Publications by authors named "Luciana Giardino"

53 Publications

Time-Course Changes of Extracellular Matrix Encoding Genes Expression Level in the Spinal Cord Following Contusion Injury-A Data-Driven Approach.

Int J Mol Sci 2021 Feb 9;22(4). Epub 2021 Feb 9.

Interdepartmental Center for Industrial Research in Life Sciences and Technologies, University of Bologna, Ozzano dell'Emilia, 40064 Bologna, Italy.

The involvement of the extracellular matrix (ECM) in lesion evolution and functional outcome is well recognized in spinal cord injury. Most attention has been dedicated to the "core" area of the lesion and scar formation, while only scattered reports consider ECM modification based on the temporal evolution and the segments adjacent to the lesion. In this study, we investigated the expression profile of 100 genes encoding for ECM proteins at 1, 8 and 45 days post-injury, in the spinal cord segments rostral and caudal to the lesion and in the scar segment, in a rat model. During both the active lesion phases and the lesion stabilization, we observed an asymmetric gene expression induced by the injury, with a higher regulation in the rostral segment of genes involved in ECM remodeling, adhesion and cell migration. Using bioinformatic approaches, the metalloproteases inhibitor and the hyaluronan receptor emerged as the hub genes at all post-lesion times. Results from the bioinformatic gene expression analysis were then confirmed at protein level by tissue analysis and by cell culture using primary astrocytes. These results indicated that ECM regulation also takes place outside of the lesion area in spinal cord injury.
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http://dx.doi.org/10.3390/ijms22041744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916102PMC
February 2021

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

Int J Mol Sci 2020 Nov 25;21(23). Epub 2020 Nov 25.

Department of Veterinary Medical Science, University of Bologna, Via Tolara di Sopra, 50, 40064 Ozzano Emilia, BO, Italy.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.
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http://dx.doi.org/10.3390/ijms21238945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728128PMC
November 2020

Oligodendrocytes in a dish for the drug discovery pipeline: the risk of oversimplification.

Neural Regen Res 2021 Feb;16(2):291-293

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna; Department of Pharmacy and Biotechnology, University of Bologna; Montecatone Rehabilitation Institute, Bologna, Italy.

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http://dx.doi.org/10.4103/1673-5374.290888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896220PMC
February 2021

Improved Functional Recovery in Rat Spinal Cord Injury Induced by a Drug Combination Administered with an Implantable Polymeric Delivery System.

J Neurotrauma 2020 08 14;37(15):1708-1719. Epub 2020 May 14.

Health Sciences and Technologies (HST) CIRI-SDV, Alma Mater Studiorum-University of Bologna, Bologna, Italy.

Spinal cord injury (SCI) is an incurable condition, in which a cascade of cellular and molecular events triggered by inflammation and excitotoxicity impairs endogenous regeneration, namely remyelination and axonal outgrowth. We designed a treatment solution based on an implantable biomaterial (electrospun poly (l-lactic acid) [PLLA]) loaded with ibuprofen and triiodothyronine (T3) to counteract inflammation, thus improving endogenous regeneration. efficacy was tested by implanting the drug-loaded PLLA in the rat model of T8 contusion SCI. We observed the expected recovery of locomotion beginning on day 7. In PLLA-implanted rats (i.e., controls), the recovery stabilized at 21 days post-lesion (DPL), after which no further improvement was observed. On the contrary, in PLLA + ibuprofen (Ibu) + T3 (PLLA-Ibu-T3) rats a further recovery and a significant treatment effect were observed, also confirmed by the gait analysis on 49 DPL. Glutamate release at 24 h and 8 DPL was reduced in PLLA-Ibu-T3- compared to PLLA-implanted rats, such as the estimated lesion volume at 60 DPL. The myelin- and 200-neurofilament-positive area fraction was higher in PLLA-Ibu-T3-implanted rats, where the percentage of astrocytes was significantly reduced. The implant of a PLLA electrospun scaffold loaded with Ibu and T3 significantly improves the endogenous regeneration, leading to an improvement of functional locomotion outcome in the SCI.
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http://dx.doi.org/10.1089/neu.2019.6949DOI Listing
August 2020

White Matter and Neuroprotection in Alzheimer's Dementia.

Molecules 2020 Jan 23;25(3). Epub 2020 Jan 23.

Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano Emilia (BO), Italy.

Myelin is the main component of the white matter of the central nervous system (CNS), allowing the proper electrical function of the neurons by ensheathing and insulating the axons. The extensive use of magnetic resonance imaging has highlighted the white matter alterations in Alzheimer's dementia (AD) and other neurodegenerative diseases, alterations which are early, extended, and regionally selective. Given that the white matter turnover is considerable in the adulthood, and that myelin repair is currently recognized as being the only true reparative capability of the mature CNS, oligodendrocyte precursor cells (OPCs), the cells that differentiate in oligodendrocyte, responsible for myelin formation and repair, are regarded as a potential target for neuroprotection. In this review, several aspects of the OPC biology are reviewed. The histology and functional role of OPCs in the neurovascular-neuroglial unit as described in preclinical and clinical studies on AD is discussed, such as the OPC vulnerability to hypoxia-ischemia, neuroinflammation, and amyloid deposition. Finally, the position of OPCs in drug discovery strategies for dementia is discussed.
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http://dx.doi.org/10.3390/molecules25030503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038211PMC
January 2020

Differential effects of glucose deprivation on the survival of fetal versus adult neural stem cells-derived oligodendrocyte precursor cells.

