Objetivos: Verificar eficácia da cetamina intranasal na sedação de crianças para punção venosa. Métodos: Estudo randomizado, duplo-cego, placebo controlado realizado no Hospital de Clínicas de Porto Alegre entre janeiro e agosto de 2016. Estudo aprovado pela comissão de ética em pesquisa da instituição. Incluídas crianças que necessitasse de punção venosa, sendo randomizadas a receber cetamina IN 4mg/Kg ou solução fisiológica no grupo Placebo. Os grupos foram comparados quanto: tempo de punção, facilidade do Enfermeiro para realizar o procedimento, eventos adversos, alterações dos sinais vitais e percepção do acompanhante. Resultados: Foram incluídas 39 crianças (21 Intervenção vs 18 Placebo) sem diferenças quanto à idade, sexo, peso, motivo da internação e experiência profissional. A mediana da idade foi 19,8 vs 15,8 meses (Intervenção vs. Placebo) e a do peso foi 10 vs. 11,3Kg. A Cetamina reduziu o tempo de punção (23,0 vs 67,5 segundos; p=0,01), deu maior facilidade ao Enfermeiro para realizar o procedimento (p=0,00009). A cetamina induziu uma maior sonolência 15 minutos após (p=0,003) e reduziu o número de pessoas para contenção da criança (p=0,025). Sem diferença entre os grupos nas alterações dos sinais vitais e eventos adversos. Evento adverso observou-se em 29% das crianças do grupo cetamina e 17% do grupo placebo, sendo irritabilidade o mais comum em ambos. Em 81% do grupo Intervenção, o acompanhante afirmou que a criança ficou mais calma (p=0,0003). Conclusões: Cetamina intranasal (4mg/Kg) reduz o tempo de punção venosa, facilitando o procedimento para o enfermeiro, diminuindo o número de pessoas envolvidas e permitindo um ambiente tranquilo. / Objectives: To verify the efficacy of intranasal ketamine as sedative agent for venous access in children. Method: Randomized, double blind, placebo controlled study conducted at Hospital de Clínicas de Porto Alegre (Brazil) between January and August 2016. Children needing venous access were randomized to receive intranasal ketamine (4mg/Kg) or normal saline solution (Placebo group). Groups were compared regarding the time for venous access, facility for performing the procedure, adverse events, disturbances in vital signs and perception of the accompanying adult. The study was approved by the Local Ethics Committee. Results: 39 children (21 Ketamine; 18 Placebo) were included without differences regarding to age, sex, weight, reason for hospitalization and professional experience. The median age was similar (19.8 vs 15.8 months), as well as the median weight (10.0 vs 11.3Kg). Ketamine reduced the length for venous access (23.0 vs 67.5 seconds; p=0.01), and facilitated the procedure (p=0.00009). Ketamine induced sleepiness 15 minutes after its administration (p=0.003) and reduced the number of people for the child’s restraint (p=0.025). No difference was verified between groups regarding adverse effects or vital signs disturbance´s. Side effects were observed in 29% of the children in the Ketamine group and 17% in the Placebo group, irritability being the most common for both. The accompanying adult reported that 81% of children in ketamine group were calm and quiet (p=0.0003). Conclusions: Intranasal ketamine (4mg/Kg) reduces the time for venous puncture, facilitates the procedure to the nurse, decreases the number of people involved and provides a tranquil environment.

Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T06:15:03Z (GMT). No. of bitstreams: 1 ZanellatoFabbri_Natalia_M.pdf: 1108663 bytes, checksum: 3362aa87243ab7742f6bb55df1f964e6 (MD5) Previous issue date: 2009 / Resumo: Rinite alérgica é uma doença comum com ampla morbidade, que gera aumento considerável nos custos de tratamento médico, redução da produtividade no trabalho e absenteísmo escolar. A aplicação tópica de corticosteróides intranasal é amplamente reconhecida como primeira linha de tratamento antiinflamatório. Espera-se que a maioria dos sprays nasais prescritos como drogas de ação local, ainda não possuam patente permitindo o aumento de cópias genéricas desses medicamentos, levando a concorrência e redução de preço. Estudos de bioequivalência para sprays nasais estão ainda em discussão. Geralmente, os estudos para essa finalidade usa modelos de intervenção terapêutica a longo prazo, com altos custos para o paciente e longo tempo de duração. O objetivo deste trabalho foi mostrar a aplicabilidade da provocação nasal com histamina e rinomanometria em estudos de bioequivalência para sprays nasais. Trata-se de um estudo aberto, cruzado aleatorizado, utilizando dois períodos e duas seqüências para avaliar a equivalência farmacodinâmica entre duas formulações de sprays de dipropionato de beclometasona de manufaturamento distinto. Após estímulo nasal com histamina (0,5 mg/ml em ambas narinas), 25 voluntários saudáveis foram submetidos a rinomanometria anterior nos tempos 0; 15; 30 e 60 minutos para estabelecimento do fluxo, pressão e resistência basal de cada câmara nasal. Os voluntários foram submetidos à utilização do spray nasal com a droga teste (T) ou referência (R) de acordo com o esquema de randomização, e a área sobre a curva foi analisada. De acordo com os parâmetros estudados os resultados obtidos mostraram diferença significativa entre as duas formulações (p= 0,31) indicando a equivalência terapêutica entre as drogas T e R. Assim, estes resultados sugerem que