The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.

Anandamide (AEA), a well characterized endocannabinoid that has actions at multiple targets in the eye, may have potential as a novel therapeutic in the treatment of retinal disease. However, AEA is rapidly degraded by fatty acid amide hydrolase (FAAH). Therefore this study examined the drug URB597, that inhibits FAAH degradation of AEA, to assess AEA effects in experimental models of retinal damage. The objectives were to: 1) evaluate changes present in the aging retina, 2) determine whether the aging retina is more susceptible to tissue damage, and 3) investigate whether increasing AEA can provide retinal neurovascular protection in young and aged retina following damage. The results from this study showed that URB597 had protective effects on retinal ganglion cells and retinal capillaries and inhibited phagocytotic MG in models of retinal damage in young, but not the aged retina.

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

Brain injury, either surgically induced or as a result of trauma or stroke, is one of the leading causes of death and disability worldwide. Since transplantable stem cell sources are showing a great deal of promise and are actively being pursued to provide neuroprotection post-injury, in this body of work, we set out to characterize and examine the therapeutic potential of amniotic fluid derived (AF) cells as a potential cell source for cell-based therapies in mediating neuroprotection post-injury. Despite their heterogeneity, we found that AF cells are mainly epithelial in origin and express various genes involved in stem cell maintenance and neural commitment. A very small subset of AF cells also express pluripotency markers OCT4a, SOX2 and NANOG, which can be enriched for by single cell cloning. SOX2 positive clones have the capacity to give rise to a neuronal phenotype, in neural induction conditions, which can be used to examine the neural differentiation capabilities of AF cells. Subsequently, we examined the ability of AF cells to mediate a neuroprotective effect in a surgically induced brain injury model through gap junctional-mediated direct cell-cell communication and as a vehicle for GDNF delivery post-injury. AF cells express high levels of CX43 and are able to establish functional gap junctional intercellular communication (GJIC) with cortical astrocytes. We report an induction of Cx43 expression in astrocytes following injury and demonstrate, for the first time, CX43 expression at the interface between implanted AF cells and host astrocytes. In an effort to boost host endogenous neuroprotective mechanisms post-injury, via neurotrophic factor delivery, we engineered AF cells to secrete GDNF (AF-GDNF). GDNF pre-treatment significantly increased AF cell and cortical neuron survival rates following exposure to hydrogen peroxide. AF-GDNF cells, seeded on polyglycolic acid (PGA) scaffolds, survived longer in serum-free conditions and continued to secrete GDNF post-implantation activating the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area. Despite some promising trends, we did not observe significant behavioural improvements following AF-GDNF/PGA implantation nor reduced lesion volume during the 7 day time-frame. In conclusion, through GJIC with cortical astrocytes and delivery of exogenous neurotrophic factors, AF cells hold great promise in mediating neuroprotection post-injury.

Spinal cord injury (SCI) is a debilitating condition accompanied by motor and sensory deficits and a reduced quality of life. Current treatment options are limited and are associated with variable efficacy and a risk of adverse effects. The pathophysiology of SCI is initiated by a primary mechanical insult to the spinal cord, followed by a complex series of deleterious events known as secondary injury. Secondary injury processes include free radical formation, glutamate excitotoxicity, inflammation and cell death. Apoptotic cell death in particular plays a key role in the secondary injury processes and exacerbates tissue degradation and loss of function. The role of Fas-mediated apoptosis in SCI pathophysiology is poorly defined in the literature to date. Correlative evidence suggests that this form of cell death is delayed and occurs in white matter adjacent to sites of primary damage. The cellular and temporal mechanisms of Fas-mediated apoptosis following experimental SCI were evaluated using a clinically relevant clip compression SCI model in the rat. Furthermore, therapeutic manipulation of Fas activation using a soluble form of the Fas receptor (sFasR) was carried out to establish the efficacy and clinical relevance of targeting this aspect of secondary injury. This work shows that Fas-mediated apoptosis is an important contributor to secondary SCI pathology. Oligodendrocytes are targeted by this form of cell death in a delayed fashion post-injury, providing an opportunity for therapeutic intervention. Intrathecal administration of sFasR following SCI reduced post-traumatic apoptosis, improved cell survival, enhanced tissue preservation and resulted in an improved motor recovery. Administration of sFasR was effectively delayed by up to 24 hours post-injury, however a shorter delay of 8 hours post-injury was most efficacious. A surprising result emerged from this work. Delayed intrathecal administration of IgG following SCI showed significant efficacy in both cellular and tissue level outcomes, as well as at the functional level. Fas-mediated apoptosis is an important aspect of secondary SCI pathophysiology and is an attractive therapeutic target. The beneficial outcomes of manipulating Fas activation using sFasR provide further evidence for this. Future work will refine this treatment strategy, bringing it into the SCI patient population.

