Neuroprotective effects of VCE-004.8 in a rat model of neonatal stroke.

BACKGROUND: Currently there is no effective treatment for neonatal stroke, an acute neurologic syndrome with sequelae, due to focal ischemic, thrombotic, or hemorrhagic event occurring in the perinatal period. VCE-004.8, an aminoquinone exhibiting activity on CB2 and PPARγ receptors, is neuroprotective in adult mice models of acute and chronic brain damaging conditions. We hereby aimed to study VCE-004.8 neuroprotection in a rat model of neonatal stroke. METHODS: 7-day-old (P7) Wistar rats of both sexes were submitted to Middle Cerebral Artery Occlusion (MCAO), receiving i.p. 30 min after vehicle (MCAO + VEH) or VCE-004.8 5 mg/kg (MCAO + VCE). Non-occluded rats served as controls (SHAM). MCAO consequences were assessed at P14 by MRI, histological (TUNEL staining), biochemical (lactate/n-acetyl aspartate ratio by 1H-NMR spectroscopy) and motor studies (grasp test), and at P37 assessing myelination (MBP signal), hemiparesis and hyperlocomotion. Effects of VCE-004.8 on excitotoxicity (glutamate/n-acetyl aspartate, 1H-NMR), oxidative stress (protein nitrosylation, Oxyblot) and neuroinflammation (Toll-like receptor 4 and TNFa expression, Western blot) were assessed at P14. Therapeutic window was assessed by delaying drug administration for 12 or 18 h. RESULTS: Post-MCAO administration of VCE-004.8 reduced the volume of infarct and histological and biochemical brain damage, reducing hyperlocomotion, restoring motor performance and preserving myelination, in a manner linked to the modulation of excitotoxicity, oxidative stress and neuroinflammation. VCE-004.8 was still effective being administered 12-18 h post-insult. CONCLUSIONS: These data suggest that this drug could be effective for the treatment of stroke in newborns.

VCE-005.1, an hypoxia mimetic betulinic acid derivative, induces angiogenesis and shows efficacy in a murine model of traumatic brain injury.

One of the main global causes of mortality and morbidity is traumatic brain injury (TBI). Neuroinflammation and brain-blood barrier (BBB) disruption play a pivotal role in the pathogenesis of acute and chronic TBI onset. The activation of the hypoxia pathway is a promising approach for CNS neurodegenerative diseases, including TBI. Herein, we have studied the efficacy of VCE-005.1, a betulinic acid hydroxamate, against acute neuroinflammation in vitro and on a TBI mouse model. The effect of VCE-005.1 on the HIF pathway in endothelial vascular cells was assessed by western blot, gene expression, in vitro angiogenesis, confocal analysis and MTT assays. In vivo angiogenesis was evaluated through a Matrigel plug model and a mouse model of TBI induced by a controlled cortical impact (CCI) was used to assess VCE-005.1 efficacy. VCE-005.1 stabilized HIF-1α through a mechanism that involved AMPK and stimulated the expression of HIF-dependent genes. VCE-005.1 protected vascular endothelial cells under prooxidant and pro-inflammatory conditions by enhancing TJ protein expression and induced angiogenesis both in vitro and in vivo. Furthermore, in CCI model, VCE-005.1 greatly improved locomotor coordination, increased neovascularization and preserved BBB integrity that paralleled with a large reduction of peripheral immune cells infiltration, recovering AMPK expression and reducing apoptosis in neuronal cells. Taken together, our results demonstrate that VCE-005.1 is a multitarget compound that shows anti-inflammatory and neuroprotective effects mainly by preventing BBB disruption and has the potential to be further developed pharmacologically in TBI and maybe other neurological conditions that concur with neuroinflammation and BBB disruption.

Neuroprotective Efficacy of Betulinic Acid Hydroxamate, a B55α/PP2A Activator, in Acute Hypoxia-Ischemia-Induced Brain Damage in Newborn Rats.

