Introdução: Diabete Melito Pós-Transplante (DMPT) é considerado uma séria complicação do transplante de rim, podendo diminuir a sobrevida do enxerto e do paciente. O imunossupressor tacrolimo (TAC) pode aumentar o risco de desenvolvimento de DMPT. Objetivos: Estimar o risco de DMPT em pacientes tratados com TAC no Serviço de Transplante Renal da Irmandade da Santa Casa de Misericórdia de Porto Alegre, identificar os demais fatores de risco para o DMPT e suas conseqüências. Métodos: Foram analisados 413 pacientes sem diabetes prévia ao transplante, com 18 anos ou mais, tratados com TAC, ciclosporina (CsA) ou sirolimo (SIR), em uso de corticoterapia e com período de seguimento póstransplante maior de 6 meses. O estudo seguiu o modelo observacional–coorte retrospectivo. Os critérios da Associação Americana de Diabete foram utilizados para o diagnóstico de DMPT. Os dados foram coletados de prontuários. Resultados: Dos 413 pacientes analisados, 171(41,4%) receberam como imunossupressão inicial TAC, 221(53,5%) CsA e 21(5,1%) SIR. DMPT ocorreu em 20,6% dos pacientes da coorte (85 de 413). A mediana do tempo para o desenvolvimento de DMPT foi de 54 dias. A incidência cumulativa de DMPT foi de 24,6% e 17,2% para os grupos de tratamento TAC e CsA, respectivamente. Na análise por intenção de tratamento, o risco para o desenvolvimento de DMPT, ao comparar os grupos TAC vs. CsA foi de HR=1,563, (IC:95%=1,008–2,425), P=0,006. Pelo método de Kaplan-Meier, 78,9% dos pacientes em uso de TAC estavam livres de DMPT no mês 6 vs. 87,8% dos pacientes em uso de CsA (P= 0,044). Os demais fatores de risco independentes identificados foram: índice de massa corporal (IMC) pré-transplante (P<0,0001), idade do receptor (P<0,0001) e episódio de rejeição aguda (RA) (P=0,013). Não houve diferença estatística significativa para anti-HCV reagente do receptor. Em 3 anos, a sobrevida do enxerto, ao comparar os pacientes com diagnóstico de DMPT vs. ausência de DMPT foi de 85,5% e 93,3%, respectivamente (P=0,021) e a sobrevida do paciente foi de 88,9% e 96,7%, respectivamente (P=0,017). Conclusão: A incidência de DMPT está associada com o tipo de imunossupressão utilizada, idade do receptor, IMC pré-transplante e episódio de RA. DMPT é um importante fator de risco para perda do enxerto e mortalidade. Medidas para minimizar o risco dessa complicação devem ser tomadas, como a individualização da terapia imunossupressora. / Introduction: Post-transplant diabetes mellitus (PTDM) is considered a serious complication of kidney transplantation and may reduce the patient and graft survival. The immunosuppressive Tacrolimus (TAC) may increase the risk of developing PTDM. Purpose: To estimate the risk of PTDM in renal transplant recipients treated with TAC in our center, identify all risk factors for PTDM and its consequences. Methodology: We analyzed 413 patients without diabetes prior to transplantation, with age ≥ 18 years, who were treated with tacrolimus (TAC), cyclosporine (CyA) or sirolimus (SIR), under steroids therapy and with a follow-up post-transplant period more than 6 months. We performed a retrospective review – cohort study. PTDM was diagnosed according to American Diabetes Association guidelines. Data were collected from medical records. Results: We examined 413 renal allograft recipients, these, 171 (41.4%) received initial immunosuppresion with TAC, 221 (53.5%) CyA and 21 (5.1%) SIR. PTDM occurred in 20.6% of patients in the cohort (85 of 413). The median time to the development of PTDM was 54 days post transplant. The cumulative incidence of PTDM was 24.6% and 17.2% for groups TAC and CyA treatment, respectively. In the analysis by intention to treat, the proportion of patients receiving TAC who developed PTDM was significantly higher than patients receiving CyA (HR=1,563, (CI:95%=1,008–2,425), P=0,006. The Kaplan-Meier method showed that 78,9% patients taking TAC were free of PTDM at six months compared to 87,8% of patients taking CsA (P= 0.044). The other independent risk factors identified were: body mass index (BMI) (P<0,0001), recipient age (P<0,0001) and acute rejections episodes (AR) (P=0,013). There was no statistically significant difference for patients with hepatitis C. Three years actuarial graft survival was 85,5% in patients with PTDM compared with 93,3% for those without diabetes (P=0,021) and patient survival was 88,9% e 96,7%, respectively (P=0,017). Conclusions: The incidence of PTDM is associated with TAC use, the recipient age, BMI and acute rejections episodes. PTDM is an important risk factor for graft loss and mortality. Measures to minimize the risk of this complication should be taken, such as the individualization of immunosuppresive therapy.