Glia 2020 05 22;68(5):898-917. Epub 2019 Nov 22.

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Bologna, Italy.

Impaired myelination is a key feature in neonatal hypoxia/ischemia (HI), the most common perinatal/neonatal cause of death and permanent disabilities, which is triggered by the establishment of an inflammatory and hypoxic environment during the most critical period of myelin development. This process is dependent on oligodendrocyte precursor cells (OPCs) and their capability to differentiate into mature oligodendrocytes. In this study, we investigated the vulnerability of fetal and adult OPCs derived from neural stem cells (NSCs) to inflammatory and HI insults. The resulting OPCs/astrocytes cultures were exposed to cytokines to mimic inflammation, or to oxygen-glucose deprivation (OGD) to mimic an HI condition. The differentiation of both fetal and adult OPCs is completely abolished following exposure to inflammatory cytokines, while only fetal-derived OPCs degenerate when exposed to OGD. We then investigated possible mechanisms involved in OGD-mediated toxicity: (a) T3-mediated maturation induction; (b) glutamate excitotoxicity; (c) glucose metabolism. We found that while no substantial differences were observed in T3 intracellular content regulation and glutamate-mediated toxicity, glucose deprivation lead to selective OPC cell death and impaired differentiation in fetal cultures only. These results indicate that the biological response of OPCs to inflammation and demyelination is different in fetal and adult cells, and that the glucose metabolism perturbation in fetal central nervous system (CNS) may significantly contribute to neonatal pathologies. An understanding of the underlying molecular mechanism will contribute greatly to differentiating myelination enhancing and neuroprotective therapies for neonatal and adult CNS white matter lesions.
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http://dx.doi.org/10.1002/glia.23750DOI Listing
May 2020

A dynamic culture platform enhances the efficiency of the 3D HUVEC-based tube formation assay.

Biotechnol Bioeng 2020 03 28;117(3):789-797. Epub 2019 Nov 28.

Health Sciences and Technologies-Interdepartmental Center for Industrial Research, University of Bologna, Bologna, Italy.

Cell-based in vitro biological models traditionally use monolayer cell cultures grown over plastic surfaces bathing in static media. Higher fidelity to a natural biological tissue is expected to result from growing the cells in a three-dimensional (3D) matrix. However, due to the decreased rate of diffusion inherent to increased distances within a tridimensional space, proper fluidic conditions are needed in this setting to better approximate a physiological environment. To this aim, we here propose a prototypal dynamic cell culture platform for the automatic medium replacement, via periodic perfusion flow, in a human umbilical vein endothelial cell (HUVECs) culture seeded in a Geltrex™ matrix. A state-of-the-art angiogenesis assay performed in these dynamic conditions showed sizable effects with respect to conventional static control cultures, with significantly enhanced pro-(dual antiplatelet therapy [DAPT]) and anti-(EDTA) angiogenic compound activity. In particular, dynamic culture conditions (a) enhance the 3D-organization of HUVECs into microtubule structure; (b) accelerate and improve endothelial tube formation by HUVECs in the presence of DAPT; (c) are able to completely revert the blocking effects of EDTA. These evidence emphasize the need of setting proper fluidic conditions for a better approximation of a physiological environment as an appropriate evolution of current cell culture paradigms.
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http://dx.doi.org/10.1002/bit.27227DOI Listing
March 2020

Potential biomarkers for neuroinflammation and neurodegeneration at short and long term after neonatal hypoxic-ischemic insult in rat.

J Neuroinflammation 2019 Oct 28;16(1):194. Epub 2019 Oct 28.

Health Science and Technologies Interdepartmental Center for Industrial Research, University of Bologna, Via Tolara di Sopra 41/E, I-40064, Ozzano Emilia, BO, Italy.

Background: Hypoxic-ischemic (HI) encephalopathy causes life-long morbidity and premature mortality in term neonates. Therapies in addition to whole-body cooling are under development to treat the neonate at risk for HI encephalopathy, but are not a quickly measured serum inflammatory or neuronal biomarkers to rapidly and accurately identify brain injury in order to follow the efficacy of therapies.

Methods: In order to identify potential biomarkers for early inflammatory and neurodegenerative events after neonatal hypoxia-ischemia, both male and female Wistar rat pups at postnatal day 7 (P7) were used and had their right carotid artery permanently doubly occluded and exposed to 8% oxygen for 90 min. Sensory and cognitive parameters were assessed by open field, rotarod, CatWalk, and Morris water maze (MWM) test. Plasma and CSF biomarkers were investigated on the acute (24 h and 72 h) and chronic phase (4 weeks). Brains were assessed for gene expression analysis by quantitative RT-PCR Array.