provocação nasal com histamina em indivíduos saudáveis e o emprego da rinomanometria como método quantitativo das alterações provocadas nas câmaras nasais possam ser aplicáveis em estudos farmacodinâmicos de bioequivalência para sprays nasais / Abstract: Allergic rhinitis is a common condition with widespread morbidity, increased medical treatment costs, reduced work productivity and lost school days. Topically delivered intranasal corticosteroids are widely recognized to be the first-line anti-inflammatory treatment. It is expected that many of the most-prescribed nasal sprays for local action drugs will go off patent, allowing the increase of the generic copies of these medications, with more products competition and price reduction. The bioequivalence studies for nasal sprays are still in discussion. Usually the studies designs for this purpose use a long-term therapeutic intervention models using patients with high costs and time duration. This study was designed to demonstrate the feasibility of rhinomanometry in bioequivalence studies for nasal sprays. Study design, an open, randomized, crossover study, using two periods and two sequences to evaluate pharmacodynamic equivalence between two formulations of beclometasone dipropionate spray. After nasal challenge with histamine (0.5mg/ml, in both nostrils), 25 healthy volunteers were submited to an anterior rhinomanometry at the time 0; 15; 30 and 60 minutes building a baseline of flow, pression and resistance of nasal chamber. Then, the vontuteers were submited to nasal drug spray (Test (T) or Reference(R)), according to randomized schedule and the Are Under Curve (AUC0-t) analyzed. The results suggest the potential use of nasal histamine challenge and rhinomanometry evaluation in healthy subjects in bioequivalence studies for nasal sprays / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Devido à intensa oscilação de funções e mecanismos de defesa respiratórios evidenciados nos primeiros meses de vida dos bezerros, o discernimento do limiar etário para a resposta vacinal intranasal efetiva, atrelado ao risco de infecção e perfil da vacina são fundamentais para a pecuária racional. Diante disto, o estudo buscou comparar a resposta imune humoral e mediada por células, local e sistêmica, entre bezerros vacinados intranasalmente aos 15 ou 45 dias de vida com vacina polivalente contendo vírus sincicial respiratório bovino vivo modificado, cepas quimicamente alteradas de herpesvirus bovino tipo 1 e vírus da parainfluenza tipo 3, vírus da diarreia viral bovina tipos 1 e 2 inativados, além de culturas inativadas dos cinco sorotipos de Leptospira e adjuvante Quil-A. Foram utilizados 16 bezerros holandeses alocados em três grupos, o grupo GA composto por seis animais primovacinados aos 15 dias de idade, o grupo GB composto por cinco animais primovacinados aos 45 dias de idade e o grupo controle composto por cinco animais não vacinados. Os animais vacinados receberam uma segunda dose vacinal 21 dias após a primeira. Para avaliação da interferência da vacinação intranasal no estado geral dos bezerros, na expressão das populações leucocitárias, na capacidade fagocítica e microbicida, além da investigação de anticorpos humorais e locais, foram realizados exames clínicos e colhidas amostras de sangue e lavado broncoalveolar (LBA) antes da primeira e da segunda dose vacinal, bem como uma terceira colheita 21 dias após a segunda dose. Foi observado que após a vacinação a maioria dos animais vacinados apresentou manifestações clínicas respiratórias, acompanhadas de leucograma com declínio de leucócitos totais e linfócitos nos animais do GA em relação ao CTLA. Nos animais do GB além das manifestações clínicas, foi encontrada somente uma tendência ao aumento de neutrófilos em relação ao CTLB. Houve diminuição de macrófagos alveolares e aumento de neutrófilos sanguíneos e alveolares tanto na citologia do LBA quanto na imunofenotipagem dos animais do GA em relação ao CTLA e diminuição de macrófagos na citologia do LBA ao longo dos momentos do GA, enquanto que nos animais do GB houve diminuição de monócitos-like CD14+ ao longo dos momentos. Simultaneamente ao aumento de monócitos CD14+ ao longo dos momentos, foi notado o aumento de linfócitos TCD3+ CD4- CD8- nos animais do GA ao longo dos momentos e em relação ao CTLA. Além disso, a porcentagem de linfócitos sanguíneos TCD3+ aumentou no GA em relação ao CTLA, porém não acompanhada pelo aumento na porcentagem de células TCD4+ ou TCD8+ mas sim pelas populações TCD3+CD4-CD8- ao longo dos momentos e em relação ao CTLA e TCD3+ CD4+ CD8+ em relação ao CTLA. No LBA a porcentagem de células CD4+ diminuiu em relação ao CTLA. Nos animais do GB não houve diferença entre essas populações celulares, somente a diminuição de células CD4+ CD8+ em relação ao CTLB no LBA. Ao se avaliar as funções de fagocitose e microbicida dos grupos vacinados, foi constatada diminuição expressiva na maioria das avaliações após a vacinação, principalmente em relação ao GA. Em relação à resposta humoral por anticorpos, o estudo demonstrou que a resposta vacinal por via intranasal não sofreu influência de anticorpos colostrais, tampouco induziu imunidade sérica específica por anticorpos, no entanto, no LBA foi capaz de induzir aumento de IgA nos animais do GA e uma tendência ao aumento em animais do GB. Diante dos resultados, conclui-se que: a