Development of a local controlled release system for therapeutic proteins in the treatment of skeletal muscle injuries and diseases.

The present study aims to develop and characterize a controlled-release delivery system for protein therapeutics in skeletal muscle regeneration following an acute injury. The therapeutic protein, a membrane-GPI anchored protein called Cripto, was immobilized in an injectable hydrogel delivery vehicle for local administration and sustained release. The hydrogel was made of poly(ethylene glycol)-fibrinogen (PEG-Fibrinogen, PF), in the form of injectable microspheres. The PF microspheres exhibited a spherical morphology with an average diameter of approximately 100 micrometers, and the Cripto protein was uniformly entrapped within them. The release rate of Cripto from the PF microspheres was controlled by tuning the crosslinking density of the hydrogel, which was varied by changing the concentration of poly(ethylene glycol) diacrylate (PEG-DA) crosslinker. In vitro experiments confirmed a sustained-release profile of Cripto from the PF microspheres for up to 27 days. The released Cripto was biologically active and promoted the in vitro proliferation of mouse myoblasts. The therapeutic effect of PF-mediated delivery of Cripto in vivo was tested in a cardiotoxin (CTX)-induced muscle injury model in mice. The Cripto caused an increase in the in vivo expression of the myogenic markers Pax7, the differentiation makers eMHC and Desmin, higher numbers of centro-nucleated myofibers and greater areas of regenerated muscle tissue. Collectively, these results establish the PF microspheres as a potential delivery system for the localized, sustained release of therapeutic proteins toward the accelerated repair of damaged muscle tissue following acute injuries.

Methacrylated fibrinogen hydrogels for 3D cell culture and delivery.

Methacrylation was performed on fibrinogen to design a new biomedical hydrogel for 3D cell culture or as a biodegradable delivery matrix for in vivo implantation. The methacrylation of denatured fibrinogen in solution was performed using methacrylic anhydride (MAA). The extent of fibrinogen methacrylation was quantified by proton NMR and controlled using stochiometric quantities of MAA during the reaction. The methacrylated fibrinogen (FibMA) hydrogels were formed by light-activated free-radical polymerization in the presence of macromolecular cross-linking polymers made from acrylated poly(ethylene glycol) (PEG). The biocompatibility and biodegradability of the FibMA hydrogels were characterized by in vitro assays and in vivo implantation experiments using quantitative magnetic resonance imaging (MRI) of the implant volume. The FibMA supported the growth and metabolic activity of human dermal fibroblasts in both 2D and 3D cultures. The methacrylation did not alter important biological attributes of the fibrinogen, including the ability to support cell adhesion and 3D cell culture, as well as to undergo proteolysis. Animal experiments confirmed the biodegradability of the FibMA for potential use as a scaffold in tissue engineering, as a bioink for 3D bioprinting, or as a biodegradable matrix for in vivo sustained delivery of bioactive factors. STATEMENT OF SIGNIFICANCE: This paper describes methacrylated fibrinogen (FibMA) and the formation of a biomedical hydrogel from FibMA for cell culture and other biomedical applications. Inspired from methacrylated gelatin (GelMA), the FibMA is made from blood-derived fibrinogen which is more suitable for clinical use. Sharing similar properties to other hydrogels made from methacrylated proteins, the FibMA has yet to be reported in the literature. In this manuscript, we provide the methodology to produce the FibMA hydrogels, we document the mechanical versatility of this new biomaterial, and we show the biocompatibility using 3D cell culture studies and in vivo implantations.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2'-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD.

The novel multitarget iron chelating and propargylamine drug M30 affects APP regulation and processing activities in Alzheimer's disease models.

In many of the neurodegenerative diseases, such as Alzheimer's disease (AD) and AD-related disorders, as well as in the regular ageing process, excessive generation of oxidative stress (OS) and accumulation of iron levels and deposition have been observed in specific affected-brain regions and thus, regarded as contributing factors to the pathogenesis of the diseases. In AD, iron promotes amyloid ß (Aß) neurotoxicity by producing free radical damage and OS in brain areas affected by neurodegeneration, presumably by facilitating the aggregation of Aß. In addition, it was shown that iron modulates intracellular levels of the holo amyloid precursor protein (APP) by iron-responsive elements (IRE) RNA stem loops in the 5' untranslated region (5'UTR) of the APP transcript. As a consequence of these observations, iron chelation is one of the major new therapeutic strategies for the treatment of AD. This review describes the benefits and importance of the multimodal brain permeable chimeric iron-chelating/propargylamine drug M30, concerning its neuroprotective/neurorestorative inter-related activities relevant of the pathological features ascribed to AD, with a special focus on the effect of the drug on APP regulation and processing.