There is an increasing evidence of the neuroprotective effects of hypoxia inducing factor prolyl-hydroxylase inhibitors (HIF-PHDi) after hypoxic-ischemic (HI) brain damage (HIBD). We studied the neuroprotective effects of betulinic hydroxamate (BAH), a novel B55α/PP2A activator that dephosphorylates and inhibits PHD2 activity, in a rat model of neonatal HIBD. Seven-day-old (P7) Wistar rats were exposed to hypoxia after left carotid artery electrocoagulation and then received vehicle (HI + VEH) or BAH 3 mg/kg i.p. 30 min post-insult. Brain damage was assessed by magnetic resonance imaging (MRI) and neurobehavioral studies testing motor and cognitive performance at P14 and P37, as well as immunohistochemical studies (TUNEL and myelin basic protein (MBP) signal) at P37. Mechanisms of damage were assessed at P14 determining excitotoxicity (glutamate/N-acetylaspartate ratio by H+-magnetic resonance spectroscopy), oxidative stress (protein nitrosylation by Oxyblot), and inflammation (cytokine and chemokine concentration). BAH reduced brain damage volume and cell death, preventing the development of motor and working memory deficits. BAH showed a robust protective effect on myelination, restoring MBP expression at P37. BAH modulated excitotoxicity, oxidative stress, and inflammation. Most neuroprotective effects were still present despite BAH administration was delayed for 12 h, whereas beneficial effects on motor strength at P14 and on cell death and myelination at P37 were preserved even when BAH administration was delayed for 24 h. In conclusion, BAH appears as an effective neuroprotective treatment for neonatal HIBD in a manner associated with the modulation of excitotoxicity, oxidative stress, and inflammation, showing a broad therapeutic window.

Neuroprotective Effects of Betulinic Acid Hydroxamate in Intraventricular Hemorrhage-Induced Brain Damage in Immature Rats.

Intraventricular hemorrhage (IVH) is an important cause of long-term disability in extremely preterm infants, with no current treatment. We aimed to study in an IVH model in immature rats the neuroprotective effect of betulinic acid hydroxamate (BAH), a B55α/PP2A activator that inhibits the activity of the hypoxia-inducing factor prolyl-hydroxylase type 2. IVH was induced in 1-day-old (P1) Wistar rats by the left periventricular injection of Clostridial collagenase. Then, pups received i.p. vehicle or BAH 3 mg/kg single dose. At P6, P14 and P45, brain damage (area of damage, neurobehavioral deficits, Lactate/N-acetylaspartate ratio), white matter injury (WMI: corpus callosum atrophy and myelin basic protein signal reduction) and inflammation (TLR4, NF-κB and TNFα expression), excitotoxicity (Glutamate/N-acetylspartate) and oxidative stress (protein nitrosylation) were evaluated. BAH treatment did not reduce the volume of brain damage, but it did reduce perilesional tissue damage, preventing an IVH-induced increase in Lac/NAA. BAH restored neurobehavioral performance at P45 preventing WMI. BAH prevented an IVH-induced increase in inflammation, excitotoxicity and oxidative stress. In conclusion, in immature rats, BAH reduced IVH-induced brain damage and prevented its long-term functional consequences, preserving normal myelination in a manner related to the modulation of inflammation, excitotoxicity and oxidative stress.

Isomeric O-methyl cannabidiolquinones with dual BACH1/NRF2 activity.

Oxidative stress and inflammation in the brain are two key hallmarks of neurodegenerative diseases (NDs) such as Alzheimer's, Parkinson's, Huntington's and multiple sclerosis. The axis NRF2-BACH1 has anti-inflammatory and anti-oxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Activation of NRF2 or inhibition of BACH1 are, individually, promising therapeutic approaches for NDs. Compounds with dual activity as NRF2 activators and BACH1 inhibitors, could therefore potentially provide a more robust antioxidant and anti-inflammatory effects, with an overall better neuroprotective outcome. The phytocannabinoid cannabidiol (CBD) inhibits BACH1 but lacks significant NRF2 activating properties. Based on this scaffold, we have developed a novel CBD derivative that is highly effective at both inhibiting BACH1 and activating NRF2. This new CBD derivative provides neuroprotection in cell models of relevance to Huntington's disease, setting the basis for further developments in vivo.