A parvovirose canina é uma doença causada pelo parvovírus canino, um vírus pertencente à família Parvoviridae. A doença causa quadros agudos de gastroenterite hemorrágica altamente contagiosa, e é responsável por altas taxas de morbidade e mortalidade, principalmente, em cães jovens. O principal agente etiológico desencadeador da doença é o parvovírus canino tipo 2 (CPV 2). As vacinas parenterais comercializadas contra esse vírus não são adequadas para filhotes com menos de 45 dias de idade. Além disso, essa doença não apresenta um tratamento especifico, sendo a profilaxia de grande importância. O objetivo desse trabalho foi desenvolver um antígeno de entrega vacinal de modo a proteger o animal antes do seu desenvolvimento imunológico. Através do aumento da produção de IgA total a partir da primeira imunização e da resposta sistêmica de IgG especifica a partir da segunda imunização em camundongos, foi possível verificar que as superfícies de mucosa são ativas imunologicamente, desde o nascimento do animal como também capazes de estimular tanto a resposta local quanto a sistêmica em camundongos imaturos e adultos. No intuito de proteger também esses jovens animais, a imunização por via sublingual mostrou-se uma técnica promissora. Em comparação com os grupos de camundongos imunizados com a amostra vacinal e o hidrogel líquido, o grupo que recebeu o hidrogel liofilizado teve uma melhor resposta imunológica, uma vez que a técnica de liofilização aumentou a característica de mucoadesividade da quitosana e consequentemente aumentou o tempo de permanência do hidrogel na mucosa sublingual. / Canine parvovirus is a virus that belongs to the Parvoviridae family. The disease causes acute hemorrhagic gastroenteritis, which is highly contagious and responsible for high rates of morbidity and mortality, especially in puppies. The main etiologic agent of this disease is canine parvovirus type 2 (CPV 2). Nowadays, there is a commercial parenteral vaccine against this virus, but it is not suitable for puppies under 45 days old. This disease has no specific treatment and the prophylaxis has a great importance. The aim of this study was to develop a sublingual delivery vaccine that protect the animals before their 45 days. By increasing the total IgA production from the first immunization and systemic specific IgG response after the second immunization in mice, it demonstrated that mucosal surfaces are immunologically active since birth and capable of stimulating both systemic and local response in puppies and adults. In order to protect these puppies, mucosal immunization is a promising technique. The group that received lyophilized hydrogel had a better immune response, since the lyophilization technique increased the mucoadhesive of chitosan and consequently the residence time of the lyophilized hydrogel at sublingual mucosa.

A parvovirose canina é uma doença causada pelo parvovírus canino, um vírus pertencente à família Parvoviridae. A doença causa quadros agudos de gastroenterite hemorrágica altamente contagiosa, e é responsável por altas taxas de morbidade e mortalidade, principalmente, em cães jovens. O principal agente etiológico desencadeador da doença é o parvovírus canino tipo 2 (CPV 2). As vacinas parenterais comercializadas contra esse vírus não são adequadas para filhotes com menos de 45 dias de idade. Além disso, essa doença não apresenta um tratamento especifico, sendo a profilaxia de grande importância. O objetivo desse trabalho foi desenvolver um antígeno de entrega vacinal de modo a proteger o animal antes do seu desenvolvimento imunológico. Através do aumento da produção de IgA total a partir da primeira imunização e da resposta sistêmica de IgG especifica a partir da segunda imunização em camundongos, foi possível verificar que as superfícies de mucosa são ativas imunologicamente, desde o nascimento do animal como também capazes de estimular tanto a resposta local quanto a sistêmica em camundongos imaturos e adultos. No intuito de proteger também esses jovens animais, a imunização por via sublingual mostrou-se uma técnica promissora. Em comparação com os grupos de camundongos imunizados com a amostra vacinal e o hidrogel líquido, o grupo que recebeu o hidrogel liofilizado teve uma melhor resposta imunológica, uma vez que a técnica de liofilização aumentou a característica de mucoadesividade da quitosana e consequentemente aumentou o tempo de permanência do hidrogel na mucosa sublingual. / Canine parvovirus is a virus that belongs to the Parvoviridae family. The disease causes acute hemorrhagic gastroenteritis, which is highly contagious and responsible for high rates of morbidity and mortality, especially in puppies. The main etiologic agent of this disease is canine parvovirus type 2 (CPV 2). Nowadays, there is a commercial parenteral vaccine against this virus, but it is not suitable for puppies under 45 days old. This disease has no specific treatment and the prophylaxis has a great importance. The aim of this study was to develop a sublingual delivery vaccine that protect the animals before their 45 days. By increasing the total IgA production from the first immunization and systemic specific IgG response after the second immunization in mice, it demonstrated that mucosal surfaces are immunologically active since birth and capable of stimulating both systemic and local response in puppies and adults. In order to protect these puppies, mucosal immunization is a promising technique. The group that received lyophilized hydrogel had a better immune response, since the lyophilization technique increased the mucoadhesive of chitosan and consequently the residence time of the lyophilized hydrogel at sublingual mucosa.