A Novel Iron Chelator-Radical Scavenger Ameliorates Motor Dysfunction and Improves Life Span and Mitochondrial Biogenesis in SOD1G93A ALS Mice.

The aim of the present study was to evaluate the therapeutic effect of the novel neuroprotective multitarget brain permeable monoamine oxidase inhibitor/iron chelating-radical scavenging drug, VAR10303 (VAR), co-administered with high-calorie/energy-supplemented diet (ced) in SOD1G93A transgenic amyotrophic lateral sclerosis (ALS) mice. Administration of VAR-ced was initiated after the appearance of disease symptoms (at day 88), as this regimen is comparable with the earliest time at which drug therapy could start in ALS patients. Using this rescue protocol, we demonstrated in the current study that VAR-ced treatment provided several beneficial effects in SOD1G93A mice, including improvement in motor performance, elevation of survival time, and attenuation of iron accumulation and motoneuron loss in the spinal cord. Moreover, VAR-ced treatment attenuated neuromuscular junction denervation and exerted a significant preservation of myofibril regular morphology, associated with a reduction in the expression levels of genes related to denervation and atrophy in the gastrocnemius (GNS) muscle in SOD1G93A mice. These effects were accompanied by upregulation of mitochondrial DNA and elevated activities of complexes I and II in the GNS muscle. We have also demonstrated that VAR-ced treatment upregulated the mitochondrial biogenesis master regulator, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and increased PGC-1α-targeted metabolic genes and proteins, such as, PPARγ, UCP1/3, NRF1/2, Tfam, and ERRα in GNS muscle. These results provide evidence of therapeutic potential of VAR-ced in SOD1G93A mice with underlying molecular mechanisms, further supporting the importance role of multitarget iron chelators in ALS treatment.

Neurorescue by a ROS Decomposition Catalyst.

The effect of the bis-sulfonated iron(III) corrole (1-Fe), a potent decomposition catalyst of reactive oxygen species, on rescuing SN4741 cells that were damaged by 6-hydroxydopamine (6-OHDA) was investigated as an in vitro model system for studying cell death of dopaminergic neurons in the substantia nigra. Important findings that accompanied the ability to rescue dopaminergic neurons were increased expression of phenotypic dopaminergic proteins, such as tyrosine hydroxylase (TH) and dopamine transporter (DAT), which were significantly depleted upon 6-OHDA-mediated damage. 1-Fe also elevated expression levels of aldehyde dehydrogenase 1 (ALDH-1), previously disclosed as a cardinal protein in the pathogenesis of Parkinson's disease. Since these findings suggested that 1-Fe affects quite a wide range of intracellular mechanisms, vital intracellular pathways that involve neuroplasticity, growth, differentiation and survival of neurons, were examined. Phosphatidylinositol 3-kinase (PI3K) and protein kinase c (PKC) were found to be involved, as pharmacological inhibitors of these kinases abolished the neurorescue effect of 1-Fe. 1-Fe also elevated the expression of antiapoptotic protein Bcl-2, which is essential for proper mitochondrial function and cellular survival. The overall conclusion is that 1-Fe is capable of rescuing already damaged neuronal cells by a variety of mechanisms that are beyond its antioxidant activity.

Neuroprotective effects of multifaceted hybrid agents targeting MAO, cholinesterase, iron and ß-amyloid in ageing and Alzheimer's disease.

UNLABELLED: Alzheimer's disease (AD) is accepted nowadays as a complex neurodegenerative disorder with multifaceted cerebral pathologies, including extracellular deposition of amyloid ß peptide-containing plaques, intracellular neurofibrillary tangles, progressive loss of cholinergic neurons, metal dyshomeostasis, mitochondrial dysfunction, neuroinflammation, glutamate excitoxicity, oxidative stress and increased MAO enzyme activity. This may explain why it is currently widely accepted that a more effective therapy for AD would result from the use of multifunctional drugs, which may affect more than one brain target involved in the disease pathology. The current review will discuss the potential benefits of novel multimodal neuroprotective, brain permeable drugs, recently developed by Youdim and collaborators, as a valuable therapeutic approach for AD treatment. The pharmacological and neuroprotective properties of these multitarget-directed ligands, which target MAO enzymes, the cholinergic system, iron accumulation and amyloid ß peptide generation/aggregation are described, with a special emphasis on their potential therapeutic value for ageing and AD-associated cognitive functions. This review is conceived as a tribute to the broad neuropharmacology work of Professor Moussa Youdim, Professor Emeritus in the Faculty of Medicine and Director of Eve Topf Center of Excellence in Technion-Israel Institute of Technology, and Chief Scientific Officer of ABITAL Pharma Pipeline Ltd., at the occasion of his 75th birthday. LINKED ARTICLES: This article is part of a themed section on Updating Neuropathology and Neuropharmacology of Monoaminergic Systems. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.13/issuetoc.