resposta à vacina utilizada por via intranasal em bezerros com 15 dias de idade é diferente de quando utilizada aos 45 dias, e que causou alterações de exame clínico, leucograma e citologia do LBA mais intensas nos animais vacinados aos 15 dias de idade. Embora não apresente resposta anticorpo específica sistêmica em ambos os casos, aos 15 dias apresentou resposta neutrofílica e monocítica sistêmica acompanhada de aumento de linfócitos T duplo negativos e duplo positivos, enquanto a resposta local celular foi pobre em macrófagos e rica em neutrófilos e IgA, porém com comprometimento das funções celulares. Dessa forma, a vacina utilizada nesse período, por via intranasal, em especial aos 15 dias de idade pode predispor o animal a infecções bacterianas secundárias. No entanto, deve-se ficar claro que este resultado se aplica somente a estas condições de vacina, via e idade / Due to the intense oscillating of functions and respiratory defense mechanisms evidenced in the first months of life of calves, the discernment about the age threshold for the effective intranasal vaccine response, linked to the risk of infection and vaccines profile are fundamental to rational livestock. In view of this, the study aimed to compare the humoral immune response and local and systemic cell-mediated among calves vaccinated intranasally to 15 or 45 days of life with polyvalent vaccine containing bovine respiratory syncytial modified live virus, chemically altered strains of bovine herpesvirus type 1 and parainfluenza virus type 3, inactivated bovine viral diarrhea types 1 and 2, inactivated cultures of five Leptospira serovars and Quil-A adjuvant. 16 Holstein calves were allocated in three groups, the GA group consisting of six animals vaccinated at 15 days of age, the GB group consisting of five animals vaccinated at 45 days of age and the control group of five animals not vaccinated. Vaccinated animals received a second immunization dose 21 days after the first one. To evaluate the impact of intranasal vaccination in the general condition of the calves, in the expression of leukocyte populations, phagocytic and microbicide capacity, as well as research of humoral and local antibodies, clinical examinations were performed and blood and bronchoalveolar lavage (BAL) samples were collected before the first and second vaccine vaccination, as well as a third sample 21 days after the second dose. It was observed that after vaccination most vaccinated animals showed respiratory clinical manifestations, accompanied by white blood cell count with decline of total leukocytes and lymphocytes in GA animals compared to CTLA. In GB animals beyond the clinical manifestations, it was only found a tendency to increase in neutrophils in relation to CTLB. There was a decrease in alveolar macrophages and increased blood and alveolar neutrophils in both BAL cytology and immunophenotyping of GA animals compared to the CTLA and reduction of macrophages in BAL cytology over the times in GA animals, whereas in GB animals there was a decreased in CD14+ monocyte-like along time. Simultaneously with the increase in CD14+ monocytes over the time, it was noted an increase in TCD3+CD4-CD8- lymphocytes in GA animals along time and compared to CTLA. Furthermore, the percentage of TCD3+ blood lymphocytes increased in GA compared to the CTLA but it was not accompanied by an increase in the percentage of CD4+ or CD8+ T cells but by TCD3+ CD4- CD8- population along time and compared to the CTLA and TCD3+ CD4+ CD8+ cells compared to CTLA. In BAL the percentage of CD4+ cells decreased compared to CTLA. In animals GB there was no difference between these cell populations, only the reduction of CD4+ CD8+ BAL cells compared to CTLB. When evaluating the phagocytosis and microbicidal functions of the vaccinated groups it was observed significant decrease in most evaluations after vaccination, especially in relation to GA. Regarding the humoral antibody response, the study demonstrated that the vaccine response by intranasally rout was not influenced by colostral antibodies, either induced serum specific immunity antibodies, however, in BAL was able to induce increase of IgA in GA animals and a tendency to increase in GB animals. Given the results, it is concluded that: the response to the vaccine used intranasally in 15 days old calves is different than when used at 45 days, and it caused clinical examination, WBC and BAL fluid cytology more intense changes in animals vaccinated at 15 days of age. Although it has not systemic specific antibody response in both cases, after 15 days showed neutrophilic and monocytic systemic response accompanied by increase of double negative and double positive T lymphocytes, while the local cell response was poor in macrophages and rich in neutrophils and IgA, but with impairment of cellular functions. Thus, the vaccine used intranasally in that period, particularly at 15 days of age may predispose an animal to secondary bacterial infections. However, it should be clear that this result applies only to these conditions of vaccine, route and age