Results: We found a delay of neurological reflex maturation in HI rats. We observed anxiolytic-like baseline behavior in males more than females following HI injury. HI rats held on the rotarod for a shorter time comparing to sham. HI injury impaired spatial learning ability on MWM test. The CatWalk assessment demonstrated a long-term deficit in gait parameters related to the hind paw. Proinflammatory biomarkers such as IL-6 in plasma and CCL2 and TNF-α in CSF showed an upregulation at 24 h after HI while other cytokines, such as IL-17A and CCL5, were upregulated after 72 h in CSF. At 24 h post-injury, we observed an increase of Edn1, Hif1-α, and Mmp9 mRNA levels in the ipsilateral vs the contralateral hemisphere of HI rats. An upregulation of genes involved with clotting and hematopoietic processes was observed 72 h post-injury.

Conclusions: Our work showed that, in the immature brain, the HI injury induced an early increased production of several proinflammatory mediators detectable in plasma and CSF, followed by tissue damage in the hypoxic hemisphere and short-term as well as long-lasting neurobehavioral deficits.
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http://dx.doi.org/10.1186/s12974-019-1595-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819609PMC
October 2019

Possible Strategies to Optimize a Biomarker Discovery Approach to Correlate with Neurological Outcome in Patients with Spinal Cord Injury: A Pilot Study.

J Neurotrauma 2020 02 1;37(3):431-440. Epub 2019 Aug 1.

Department of Health Sciences and Technologies - Interdepartmental Center for Industrial Research-CIRI-SDV, University of Bologna, Bologna, Italy.

The lack of reliable diagnostic and prognostic markers for spinal cord injured (SCI) patients is a severe obstacle in development and testing of new therapies, and it also impairs appropriate rehabilitation care. The sparse available data on the biochemical composition of cerebrospinal fluid (CSF) during the acute and/or chronic phase of the lesion provide, up until now, inconsistent results. In this pilot study, we then explored the possibility of combining a multi-parametric and bioinformatic analysis of CSF for its biological properties tested on different cells types, suitable for investigating inflammation and re-myelination. The patient enrollment was based on stringent inclusion criteria; that is, cervical and thoracic SCI trauma, CSF collection within 24 h of trauma, type of surgical approach for spine stabilization, and absence of steroid therapy before CSF collection. Eleven SCI patients and four healthy controls were included, and in three patients, CSF was also collected at 3 months after lesion. We identified 19 proteins among the 60 investigated cytokines, chemokines, growth factors, and structural biomarkers, which are transiently regulated 24 h after SCI. A bioinformatic analysis indicated that interleukin (IL)-6 and IL-10 are in the core of the interconnected net of activated proteins. Cell-based experiments indicate that CSF from SCI patients stimulates astroglia derivation from neural precursor cells, and an inverse correlation between IL-8 CSF level and oligodendrocyte precursor cells generated from neural stem cells was also observed. Results from this pilot study suggest that using a combined bioanalytic and biological approach to analyze SCI CSF at different times after injury could be a useful approach for identifying reliable diagnostic and prognostic markers in SCI.
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http://dx.doi.org/10.1089/neu.2018.6362DOI Listing
February 2020

The role of nuclear receptors in the differentiation of oligodendrocyte precursor cells derived from fetal and adult neural stem cells.

Stem Cell Res 2019 05 17;37:101443. Epub 2019 Apr 17.

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Italy; Department of Pharmacy and Biotechnology, University of Bologna, Italy; IRET Foundation, Ozzano Emilia, Italy.

Oligodendrocyte precursor cells (OPCs) differentiation from multipotent neural stem cells (NSCs) into mature oligodendrocytes is driven by thyroid hormone and mediated by thyroid hormone receptors (TRs). We show that several nuclear receptors display strong changes in expression levels between fetal and adult NSCs, with an overexpression of TRβ and a lower expression of RXRγ in adult. Such changes may determine the reduced capacity of adult OPCs to differentiate as supported by reduced yield of maturation and compromised mRNA expression of key genes. RXRγ may be the determinant of these differences, on the evidence of reduced number of mature oligodendrocytes and increased number of proliferating OPCs in RXRγ-/- cultures. Such data also points to RXRγ as an important regulator of the cell cycle exit, as proved by the dysregulation of T3-induced cell cycle exit-related genes. Our data highlight the biological differences between fetal and adult OPCs and demonstrate the essential role of RXRγ in the T3-mediated OPCs maturation process.
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http://dx.doi.org/10.1016/j.scr.2019.101443DOI Listing
May 2019

Age-Related Changes of the Neurovascular Unit in the Cerebral Cortex of Alzheimer Disease Mouse Models: A Neuroanatomical and Molecular Study.

J Neuropathol Exp Neurol 2019 02;78(2):101-112

Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy.