Este trabalho centrou-se no desenvolvimento de nanocápsulas de núcleo lipídico contendo tacrolimus com revestimentos não iônico de polissorbato 80, aniônico de lecitina e catiônico de quitosana para posterior avaliação in vivo das atividades antiedematogênica e imunossupressora das formulações. As suspensões foram preparadas pelo método de deposição interfacial do polímero pré-formado utilizando a poli(ɛ-caprolactona) como polímero biodegradável. Para obtenção das nanocápsulas de núcleo lipídico de superfície catiônica, a lecitina foi adicionada à fase orgânica das suspensões e o revestimento procedeu-se por meio da incubação das partículas em solução aquosa de quitosana. De forma geral, todas as formulações apresentaram distribuição monomodal de partículas apenas na faixa nanométrica e baixa polidispersão. Os valores de potencial zeta obtidos para as formulações não iônicas e aniônicas foram negativos, enquanto que para as formulações catiônicas foram positivos. O teor de fármaco foi próximo ao teórico (0,6 ou 0,8 mg/mL) com alta eficiência de encapsulação (> 99%) e os estudos de liberação in vitro mostraram perfis de liberação sustentada do tacrolimus com mecanismo de transporte anômalo para todas as formulações. Além disso, as suspensões apresentaram estabilidade física adequada por meio de análises de retroespalhamento de luz e partículas de formato esferoide por microscopia eletrônica de transmissão. No modelo de artrite reumatóide induzida por adjuvante completo de Freund, o tacrolimus encapsulado em nanocápsulas de núcleo lipídico revestidas com polissorbato 80 mostrou atividade antiedematogênica superior ao fármaco em solução na dose de 2,0 mg/kg/dia, após administração intraperitoneal das formulações em ratos Wistar. Para avaliar o potencial de nanocápsulas de núcleo lipídico em aumentar a eficácia imunossupressora do tacrolimus pela via oral, realizou-se um estudo com camundongos CF1, o qual foi conduzido em duas etapas. Primeiramente, buscando avaliar o efeito da nanoencapsulação independente da barreira gastrointestinal, o fármaco em solução ou nanoencapsulado foi administrado pela via intraperitoneal nas doses de 4,0 e 6,0 mg/kg/dia, durante 10 dias. Nos tempos 0, 5 e 10 dias, amostras de sangue foram coletadas para posterior contagem do número percentual de linfócitos. Os resultados mostraram que o tacrolimus nanoencapsulado provocou maior redução linfocitária na dose de 4,0 mg/kg/dia em relação ao fármaco livre, enquanto que na dose de 6,0 mg/kg/dia, o efeito farmacodinâmico foi similar para o tacrolimus em solução ou nanoencapsulado. Continuando o estudo, as formulações foram administradas nas doses de 6,0 e 10,0 mg/kg/dia pela via oral e os resultados mostraram que o fármaco em solução na dose de 6,0 mg/kg/dia não apresentou efeito sobre o número percentual de linfócitos durante todo o experimento, enquanto que na dose de 10,0 mg/kg/dia, o efeito da solução foi observado somente no último dia de tratamento. Quando o fármaco foi nanoencapsulado, a redução linfocitária foi significativa nas duas doses testadas (6,0 e 10,0 mg/kg/dia), a qual se iniciou no quinto dia e manteve-se até o final do experimento. Em uma análise comparativa, somente dos grupos tratados com as formulações na mesma dose (6,0 mg/Kg/dia), pelas diferentes vias de administração, observou-se que os animais tratados com o fármaco livre apresentaram diferença na redução linfocitária entre as vias oral e intraperitoneal, enquanto que os animais tratados com as nanocápsulas núcleo lipídico revestidas com polissorbato 80 apresentaram equivalência farmacodinâmica entre as vias de administração avaliadas. Nesse mesmo estudo, constatou-se que as nanocápsulas na dose de 6,0 mg/kg/dia evitaram o aumento das concentrações séricas de glicose após administração oral das formulações. Prosseguindo com o mesmo modelo animal e buscando estudar a influência do tipo de revestimento das partículas (não iônico e catiônico) após administração oral do tacrolimus, foram desenvolvidas nanocápsulas de núcleo lipídico estabilizadas com lecitina e polissorbato 80 revestidas ou não com quitosana. As formulações foram administradas na dose de 6,0 mg/kg/dia e os resultados mostraram maior redução do número percentual de linfócitos para os animais tratados com a formulação de superfície catiônica, a qual provocou efeito imunossupressor já no quinto dia de tratamento enquanto que a formulação aniônica mostrou efeito somente no décimo dia. Os animais tratados com a dispersão do fármaco que continha quitosana não apresentaram redução do número de linfócitos durante o experimento. Além disso, a formulação catiônica foi a única capaz de evitar alterações nas concentrações séricas de glicose, creatinina e fosfatase alcalina dos animais. O conjunto destes estudos demonstra que as nanocápsulas de núcleo lipídico apresentam-se como uma estratégia tecnológica promissora, visando o aumento da eficácia farmacológica e redução de efeitos adversos do tacrolimus. / This work was based on the development of tacrolimus-loaded lipid-core nanocapsules employing the nonionic polysorbate 80, anionic lecithin and cationic chitosan coatings aiming the in vivo evaluation of antiedematogenic and immunosuppressive activities. The suspensions were prepared by interfacial deposition of preformed polymer using the poly(ɛ-caprolactone) as biodegradable polymer. In order to obtain the lipid-core nanocapsules with cationic surface, lecithin was added to the organic phase of the suspensions and the coating proceeded through the incubation of the particles in aqueous solutions of chitosan. In general, all formulations showed unimodal size distributions only at nanoscale and low polydispersity. The zeta potential values obtained for the nonionic and anionic formulations were negative whereas for the cationic formulations were positive. The drug contents were close to the theoretical values (0.6 or 0.8 mg mL-1) with high encapsulation efficiency (> 99%). The colloidal suspensions presented appropriate physical stability by light backscattered analysis and spheroid shaped particles by transmission electron microscopy. Moreover, drug release studies using the dialysis bag method showed sustained release profiles of tacrolimus with anomalous transport mechanism for all formulations. In the Freund's complete adjuvant–induced arthritis model, the tacrolimus-loaded lipid-core nanocapsules coated with polysorbate 80 showed higher antiedematogenic activity compared to the drug in solution after intraperitoneal administration of 2.0 mg kg-1 day-1 in Wistar rats. Proceeding the work, a study using CF1 mice was conducted to evaluate the potential of lipid-core nanocapsules in increasing the immunosuppressive efficacy of tacrolimus. The first evaluation was carried out to determine the influence of encapsulation of tacrolimus on the pharmacodynamic effect of the drug, independently of the oral absorption barrier. In this case, the tacrolimus-loaded lipid-core nanocapsules were compared to the drug in solution after intraperitoneal administration at 4.0 and 6.0 mg kg-1 day-1. Blood samples were collected at times 0, 5 and 10 days for subsequent determination of lymphocytes count percent. The results showed that drug nanoencapsulation caused greater reduction lymphocyte count (%) at the dose of 4.0 mg kg-1 day-1 compared to tacrolimus free, whereas at the dose of 6.0 mg kg-1 day-1, the pharmacodynamic effect was similar for drug encapsulated or nonencapsulated. Continuing the study, the formulations were administered by oral route at 6.0 and 10.0 mg kg-1 day-1. The results showed that the treatment with free drug at 6.0 mg kg-1 day-1 led to no significant difference in lymphocytes percent during the whole experiment while the treatment with drug in solution at 10.0 mg kg-1 day-1 caused exclusively a significant decrease after 10 days. On the other hand, after 5 days of treatment with tacrolimus-loaded lipid-core nanocapsules at both doses of 6.0 and 10 mg kg-1 day-1 had caused significant decreases in lymphocyte count (%); and the pharmacodynamic effect continued until the end of the experiment. In a comparative analysis only those groups treated with the formulations at the same dose (6.0 mg kg-1 day-1), the animals treated with the tacrolimus solution showed different percent reduction in lymphocyte between oral and intraperitoneal routes whereas the animals treated with the lipid core nanocapsules coated with polysorbate 80 showed equivalent pharmacodynamic effect among the administration routes evaluated. The influence of particle coatings (cationic and nonionic) after oral administration of tacrolimus was also investigated using the same animal model. For this, lipid-core nanocapsules stabilized with polysorbate 80-lecithin uncoated (anionic) or coated with chitosan were developed. The formulations were administered at the dose of 6.0 mg kg-1 day-1 and the results showed higher reduction in lymphocytes count percent for the formulation of cationic surface, which caused immunosuppressive effect on the fifth day while uncoated formulation showed the immunosuppressive effect only on the tenth day. The animals treated with the drug dispersion containing chitosan showed no reduction in lymphocytes count (%) during the experiment. Moreover, the formulation of lipid-core nanocapsules with cationic surface was the unique able to prevent alterations in serum levels of glucose, creatinine and alkaline phosphatase. This set of studies showed that the lipid-core nanocapsules are a promising technology strategy aiming at increasing of the pharmacological efficacy and reduction of adverse effects of tacrolimus.