Sedan början av 2000-talet har ambulanssjukvården haft en snabb utveckling inom flera områden. Möjligheten att administrera smärtlindring har utvecklats och dagens ambulanser är nu mer välutrustade med ett flertal smärtlindrande preparat att välja mellan. Detta ställer stora krav på den specialistsjuksköterska som vårdar patienten. År 2015 infördes nya läkemedel och även ett, för ambulanssjukvården nytt administreringssätt: intranasalt. En stor andel av patienterna i ambulanssjukvården lider av akut smärta och är således i behov av smärtlindring. Sufentanil är en stark opioid med egenskaper lämpade för intranasal administrering. Det finns dock få prehospitala studier om intranasal smärtlindring generellt och än färre gällande sufentanil. Dessutom har ingen tidigare forskning har jämfört effekten av intranasalt administrerat sufentanil mellan barn, vuxna och äldre.   Syftet med denna studie var att beskriva användningen av intranasalt administrerat sufentanil i en prehospital kontext. Vidare studerades frågeställningar gällande grad av smärtlindring, förekomst av biverkningar, etablering av perifer infart, i vilken miljö patienten befann sig samt personalens upplevelser.   Författarna analyserade ett antal utvärderingsprotokoll som fyllts i av sjuksköterskor som administrerat intranasalt sufentanil. Studiens data delades upp i tre grupper utifrån deltagarnas ålder. De tre grupperna var barn, vuxna och äldre. Kvantitativ ansats användes och datan analyserades med hjälp av ANOVA och chi2test beroende på datanivå. Jämförande analyser gällande skillnad i medelvärde samt förekomst genomfördes mellan grupperna. Till exempel jämfördes grad av smärtlindring, mängd givet läkemedel samt förekomst av biverkningar mellan grupperna.   Resultatet visade att samtliga grupper erhåller en adekvat smärtlindring, vilket definierades som tre poäng eller lägre mätt med visuell analog skala [VAS] vid avlämning på akutmottagningen. Barn erhöll signifikant högre doser jämfört med både vuxna och äldre samt hade en lägre VAS vid avlämning på akutmottagning. Andningsdepression förekom i två fall och då i gruppen äldre. Illamående och kräkningar var den vanligaste biverkan hos vuxna och äldre medan det hos barn var slöhet. I barngruppen så etablerades signifikant färre antal perifera venkatetrar [PVK] jämfört med övriga grupper. Sjuksköterskorna uppskattade att behandlingen hade bäst i effekt på barn och därefter äldre samt vuxna.   Slutsatsen var att prehospital administrering av sufentanil tycks ge en bra smärtlindring som ligger inom behandlingsmålet, andelen biverkningar är inte större än i tidigare studier. Däremot visade vårt resultat att barn i viss utsträckning blir slöa vid behandling med intranasalt sufentanil. / Since the beginning of the 2000s, Emergency Medical Services has had rapid development in several areas. The possibility to administer pain relief has been developed and today's ambulances are well-equipped with a variety of pain relief preparations to choose from. This places great demands on the specialist nurse who cares for the patient. In 2015, new medicines were introduced alongside with a, for the ambulance service, new route of administration: Intranasal. A large proportion of the patients in the ambulance service are suffering from acute pain and are therefore in need of adequate pain relief. Sufentanil is a strong opioid that has properties to be the optimal intranasal drug for pain management. However, there are few studies on intranasal pain management in a pre-hospital context. In addition, no previous studies have compared the effect of intranasally administered sufentanil between children, adults and the elderly.   The aim of the study was to describe the use of intranasally administered sufentanil in the pre hospital environment. More specifically we studied: the degree of pain relief, the occurrence of side effects, the establishment of peripheral lines, the environment in which the patient was and also the experience of the staff.   The authors analyzed evaluation protocol witch had been filled in by nurses, who had administered intranasal sufentanil. The protocols were divided into three groups based on age. The groups where children, adults and elderly. Quantitative approach where used and data were analyzed according to ANOVA and chi2test depending on the data grade. Both nominal and ordinal data were included in the analysis. Data presented in visual analogue scale (VAS) before pain management and at arrival at emergency department (ED), dose and incidence of adverse effects were analyzed and compared between the groups.   The results showed that all groups receive adequate pain relief, which was defined as three points or lower measured by visual analogue scale [VAS] on handoff at the emergency department [ED]. Children received significantly higher doses compared to adults and elderly and they also scored a lower VAS at arrival to the ED. Respiratory depression occurred in two cases in the elderly group. The most common adverse effects were nausea and vomiting except in children where sedation was the dominant adverse effect. In children significantly lower number of peripheral lines were established compared to adults and elderly. Nurses appreciated the effect of the intervention as good but with greater contented in the children.   The conclusion was that pre-hospital administration of sufentanil appears to provide a good pain relief within the treatment target, the proportion of adverse reactions is not greater than in previous studies. On the other hand, our results showed that children to a certain extent become drowsy in treatment with intranasal sufentanil.