We describe age-related histological structure and molecular changes of the neurovascular unit (NVU) in the cerebral cortex of Tg2576 and age-matched wild-type (WT) mice. Major results can be summarized as follows: (i) β-amyloid (6E10)-immunoreactivity progressively increases in neurons and astrocytes of Tg2576 mice, reaching the highest concentration at 5 months and then decreasing as soon as extracellular plaque deposition begins; (ii) the synaptic puncta density of glutamatergic and GABAergic neurons in Tg2576 mice is unbalanced versus WT at all investigated ages, with a decrease in synaptophysin and VGLUT1; density of VGAT contacts is higher in 27-month-old Tg2576 versus WT mice; (iii) capillary density is higher in 5-month-old Tg2576 versus WT mice, then decreases to a lower density at 27 months, when the capillary-astrocyte interface is lower; and (iv) mRNA expression of genes involved in microvessel dynamics indicates age- and genotype-dependent changes in the expression levels of hypoxia-related genes, i.e. the highest level is in 5-month-old animals and there is impaired regulation in Tg2576. We conclude that at 5 months, when learning and memory impairment is already present in the absence of extracellular amyloid plaque deposition, Tg2576 mice display alterations in the structure and molecular regulation of the NVU.
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http://dx.doi.org/10.1093/jnen/nly125DOI Listing
February 2019

Thyroid Hormone and the White Matter of the Central Nervous System: From Development to Repair.

Vitam Horm 2018 12;106:253-281. Epub 2017 Jun 12.

CIRI-SDV, University of Bologna, Bologna, Italy; IRET Foundation, Ozzano Emilia, Italy; DIMEVET, University of Bologna, Bologna, Italy.

The role of thyroid hormone (TH) on brain development, and particularly in myelination, is well known since many decades, as testified by the severe structural and functional consequences of congenital hypothyroidism. This role during development, the consideration that the early TH supplementation restores myelination capability, and the fact the cell responsible for developmental myelination and remyelination is the same, i.e., the oligodendrocyte precursor cell (OPC), claimed the attempt to improve myelin repair in the adulthood via TH supplementation. In this chapter, the impact of TH on development, homeostasis, and repair of the myelin in the CNS will be reviewed, focusing on the regulation of the TH tissue signaling during physiological and pathological conditions affecting myelination and/or myelin repair during early postnatal age and during the adulthood. The impact of the tissue inflammation on molecular mediators of the TH cellular action and metabolism will be discussed, with regard to the consequences on the biology of the OPC.
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http://dx.doi.org/10.1016/bs.vh.2017.04.003DOI Listing
October 2018

Growth and Neurotrophic Factors in Embryonic Stem Cells.

Methods Mol Biol 2018 ;1727:275-294

Department of Veterinary Medical Science, University of Bologna, Ozzano Emilia, Bologna, Italy.

In this chapter we illustrate protocols to investigate growth and neurotrophic factors in human and rodent (rat and mouse)-derived embryonic stem cells. The conventional two-dimensional cell monolayer system to grow embryonic stem cells is presented, focusing on the coating strategies also using extracellular matrix components. Then, different approaches for three-dimensional stem cell culture are presented, using hydrogels and scaffolds. Quantitative polymerase chain reaction, immunocytochemistry, immunoenzymatic ELISA assay, and multiparametric assays to quantify growth and neurotrophic factor production are presented.
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http://dx.doi.org/10.1007/978-1-4939-7571-6_21DOI Listing
July 2018

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

Stem Cell Res 2017 07 30;22:54-60. Epub 2017 May 30.

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Italy; Department of Pharmacy and Biotechnology, University of Bologna, Italy; IRET Foundation, Ozzano Emilia, Italy. Electronic address:

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy.
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http://dx.doi.org/10.1016/j.scr.2017.05.011DOI Listing
July 2017

Vulnerability of primary neurons derived from Tg2576 Alzheimer mice to oxygen and glucose deprivation: role of intraneuronal amyloid-β accumulation and astrocytes.

Dis Model Mech 2017 05 24;10(5):671-678. Epub 2017 Feb 24.

Interdepartmental Centre for Industrial Research in Health Science and Technologies (ICIR - HST), University of Bologna, 40064 Ozzano Emilia, Bologna, Italy

Microvascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium.
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http://dx.doi.org/10.1242/dmm.028001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451168PMC
May 2017

Retrospective evaluation of circulating thyroid hormones in critically ill dogs with systemic inflammatory response syndrome.

J Vet Sci 2017 Dec;18(4):471-477

Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, 40126 Bologna, Italy.

Critical illness can be associated with transient alterations in circulating thyroid hormone concentrations, indicating the presence of non-thyroidal illness (NTI). NTI is well described in humans, but there are few reports on its occurrence and prognostic significance in dogs. This retrospective study assessed the occurrence of NTI in a population of dogs with systemic inflammatory response syndrome (SIRS) and investigated its association with disease severity (APPLE scores). A total of 41 SIRS dogs were included and were divided by SIRS origin (non-septic SIRS, n = 10; septic SIRS, n = 41) and final outcome (survivors, n = 37; non-survivors, n = 4). Healthy, age-matched dogs (n = 15) were included as controls. Serum thyroid hormone levels including total T3, free T3, total T4, and reverse T3 were measured upon admission. Compared to controls, there were significant changes in serum thyroid hormone concentrations in SIRS dogs, suggesting the presence of NTI. Septic SIRS dogs had higher APPLE scores and lower serum thyroid hormones concentrations than those in non-septic SIRS and control dogs. In conclusion, NTI was frequent in dogs with SIRS and may be associated with the presence of sepsis or high illness severity.
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http://dx.doi.org/10.4142/jvs.2017.18.4.471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746440PMC
December 2017

Cytokine and chemokine alterations in tissue, CSF, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (EAE), in a rat model of multiple sclerosis.