Njurtransplantation är det enda botande behandlingsalternativet för patienter som befinner sig i kronisk njursviktsstadium 5. Dagens mest använda immunsuppressiva kombinationsbehandling i klinisk praxis består av calcineurinhämmaren takrolimus, mykofenolatmofetil och kortikosteroider i form av prednisolon. Belatacept (Nulojix®) är ett fusionsprotein som introducerades på marknaden 2011 och ska ses som ett alternativ för primär immunsuppressiv behandling. Verkningsmekanismen för belatacept är att hämma aktiveringen av T-celler genom blockera co-stimulatoriska signaler från antigenpresenterande celler. Teorin bakom belatacept är att man genom en mer specifik immunsuppression ska kunna undvika de nefrotoxiska biverkningar som calcineurinhämmarna takrolimus(Prograf®) och ciklosporin(Sandimmun®) ofta ger. Nefrotoxicitet kan på långsikt leda till en försämring av njurfunktion vilket på sikt kan leda till förlust av transplantatet. Syftet med detta arbete var att undersöka effektiviteten av belatacept jämfört med calcineurinhämmare med avseende på graftöverlevnad, njurfunktion och förekomsten av akuta rejektioner. Detta arbete är en litteraturstudie som gjorts genom att utvärdera fem studier som hittades på sökdatabasen PubMed. Samtliga studier som utvärderades i detta arbete påvisade inga signifikanta skillnader i graftöverlevnad mellan de patienter som behandlades med belatacept och de som behandlades med en calcineurinhämmare. Samtliga studier påvisade en signifikant högre njurfunktion mätt i cGFR, Calculated Glomerular Filtration Rate, hos patienter som behandlades med belatacept. Hos dessa patienter ökade njurfunktionen över tid vilket bekräftar teorin bakom belatacept som säger att man genom att undvika nefrotoxicitet ska kunna behålla en stabil nivå i njurfunktion.  I samtliga studier förutom i studie 2 så har patientgrupperna som mottagit belatacept drabbats av en högre incidens av akuta rejektioner där nästan alla skedde inom de första sex månader efter transplantation vilka oftast ger lindriga komplikationer. Slutsatserna som kan dras är att belatacept ger en högre njurfunktion på lång sikt vilket gör det mycket fördelaktigt framför calcineurinhämmare. Belatacept är förenat med ökad förekomst av akuta rejektioner men fördelen med den höga njurfunktionen kan anses väga tyngre då akuta rejektioner oftast ger lindriga komplikationer. Då belatacept är ett nytt läkemedel så kommer det behövas längre studier framöver för att påvisa en högre graftöverlevnad. / Kidney transplant is the only curing treatment for patients who have chronic kidney disease stage 5. Today’s most used immunosuppressive treatment after kidney transplant in Sweden and worldwide is the combination of the calcineurin inhibitor tacrolimus, mycophenolate mofetil and corticosteroids. Belatacept (Nulojix®) is a fusion protein which was introduced on the pharmaceutical market 2011 and should be viewed as an alternative for primary immunosuppressive treatment after kidney transplant. The mechanism of action for belatacept is to inhibit the activation of T-cells by blocking co-stimulatory signals provided by antigen-presenting cells. The theory behind belatacept is to avoid the nephrotoxic adverse events through a more specific immunosuppression. Nephrotoxicity is often seen with the calcineurin inhibitors tacrolimus (Prograf®) and cyclosporine (Sandimmune®). The consequence of nephrotoxicity is a deterioration in renal function which in a long-term can lead to graft loss. The aim of this study is to evaluate the efficacy of belatacept in comparison to calcineurininhibitors regarding graft survival, renal function and the occurrence of acute rejections. This literature study was conducted by evaluating five studies found in the PubMed database. None of the studies that were evaluated in this study showed any significant differences in graft survival of the patients treated with belatacept compared to calcineurin inhibitors. All studies demonstrated a significantly higher renal function measured in cGFR among patients treated with belatacept. The renal function increased over time which confirms the theory behind belatacept, i.e., that you can keep a more stable renal function over time by avoiding nephrotoxicity. All studies except study 2 demonstrated a higher incidence of acute rejection among patients who received belatacept as treatment. Almost all acute rejections in each study occurred within the first 6 months of the study which most of the time give minor complications.The conclusion that can be drawn from this literature study is that treatment with belatacept results in a higher renal function which makes it favorable to calcineurin inhibitors. Treatment with belatacept also results in a higher incidence of acute rejections but the benefit of a higher renal function can be considered to outweigh the risk of acute rejection.4In order to observe a significant difference in graft survival between patients receiving belatacept and those receiving calcineurin inhibitors several and longer studies, including more patients, need to be conducted.