Neuroprotective and neurorestorative potential of propargylamine derivatives in ageing: focus on mitochondrial targets.

The mitochondrial theory of ageing proposes that accumulation of damage to mitochondrial function and DNA mutation lead to ageing of humans and animals. It has been suggested that mitochondria play dynamic roles in regulating synaptogenesis and morphological/functional responses of synaptic activity, and thus, deteriorating of mitochondrial function (e.g., deficits of the mitochondrial respiratory enzymes, reduced calcium influx, increased accumulation of mitochondrial DNA defects/apoptotic proteins and impairment of mitochondrial membrane potential) can lead to severe neuronal energy deficit, and in the long run, to modifications in neuronal synapses and neurodegeneration in the ageing brain. Hence, considering the mechanisms by which mitochondrial impairment can lead to neuronal death, the development of neuroprotective molecules that target various mitochondrial pathogenic processes can be effective in the treatment of ageing and age-related neurodegenerative diseases. This review addresses several aspects of the neuroprotective effects of propargylamine derivatives (e.g., the monoamine oxidase-B inhibitors, selegiline and rasagiline and the multifunctional drugs, ladostigil, M30 and VAR10303) in ageing with a special focus on mitochondrial molecular protective mechanisms.

Effect of long-term treatment with rasagiline on cognitive deficits and related molecular cascades in aged mice.

The present study aimed to investigate the protective effects of prolonged treatment with the selective, irreversible monoamine oxidase-B inhibitor, novel anti-parkinsonian drug, rasagiline (Azilect) in aged animals. Our findings from behavioral experiments demonstrated that long-term treatment of aged mice with rasagiline (0.2 mg/kg) exerted significant beneficial effects on mood-related dysfunction and spatial learning and memory functions. At this dose of rasagiline, chronic drug administration significantly inhibited monoamine oxidase-B activity and caused an increase in striatal dopamine and serotonin levels, while decreasing their metabolism. In addition, rasagiline treatment elevated striatal mRNA expression levels of dopamine receptors D1 and D2. Furthermore, we found that rasagiline upregulated expression levels of the synaptic plasticity markers brain-derived neurotrophic factor, tyrosine kinase-B receptor, and synapsin-1, increased Bcl-2 to Bax antiapoptotic ratio and the activity of the antioxidant enzyme, catalase in brain of aged mice. The present study demonstrated that long-term treatment with rasagiline could affect behavioral deficits in aged mice and upregulate various neuroprotective parameters in the aging brain, indicating that the drug may have therapeutic potential for treatment of age-associated neurodegenerative disorders.

Beneficial behavioral, neurochemical and molecular effects of 1-(R)-aminoindan in aged mice.

Previous neuroprotective studies demonstrated that 1-(R)-aminoindan (AI), which is the major metabolite of the anti-Parkinsonian drug rasagiline, possesses beneficial pharmacological effects in various cell culture and animal models of neurodegeneration. The present study was aimed at investigating the possible neuroprotective effects of AI on cognitive impairments and neurochemical alterations in aged mice. Our findings provide evidence that following chronic systemic treatment with AI (5 mg/kg; daily; 3 months) of aged mice (24 months old), the compound exerted a significant positive impact on neuropsychiatric functions and cognitive behavior deficits, assessed in a variety of tasks (spatial learning and memory retention, working memory, learning abilities and nest building behavior) and produced an antidepressant-like effect. In addition, chronic AI treatment significantly enhanced expression levels of neurotrophins, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), tyrosine kinase- B (Trk-B) receptor and synaptic plasticity markers, such as synapsin-1 and growth-associated protein-43 (GAP-43) in the striatum and hippocampus in aged mice. Our results also indicate that AI treatment up-regulated the expression levels of the pro-survival Bcl-2 mRNA, increased the anti-apoptotic index Bcl-2/Bax and enhanced the activity of the antioxidant enzyme catalase in the brain of aged mice. These effects of AI were also confirmed in aged rats (24 months old). Altogether, the present findings indicate that AI can induce neuroprotective effects on age-related alterations in neurobehavioral functions and exerts neurotrophic up-regulatory and anti-apoptotic properties in aged animals.