One of the major limitations in drug development and gene therapy for brain diseases is the natural defensive structure called the blood brain barrier (BBB), which prevents therapeutic polypeptide drugs and viral vectors from entering the brain. Intranasal delivery of therapeutic gene products into the brain offers a non-invasive alternative towards a feasible gene and protein therapy for neurological diseases. From recent studies involving axonal transport, it is tempting to speculate that therapeutic macromolecules including neurotrophic factors and viral vectors can be delivered into the brain by peripheral neurons, such as olfactory receptor neurons (ORNs), which span the BBB. It is thought that the nasal pathway into the brain involves two general mechanisms; intracellular (intraneuronal) or extracellular routes of transport. However the pathways involved have not yet been fully characterized. In this study I firstly investigated the temporal and spatial localisation pattern of both biotinylated and I125 labelled ciliary neurotrophic factor (CNTF) following nasal delivery into Sprague-Dawley rats. Results showed that intranasal delivered CNTF was transported to several brain regions by both intracellular axonal pathway through ORNs and the extracellular trigeminal pathway. Excess unlabelled CNTF competed for receptor binding in the olfactory mucosa confirming receptor mediated intracellular transport to the olfactory bulb via ORNs. Denervation of the olfactory mucosa prior to CNTF delivery failed to prevent CNTF transport to trigeminal and hypothalamic brain regions. Intranasal delivered CNTF was biologically active, resulting in activation of the STAT3 signalling pathway in the thalamus and hypothalamus. To examine the functional activity of intranasal delivered CNTF, I conducted a weight loss trial using an obese Zucker rat (OZR) model to test whether CNTF treatment caused body weight loss. Intranasal administration of CNTF resulted in reduced body weight in the CNTF treated OZR group compared to the BSA control group during the 12 day trial and for 3 days after. Intranasal delivery of CNTF may be a valuable method for the treatment of obesity. In the second study, I investigated the temporal and spatial expression of Enhanced Green Fluorescent Protein (EGFP) transferred by a single nasal delivery of either a recombinant adenovirus vector (Ad5CMV-EGFP) or an adeno-associated virus vector (AAV2-EGFP) into Sprague-Dawley rats. Adenovirus mediated EGFP expression was localized in ORNs throughout the olfactory epithelium after 24 hours. EGFP in the ORNs appeared to be anterogradely transported along their axons to the olfactory bulb and transferred in glomeruli to second-order neurons. EGFP was transferred to several brain regions including the cortex, hippocampus, and brainstem after 7 days. EGFP expression co-localized with Olfactory Marker Protein and was confirmed with EGFP immunofluorescence labelling and western blotting. AAV expressed EGFP localized in similar olfactory and brain regions 6 weeks after delivery. mRNA levels suggested that the AAV-EGFP construct was only incorporated into olfactory mucosa cells and the viral vector was not present in olfactory bulb and brain regions. In conclusion, this simple and non-invasive polypeptide and gene delivery method provides ubiquitous macromolecule distribution throughout the rodent brain and may be useful for the treatment of neurological disorders.

A comprehensive review of the nasal route of administration, in particular the nasal drug delivery system has been presented. The physicochemical properties, mode of action and pharmacology of H1-receptor antagonists, in particular cyclizine HCl, have been highlighted. The techniques for the assessment of toxicity (in-vitro ciliary beat frequency (CBF) studies for human nasal explants and morphology studies of the rat nasal mucosa), synthesis of cyclizine lactate, solubility studies of both cyclizine HCI and cyclizine lactate, viscosity determination of the gel formulated and assessment of the deposition and distribution of the hydroxypropylmethyl cellulose (HPMC) dispersions within the human nasal cavity model were conducted. In this study, preliminary studies on the toxicity of the various formulation components (excipients and active ingredient) were carried out. Results from these studies indicated that for both the excipients and the drug, pH significantly affects the ciliary motility hence all ciliary beat frequency determinations were conducted at nasal pH. Furthermore, effects of the various concentrations (0.0625%(w/v), 0.125%(w/v), 0.25%(w/v), 0.5%(w/v) and l%(w/v)) of the excipients on ciliary motility were investigated. Transmission electron microscopy (TEM) studies proved useful in evaluating the integrity and changes in the surface morphology of the rat nasal mucosa post treatment with the various excipients (carboxymethyl cellulose, hydroxypropylmethyl cellulose, trimethyl chitosan 36.3% DQ, Carbopol P934 and polysorbate-80) at varying concentrations. Of the excipients investigated, hydroxypropylmethyl cellulose (HPMC) showed ciliofriendliness since there was no apparent ultra structural damage, although a slight decrease in ciliary beat frequency (CBF) was observed at the highest viscosity. Moreover, hydroxypropylmethyl cellulose (HPMC) is said to be a bioadhesive excipient, which would therefore confer its bioadhesive properties to the intranasal preparation to enhance the retention time between the absorbing mucosa and the drug and hence increase nasal drug absorption. This excipient was therefore selected as the ideal for use in the formulation of the intranasal preparation. The aqueous solubility of a drug plays an important role in nasal administration since it is required that the drug component be applied in a limited volume of about 200pl. To enhance the aqueous solubility of the sparingly water-soluble cyclizine HCl, a lactate salt was synthesised and characterised. This compound was found to be highly soluble in water. The intranasal preparation was therefore manufactured using the lactate form of cyclizine. A single blind study was conducted to determine and compare the pharmacokinetic parameters for both Valoid oral tablets containing 100mg cyclizine HCl (reference drug) and cyclizine lactate intranasal preparation 125mglml (study drug). The results obtained indicated a significant improvement in the bioavailability of cyclizine. For oral administration Cmax = 200.79ng/ml at tmax = 5.57h and for the intranasal preparation Cmax = 5354.22ng/ml at tmax = 1.59h. A 19.2-fold increase in drug bioavailability was observed after intranasal administration (AUCin = 122860.70ng/ml/h) compared with oral administration (AUCpo = 5943.48ng/ml/h). This enhanced bioavailability through nasal administration indicated that enhanced nasal drug absorption and hence increased bioavailability not only depends on the favourable anatomical and physiological characteristics of the nasal mucosa but possibly on the inherent physico-chemical characteristics of the drug molecule and the formulation components. Thus chemical modification of the sparingly water-soluble cyclizine HCl to the highly water-soluble cyclizine lactate facilitated the dissolution of more solute in a limited volume of solvent. This new feature therefore may have impacted positively to the transport of cyclizine across the nasal mucosa. Furthermore, the hydroxypropylmethyl cellulose (HPMC), component of the formulation, could have conferred its mucoadhesive properties to the preparation. Perhaps it increased the retention time of the dosage form within the nasal passages through bond formation with the nasal mucosa thereby increasing the contact time between the absorbing mucosa and the dosage form. This interaction between the mucoadhesive and the nasal mucosa may have resulted in the modification of tissue permeability (possibly transient opening of the tight junctions) and eventual increase in the drug penetration/absorption. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

A comprehensive review of the nasal route of administration, in particular the nasal drug delivery system has been presented. The physicochemical properties, mode of action and pharmacology of H1-receptor antagonists, in particular cyclizine HCl, have been highlighted. The techniques for the assessment of toxicity (in-vitro ciliary beat frequency (CBF) studies for human nasal explants and morphology studies of the rat nasal mucosa), synthesis of cyclizine lactate, solubility studies of both cyclizine HCI and cyclizine lactate, viscosity determination of the gel formulated and assessment of the deposition and distribution of the hydroxypropylmethyl cellulose (HPMC) dispersions within the human nasal cavity model were conducted. In this study, preliminary studies on the toxicity of the various formulation components (excipients and active ingredient) were carried out. Results from these studies indicated that for both the excipients and the drug, pH significantly affects the ciliary motility hence all ciliary beat frequency determinations were conducted at nasal pH. Furthermore, effects of the various concentrations (0.0625%(w/v), 0.125%(w/v), 0.25%(w/v), 0.5%(w/v) and l%(w/v)) of the excipients on ciliary motility were investigated. Transmission electron microscopy (TEM) studies proved useful in evaluating the integrity and changes in the surface morphology of the rat nasal mucosa post treatment with the various excipients (carboxymethyl cellulose, hydroxypropylmethyl cellulose, trimethyl chitosan 36.3% DQ, Carbopol P934 and polysorbate-80) at varying concentrations. Of the excipients investigated, hydroxypropylmethyl cellulose (HPMC) showed ciliofriendliness since there was no apparent ultra structural damage, although a slight decrease in ciliary beat frequency (CBF) was observed at the highest viscosity. Moreover, hydroxypropylmethyl cellulose (HPMC) is said to be a bioadhesive excipient, which would therefore confer its bioadhesive properties to the intranasal preparation to enhance the retention time between the absorbing mucosa and the drug and hence increase nasal drug absorption. This excipient was therefore selected as the ideal for use in the formulation of the intranasal preparation. The aqueous solubility of a drug plays an important role in nasal administration since it is required that the drug component be applied in a limited volume of about 200pl. To enhance the aqueous solubility of the sparingly water-soluble cyclizine HCl, a lactate salt was synthesised and characterised. This compound was found to be highly soluble in