J Neuroinflammation 2016 11 15;13(1):291. Epub 2016 Nov 15.

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 41/E, Bologna, Ozzano Emilia I, 40064, Italy.

Background: Experimental allergic encephalomyelitis (EAE) is the most commonly used experimental animal model for human multiple sclerosis (MS) that has been used so far to study the acute and remission-relapsing phases of the disease. Despite the vast literature on neuroinflammation onset and progression in EAE, important questions are still open regarding in particular the early asymptomatic phase between immunization and clinical onset.

Methods: In this study, we performed a time-course investigation of neuroinflammation and demyelination biomarkers in the spinal cord (SC), cerebrospinal fluid (CSF), and blood in EAE induced in dark agouti (DA) female rats compared to the controls and adjuvant-injected rats, using high-throughput technologies for gene expression and protein assays and focusing on the time-course between immunization, clinical onset (1, 5, 8 days post-immunization (DPI)), and progression (11 and 18 DPI). The expression profile of 84 genes related to T cell activation/signaling, adaptive immunity, cytokine/chemokine inflammation, demyelination, and cellular stress were analyzed in the tissue; 24 cytokines were measured in the CSF and plasma.

Results: The macrophage colony-stimulating factor (CSF1) was the first up-regulated protein as far as 1 DPI, not only in blood but also in CSF and SC. A treatment with GW2580, a selective CSF1R inhibitor, slowed the disease progression, significantly reduced the severity, and prevented the relapse phase. Moreover, both pro-inflammatory (IL-1β, TNF-α) and anti-inflammatory cytokines (IL-5, IL-10, VEGF) were up-regulated starting from 8 DPI. Myelin genes were down-regulated starting from 8 DPI, especially MAL, MBP, and PMP22 while an opposite expression profile was observed for inflammation-related genes, such as CXCL11 and CXCL10.

Conclusions: This early cytokine and chemokine regulation indicates that novel biomarkers and therapeutic options could be explored in the asymptomatic phase of EAE. Overall, our findings provide clear evidence that CSF1R signaling regulates inflammation in EAE, supporting therapeutic targeting of CSF1R in MS.
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http://dx.doi.org/10.1186/s12974-016-0757-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111339PMC
November 2016

REAC technology modifies pathological neuroinflammation and motor behaviour in an Alzheimer's disease mouse model.

Sci Rep 2016 10 24;6:35719. Epub 2016 Oct 24.

IRET Foundation, Ozzano Emilia, Italy.

The search for new therapeutic approaches to Alzheimer disease (AD) is a major goal in medicine and society, also due to the impressive economic and social costs of this disease. In this scenario, biotechnologies play an important role. Here, it is demonstrated that the Radio Electric Asymmetric Conveyer (REAC), an innovative technology platform for neuro- and bio-modulation, used according to the neuro-regenerative protocol (RGN-N), significantly increases astroglial reaction around the amyloid plaques in an AD mouse model, as evaluated by GFAP-immunoreactivity, and reduces microglia-associated neuroinflammation markers, as evaluated by Iba1-immunoreactivity and mRNA expression level of inflammatory cytokines TREM. IL1beta, iNOS and MRC1 were not affected neither by the genotype or by REAC RGN-N treatment. Also observed was an increase in locomotion in treated animals. The study was performed in 24-month-old male Tg2576 mice and age-matching wild-type animals, tested for Y-maze, contextual fear conditioning and locomotion immediately after the end of a specific REAC treatment administered for 15 hours/day for 15 days. These results demonstrated that REAC RGN-N treatment modifies pathological neuroinflammation, and mitigates part of the complex motor behaviour alterations observed in very old Tg2576 mice.
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http://dx.doi.org/10.1038/srep35719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075930PMC
October 2016

In Vitro Testing of Biomaterials for Neural Repair: Focus on Cellular Systems and High-Content Analysis.

Biores Open Access 2016 1;5(1):201-11. Epub 2016 Aug 1.

Health Sciences and Technologies-Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Bologna, Italy.; Department of Pharmacy and Biotechnology (FaBit), University of Bologna, Bologna, Italy.

Biomimetic materials are designed to stimulate specific cellular responses at the molecular level. To improve the soundness of in vitro testing of the biological impact of new materials, appropriate cell systems and technologies must be standardized also taking regulatory issues into consideration. In this study, the biological and molecular effects of different scaffolds on three neural systems, that is, the neural cell line SH-SY5Y, primary cortical neurons, and neural stem cells, were compared. The effect of poly(L-lactic acid) scaffolds having different surface geometry (conventional two-dimensional seeding flat surface, random or aligned fibers as semi3D structure) and chemical functionalization (laminin or ECM extract) were studied. The endpoints were defined for efficacy (i.e., neural differentiation and neurite elongation) and for safety (i.e., cell death/survival) using high-content analysis. It is demonstrated that (i) the definition of the biological properties of biomaterials is profoundly influenced by the test system used; (ii) the definition of the in vitro safety profile of biomaterials for neural repair is also influenced by the test system; (iii) cell-based high-content screening may well be successfully used to characterize both the efficacy and safety of novel biomaterials, thus speeding up and improving the soundness of this critical step in material science having medical applications.
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http://dx.doi.org/10.1089/biores.2016.0025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991583PMC
September 2016

Inflammation severely alters thyroid hormone signaling in the central nervous system during experimental allergic encephalomyelitis in rat: Direct impact on OPCs differentiation failure.