The lithium/pilocarpine model of epilepsy mimics mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) in humans. Systemic injection of pilocarpine in lithium chloride (LiCL) pretreated adult rats results in an acute episode of severe continuous seizure activity (status epilepticus, SE). SE causes a latent period, whereby the animal appears neurologically normal, with subsequent development of spontaneous recurrent seizures (SRSs). Neuropathological changes that occur during the latent period are believed to contribute to the epileptic condition. The present thesis characterized the development of neuronal death and behavioural alterations in rats after SE induced by the repeated low-dose pilocarpine procedure (RLDP), and investigated the causal relationship between these two processes. Our data demonstrated that the RLDP procedure for the induction of SE results in widespread neurodegeneration and behavioural alterations comparable to the pilocarpine and low-dose pilocarpine (LDP) procedures. However, the advantage to using this protocol was strain-dependent as it reduced mortality in Wistar, but not in Long Evans Hooded (LEH), rats. Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various times (1 hr to 3 months) following SE showed that different brain regions within the hippocampus, amygdala, thalamus and piriform cortex exhibited differential rates of neuronal loss, with the majority of SE-induced neuronal death present by 24 hours. SE resulted in decreased exploratory behavior as assessed in the open field test, increased aggression to handling, increased hyperreactivity as assessed in the touch-response test, and anxiolytic effects as measured in the elevated-plus maze. Furthermore, deficits in search strategies used, as well as impaired spatial learning and memory, contributed to poor Morris water maze (MWM) performance. Partial neuroprotection within the hippocampus (by tat-NR2B9c) had no effect on the number of rats developing SRSs or on behavioural alterations; this argues against a causal relationship between neurodegeneration within this region, genesis of SRSs, and behavioural morbidity.

Traumatic Brain Injury (TBI) is a leading cause of death and disability in the United States for which there are no federally approved pharmacological treatments. Preclinical trials with nicotinamide (NAM) and progesterone (Prog) treatment demonstrate beneficial neuroprotection and recovery of function following TBI. The primary goal of this study was to assess both neuroprotection and recovery of function in an animal model of TBI after combination treatment of both NAM and Prog. Animals received a cortical contusion injury over the sensorimotor cortex and were treated with either nicotinamide (75 mg/kg, i.p. NAM loading dose, 12 mg/kg/hr NAM, s.c. over 72 hrs), Prog (10 mg/kg Prog, i.p. over 72 hrs), NAM and Prog(75 mg/kg, i.p. NAM loading dose, followed by continuous infusion of 12 mg/kg/hr NAM, s.c. over 72 hrs; 10 mg/kg Prog, i.p. over 72 hrs) or Vehicle (75 mg/kg, i.p. sterile saline loading dose, followed by continuous infusion 12 mg/kg/hr sterile saline, s.c. over 72 hrs; 10 mg/kg peanut oil, i.p. over 72 hrs), and compared to a craniotomy only (Sham) group. Following this regimen they were assessed in a battery of behavioral (fine and gross motor, sensory, and cognitive) tasks or a histological assessment at 24 hrs post-injury assessing lesion cavity size, degenerating neurons, and reactive astrocytes. Our results replicate the beneficial effects of treatment with either NAM or Prog demonstrating significant improvements in recovery of function, and a reduction in lesion cavitation, degenerating neurons and reactive astrocytes 24 hours post-injury. The combination treatment of NAM and Prog led to a significant improvement in both neuroprotection at 24 hrs post-injury and recovery of function in sensorimotor related tasks when compared to each individual treatment (NAM or Prog). It is suggested here that further preclinical trials using NAM and Prog as a combination treatment should be done to identify any drug interactions, pharmacokinetics, and a new window of opportunity and proper dosing of this combination treatment.