Bien que le Tacrolimus soit un immunosuppresseur largement prescrit en transplantation rénale, ses effets néphrotoxiques limitent son utilisation. En effet, le Tacrolimus contribue au développement de lésions de fibrose interstitielle rénale et d’atrophie tubulaire à plus ou moins brève échéance. Parmi les mécanismes qui interviennent dans le processus de fibrogenèse, la transition épithélio-mésenchymateuse (TEM) a été évoquée. Dans ce processus, une cellule épithéliale polarisée perd l’expression de certains de ses marqueurs épithéliaux (E-cadhérine, cytokératine, &#946;-caténine membranaire...) au profit d’un phénotype mésenchymateux (expression de marqueurs mésenchymateux tels que vimentine ou translocation nucléaire de la &#946;-caténine, sécrétion de composants de la matrice extracellulaire). Ainsi, afin d’identifier des déterminants moléculaires de la néphrotoxicité induite par le Tacrolimus et d’évaluer la contribution du processus de TEM, plusieurs approches in vitro et in vivo ont été combinées.Tout d’abord, un modèle de culture primaire de cellules tubulaires proximales rénales (CTP), cibles privilégiées des xénobiotiques au niveau rénal a été développé à partir de pièces opératoires. Ce modèle a été caractérisé : analyse de la stabilité sur 5 passages, de l’expression de marqueurs épithéliaux proximaux et mésenchymateux, et de la résistance trans-épithéliale... De plus, la comparaison de la capacité métabolique des échantillons de tissu rénal sain à différents modèles cellulaires (HEK293, HK-2, CTP) nous a permis de montrer que les CTP est le modèle le plus pertinent. De plus, l’exposition de cellules rénales au Tacrolimus induit une modification du phénotype des cellules.De plus, le développement d’un modèle murin de néphrotoxicité induite par le Tacrolimus a été réalisé (exposition durant 28 jours à une dose de 1 mg/kg/j de Tacrolimus par implantation sous-cutanée de pompes Alzet® ou par injection intra-péritonéale). Les données histologiques et d’expression génique montrent que les reins de souris traitées par Tacrolimus présentent des zones localisées d’expression des marqueurs mésenchymateux (vimentine) et de fibrose (collagène, &#945;-SMA, miR-21). Par ailleurs, la variabilité interindividuelle des effets néphrotoxiques du Tacrolimus étant potentiellement associée à la présence de polymorphismes génétiques (SNP), les ADN de patients transplantés rénaux et de leur greffon ont été génotypés pour des SNP (i) affectant les CYP3A5 et ABCB1, intervenant dans la prise en charge du Tacrolimus, (ii) affectant la cavéoline-1, impliquée dans le processus de fibrose. Nos résultats montrent que deux SNP affectant le donneur (CYP3A5 6986A>G et ABCB1 3435C>T) sont significativement associés à une plus faible expression des marqueurs de TEM (expression de novo de la vimentine et translocation nucléaire de la &#946;-caténine) et à un nombre moins important de lésions de fibrose rénale sur les biopsies de greffons à 3 mois post-greffe. Enfin, les patients porteurs d’un greffon de génotype CAV1 rs4730751AA ont une perte de fonction rénale plus rapide. Ces patients semblent développer plus fréquemment des lésions de fibrose. Dans le cadre de la transplantation rénale, ces résultats suggèrent que certains SNP du donneur influencent la néphrotoxicité du Tacrolimus et que son métabolisme in situ est un élément clé dans la compréhension de la fibrogenèse du greffon.Au total, les résultats obtenus nous ont permis d’identifier des facteurs individuels de vulnérabilité à la toxicité du Tacrolimus. De telles données, utilisées comme outil prédictif d’une néphrotoxicité plus ou moins importante, aideraient à un meilleur choix de traitement immunosuppresseur. A terme, sur le plan médico-économique, notre étude pourrait permettre d’améliorer la prise en charge de la néphrotoxicité des immunosuppresseurs, et ainsi réduire les coûts de traitement liés aux pertes de greffons induites par la toxicité du Tacrolimus. / Although widely prescribed in kidney transplantation, Tacrolimus use is limited by its nephrotoxic effects. Indeed, Tacrolimus contributes to the development of renal interstitial fibrosis lesions and tubular atrophy with a large variability between patients. Among the mechanisms involved in fibrogenesis, the epithelial-mesenchymal transition (EMT) has been proposed. EMT is a dynamic process by which a polarized epithelial cell loses its epithelial markers (E-cadherin, cytokeratin, membrane &#946;-catenin...) and acquires a mesenchymal phenotype (de novo expression of vimentin or nuclear translocation of &#946;-catenin, secretion of extracellular matrix components). Thus, to identify molecular determinants of Tacrolimus-induced nephrotoxicity and to evaluate the contribution of EMT, several in vitro and in vivo approaches were combined.First, a model of primary culture of renal proximal tubular cells (PT cells), the main target of xenobiotics in kidney, has been developed and characterized: phenotypic stability, functional properties, expression of proximal and mesenchymal markers and transepithelial resistance. In addition, the comparison of the metabolic capacity of the healthy renal tissue samples to different cell models (HEK293, HK-2 CTP) has revealed that PT cells is the most appropriate model. Furthermore, renal cells exposure to Tacrolimus induced a modification of the cell phenotype.Moreover, the development of a murine model of Tacrolimus-induced nephrotoxicity has been performed (28 days-exposure at 1 mg/kg/day by subcutaneous implantation of Alzet® pumps or by intra-peritoneal injection). Histological and gene expression data indicated that kidney of Tacrolimus-treated mice exhibited localized expression of mesenchymal markers (vimentin) and fibrosis areas (collagen, &#945;-SMA, miR-21).Furthermore, as the interindividual variability of Tacrolimus nephrotoxic effects is potentially associated with genetic polymorphisms (SNPs), renal transplant recipients and their corresponding graft were genotyped for (i) CYP3A5 and ABCB1 SNPs, involved in Tacrolimus cellular processing, (ii) caveolin-1 SNP, involved in fibrosis. Our results showed that two SNPs affecting the donor (CYP3A5 6986A> G and ABCB1 3435C> T) were significantly associated with a lower expression of EMT markers (vimentine de novo expression and nuclear translocation of &#946;-catenin) together with less fibrosis lesions evaluated on renal graft biopsies performed at 3 months post-transplant. Finally, patients with a CAV1 rs4730751AA graft displayed a more severe renal function decrease. These patients also developed more frequently fibrotic lesions. In the context of renal transplantation, these results suggest that some donor SNPs modulate Tacrolimus-induced nephrotoxicity and that its in situ metabolism is a key element in the graft fibrogenesis understanding.Overall, these data allowed us to identify some molecular determinants of Tacrolimus-induced nephrotoxicity. Early identification of patients at high risk of Tacrolimus renal toxicity represents one of the most important and future challenges in kidney transplantation to tailor treatment before the development of irreversible lesions. Although preliminary, our data suggest that the genetic make-up of donors as well as the early detection of nephrotoxicity markers such as mesenchymal markers, may improve to the medical management of renal transplant patients.