Glia 2016 09 12;64(9):1573-89. Epub 2016 Jul 12.

Health Science and Technology Interdepartmental Center for Industrial Research, University of Bologna, Bologna, Italy.

Differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is severely impaired by inflammatory cytokines and this could lead to remyelination failure in inflammatory/demyelinating diseases. Due to the role of thyroid hormone in the maturation of OPCs and developmental myelination, in this study we investigated (i) the possible occurrence of dysregulation of thyroid hormone signaling in the CNS tissue during experimental neuroinflammation; (ii) the possible impact of inflammatory cytokines on thyroid hormone signaling and OPCs differentiation in vitro. The disease model is the experimental allergic encephalomyelitis in female Dark-Agouti rats, whereas in vitro experiments were carried out in OPCs derived from neural stem cells. The main results are the following: (i) a strong upregulation of cytokine mRNA expression level was found in the spinal cord during experimental allergic encephalomyelitis; (ii) thyroid hormone signaling in the spinal cord (thyroid hormone receptors; deiodinase; thyroid hormone membrane transporter) is substantially downregulated, due to the upregulation of the thyroid hormone inactivating enzyme deiodinase 3 and the downregulation of thyroid hormone receptors, as investigated at mRNA expression level; (iii) when exposed to inflammatory cytokines, deiodinase 3 is upregulated in OPCs as well, and OPCs differentiation is blocked; (iv) deiodinase 3 inhibition by iopanoic acid recovers OPCs differentiation in the presence on inflammatory cytokines. These data suggest that cellular hypothyroidism occurs during experimental allergic encephalomyelitis, possibly impacting on thyroid hormone-dependent cellular processes, including maturation of OPCs into myelinating oligodendrocytes. GLIA 2016;64:1573-1589.
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http://dx.doi.org/10.1002/glia.23025DOI Listing
September 2016

Role of the Thyroid System in Myelination and Neural Connectivity.

Compr Physiol 2015 Jul;5(3):1405-21

Department of Pharmacy and Biotechnology, University of Bologna, Italy.

The role of thyroid hormone on brain development is dramatically illustrated by "cretinism," a severe mental retardation due to iodine deficiency and maternal hypothyroidism during gestation. In the last decades, the molecular bases of the cellular action of thyroid hormone in the nervous tissue have been at least partially elucidated, and the emerged picture is much more complex than expected. In this article, the main mechanisms determining thyroid hormone availability, nuclear and membrane receptor occupancy and downstream action, gene expression, and nongenomic mechanism are reviewed, focusing on myelination and myelin turnover.
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http://dx.doi.org/10.1002/cphy.c140035DOI Listing
July 2015

In vitro exposure to very low-level laser modifies expression level of extracellular matrix protein RNAs and mitochondria dynamics in mouse embryonic fibroblasts.

BMC Complement Altern Med 2015 Mar 24;15:78. Epub 2015 Mar 24.

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Bologna, Italy.

Background: Low-level lasers working at 633 or 670 nm and emitting extremely low power densities (Ultra Low Level Lasers - ULLL) exert an overall effect of photobiostimulation on cellular metabolism and energy balance. In previous studies, it was demonstrated that ULLL pulsed emission mode regulates neurite elongation in vitro and exerts protective action against oxidative stress.

Methods: In this study the action of ULLL supplied in both pulsed and continuous mode vs continuous LLL on fibroblast cultures (Mouse Embryonic Fibroblast-MEF) was tested, focusing on mitochondria network and the expression level of mRNA encoding for proteins involved in the cell-matrix adhesion.

Results: It was shown that ULLL at 670 nm, at extremely low average power output (0.21 mW/ cm(2)) and dose (4.3 mJ/ cm(2)), when dispensed in pulsed mode (PW), but not in continuous mode (CW) supplied at both at very low (0.21 mW/cm(2)) and low levels (500 mW/cm(2)), modifies mitochondria network dynamics, as well as expression level of mRNA encoding for selective matrix proteins in MEF, e.g. collagen type 1α1 and integrin α5.

Conclusions: We suggest that pulsatility, but not energy density, is crucial in regulating expression level of collagen I and integrin α5 in fibroblasts by ULLL.
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http://dx.doi.org/10.1186/s12906-015-0593-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387590PMC
March 2015

Radio electric asymmetric conveyer: a novel neuromodulation technology in Alzheimer's and other neurodegenerative diseases.

Front Psychiatry 2015 17;6:22. Epub 2015 Feb 17.