Neuroprotective and neurorestorative activities of a novel iron chelator-brain selective monoamine oxidase-A/monoamine oxidase-B inhibitor in animal models of Parkinson's disease and aging.

Recently, we have designed and synthesized a novel multipotent, brain-permeable iron-chelating drug, VAR10303 (VAR), possessing both propargyl and monoamine oxidase (MAO) inhibitory moieties. The present study was undertaken to determine the multiple pharmacological activities of VAR in neurodegenerative preclinical models. We demonstrate that VAR affords iron chelating/iron-induced lipid-peroxidation inhibitory potency and brain selective MAO-A and MAO-B inhibitory effects, with only limited tyramine-cardiovascular potentiation of blood pressure. The results show that in 6-hydroxydopamine rat (neuroprotection) and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse (neurorescue) Parkinson's disease models, VAR significantly attenuated the loss of striatal dopamine levels, markedly reduced dopamine turnover, and increased tyrosine-hydroxylase levels. Furthermore, chronic systemic treatment of aged rats with VAR improved cognitive behavior deficits and enhanced the expression levels of neurotrophic factors (e.g., brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and nerve growth factor), Bcl-2 family members and synaptic plasticity in the hippocampus. Our study indicates that the multitarget compound VAR exerted neuroprotective and neurorestorative effects in animal models of Parkinson's disease and aging, further suggesting that a drug that can regulate multiple brain targets could be an ideal treatment-strategy for age-associated neurodegenerative disorders.

Molecular targets of the multifunctional iron-chelating drug, M30, in the brains of mouse models of type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Neurodegenerative diseases are now recognized to be multifunctional, whereby a heterogeneous set of reactions acts independently or cooperatively, leading eventually to the demise of neurons. This has led our group to design and synthesize the multifunctional, nontoxic, brain-permeable, iron chelator compound M30 with a range of pharmacological properties. Here, we have characterized the molecular targets of M30 in the brains of animal models of type 2 diabetes mellitus (T2DM). EXPERIMENTAL APPROACH: Effects of M30 on molecular mechanisms associated with neuroprotection in the CNS were investigated-in the high-fat diet (HFD) and ob/ob transgenic mouse models of T2DM, using real-time PCR and Western blotting analyses. Brain monoamine oxidase (MAO) activity and catecholamine levels, and peripheral glucose tolerance were assayed after treatment in vivo. KEY RESULTS: M30 increased cerebral levels of insulin and insulin receptor and phosphorylated-GSK-3ß in HFD mice, compared with vehicle-treated HFD mice. In both T2DM mice models, M30 treatment significantly up-regulated cerebral hypoxia-inducible factor (HIF)-1α protein levels and induced the expression of several HIF-1 target genes involved in neuroprotection, glycolysis, neurogenesis, oxidative stress and anti-inflammation. Additionally, M30 inhibited MAO-A and -B activities in the cerebellum. Accordingly, M30 administration significantly reduced brain levels of dopamine metabolites and increased levels of 5-HT and noradrenaline. Glucose tolerance was also improved after M30 treatment in both models of T2DM. CONCLUSIONS AND IMPLICATIONS: In the brain of HFD and ob/ob transgenic mice, M30 exerted a variety of beneficial neuroprotective regulatory effects that may act synergistically to delay or prevent neurodegenerative processes associated with T2DM.

The novel multi-target iron chelator, M30 modulates HIF-1α-related glycolytic genes and insulin signaling pathway in the frontal cortex of APP/PS1 Alzheimer's disease mice.

