New clinical insight in amyotrophic lateral sclerosis and innovative clinical development from the non-profit repurposing trial of the old drug guanabenz.

Drug repurposing is considered a valid approach to accelerate therapeutic solutions for rare diseases. However, it is not as widely applied as it could be, due to several barriers that discourage both industry and academic institutions from pursuing this path. Herein we present the case of an academic multicentre study that considered the repurposing of the old drug guanabenz as a therapeutic strategy in amyotrophic lateral sclerosis. The difficulties encountered are discussed as an example of the barriers that academics involved in this type of study may face. Although further development of the drug for this target population was hampered for several reasons, the study was successful in many ways. Firstly, because the hypothesis tested was confirmed in a sub-population, leading to alternative innovative solutions that are now under clinical investigation. In addition, the study was informative and provided new insights into the disease, which are now giving new impetus to laboratory research. The message from this example is that even a repurposing study with an old product has the potential to generate innovation and interest from industry partners, provided it is based on a sound rationale, the study design is adequate to ensure meaningful results, and the investigators keep the full clinical development picture in mind.

The positive impact on translational research of Fondazione italiana di ricerca per la Sclerosi Laterale Amiotrofica (AriSLA), a non-profit foundation focused on amyotrophic lateral sclerosis. Convergence of ex-ante evaluation and ex-post outcomes when goals are set upfront.

Charities investing on rare disease research greatly contribute to generate ground-breaking knowledge with the clear goal of finding a cure for their condition of interest. Although the amount of their investments may be relatively small compared to major funders, the advocacy groups' clear mission promotes innovative research and aggregates highly motivated and mission-oriented scientists. Here, we illustrate the case of Fondazione italiana di ricerca per la Sclerosi Laterale Amiotrofica (AriSLA), the main Italian funding agency entirely dedicated to amyotrophic lateral sclerosis research. An international benchmark analysis of publications derived from AriSLA-funded projects indicated that their mean relative citation ratio values (iCite dashboard, National Institutes of Health, U.S.) were very high, suggesting a strong influence on the referring international scientific community. An interesting trend of research toward translation based on the "triangle of biomedicine" and paper citations (iCite) was also observed. Qualitative analysis on researchers' accomplishments was convergent with the bibliometric data, indicating a high level of performance of several working groups, lines of research that speak of progression toward clinical translation, and one study that has progressed from the investigation of cellular mechanisms to a Phase 2 international clinical trial. The key elements of the success of the AriSLA investment lie in: (i) the clear definition of the objectives (research with potential impact on patients, no matter how far), (ii) a rigorous peer-review process entrusted to an international panel of experts, (iii) diversification of the portfolio with ad hoc selection criteria, which also contributed to bringing new experts and younger scientists to the field, and (iv) a close interaction of AriSLA stakeholders with scientists, who developed a strong sense of belonging. Periodic review of the portfolio of investments is a vital practice for funding agencies. Sharing information between funding agencies about their own policies and research assessment methods and outcomes help guide the international debate on funding strategies and research directions to be undertaken, particularly in the field of rare diseases, where synergy is a relevant enabling factor.

Impact of lower limb movement on the hemodynamics of femoropopliteal arteries: A computational study.

Femoropopliteal arteries (FPAs) are subjected to a wide range of deformations, mainly determined by leg movement. FPAs are often affected by atherosclerotic plaque development, presumably influenced by the biomechanics of surrounding tissues. Although abnormal hemodynamics in FPAs appears to be an important factor in driving plaque development, to date it has been investigated in few studies, in which the leg was modeled in either fixed straight or bent configuration. Hence, the current work investigates the impact of leg movement on FPA hemodynamics. An idealized model of FPA was created to perform moving-boundary computational fluid dynamics analyses. By mimicking hip rotation, knee flexion and complete movement of walking, the hemodynamics was compared between moving- and fixed-boundary models. Moreover, additional features affecting the hemodynamics (e.g. flow-rate curve amplitude, walking speed) were examined. Significant hemodynamic differences were found between the moving- and fixed-boundary models, with the leg movement inducing higher time-averaged wall shear stress (TAWSS) (up to 66%). The flow-rate amplitude and walking period were the most influential parameters (differences in TAWSS up to 68% and 74%, respectively). In conclusion, this numerical approach highlighted the importance of considering leg movement to investigate FPA hemodynamics, and it could be employed in future patient-specific analyses.

Comparing the different response of PNS and CNS injured neurons to mesenchymal stem cell treatment.

Mesenchymal stem cells (MSCs) are adult bone marrow-derived stem cells actually proposed indifferently for the therapy of neurological diseases of both the Central (CNS) and the Peripheral Nervous System (PNS), as a panacea able to treat so many different diseases by their immunomodulatory ability and supportive action on neuronal survival. However, the identification of the exact mechanism of MSC action in the different diseases, although mandatory to define their real and concrete utility, is still lacking. Moreover, CNS and PNS neurons present many different biological properties, and it is still unclear if they respond in the same manner not only to MSC treatment, but also to injuries. For these reasons, in this study we compared the susceptibility of cortical and sensory neurons both to toxic drug exposure and to MSC action, in order to verify if these two neuronal populations can respond differently. Our results demonstrated that Cisplatin (CDDP), Glutamate, and Paclitaxel-treated sensory neurons were protected by the co-culture with MSCs, in different manners: through direct contact able to block apoptosis for CDDP- and Glutamate-treated neurons, and by the release of trophic factors for Paclitaxel-treated ones. A possible key soluble factor for MSC protection was Glutathione, spontaneously released by these cells. On the contrary, cortical neurons resulted more sensitive than sensory ones to the toxic action of the drugs, and overall MSCs failed to protect them. All these data identified for the first time a different susceptibility of cortical and sensory neurons, and demonstrated a protective action of MSCs only against drugs in peripheral neurotoxicity.