Este trabalho centrou-se no desenvolvimento de nanocápsulas de núcleo lipídico contendo tacrolimus com revestimentos não iônico de polissorbato 80, aniônico de lecitina e catiônico de quitosana para posterior avaliação in vivo das atividades antiedematogênica e imunossupressora das formulações. As suspensões foram preparadas pelo método de deposição interfacial do polímero pré-formado utilizando a poli(ɛ-caprolactona) como polímero biodegradável. Para obtenção das nanocápsulas de núcleo lipídico de superfície catiônica, a lecitina foi adicionada à fase orgânica das suspensões e o revestimento procedeu-se por meio da incubação das partículas em solução aquosa de quitosana. De forma geral, todas as formulações apresentaram distribuição monomodal de partículas apenas na faixa nanométrica e baixa polidispersão. Os valores de potencial zeta obtidos para as formulações não iônicas e aniônicas foram negativos, enquanto que para as formulações catiônicas foram positivos. O teor de fármaco foi próximo ao teórico (0,6 ou 0,8 mg/mL) com alta eficiência de encapsulação (> 99%) e os estudos de liberação in vitro mostraram perfis de liberação sustentada do tacrolimus com mecanismo de transporte anômalo para todas as formulações. Além disso, as suspensões apresentaram estabilidade física adequada por meio de análises de retroespalhamento de luz e partículas de formato esferoide por microscopia eletrônica de transmissão. No modelo de artrite reumatóide induzida por adjuvante completo de Freund, o tacrolimus encapsulado em nanocápsulas de núcleo lipídico revestidas com polissorbato 80 mostrou atividade antiedematogênica superior ao fármaco em solução na dose de 2,0 mg/kg/dia, após administração intraperitoneal das formulações em ratos Wistar. Para avaliar o potencial de nanocápsulas de núcleo lipídico em aumentar a eficácia imunossupressora do tacrolimus pela via oral, realizou-se um estudo com camundongos CF1, o qual foi conduzido em duas etapas. Primeiramente, buscando avaliar o efeito da nanoencapsulação independente da barreira gastrointestinal, o fármaco em solução ou nanoencapsulado foi administrado pela via intraperitoneal nas doses de 4,0 e 6,0 mg/kg/dia, durante 10 dias. Nos tempos 0, 5 e 10 dias, amostras de sangue foram coletadas para posterior contagem do número percentual de linfócitos. Os resultados mostraram que o tacrolimus nanoencapsulado provocou maior redução linfocitária na dose de 4,0 mg/kg/dia em relação ao fármaco livre, enquanto que na dose de 6,0 mg/kg/dia, o efeito farmacodinâmico foi similar para o tacrolimus em solução ou nanoencapsulado. Continuando o estudo, as formulações foram administradas nas doses de 6,0 e 10,0 mg/kg/dia pela via oral e os resultados mostraram que o fármaco em solução na dose de 6,0 mg/kg/dia não apresentou efeito sobre o número percentual de linfócitos durante todo o experimento, enquanto que na dose de 10,0 mg/kg/dia, o efeito da solução foi observado somente no último dia de tratamento. Quando o fármaco foi nanoencapsulado, a redução linfocitária foi significativa nas duas doses testadas (6,0 e 10,0 mg/kg/dia), a qual se iniciou no quinto dia e manteve-se até o final do experimento. Em uma análise comparativa, somente dos grupos tratados com as formulações na mesma dose (6,0 mg/Kg/dia), pelas diferentes vias de administração, observou-se que os animais tratados com o fármaco livre apresentaram diferença na redução linfocitária entre as vias oral e intraperitoneal, enquanto que os animais tratados com as nanocápsulas núcleo lipídico revestidas com polissorbato 80 apresentaram equivalência farmacodinâmica entre as vias de administração avaliadas. Nesse mesmo estudo, constatou-se que as nanocápsulas na dose de 6,0 mg/kg/dia evitaram o aumento das concentrações séricas de glicose após administração oral das formulações. Prosseguindo com o mesmo modelo animal e buscando estudar a influência do tipo de revestimento das partículas (não iônico e catiônico) após administração oral do tacrolimus, foram desenvolvidas nanocápsulas de núcleo lipídico estabilizadas com lecitina e polissorbato 80 revestidas ou não com quitosana. As formulações foram administradas na dose de 6,0 mg/kg/dia e os resultados mostraram maior redução do número percentual de linfócitos para os animais tratados com a formulação de superfície catiônica, a qual provocou efeito imunossupressor já no quinto dia de tratamento enquanto que a formulação aniônica mostrou efeito somente no décimo dia. Os animais tratados com a dispersão do fármaco que continha quitosana não apresentaram redução do número de linfócitos durante o experimento. Além disso, a formulação catiônica foi a única capaz de evitar alterações nas concentrações séricas de glicose, creatinina e fosfatase alcalina dos animais. O conjunto destes estudos demonstra que as nanocápsulas de núcleo lipídico apresentam-se como uma estratégia tecnológica promissora, visando o aumento da eficácia farmacológica e redução de efeitos adversos do tacrolimus. / This work was based on the development of tacrolimus-loaded lipid-core nanocapsules employing the nonionic polysorbate 80, anionic lecithin and cationic chitosan coatings aiming the in vivo evaluation of antiedematogenic and immunosuppressive activities. The suspensions were prepared by interfacial deposition of preformed polymer using the poly(ɛ-caprolactone) as biodegradable polymer. In order to obtain the lipid-core nanocapsules with cationic surface, lecithin was added to the organic phase of the suspensions and the coating proceeded through the incubation of the particles in aqueous solutions of chitosan. In general, all formulations showed unimodal size distributions only at nanoscale and low polydispersity. The zeta potential values obtained for the nonionic and anionic formulations were negative whereas for the cationic formulations were positive. The drug contents were close to the theoretical values (0.6 or 0.8 mg mL-1) with high encapsulation efficiency (> 99%). The colloidal suspensions presented appropriate physical stability by light backscattered analysis and spheroid shaped particles by transmission electron microscopy. Moreover, drug release studies using the dialysis bag method showed sustained release profiles of tacrolimus with anomalous transport mechanism for all formulations. In the Freund's complete adjuvant–induced arthritis model, the tacrolimus-loaded lipid-core nanocapsules coated with polysorbate 80 showed higher antiedematogenic activity compared to the drug in solution after intraperitoneal administration of 2.0 mg kg-1 day-1 in Wistar rats. Proceeding the work, a study using CF1 mice was conducted to evaluate the potential of lipid-core nanocapsules in increasing the immunosuppressive efficacy of tacrolimus. The first evaluation was carried out to determine the influence of encapsulation of tacrolimus on the pharmacodynamic effect of the drug, independently of the oral absorption