Increasing evidence suggests that dysregulation of brain insulin/insulin receptor (InsR) and insulin signaling cascade are associated with the pathogenesis of Alzheimer's disease (AD). Our group has designed and synthesized a series of multi-target iron chelating, brain permeable compounds for AD. One leading multi-target compound, M30 possesses the neuroprotective N-propargyl moiety of the anti-Parkinsonian, monoamine oxidase (MAO)-B inhibitor, rasagiline (Azilect®) and the antioxidant-iron chelating moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Positive outcomes for the behavioral/cognitive and neuroprotective effects of M30 were recently obtained in preclinical experimental studies, regarding pathological aspects relevant to ageing and AD. We report that chronic treatment with M30 (1 and 5 mg/kg p.o; three times a week for 9 months) significantly elevated cortical insulin and InsR transcript and protein expression, respectively and increased the phosphorylated form of glycogen synthase kinase-3ß in the frontal cortex of amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice. In addition, M30 treatment upregulated the levels of hypoxia-inducible factor (HIF)-1α and expression of its target genes involved in glycolysis including, aldolase A, enolase-1 and glucose transporter-1 (Glut-1), in the frontal cortex of APP/PS1 mice. Treatment with M30 also lead to an increase in the hepatic protein expression levels of InsR and Glut-1 and lowered the increase in blood glucose levels following glucose tolerance test. The present findings indicate that the multifunctional iron chelating drug, M30 regulates major brain glucose metabolism parameters and thus, might be beneficial for AD, in which impaired neuronal insulin signaling and Glut expression have been implicated.

Effects of novel neuroprotective and neurorestorative multifunctional drugs on iron chelation and glucose metabolism.

Iron accumulation and iron-related oxidative stress are involved in several pathological conditions and provide a rationale for the development of iron chelators as novel promising therapeutic strategies. Thus, we have recently synthesized multifunctional non-toxic, brain permeable iron chelating compounds, M30 and HLA20, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase (MAO)-B inhibitor, rasagiline and the antioxidant-iron chelating moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Here, we examined the hepatic regulatory effects of these novel compounds using two experimental approaches: chelation activity and glucose metabolism parameters. The present study demonstrated that M30 and HLA20 significantly decreased intracellular iron content and reduced ferritin expression levels in iron-loaded hepatoma Hep3B cells. In electron microscopy analysis, M30 was shown to reduce the electron-dense deposits of siderosomes by ~30 %, as well as down-regulate cytosolic ferritin particles observed in iron-overloaded cells. In vivo studies demonstrated that M30 administration (1 mg/kg, P.O. three times a week) reduced hepatic ferritin levels; increased hepatic insulin receptor and glucose transporter-1 levels and improved glucose tolerance in C57BL/6 mice and in a mouse model of type-2 diabetes, the ob/ob (leptin(-/-)). The results clearly indicate that the novel multifunctional drugs, especially M30, display significant capacity of chelating intracellular iron and regulating glucose metabolism parameters. Such effects can have therapeutic significance in conditions with abnormal local or systemic iron metabolism, including neurological diseases.

I1 imidazoline receptor: novel potential cytoprotective target of TVP1022, the S-enantiomer of rasagiline.

TVP1022, the S-enantiomer of rasagiline (Azilect®) (N-propargyl-1R-aminoindan), exerts cyto/cardio-protective effects in a variety of experimental cardiac and neuronal models. Previous studies have demonstrated that the protective activity of TVP1022 and other propargyl derivatives involve the activation of p42/44 mitogen-activated protein kinase (MAPK) signaling pathway. In the current study, we further investigated the molecular mechanism of action and signaling pathways of TVP1022 which may account for the cyto/cardio-protective efficacy of the drug. Using specific receptor binding and enzyme assays, we demonstrated that the imidazoline 1 and 2 binding sites (I(1) & I(2)) are potential targets for TVP1022 (IC(50) =9.5E-08 M and IC(50) =1.4E-07 M, respectively). Western blotting analysis showed that TVP1022 (1-20 µM) dose-dependently increased the immunoreactivity of phosphorylated p42 and p44 MAPK in rat pheochromocytoma PC12 cells and in neonatal rat ventricular myocytes (NRVM). This effect of TVP1022 was significantly attenuated by efaroxan, a selective I(1) imidazoline receptor antagonist. In addition, the cytoprotective effect of TVP1022 demonstrated in NRVM against serum deprivation-induced toxicity was markedly inhibited by efaroxan, thus suggesting the importance of I(1)imidazoline receptor in mediating the cardioprotective activity of the drug. Our findings suggest that the I(1)imidazoline receptor represents a novel site of action for the cyto/cardio-protective efficacy of TVP1022.

Multi-target, neuroprotective and neurorestorative M30 improves cognitive impairment and reduces Alzheimer's-like neuropathology and age-related alterations in mice.