water. The intranasal preparation was therefore manufactured using the lactate form of cyclizine. A single blind study was conducted to determine and compare the pharmacokinetic parameters for both Valoid oral tablets containing 100mg cyclizine HCl (reference drug) and cyclizine lactate intranasal preparation 125mglml (study drug). The results obtained indicated a significant improvement in the bioavailability of cyclizine. For oral administration Cmax = 200.79ng/ml at tmax = 5.57h and for the intranasal preparation Cmax = 5354.22ng/ml at tmax = 1.59h. A 19.2-fold increase in drug bioavailability was observed after intranasal administration (AUCin = 122860.70ng/ml/h) compared with oral administration (AUCpo = 5943.48ng/ml/h). This enhanced bioavailability through nasal administration indicated that enhanced nasal drug absorption and hence increased bioavailability not only depends on the favourable anatomical and physiological characteristics of the nasal mucosa but possibly on the inherent physico-chemical characteristics of the drug molecule and the formulation components. Thus chemical modification of the sparingly water-soluble cyclizine HCl to the highly water-soluble cyclizine lactate facilitated the dissolution of more solute in a limited volume of solvent. This new feature therefore may have impacted positively to the transport of cyclizine across the nasal mucosa. Furthermore, the hydroxypropylmethyl cellulose (HPMC), component of the formulation, could have conferred its mucoadhesive properties to the preparation. Perhaps it increased the retention time of the dosage form within the nasal passages through bond formation with the nasal mucosa thereby increasing the contact time between the absorbing mucosa and the dosage form. This interaction between the mucoadhesive and the nasal mucosa may have resulted in the modification of tissue permeability (possibly transient opening of the tight junctions) and eventual increase in the drug penetration/absorption. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

O estudo de metodologias que possibilitem o acesso de moléculas terapêuticas ao cérebro evitando a barreira hematoencefálica (blood-brain barrier, BBB), é extremamente importante para o tratamento das doenças que afetam o sistema nervoso central. Os estudos se concentram na busca por vias seguras e pouco invasivas. Recentemente a via nasal tem mostrado resultados positivos na administração de fármacos ao sistema nervoso central de maneira segura, sugerindo a utilização desta via para a terapia gênica. Neste trabalho, foi estudada a administração intranasal de um vetor não viral, que codifica o gene da proteína verde fluorescente otimizada (enhanced green fluorescent proten, EGFP), diluído em água ou em solução de sulfato de zinco em camundongos adultos da linhagem BALB/c. Os resultados de RT-PCR mostraram que o vetor atingiu o cérebro em ambos tratamentos, e sua expressão foi mantida até 8 semanas após o tratamento. A expressão da EGPF em diferentes regiões cerebrais foi analisada 1, 2, 4 e 8 semanas após o tratamento, através de imunohistoquímica. Nossos resultados mostraram que um número muito pequeno de células foi transfectado em ambos tratamentos e que a freqüência destas células nas regiões cerebrais estudadas varia muito mesmo entre animais do mesmo grupo. O tratamento com sulfato de zinco não aumentou a eficiência do vetor. Os maiores números de células transfectadas encontradas foram no bulbo olfatório e hipocampos uma semana após o tratamento. Nossos resultados mostram pela primeira vez a expressão prolongada de um gene administrados pela via nasal no cérebro. / The investigation of methods that allow the access of therapeutic molecules to the brain, overcoming the blood-brain barrier (BBB), is of great importance for the treatment of diseases reaching the central nervous system. Studies concentrate mainly on the search for safer and less aggressive routes. Recent studies have suggested that pharmaceutical agents can reach the brain by the nasal route, that may thus be also appropriate for gene therapy. Here, we investigated brain targeting by intranasal administration of a plasmid vector (pREGFP) with the reporter gene EGFP, diluted in water or in a zinc sulfate/water solution, to adult BALB/c mice. Brain regions were examined by RT-PCR or immunohistochemistry 1, 2, 4 or 8 weeks after the treatment. RT-PCR results showed that the plasmid DNA reached the brain similarly with the two types of treatment. Expression of egfp was observed in all groups, and maintained even 8 weeks after treatment. Brain sections submitted to immunohistochemistry (IHC) were analyzed for the detection of EGFP-positive cells, that could be observed in brain samples from mice treated by intranasal administration of pREGFP. The frequency of EGFP-positive cells was very low, and presented great variation even among mice from the same experimental group. The supplementation of the transfection medium with zinc sulfate did not induce higher numbers of EGFP-positive cells. A higher frequency of these cells was observed in the olfactory bulb one week after treatment, and in the hippocampus as compared to other brain regions. Our results show for the first time the long-term expression of a gene carried by a nonviral vector to the central nervous system.