Human Mesenchymal Stem Cells Protect Dorsal Root Ganglia from the Neurotoxic Effect of Cisplatin.

BACKGROUND/AIM: Peripheral neurotoxicity is a dose-limiting factor of many chemotherapeutic agents, including cisplatin. Mesenchymal stem cells are promising for the treatment of several neurological disorders, and our aim was to verify the neuroprotective potential of human mesenchymal stem cells (hMSCs) on dorsal root ganglia (DRG) exposed to cisplatin. MATERIALS AND METHODS: DRG were exposed to different cisplatin concentrations and then co-cultured with hMSCs or with hMSC-conditioned medium. RESULTS: hMSCs showed a neuroprotective effect on cisplatin-induced death of DRG, mediated by direct contact. Moreover, DRG exhibited an MSC-dependent promotion of neurite outgrowth, in particular at early time points. For this effect, the expression of Neurite Outgrowth Inhibitor (NOGO) and Myelin Associated Glycoprotein (MAG) by hMSCs was pivotal. CONCLUSION: hMSCs are a promising tool for reducing the neurotoxic effect of cisplatin.

Neurobasal medium toxicity on mature cortical neurons.

Neurobasal medium (NBM) is a widely used medium for neuronal cultures, originally formulated to support survival of rat hippocampal neurons, but then optimized for several other neuronal subtypes. In the present study, the toxic effect of NBM on long-term cortical neuron cultures has been reported and investigated. A significant neuronal cell loss was observed 24 h after the total medium change performed at days in vitro 10. The neurotoxic effect was specifically because of NBM-A, a commercially derived modification of classic NBM, as neurons exposed to minimum essential medium for 24 h did not show the same mortality rate. We showed that the toxic effect was mediated by the N-methyl-D-aspartate receptor (NMDAr) as its inactivation partly prevented NBM-induced neuronal loss, and the addition of NMDAr activators, such as L-cysteine or glycine to minimum essential medium, reproduced the same toxicity rate observed in NBM. Besides the toxicity associated with NMDAr activation, the decreased antioxidative defenses also worsen (because of glutathione depletion) neuronal death, thus amplifying the effect of excitotoxic amino acids. Indeed, glutathione supplementation by the addition of its precursor N-acetyl-cysteine resulted in an increase in neuronal survival that partially prevented NBM-A toxicity. These results evidenced, on the one hand, the unsuitability of NBM-A for long-term neuronal culture, and on the other, they highlight the importance of selection of more suitable culture conditions.

A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets.

The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.

Undifferentiated MSCs are able to myelinate DRG neuron processes through p75.

Over the last few years the therapeutic approach to demyelinating diseases has radically changed, strategies having been developed aimed at partnering the classic symptomatic treatments with the most advanced regenerative medicine tools. At first, the transplantation of myelinogenic cells, Schwann cells or oligodendrocytes was suggested, but the considerable technical difficulties, (poor availability, difficulties in harvesting and culturing, and the problem of rejection in the event of non-autologous sources), shifted attention towards more versatile cellular types, such as Mesenchymal Stem Cells (MSCs). Recent studies have already demonstrate both in vitro and in vivo that glially-primed MSCs (through exposure to chemical cocktails) have myelogenic abilities. In spite of a large number of papers on glially-differentiated MSCs, little is known about the ability of undifferentiated MSCs to myelinate axons and processes. Here we have demonstrated that also undifferentiated MSCs have the ability to myelinate, since they induce the myelination of rat DRG neuron processes after direct co-culturing. In this process a pivotal role is performed by the p75 receptor.

NGF protects dorsal root ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2.

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.

Role of MAPKs in platinum-induced neuronal apoptosis.

An unsolved question is how platinum derivatives used for solid cancer therapy cause peripheral neuropathy in patients and apoptosis in "in vitro" models of chemotherapy-induced peripheral neuropathy. DRG neurons from E15 rat embryos were treated with toxic doses of oxaliplatin or cisplatin. Here, the role of MAPKs in neuronal apoptosis was studied. Both oxaliplatin and cisplatin induced a dose-dependent neuronal apoptosis, modulated by the proteins of Bcl-2 family. Regarding MAPKs, platinum derivatives activated p38 while they reduced the active form and the total amount of JNK/Sapk. Both oxaliplatin and cisplatin activated ERKs at early stages, although they behaved differently at later stages. By using specific inhibitors of the various MAPKs it was demonstrated that the platinum-induced neuronal apoptosis is mediated by early p38 and ERK1/2 activation, while JNK/Sapk has a neuroprotective role. These results suggest a role for the different MAPKs in peripheral neuropathies characterized by apoptosis of DRG neurons.

Adult mesenchymal stem cells support cisplatin-treated dorsal root ganglion survival.

Mesenchymal stem cells (MSCs) have been found to be useful in the management of different models of neurological diseases. In the present study, we tested the possible protective effect of MSCs on sensory dorsal root ganglia (DRG) explants exposed to the toxic effect of CDDP, a widely used anticancer drug. DRG explants cultured on a collagen layer and exposed to NGF for 2h (differentiating neurons) or for 5 days (fully differentiated neurons) were treated with CDDP and subsequently co-cultured with MSCs. MSCs were able to support the survival of both differentiating and fully differentiated DRG neurons up to 2 months after the drug treatment, reducing the CDDP-induced death of DRG neurons. MSCs were, however, unable to restore the correct length of DRG neurites compromised by CDDP treatment. The positive effect on neuronal survival was exerted through the contact between DRG and MSCs, and not mediated by neurotrophic factors released by the MSCs. Our observations could represent a starting point for designing a neuroprotective strategy to limit CDDP induced neuropathy without interfering with its anticancer properties.