barrier. In this case, the tacrolimus-loaded lipid-core nanocapsules were compared to the drug in solution after intraperitoneal administration at 4.0 and 6.0 mg kg-1 day-1. Blood samples were collected at times 0, 5 and 10 days for subsequent determination of lymphocytes count percent. The results showed that drug nanoencapsulation caused greater reduction lymphocyte count (%) at the dose of 4.0 mg kg-1 day-1 compared to tacrolimus free, whereas at the dose of 6.0 mg kg-1 day-1, the pharmacodynamic effect was similar for drug encapsulated or nonencapsulated. Continuing the study, the formulations were administered by oral route at 6.0 and 10.0 mg kg-1 day-1. The results showed that the treatment with free drug at 6.0 mg kg-1 day-1 led to no significant difference in lymphocytes percent during the whole experiment while the treatment with drug in solution at 10.0 mg kg-1 day-1 caused exclusively a significant decrease after 10 days. On the other hand, after 5 days of treatment with tacrolimus-loaded lipid-core nanocapsules at both doses of 6.0 and 10 mg kg-1 day-1 had caused significant decreases in lymphocyte count (%); and the pharmacodynamic effect continued until the end of the experiment. In a comparative analysis only those groups treated with the formulations at the same dose (6.0 mg kg-1 day-1), the animals treated with the tacrolimus solution showed different percent reduction in lymphocyte between oral and intraperitoneal routes whereas the animals treated with the lipid core nanocapsules coated with polysorbate 80 showed equivalent pharmacodynamic effect among the administration routes evaluated. The influence of particle coatings (cationic and nonionic) after oral administration of tacrolimus was also investigated using the same animal model. For this, lipid-core nanocapsules stabilized with polysorbate 80-lecithin uncoated (anionic) or coated with chitosan were developed. The formulations were administered at the dose of 6.0 mg kg-1 day-1 and the results showed higher reduction in lymphocytes count percent for the formulation of cationic surface, which caused immunosuppressive effect on the fifth day while uncoated formulation showed the immunosuppressive effect only on the tenth day. The animals treated with the drug dispersion containing chitosan showed no reduction in lymphocytes count (%) during the experiment. Moreover, the formulation of lipid-core nanocapsules with cationic surface was the unique able to prevent alterations in serum levels of glucose, creatinine and alkaline phosphatase. This set of studies showed that the lipid-core nanocapsules are a promising technology strategy aiming at increasing of the pharmacological efficacy and reduction of adverse effects of tacrolimus.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Atopic Dermatitis (AD) is a chronic inflammatory skin disease that affects patients with disabilities in the skin barrier and low immune response. Tacrolimus ointment for topical use is an immunomodulator that has shown to be a good alternative for the treatment of AD. However, it also has adverse effects such as low and variable bioavailability, burning sensation and itching at the application site. Thus, the development of new dosage forms that overcome these drawbacks is crucial to the success of the therapy. The objective of this study was to obtain lipid nanoparticles (LN) of tacrolimus for future application in AD therapy. The LN were obtained by solvent diffusion method associated with ultrasonication using stearic acid (SA) and beeswax as solid lipid in the solid lipid nanoparticles (SLN) and oleic acid (OA) incorporated into the solid lipid matrix in the carriers lipid nanostructured (NLC). Lipid dispersions were characterized by determining the particle size, Polydispersity Index (PDI), Zeta Potential (ZP) and and drug content in the NL. Evaluation by Transmission Electron Microscopy (TEM), analysis by Differential Scanning Calorimetry (DSC), X-ray diffraction (XRD) and Infrared Spectroscopy in the region Fourier Transform (FT-IR). As results, LN presented nano-sized spherical shape with an average diameter ranging from 139 – 274, 9 nm and IPD from 0.3 - 0.5. PZ of LN was higher than -|25 mV|, which ensures the stability of the dispersion. In the results of DSC, it was observed that the endothermic event in the NLC was shifted to lower temperatures. Drug loading of 2,3 a 3,2%. The results of XRD also showed low crystallinity when NLC was compared with SLN. The results of FTIR was not observed interaction between the drug and the lipid components of the matrix. Then, it can be concluded that NL develop were successfully obtained and may represent promising system for the placement of tacrolimus in topical formulations. / A Dermatite Atópica (DA) é uma doença inflamatória cutânea crônica que acomete principalmente pacientes com deficiência na barreira cutânea e com baixa resposta imunológica. O tacrolimo para uso tópico em pomada é um imunomodulador que tem mostrado ser uma boa alternativa para o tratamento da DA. Porém, ele também apresenta efeitos adversos, tais como baixa e variável biodisponibilidade cutânea, sensação de queimação e prurido no sítio de aplicação. Assim, o desenvolvimento de novas formas farmacêuticas que contornem estas desvantagens é crucial para o sucesso da terapia. Desta forma, o objetivo deste trabalho foi obter nanopartículas lipídicas (NL) contendo tacrolimo para uso tópico. As NL foram obtidas pelo método de difusão do solvente associado a ultrassonicação, utilizando o ácido esteárico (AE) e a cera de abelha (CA) como lipídios sólidos nas nanopartículas lipídicas sólidas (NLS) e ácido oléico (AO) incorporado à matriz lipídica sólida nos carreadores lipídicos nanoestruturados (CLN). As dispersões lipídicas foram caracterizadas através da determinação do tamanho de partícula, Índice de Polidispersidade (IPD), Potencial Zeta (PZ) e teor de fármaco nas NL, avaliação por Microscopia Eletrônica de Transmissão (MET), análises através de Calorimetria Exploratória Diferencial (DSC), Difração de raio-X (DRX) e Espectroscopia na região do Infravermelho com Transformada de Fourier (FTIR). Como resultados, as NL apresentaram tamanho nanométrico, com um diâmetro médio variando de 139 – 274,9 nm e IPD de 0,3 - 0,5. O PZ das NL foi maior do que - |25 mV|. O teor de fármaco encontrado nas NL foi de 2,3 a 3,2 %. Nos resultados de DSC foi observado que o evento endotérmico nas CLN e nas NLS com fármaco foi deslocado para temperaturas menores, sugerindo maior desorganização da estrutura assim como os resultados de DRX, que também revelaram menor cristalinidade. Nos resultados de FTIR não foi observado interação entre o fármaco e os componentes da matriz lipídica. Então, pode ser concluído que as NL desenvolvidas foram obtidas com sucesso e podem representar futuros sistemas promissores para a veiculação do tacrolimo em formulações tópicas.