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T11:40:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_BrunaCostabeberGuerino.pdf: 1767438 bytes, checksum: 21a9128d4c5ddfc4768c4fa4fdc8325c (MD5) / Made available in DSpace on 2018-08-17T11:40:09Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_BrunaCostabeberGuerino.pdf: 1767438 bytes, checksum: 21a9128d4c5ddfc4768c4fa4fdc8325c (MD5) Previous issue date: 2016-07-12 / The statins are drugs used to treat hypercholesterolemia because their mechanism of action is to inhibit the enzyme HMG CoA reductase what is part of cholesterol biosynthesis. Studies have shown that statins have pleiotropic effects and action in the central nervous system (CNS) simvastatin (SIN) is evaluated as a prevention strategy in the treatment of neurodegenerative diseases such as Alzheimers and Parkinson’s. SIN is metabolized rapidly in the body with nonspecifically delivery to tissues. An alternative solution to these problems is to use nanocarriers that take the drugs to their target site. The nanocapsules systems are considered vectors for the administration of lipophilic substances by having a large surface area. The objective is to evaluate the effect of simvastatin nanocoated memory and type behavior anxious rats. The suspensions of simvastatin nanocapsules (NS) were produced by the interfacial deposition technique preformed polymer described by Fessi et al (1989) adapted by Venturini et al (2011). NS were characterized by dynamic light scattering (Zetasizer), Zeta potential, pH determination, drug content and encapsulation efficiency. The rats Wistar adult young (3 months, 250g-300g), midle aged (8 months, 450-500g) and adult old (12 to 13 months, 500g-700g) were from the central University vivarium Federal de Santa Maria and all procedures were performed after approval of CEUA (nº002/ 2016 protocol). First it performed a chronic treatment at the dose of 15 mg/kg or 30 mg/kg and SIN or NS rats young adults orally (v.o.) for 21 days and 30 min before testing of the inhibitory avoidance task (EI) was administered scopolamine amnesia-inducing (ESC) at a dose of 0.4 mg/kg. After this protocol was performed an acute treatment in young-adult rats with SIN or NS at a dose of 15mg/kg and 30 min before the EI test was administered i.p ESC or 75 min the other amnesic inducer ketamine (CET). The third protocol rats middle age was performed an acute treatment with SIN or NS v.o. at a dose of 15 mg/kg and the animals were evaluated for aversive memory in EI. In the last protocol adults age animal were treated chronically v.o. with SIN or NS at a dose of 15 mg/kg were evaluated in open field task, Plus Maze and EI. The results show that NS presented the particle diameter of 226.9 ± 16.4 nm, polydispersity 0.165 ± 0.04, zeta potential (mV) -8.4 ± 1.07) 6.67 ± 0.27 pH, efficiency encapsulation 77.7% and content of 85%. These parameters are in accordance with the methodology. Chronic treatment with SIN could reverse the damage caused by the ESC in memory at both doses compared to the SAL group. Acute doses of NS reverse the damage 11 caused in the memory of both the ESC and CET as compared to SAL groups. The SIN has a tendency effect not as expressive as the NS. In the acute treatment dose with NS or SIN adult-aged rats NS could be effective in reversing the natural memory