Made available in DSpace on 2016-08-29T15:34:58Z (GMT). No. of bitstreams: 1 tese_7846_DISSERTAÇÃO FINAL - CORREÇÕES + Ficha + Parecer _Reparado_2.pdf: 1750239 bytes, checksum: 6771fcd952a5a0a0f802782437acdc1c (MD5) Previous issue date: 2014-07-11 / O dimetil dioctadecil amônio (DDA) é um composto quaternário de amônio, formado por um lípido anfifílico sintético ligado a duas cadeias hidrofóbicas de alquilo de 18 carbonos, que mostrou efetivo efeito adjuvante, promovendo respostas imunes humoral e celular contra diversos agentes patogênicos, incluindo Mycobacterium tuberculosis e Chlamydia trachomatis. Neste presente trabalho, nós investigamos a capacidade do adjuvante CAF associado a antígenos totais de Leishmania amazonenses (LaAg) em induzir respostas imunogênicas e protetoras no desafio contra L. chagasi. Assim, camundongos BALB/c foram imunizados por via intranasal com 25 mg de LaAg livre ou associado a 150 mg de CAF em 2 doses intervaladas por 15 dias. A dosagem de transaminases e creatinina no soro dos animais demonstraram a biocompatibilidade e segurança da associação LaAg/CAF. Além disso, observamos significativas produções de IFN-g e NO por esplenócitos estimulados com antígenos do parasito, além de significativas respostas linfoproliferativas e maior percentagem de células de memória (TCD4+ CD44+ CD62L+). Níveis aumentados de IgG total e das subclasses específicas (IgG1 e IgG2a) também foram observadas no grupo vacinado com LaAg associado ao CAF, quando comparado aos controles. De forma semelhante, camundongos vacinados com a formulação e desafiados 15 dias após o reforço com L. chagasi, apresentaram uma significativa diminuição da carga parasitária no baço e fígado, associada a uma maior produção de IFN-g, respostas linfoproliferativas significativamente aumentadas. Juntos, nossos dados demonstraram pela primeira vez viabilidade da vacinação intranasal com LaAg associado ao CAF, como um efetivo mecanismo para a vacinação contra a leishmaniose visceral. Palavras chave: L. chagasi, imunização intranasal, LaAg, CAF, resposta imune.

O estudo de metodologias que possibilitem o acesso de moléculas terapêuticas ao cérebro evitando a barreira hematoencefálica (blood-brain barrier, BBB), é extremamente importante para o tratamento das doenças que afetam o sistema nervoso central. Os estudos se concentram na busca por vias seguras e pouco invasivas. Recentemente a via nasal tem mostrado resultados positivos na administração de fármacos ao sistema nervoso central de maneira segura, sugerindo a utilização desta via para a terapia gênica. Neste trabalho, foi estudada a administração intranasal de um vetor não viral, que codifica o gene da proteína verde fluorescente otimizada (enhanced green fluorescent proten, EGFP), diluído em água ou em solução de sulfato de zinco em camundongos adultos da linhagem BALB/c. Os resultados de RT-PCR mostraram que o vetor atingiu o cérebro em ambos tratamentos, e sua expressão foi mantida até 8 semanas após o tratamento. A expressão da EGPF em diferentes regiões cerebrais foi analisada 1, 2, 4 e 8 semanas após o tratamento, através de imunohistoquímica. Nossos resultados mostraram que um número muito pequeno de células foi transfectado em ambos tratamentos e que a freqüência destas células nas regiões cerebrais estudadas varia muito mesmo entre animais do mesmo grupo. O tratamento com sulfato de zinco não aumentou a eficiência do vetor. Os maiores números de células transfectadas encontradas foram no bulbo olfatório e hipocampos uma semana após o tratamento. Nossos resultados mostram pela primeira vez a expressão prolongada de um gene administrados pela via nasal no cérebro. / The investigation of methods that allow the access of therapeutic molecules to the brain, overcoming the blood-brain barrier (BBB), is of great importance for the treatment of diseases reaching the central nervous system. Studies concentrate mainly on the search for safer and less aggressive routes. Recent studies have suggested that pharmaceutical agents can reach the brain by the nasal route, that may thus be also appropriate for gene therapy. Here, we investigated brain targeting by intranasal administration of a plasmid vector (pREGFP) with the reporter gene EGFP, diluted in water or in a zinc sulfate/water solution, to adult BALB/c mice. Brain regions were examined by RT-PCR or immunohistochemistry 1, 2, 4 or 8 weeks after the treatment. RT-PCR results showed that the plasmid DNA reached the brain similarly with the two types of treatment. Expression of egfp was observed in all groups, and maintained even 8 weeks after treatment. Brain sections submitted to immunohistochemistry (IHC) were analyzed for the detection of EGFP-positive cells, that could be observed in brain samples from mice treated by intranasal administration of pREGFP. The frequency of EGFP-positive cells was very low, and presented great variation even among mice from the same experimental group. The supplementation of the transfection medium with zinc sulfate did not induce higher numbers of EGFP-positive cells. A higher frequency of these cells was observed in the olfactory bulb one week after treatment, and in the hippocampus as compared to other brain regions. Our results show for the first time the long-term expression of a gene carried by a nonviral vector to the central nervous system.