Kidney transplant recipients must take immunosuppressive medications to prevent the rejection of their transplanted kidney. If female transplant recipients become pregnant, however, very limited data are available about the effects of these medications on their exposed offspring. This study specifically reviews two of the most commonly used immunosuppressive medications prescribed to transplant recipients, cyclosporine and tacrolimus, and evaluates physical and cognitive development of the recipients' children who were exposed to these medications in utero. Participants in this study (n = 71) were female kidney transplant recipients who (a) voluntarily consented to be part of the National Transplantation Pregnancy Registry, (b) took cyclosporine or tacrolimus while pregnant, (c) had a child who is under the age of 6 years at the time of the study, and (d) were reachable via phone. Participants were asked standardized assessment questions related to their child's cognition and physical abilities from the Development Assessment of Young Children (DAYC). Standard scores from the assessment were recorded and analyzed to show that children exposed to cyclosporine or tacrolimus showed higher cognitive scores on the DAYC compared to the normative population. Children exposed to cyclosporine also showed higher physical scores compared to the normative population. Children exposed to tacrolimus did not show significant differences in physical development from the normative population. When cyclosporine or tacrolimus are required during pregnancy, these results help provide reassurance to parents and medical care providers about the cognitive and physical development of their offspring. Practical implications for school psychologists, limitations of this research, and directions for future research were discussed. / School Psychology

Les immunosuppresseurs sont des médicaments ayant démontré leur efficacité dans la prévention du rejet de transplantation d’organe solide. Néanmoins, ces médicaments présentent une importante variabilité de la réponse pharmacologique notamment liée à une variation de leur pharmacocinétique. Cette variabilité peut être à l’origine d’un défaut d’efficacité du traitement ou de sur-expositions entrainant une toxicité. Le suivi thérapeutique pharmacologique (STP) des concentrations sanguines d’immunosuppresseur permet de limiter ces risques de sur ou de sous-exposition en facilitant l’adaptation de posologie de ces traitements. En dépit de l'utilisation intensive du STP des concentrations d'immunosuppresseurs chez le transplanté d'organe, la fréquence du rejet aigu a peu diminué au cours de ces dernières années et reste élevée. Le rejet aigu cellulaire peut, en outre, survenir chez le patient alors même que ses concentrations sanguines sont en zone thérapeutique. C'est pourquoi la recherche d'améliorations de ce suivi thérapeutique ainsi que la recherche de nouveaux moyens de monitoring sont des axes pertinents d'investigation en pharmacologie des immunosuppresseurs. L’objectif de ce travail de doctorat a été de développer de nouveaux outils pharmacologiques de monitoring de l’effet de deux immunosuppresseurs, l’évérolimus et le tacrolimus, dans le but de participer à la maitrise de la variabilité pharmacologique de l’effet immunosuppresseur. Pour la première fois, la pharmacocinétique de l'évérolimus chez le transplanté cardiaque a été modélisée par une approche de population. La modélisation pharmacologique est un des axes actuels d’amélioration du STP des immunosuppresseurs permettant d’évaluer l’impact de covariables démographiques, biologiques et/ou génétiques sur la pharmacocinétique de ces médicaments. L’élaboration de ce modèle doit permettre une individualisation des posologies conduisant à la limitation de la variabilité pharmacocinétique lors d’un traitement par cette molécule.Au cours de ce travail, deux méthodes analytiques ont également été développées par LC-MS/MS pour le dosage intracellulaire de l’évérolimus et du tacrolimus. La mesure des concentrations des immunosuppresseurs à l’intérieur même de la cellule, c’est à dire au niveau de son site d’action, apparait comme une mesure plus pertinente que la simple mesure des concentrations dans le sang. Ces méthodes ont ensuite été évaluées sur des petites cohortes de patients transplantés cardiaques. La faisabilité de tels dosages a été démontrée et a permis la réalisation de la dernière partie présentée au cours de ce travail. En effet, une étude clinique a été réalisée chez le patient transplanté hépatique de novo en vue de rapprocher les concentrations sanguines et intracellulaires de tacrolimus et l'effet sur la protéine cible, la calcineurine. Pour la première fois des pharmacocinétiques intracellulaires complètes ont pu être obtenues permettant la description de la cinétique intracellulaire du tacrolimus. Cette étude a également permis de mettre en évidence et de décrire la relation dose - concentration sanguine - concentration intracellulaire - effet sur la protéine cible du tacrolimus chez le transplanté hépatique. Ces travaux ont permis de jeter les bases nécessaires à la réalisation d'essais cliniques permettant d'évaluer la pertinence d'un suivi longitudinal des concentrations intracellulaires et/ou de l'activité de la calcineurine dans la prévention de rejet de transplantation. Les outils développés au cours de ce travail de doctorat visent, d’une part, à mieux appréhender la variabilité de la réponse pharmacologique au cours d’un traitement immunosuppresseur et, d’autre part, à être des outils de compréhension des mécanismes pharmacocinétiques et cellulaires de ces traitements. L'utilisation de ces outils doit concourir à la diminution de la fréquence du rejet de greffe et à l'amélioration globale de la prise en charge du patient transplanté d'organe. / Immunosuppressive drugs have proven efficacy in the prevention of acute rejection of solid organ transplantation. However, these drugs exhibit substantial variability in pharmacological response due to such a variation in their pharmacokinetics. This variability may be the cause of underexposure with a lack of efficacy or over-exposure