loss. Since SIN had no effect. Chronic treatment with NS and SIN adults-old rats could reverse the natural loss of memory in the test of EI. There was no significant difference in locomotor and exploratory activity of animals. The adult aged treated with NS for a longer time in the open arms in elevated cross maze and the number of dips was greater than salt and SIN, indicating an anxiolytic effect of NS. We conclude that the NS has a more significant effect on acute doses and an anxiolytic effect on chronic treatment in adults-old mice. / As estatinas são fármacos utilizados no tratamento da hipercolesterolemia pois seu mecanismo de ação é diminuir a síntese da enzima HMG COA-redutase que faz parte da biossíntese do colesterol. Estudos têm mostrado que as estatinas possuem efeitos pleiotrópicos como ação no sistema nervoso central (SNC) A sinvastatina (SIN) está sendo estuda como uma estratégia de prevenção no tratamento de doenças neurodegenerativas como Alzheimer e Parkinson. Um dos problemas da SIN é que ela é rapidamente metabolizada no organismo e entregue de forma inespecífica aos tecidos. Uma alternativa para solução destes problemas é utilizar nanocarreadores que levam os fármacos ao seu sítio alvo. As nanocápsulas são sistemas considerados vetores para a administração de substâncias lipofílicas por possuírem uma grande área superficial. O objetivo do trabalho foi avaliar o efeito da sinvastatina nanoencapsulada na memória e no comportamento do tipo ansioso em ratos. As suspensões de nanocápsulas de sinvastatina (NS) foram produzidas através da técnica de deposição interfacial do polímero pré-formado descrita por Fessi e colaboradores (1989) e adaptada por Venturini e colaboradores (2011). As NS foram caracterizadas através do espalhamento de luz dinâmico (Zetasizer), potencial Zeta, determinação do pH, teor de fármaco e eficiência de encapsulação. Não foi realizado tratamento com nanocápsulas branca (NB), pois em trabalho anterior do grupo de pesquisa (ALVES, 2015), não foi observada diferença significativa entre os grupos SAL, NB e Näive. Os animais ratos Wistar adultos-jovens (3 meses, 250g-300g), ratos adultos meia idade (8 meses, 450-500g) e ratos adultos-velhos (12 a 13 meses, 500g-700g) foram provenientes do biotério central da Universidade Federal de Santa Maria e todos os procedimentos foram realizados após aprovação do Conselho de Ética do uso de animais do Centro Universitário Franciscano (CEUA) (protocolo nº002/2016). Primeiramente foi realizado um tratamento crônico na dose de 15 mg/kg ou 30 mg/kg com SIN ou NS em ratos adultos-jovens via oral (v.o.) durante 21 dias e 30 min antes do teste da tarefa da Esquiva Inibitória (EI) foi administrado o indutor amnésico escopolamina (ESC) na dose de 0,4 mg/kg. Após este protocolo foi realizado um tratamento agudo em ratos adultos-jovens com SIN ou NS na dose de 15mg/kg (v.o) e 30 min antes do teste de EI foi administrado i.p ESC e 75min o outro indutor amnésico a cetamina (CET). O terceiro protocolo foi realizado um tratamento agudo 9 com SIN ou NS v.o. na dose de 15 mg/kg e os animais foram avaliados quanto a memória aversiva na EI. No último protocolo os animais adultos-velhos foram tratados cronicamente v.o. com SIN ou NS na dose de 15 mg/kg e foram avaliados nos testes de Campo Aberto, Labirinto da Cruz Elevado e EI. As NS apresentaram o diâmetro de partícula 226,9 ± 16,4 nm, índice de polidispersão 0,165 ± 0,04, potencial zeta (mV) -8,4 ± 1,07), pH 6,67 ± 0,27, eficiência de encapsulação 77,7% e teor 85%. Estes parâmetros estão de acordo com a metodologia utilizada. O tratamento crônico com a SIN conseguiu reverter o dano na memória provocado pela ESC em ambas as doses comparado ao grupo SAL. As doses agudas de NS conseguirem o reverter o dano causado