Based on a multimodal drug design strategy for age-related neurodegenerative diseases, we have synthesized a multifunctional nontoxic, brain-permeable iron-chelating compound, M30, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase-B inhibitor, rasagiline and the antioxidant-iron chelator moiety of the 8-hydroxyquinoline derivative of the iron chelator, VK28. In the present short overview, we describe the neuroprotective and the neurorestorative activity of M30, acting against multiple brain targets, including regulation on amyloid ß, neurogenesis, and activation of hypoxia inducible factor signaling pathways. The diverse pharmacological properties and several pathological targets of M30 make this drug potential valuable for therapeutic strategy of Alzheimer's-like neuropathology and aging.

Cardiovascular baroreceptor activity and selective inhibition of monoamine oxidase.

Cardiovascular baroreceptor responsiveness of conscious rats treated with selective inhibitors of monoamine oxidase (MAO) types A and B was determined by measurement of blood pressure (BP) and heart rate (HR) responses to intravenous injection of phenylephrine and sodium nitroprusside. Treatment with selegiline (1 or 5 mg/kg p.o. daily for 7 days) did not significantly modify resting levels of BP and HR, lower or upper HR plateau levels, or HR/BP gain. Treatment with clorgyline (2 mg/kg p.o. daily for 7 days) increased HR/BP gain but also did not modify resting BP or HR, or lower and upper plateau levels of HR. The results are compatible with an effect of MAO-A inhibition to modify monoamine levels in medullary areas participating in CNS control of blood pressure.

Neuroprotection by the multitarget iron chelator M30 on age-related alterations in mice.

Based on a multimodal drug design paradigm, we have synthesized a multifunctional non-toxic, brain permeable iron chelating compound, M30, possessing the neuroprotective N-propargyl moiety of the anti-Parkinsonian drug, monoamine oxidase (MAO)-B inhibitor, rasagiline and the antioxidant-iron chelator moiety of an 8-hydroxyquinoline derivative of the iron chelator, VK28. Here, we report that a chronic systemic treatment of aged mice with M30 (1 and 5mg/kg; 4 times weekly for 6 months), had a significant positive impact on neuropsychiatry functions and cognitive age-related impairment. M30 significantly reduced cerebral iron accumulation as demonstrated by Perl's staining, accompanied by a marked decrease in cerebral ß-amyloid plaques. In addition, our results demonstrate that M30 caused a significant inhibition of both MAO-A and -B activities in the cerebellum of aged mice, compared with vehicle-treated aged control mice. In summary, the present study indicates that the novel MAO inhibitor/iron chelating drug, M30, acting against multiple brain targets could reverse age-associated memory impairment and provide a potential treatment against the progression of neurodegeneration in ageing.

From anti-Parkinson's drug rasagiline to novel multitarget iron chelators with acetylcholinesterase and monoamine oxidase inhibitory and neuroprotective properties for Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial syndrome involving a complex array of different, while related, factors in its progression. Accordingly, novel approaches that can simultaneously modulate several disease-related targets hold great promise for the effective treatment of AD. This review describes the development of novel hybrid molecules with multimodal activity, including: i) M30, the brain permeable selective monoamine oxidase (MAO)-A and -B inhibitor with chelating and neuroprotective activity; ii) HLA20, a brain permeable metal chelator with neuroprotective activity; iii) HLA20A, an acetylcholinesterase (AChE) inhibitor with site-activated chelating and neuroprotective activity; iv) M30D, an AChE and MAO-A and -B inhibitor with site-activated chelating and neuroprotective activity; and v) analogs of the neuroprotective aminoacid peptide, NAPVSIPQ. HLA20A and M30D act as pro-chelators and can be activated to liberate their respective active chelators HLA20 and M30 through pseudo inhibition of AChE. We first discuss the knowledge and structure-based strategy for the rational design of these novel compounds. Then, we review our recent studies on these drug candidates, regarding their wide range in vitro and in vivo activities, with emphasis on antioxidant-chelating potency and AchE and MAO-A and -B inhibitory activity, as well as neuroprotective/neurorescue effects. Finally, we discuss the diverse molecular mechanisms of action of these compounds with relevance to AD, including modulation of amyloid-ß and amyloid-ß protein precursor expression/processing; induction of cell cycle arrest; inhibition of neuronal death markers; and upregulation of neurotrophic factors, as well as activation of protein kinase signaling pathways.