Rinaldi Fontani Foundation , Florence , Italy ; Department of Neuro Psycho Physical Optimization, Rinaldi Fontani Institute , Florence , Italy ; Department of Regenerative Medicine, Rinaldi Fontani Institute , Florence , Italy.

Global research in the field of pharmacology has not yet found effective drugs to treat Alzheimer's disease (AD). Thus, alternative therapeutic strategies are under investigation, such as neurostimulation by physical means. Radio electric asymmetric conveyer (REAC) is one of these technologies and has, until now, been used in clinical studies on several psychiatric and neurological disorders with encouraging results in the absence of side effects. Moreover, studies at the cellular level have shown that REAC technology, with the appropriate protocols, is able to induce neuronal differentiation both in murine embryonic cells and in human adult differentiated cells. Other studies have shown that REAC technology is able to positively influence senescence processes. Studies conducted on AD patients and in transgenic mouse models have shown promising results, suggesting REAC could be a useful therapy for certain components of AD.
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http://dx.doi.org/10.3389/fpsyt.2015.00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330882PMC
March 2015

Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery.

World J Stem Cells 2013 Oct;5(4):229-37

Vito Antonio Baldassarro, Mercedez Fernández, Giulia Lizzo, Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), I-40064 Ozzano Emilia, Bologna, Italy.

Aim: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer's disease and for testing new molecules.

Methods: Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software.

Results: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt.

Conclusion: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.
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http://dx.doi.org/10.4252/wjsc.v5.i4.229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812526PMC
October 2013

CHF5074 reduces biomarkers of neuroinflammation in patients with mild cognitive impairment: a 12-week, double-blind, placebo-controlled study.

Curr Alzheimer Res 2013 Sep;10(7):742-53

Memory Enhancement Center of America, 4 Industrial Way West 2nd Floor, Eatontown, NJ 07724, USA.

As neuroinflammation is an early event in the pathogenesis of Alzheimer' s disease, new selective antiinflammatory drugs could lead to promising preventive strategies. We evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of CHF5074, a new microglial modulator, in a 12-week, double-blind, placebo-controlled, parallel groups, ascending dose study involving 96 MCI patients. Subjects were allocated into three successive study cohorts to receive ascending, titrated doses of CHF5074 (200, 400 or 600 mg/day) or placebo. Vital signs, cardiac safety, neuropsychological performance and safety clinical laboratory parameters were assessed on all subjects. Plasma samples were collected throughout the study for measuring drug concentrations, soluble CD40 ligand (sCD40L) and TNF-α. At the end of treatment, cerebrospinal fluid (CSF) samples were optionally collected after the last dose to measure drug levels, β- amyloid1-42 (Aβ42), tau, phospho-tau181, sCD40L and TNF-α. Ten patients did not complete the study: one in the placebo group (consent withdrawn), two in the 200-mg/day treatment group (consent withdrawn and unable to comply) and seven in the 400-mg/day treatment group (five AEs, one consent withdrawn and one unable to comply). The most frequent treatment-emergent adverse events were diarrhea, dizziness and back pain. There were no clinically significant treatmentrelated clinical laboratory, vital sign or ECG abnormalities. CHF5074 total body clearance depended by gender, age and glomerular filtration rate. CHF5074 CSF concentrations increased in a dose-dependent manner. At the end of treatment, mean sCD40L and TNF-α levels in CSF were found to be inversely related to the CHF5074 dose (p=0.037 and p=0.001, respectively). Plasma levels of sCD40L in the 600-mg/day group were significantly lower than those measured in the placebo group (p=0.010). No significant differences between treatment groups were found in neuropsychological tests but a positive dose-response trend was found on executive function in APOE4 carriers. This study shows that CHF5074 is well tolerated in MCI patients after a 12-week titrated treatment up to 600 mg/day and dose-dependently affects central nervous system biomarkers of neuroinflammation.
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http://dx.doi.org/10.2174/13892037113149990144DOI Listing
September 2013

From the multifactorial nature of Alzheimer`s disease to multitarget therapy: the contribution of the translational approach.

Curr Top Med Chem 2013 ;13(15):1843-52

Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 41/E, 40064 Ozzano Emilia, Bologna, Italy.

The drug discovery for disease-modifying agents in Alzheimer disease (AD) is facing a failure of clinical trials with drugs based on two driving hypotheses, i.e. the cholinergic and amyloidogenic hypotheses. In this article we recapitulate the main aspects of AD pathology, focusing on possible mechanisms for synaptic dysfunction, neurodegeneration and inflammation. We then present the pharmacological and neurobiological profile of a novel compound (CHF5074) showing both anti-inflammatory and gamma-secretase modulatory activities, discussing the possible time-window for effective treatment in an AD transgenic mouse model. Finally, the concept of cognitive reserve is introduced as possible target for preventive therapies.
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http://dx.doi.org/10.2174/15680266113139990140DOI Listing
March 2014

Multi-target action of the novel anti-Alzheimer compound CHF5074: in vivo study of long term treatment in Tg2576 mice.

BMC Neurosci 2013 Apr 5;14:44. Epub 2013 Apr 5.

Department of Veterinary Medicine, University of Bologna, Bologna, Italy.