na memória tanto da ESC e CET. A SIN tem uma tendência efeito não tão expressivo quanto a NS. No tratamento de dose aguda com NS ou SIN em ratos adultos-meia idade a NS conseguiu ter efeito na reversão da perda natural da memória. Já a SIN não teve efeito. O tratamento crônico com NS e SIN nos ratos adultos-velhos conseguiu reverter a perda natural da memória no teste da EI. Não houve diferença significativa na locomoção e na atividade exploratória dos animais. Os animais tratados com NS ficaram durante mais tempo nos braços abertos no Labirinto de Cruz elevado e o número de mergulhos foi maior que o SAL e SIN indicando um efeito ansiolítico. Conclui-se que a NS possui um efeito mais expressivo em doses agudas e um efeito ansiolítico no tratamento crônico em ratos adultos-velhos.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T19:15:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MichelePereiraMoreira.pdf: 1257282 bytes, checksum: ec3939cec40b071e77b87bd241002dab (MD5) / Made available in DSpace on 2018-08-17T19:15:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MichelePereiraMoreira.pdf: 1257282 bytes, checksum: ec3939cec40b071e77b87bd241002dab (MD5) Previous issue date: 2017-03-31 / Simvastatin is used to treat hypercholesterolemia acting by inhibition of 3-hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase responsible for cholesterol synthesis. Studies carried out in humans, in animal models and in vitro observed neural damage reduction and increased glutamate uptake after simvastatin treatment. Glutamate is the major excitatory neurotransmitter in the central nervous system with crucial role in several physiological mechanisms, but at high levels it leads to cell death and participate to neurodegenerative diseases. Despite of pharmaceutical characteristics of simvastatin, as lipophilic drug that can cross cell membranes it has low bioavailability and non-specific biodistribution, simvastatin reach nervous system at low rates, limiting the pleiotropic effects described. Nanoemulsions may enhance solubility and increase biodistribution of lipophilic drugs, thus this technology can improve simvastatin performance in the neurodegenerative diseases treatment. The aim of study is producing simvastatin-loaded nanoemulsion, evaluate its physicochemical characteristics besides biological safety, and effect against glutamatergic toxicity. Blank nanoemulsion (without drug; BNE) and 1 mg/mL simvastatin nanoemulsion (SNE) were produced by spontaneous emulsification. Physicochemical characterization and stability were determined at three different temperatures (± 4 oC, ± 23 oC or ± 40 oC) for 15 days. Toxicity of formulations at 0.1 μg/mL; 1 μg/mL e 10 μg/mL was evaluated in Vero cell linage cultures and hippocampal slices from rat. Effect of formulations against glutamatergic excitotoxicity (10 mM glutamate) was evaluated at hippocampal slices from rat. All procedures with animals were previously approved by local Ethical Committee (CEUA no 05/2016). Formulation showed macroscopically homogeneous aspect and white color with Tyndall effect. Particle size was approximately 204 nm (BNE) and 139 (SNE). Zeta potential was -3 mV and -5 mV, for BNE and SNE respectively. Both of them appeared polydispersity index lower than 0.3, pH ± 6.5. Drug content was ± 1.01 mg/mL with encapsulation efficiency ± 98%. Formulations did not show any physicochemical alterations following 15 days at ± 23 oC or ± 40 oC, but simvastatin content was reduced. However, nanoemulsions exposed to ± 4 oC had constant drug content with increased particle size and polydispersity index. For all formulations, Zeta potential and pH were constant during 15 days. Cytotoxicity was no detected for all nanoemulsions. When hippocampal slices were