Administration of drugs sublingually allows direct absorption into the systemic circulation which results in quick onset of action and a higher bioavailability as a consequence of by-passing first pass metabolism. Absorption of drugs across sublingual mucosa is typically determined by means of in vitro permeation studies using excised sublingual tissue during early phases of drug development. Although in vitro set up has been designed to mimic in vivo system yet the results of in vitro studies often deviate from in vivo results. Therefore, it is not known if the in vitro studies can be used as surrogate for in vivo studies in a predictable manner. To understand the relationship between in vitro and in vivo system for sublingual drug delivery, the first objective of this dissertation research was to investigate difference/similarities between in vitro and in vivo system by performing parallel in vitro and in vivo studies and establish a correlation. Five model drugs possessing diverse physicochemical properties and New Zealand White rabbits were used for these studies. Comparison of time course of absorption revealed a significant difference in time lag between in vivo (less than 5 min) and in vitro (30-120 min) systems. However, the derived absorption parameter permeability coefficient was similar in in vitro and in vivo system for caffeine: (2.10±0.22)×10 –5 , (2.06±0.47)×10 –5 ; Naproxen: (1.91±0.44)×10 –5 , (2.34±0.26)×10 –5 ; Propranolol: (2.93±0.52)×10 –5 , (3.51±0.75)×10 –5 ; Verapamil: (3.95±0.29)×10 –5 , (4.75±0.81)×10 –5 and Atenolol: (2.01±0.68)×10 –6 , (2.95±0.32)×10 –6 cm/s, respectively (p>0.05). The discrepancy between in vitro and in vivo system was hypothesized in this study to be due to the difference in thickness and role of extensive microcirculation in the two systems. Histological evaluation revealed the presence of rich vasculature 10-20 μm below the epithelium which is responsible for quick removal of drug permeating the epithelium (100-150 μm) of sublingual mucosa and reaching systemic circulation in an in vivo system. In contrast, in in vitro system the permeated drug can only be detected after crossing the excised sublingual tissue of 250±50 μm thickness. A mathematical model based on the monolayer (epithelium) and bilayer (epithelium+connective tissue) nature of the membrane representing in vivo and in vitro system, respectively demonstrated the nature of membrane to be responsible for difference in time lag but similar permeability coefficient. To be able to predict in vivo result using in vitro data, the second objective of this dissertation research was to develop a predictive pharmacokinetic model based on the established in vitro in vivo correlation (IVIVC) of sublingual absorption parameters across two systems. Predicted plasma concentration-time profiles of propranolol, verapamil, naproxen, atenolol and caffeine were found to be in good agreement with the experimental profile with the coefficient of determination of 0.85, 0.80, 0.97, 0.98 and 0.88, respectively. The applicability of the model was further evaluated by predicting in vivo performance of Zolpidem and Propranolol following sublingual administration in human beings and comparing area under the plasma concentration-time curve. Percent prediction error was 12.02% and less than 10% (4.69, 6.69, 5.02 for 1, 1.75 and 3 mg dose, respectively) for Propranolol and Zolpidem, respectively. The final objective of this dissertation was to extend the established IVIVC to other suitable animal models such as pig for assessing sublingual absorption. Histological evaluation revealed the similarity in the structure of sublingual mucosa of pig and New Zealand White rabbit. Similar transport characteristics (p>0.05) of model drugs across sublingual mucosae of two species were observed indicating the possibility of using them interchangeably. In conclusion, a rational attempt was made in this dissertation research to identify the root cause of the discrepancy between in vitro and in vivo system and establish a correlation correcting the discrepancies. The established IVIVC and predictive pharmacokinetic model will help in rationale design and development of new sublingual formulations and will be a valuable tool in the preclinical phase of early drug development stage.