Background: Alzheimer disease is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The pathological hallmarks includes extracellular amyloid plaques and intraneuronal neurofibrillary tangles, but the primary cause is only partially understood. Thus, there is growing interest in developing agents that might target multiple mechanisms leading to neuronal degeneration. CHF5074 is a nonsteroidal anti-inflammatory derivative that has been shown to behave as a γ-secretase modulator in vitro and to inhibit plaque deposition and to reverse memory deficit in vivo in transgenic mouse models of Alzheimer's disease (AD). In the present study, the effects of a long-term (13-month) treatment with CHF5074 on indicators of brain functionality and neurodegeneration in transgenic AD mice (Tg2576) have been assessed and compared with those induced by a prototypical γ-secretase inhibitor (DAPT).

Results: To this end, plaque-free, 6-month-old Tg2576 mice and wild-type littermates were fed with a diet containing CHF5074 (125 and 375 ppm/day), DAPT (375 ppm/day) or vehicle for 13 months. The measured indicators included object recognition memory, amyloid burden, brain oligomeric and plasma Aβ levels, intraneuronal Aβ, dendritic spine density/morphology, neuronal cyclin A positivity and activated microglia. Tg2576 mice fed with standard diet displayed an impairment of recognition memory. This deficit was completely reverted by the higher dose of CHF5074, while no effects were observed in DAPT-treated mice. Similarly, amyloid plaque burden, microglia activation and aberrant cell cycle events were significantly affected by CHF5074, but not DAPT, treatment. Both CHF5074 and DAPT reduced intraneuronal Aβ content, also increasing Aβ40 and Aβ42 plasma levels.

Conclusions: This comparative analysis revealed a profoundly diverse range of clinically relevant effects differentiating the multifunctional anti-inflammatory derivative CHF5074 from the γ-secretase inhibitor DAPT and highlighted unique mechanisms and potential targets that may be crucial for neuroprotection in mouse models of AD.
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http://dx.doi.org/10.1186/1471-2202-14-44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626610PMC
April 2013

CHF5074 restores visual memory ability and pre-synaptic cortical acetylcholine release in pre-plaque Tg2576 mice.

J Neurochem 2013 Mar 28;124(5):613-20. Epub 2013 Jan 28.

Department of Veterinary Medicine and Health Science, University of Bologna, Bologna, Italy.

CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's disease transgenic mice. In this study, the effect of an acute or subacute CHF5074 treatment on in vivo novel object recognition test and on [³H]Acetylcholine (ACh) and GABA release in pre-plaque (7-month-old) Tg2576 mice have been compared with those induced by the γ-secretase inhibitor LY450139 (semagacestat). Vehicle-treated Tg2576 mice displayed an impairment of recognition memory compared with wild-type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle-treated Tg2576 mice, K⁺-evoked [³H]ACh release was lower than that measured in wild-type mice. This reduction was absent in transgenic animals subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days). There were no differences between the groups on spontaneous [³H]ACh release as well as spontaneous and K⁺-evoked GABA release. These results suggest that CHF5074 has beneficial effects on visual memory and cortical cholinergic dysfunctions in pre-plaque Tg2576 mice. Together with previous findings, these data suggest that CHF5074 could be a possible candidate for early Alzheimer's disease therapeutic regimens.
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http://dx.doi.org/10.1111/jnc.12136DOI Listing
March 2013

Pharmacokinetics and pharmacodynamics of CHF5074 after short-term administration in healthy subjects.

Alzheimer Dis Assoc Disord 2013 Jul-Sep;27(3):278-86

Research & Development, Chiesi Pharmaceuticals Inc., Rockville, MD, USA.

CHF5074 has been shown to inhibit brain β-amyloid deposition and attenuate memory deficits in different transgenic mice models of Alzheimer disease. We evaluated the safety, pharmacokinetics, and pharmacodynamics of 3 ascending dose regimens of CHF5074 (200, 400, and 600 mg/d for 14 d) in a double-blind, placebo-controlled, parallel group study involving 48 healthy subjects. Plasma, urine, and cerebrospinal fluid (CSF) samples were collected for measuring drug and main metabolite concentrations and potential biomarkers of pharmacodynamic activity (β-amyloid1-40, β-amyloid1-42, soluble CD40 ligand, and tumor necrosis factor-α). All subjects completed the study, and no serious or severe adverse events were reported. The maximum tolerated dose was close to 600 mg/d with mild diarrhea being the most frequent adverse event at this dose. CHF5074 reached peak plasma levels 2 to 3 hours after drug administration and then was slowly eliminated (t(1/2z)=30 h) in the urine as glucoronide. Systemic exposure to the drug appeared to be dose-proportional with a 2-fold accumulation ratio at steady state. Metabolite plasma levels peaked at 4 to 5 hours and accounted for about 25% of the parent compound. Drug levels in the CSF were dose-proportional. The drug dose-dependently lowered the levels of the soluble CD40 ligand, a marker of microglia activation, in both plasma and CSF samples.
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http://dx.doi.org/10.1097/WAD.0b013e3182622aceDOI Listing
March 2014