incubated with glutamate with free simvastatin or SNE, 42% of cells were died, without effect of simvastatin. At the present work, nanoemulsions were produced efficiently, without cytotoxicity at parameters assayed. Additional studies should be performed to improve nanoemulsions stability. Simvastatin-loaded nanoemulsion or free did not show effect against glutamatergic damage evaluate in hippocampal slices from rat. / A sinvastatina é um fármaco prescrito para hipercolesterolemia, que atua inibindo a 3-metil-hidróxi-glutaril coenzima A (HMG-CoA) redutase, que controla a síntese do colesterol. Estudos em humanos e modelos in vitro e/ou animais indicam que a sinvastatina é capaz de reduzir o dano neural, e também aumentar a captação do glutamato. O glutamato é o principal neurotransmissor excitatório do sistema nervoso central, sendo fundamental em diferentes mecanismos fisiológicos, mas em altas concentrações pode induzir à morte celular e doenças neurodegenerativas. Apesar da sinvastatina ser um fármaco lipofílico e atravessar facilmente as barreiras celulares, a sua baixa biodisponibilidade e biodistribuição inespecífica fazem com que ela alcance o sistema nervoso em baixas concentrações, limitando os efeitos pleiotrópicos descritos. O uso de nanoemulsões pode aumentar a solubilidade e melhorar a biodistribuição de fármacos lipofílicos e, assim, pode melhorar o desempenho da sinvastatina no tratamento de doenças neurológicas. Portanto, o objetivo deste trabalho é preparar uma nanoemulsão contendo sinvastatina, avaliar suas características físico-químicas e de segurança, e avaliar o efeito contra a excitotoxicidade glutamatérgica. As nanoemulsões branca (NEB) ou contendo sinvastatina (NES) na concentração de 1 mg/mL foram preparadas por emulsificação espontânea. Foi realizada a caracterização físico-química e a estabilidade das formulações foi determinada em três temperaturas (±4 oC, ±23 oC ou ±40 oC), por 15 dias. A toxicidade das formulações foi avaliada nas concentrações de 0,1 μg/mL; 1 μg/mL e 10 μg/mL em células Vero e em fatias de hipocampo de ratos. A avaliação de efeito contra excitotoxicidade foi realizada nas fatias de hipocampo submetidas ao glutamato 10 mM. Os procedimentos descritos foram aprovados pela Comissão de Ética no Uso de Animais (CEUA) do Centro Universitário Franciscano (protocolo 05/2016). As formulações produzidas apresentaram-se homogêneas, de cor branca levemente azulada. O tamanho de partícula foi de aproximadamente 204 nm (NEB) e 139 nm (NES). O potencial zeta foi de -3 mV e -5 mV, para NEB e NES, respectivamente. Ambas apresentaram índice de polidispersão abaixo de 0,3, e o pH ± 6,5. O teor de fármaco encontrado em NES foi de ± 1,01 mg/mL com taxa de associação de ± 98%. As nanoemulsões não apresentaram alterações nas características físico-químicas após 15 dias de armazenamento, mantidas a ± 23 oC ou ± 40 oC, mas houve redução no teor de sinvastatina. Enquanto as nanoemulsões mantidas em ± 4 oC mantiveram o teor do fármaco, mas sofreram aumento no tamanho e índice de polidispersão. O potencial zeta e o pH mantiveram-se estáveis em todas as formulações desde o preparo até após 15 dias. Não houve indicativo de toxicidade por parte das nanoemulsões nos ensaios propostos. Nas fatias de hipocampo submetidas ao glutamato 10 mM e à sinvastatina livre ou em nanoemulsão, observou-se redução em até 42% da viabilidade, sem efeito da sinvastatina. Foi possível, no presente trabalho, produzir com sucesso nanoemulsões contendo sinvastatina, que não demonstraram toxicidade através dos parâmetros avaliados. Contudo estudos adicionais para melhorar a estabilidade deverão ser realizados. A sinvastatina livre ou em nanoemulsão, não foi capaz de reverter o dano provocado pelo glutamato no modelo proposto.