and suggest that the mechanisms involved in this effect may include the modulation of reactive astrogliosis, as well as neuroinflammation and the anti-apoptotic cell signaling pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Three main types of Cuban propolis directly related to their secondary metabolite composition have been identified: brown, red and yellow propolis; the former is majoritarian and is characterized by the presence of nemorosone. In this study, brown Cuban propolis extracts were found cytotoxic against HepG2 cells and primary rat hepatocytes, in close association with the nemorosone contents. In mitochondria isolated from rat liver the extracts displayed uncoupling activity, which was demonstrated by the increase in succinate-supported state 4 respiration rates, dissipation of mitochondrial membrane potential, Ca(2+) release from Ca(2+)-loaded mitochondria, and a marked ATP depletion. As in cells, the degree of such mitotoxic events was closely correlated to the nemorosone content. The propolis extracts that do not contain nemorosone were neither cytotoxic nor mitotoxic, except R-29, whose detrimental effect upon cells and mitochondria could be mediated by its isoflavonoids and chalcones components, well known mitochondrial uncouplers. Our results at least partly unravel the cytotoxic mechanism of Cuban propolis, particularly regarding brown propolis, and raise concerns about the toxicological implication of Cuban propolis consumption., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel benzodiazepine dihydropyridine hybrid molecule, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effect of JM-20 on the functionality of the glutamatergic system has not been investigated. In this study, by using different in vitro preparations, we investigated the effects of JM-20 on (i) rat brain synaptic vesicles (L-[(3)H]-glutamate uptake, proton gradient built-up and bafilomycin-sensitive H(+)-ATPase activity), (ii) rat brain synaptosomes (glutamate release) and (iii) primary cultures of rat cortical neurons, astrocytes and astrocyte-neuron co-cultures (L-[(3)H]-glutamate uptake and glutamate release). We observed here that JM-20 impairs H(+)-ATPase activity and consequently reduces vesicular glutamate uptake. This molecule also inhibits glutamate release from brain synaptosomes and markedly increases glutamate uptake in astrocytes alone, and co-cultured neurons and astrocytes. The impairment of vesicular glutamate uptake by inhibition of the H(+)-ATPase caused by JM-20 could decrease the amount of the transmitter stored in synaptic vesicles, increase the cytosolic levels of glutamate, and will thus down-regulate neurotransmitter release. Together, these results contribute to explain the anti-excitotoxic effect of JM-20 and its strong neuroprotective effect observed in different in vitro and in vivo models of brain ischemia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Because mitochondrial oxidative stress and impairment are important mediators of neuronal damage in neurodegenerative diseases and in brain ischemia/reperfusion, in the present study, we evaluated the antioxidant and mitoprotective effect of a new promising neuroprotective molecule, JM-20, in mitochondria and synaptosomes isolated from rat brains. JM-20 inhibited succinate-mediated H₂O₂ generation in both mitochondria and synaptosomes incubated in depolarized (high K(+)) medium at extremely low micromolar concentration and with identical IC₅₀ values of 0.91 μM. JM-20 also repressed glucose-induced H₂O₂ generation stimulated by rotenone or by antimycin A in synaptosomes incubated in high sodium-polarized medium at extremely low IC₅₀ values of 0.395 μM and 2.452 μM, respectively. JM-20 was unable to react directly with H₂O₂ or with superoxide anion radicals but displayed a cathodic reduction peak at -0.71V, which is close to that of oxygen (-0.8V), indicating high electron affinity. JM-20 also inhibited uncoupled respiration in mitochondria or synaptosomes and was a more effective inhibitor in the presence of the respiratory substrates glutamate/malate than in the presence of succinate. JM-20 also prevented Ca(2+)-induced mitochondrial permeability transition pore opening, membrane potential dissipation and cytochrome c release, which are key pathogenic events during stroke. This molecule also prevented Ca(2+) influx into synaptosomes and mitochondria; the former effect was a consequence of the latter because JM-20 inhibition followed the patterns of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a classic mitochondrial uncoupler. Because the mitochondrion is considered an important source and target of neuronal cell death signaling after an ischemic insult, the antioxidant and protective effects of JM-20 against the deleterious effects of Ca(2+) observed at the mitochondrial level in this study may endow this molecule with the ability to succeed in mitochondrion-targeted strategies to combat ischemic brain damage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We previously showed that JM-20, a novel 1,5-benzodiazepine fused to a dihydropyridine moiety, possessed an anxiolytic profile similar to diazepam and strong neuroprotective activity in different cell models relevant to cerebral ischemia. Here, we investigated whether JM-20 protects against ischemic neuronal damage in vitro and in vivo. The effects of JM-20 were evaluated on hippocampal slices subjected to oxygen and glucose deprivation (OGD). For in vivo studies, Wistar rats were subjected 90 min of middle cerebral artery occlusion (MCAo) and oral administration of JM-20 at 2, 4 and 8 mg/kg 1 h following reperfusion. Twenty-four hours after cerebral blood flow restoration, neurological deficits were scored, and the infarct volume, histopathological changes in cortex, number of hippocampal and striatal neurons, and glutamate/aspartate concentrations in the cerebrospinal fluid were measured. Susceptibility to brain mitochondrial swelling, membrane potential dissipation, H2O2 generation, cytochrome c release, Ca2+ accumulation, and morphological changes in the organelles were assessed 24 h post-ischemia. In vitro, JM-20 (1 and 10 μM) administered during reperfusion significantly reduced cell death in hippocampal slices subjected to OGD. In vivo, JM-20 treatment (4 and 8 mg/kg) significantly decreased neurological deficit scores, edema formation, total infarct volumes and histological alterations in different brain regions. JM-20 treatment also protected brain mitochondria from ischemic damage, most likely by preventing Ca2+ accumulation in organelles. Moreover, an 8-mg/kg JM-20 dose reduced glutamate and aspartate concentrations in cerebrospinal fluid and the deleterious effects of MCAo even when delivered 8 h after blood flow restoration. These results suggest that in rats, JM-20 is a robust neuroprotective agent against ischemia/reperfusion injury with a wide therapeutic window. Our findings support the further examination of potential clinical JM-20 use to treat acute ischemic stroke., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca(2+) influx, stimulation of Ca(2+) efflux in Ca(2+)-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The ischemic stroke cascade is composed of several pathophysiological events, providing multiple targets for pharmacological intervention. JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel hybrid molecule, in which a benzodiazepine portion is covalently linked to a dihydropyridine ring, forming a new chemical entity with potential multisite neuroprotective activity. In the present study, JM-20 prevented PC-12 cell death induced either by glutamate, hydrogen peroxide or KCN-mediated chemical hypoxia. This molecule also protected cerebellar granule neurons from glutamate or glutamate plus pentylenetetrazole-induced damage at very low micromolar concentrations. In rat liver mitochondria, JM-20, at low micromolar concentrations, prevented the Ca2+-induced mitochondrial permeability transition, as assessed by mitochondrial swelling, membrane potential dissipation and organelle release of the pro-apoptotic protein cytochrome c. JM-20 also inhibited the mitochondrial hydrolytic activity of F1F0-ATP synthase and Ca2+ influx. Therefore, JM-20 may be a multi-target neuroprotective agent, promoting reductions in neuronal excitotoxic injury and the protection of the mitochondria from Ca2+-induced impairment as well as the preservation of cellular energy balance.

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²⁺ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells., (Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)