causing toxicity. Therapeutic drug monitoring (TDM) of immunosuppressant blood levels can limit the risk of over or underexposure facilitating dosage adjustment of these treatments. Despite the extensive use of TDM, the incidence of acute rejection has declined somewhat in recent years and remains high (in the order of 8-15%). Acute cellular rejection can further occur in patients even though blood levels are within the therapeutic range. That is why improvements in the therapeutic monitoring and new ways of monitoring are relevant lines of investigation in pharmacology.The objective of this phD work was to develop new pharmacological tools for monitoring the effect of two immunosuppressive drugs, everolimus and tacrolimus in order to control the pharmacological variability of the immunosuppressive effect .For the first time, the pharmacokinetics of everolimus in heart transplant was modeled by a population approach. Pharmacological modeling is one of the current areas of improvement of immunosuppressants TDM which allows evaluating the impact of demographic, biological and / or genetic on the pharmacokinetics of these drugs covariates. The development of this model must allow individualization of dosages leading to limit pharmacokinetic variability during treatment with this drug.During this work, two analytical methods were also developed by LC-MS/MS for assaying intracellular tacrolimus and everolimus concentrations. Measuring intracellular concentrations of immunosuppressive drugs, i.e. at its site of action, appears as a more relevant than measuring blood concentrations. These methods were then evaluated on small cohorts of heart transplant patients. The feasibility of such assays has been demonstrated and led to the completion of the last part presented in this work.Indeed, a clinical study was performed in de novo liver transplant patients to evaluate blood and intracellular concentrations of tacrolimus and their effect on the target protein, calcineurin. For the first time complete intracellular pharmacokinetics have been obtained for the description of the profile of the intracellular kinetics of tacrolimus. This study also highlights and describes the relationship dose - blood concentration - intracellular concentration - effect on the target protein of tacrolimus in liver transplant patients. This work might help conducting clinical trials to assess the relevance of a longitudinal follow-up of intracellular concentrations and / or activity of calcineurin in the prevention of transplant rejection.The tools developed in this PhD work aimed, firstly, to better understand the variability of the pharmacological response in immunosuppressive therapy and, secondly, to be tools for understanding the drug mechanisms inside of the cell. The use of these tools should contribute to the decrease in the frequency of graft rejection and the overall improvement in the management of organ transplant patients.

Made available in DSpace on 2016-01-26T18:55:32Z (GMT). No. of bitstreams: 1 Marcos_DISSERTACAOCIENCIAANIMAL_29_12.pdf: 1760249 bytes, checksum: 2221031a5e4fc9b1fd527533b56cf561 (MD5) Previous issue date: 2011-12-13 / Keratoconjunctivitis sicca is an ophthalmic disorder that affects the tear. The objective of this study was to evaluate the efficacy of tacrolimus dissolved in almond oil (OA) and linseed (OSL) in the treatment of dry eye syndrome (CCS) induced in rabbits. This study used twenty-five white male rabbits of New Zealand race. The animals were divided into five groups, Group C (control), Group TA (tacrolimus OA), Group TL (OSL tacrolimus), Group A (OA) and Group L (OSL), and were evaluated weekly by Tear Test Schirmer (TLS), Fluorescein Test (TF) and Rose Bengal Test (TRB), monthly by the exfoliative cytology of the ocular surface (CESO), and histopathology at the end of the experiment. The values were significant in TLS with an increase in all treated groups after induction of CCS, featuring improved performance, the group L. In TF, was earlier resolution of corneal ulcers with groups A and L, while in the performance of TRB groups was similar. The TA and TL groups showed less edema and degeneration of the cornea and conjunctiva. Significant increase in the number of goblet cells in all treated groups, especially in groups TA and TL. The evaluation results show that tacrolimus not only with its immunosuppressive effect, but also oily vehicles used for dilution, possibly due to their anti-inflammatory properties, were responsible in improving symptoms of dry eye induced in rabbits / A ceratoconjuntivite seca é um distúrbio oftálmico que afeta a lagrima. O objetivo deste estudo foi avaliar a eficácia do tacrolimus dissolvido em óleo amêndoa (OA) e de semente de linhaça (OSL) no tratamento da ceratoconjuntivite seca (CCS) induzida em coelhos. Neste experimento foram utilizados vinte e cinco coelhos brancos machos da raça Nova Zelândia. Os animais foram alocados em 5 grupos, Grupo C (controle), Grupo TA (tacrolimus em OA), Grupo TL (tacrolimus em OSL), Grupo A (OA) e Grupo L (OSL), sendo avaliados semanalmente pelo Teste de Lágrima de Schirmer (TLS), Teste de Fluoresceína (TF) e Teste de Rosa Bengala (TRB), mensalmente pelo o exame de citologia esfoliativa da superfície ocular (CESO), e análise histopatológica ao final do experimento. Os valores obtidos foram significativo nos TLS com aumento em todos os grupos de tratados após a indução da CCS, apresentando melhor desempenho, o grupo L. No TF, houve resolução mais precoce das úlceras de córnea com os grupos A e L, enquanto no TRB o desempenho dos grupos foi similar. Os grupos TA e TL apresentaram menos edema e degeneração de córnea e conjuntiva. Ocorreu aumento significativo no número de células caliciformes em todos os grupos de tratados, principalmente nos grupos TA e TL. A avaliação dos resultados demonstram que não somente o tacrolimus, com seu efeito imunossupressor, mas também os veículos oleosos utilizados para sua diluição, possivelmente devido às suas propriedades anti-inflamatórias, foram responsáveis na melhora dos sintomas do olho seco induzidos em coelhos.

Made available in DSpace on 2016-07-18T17:53:08Z (GMT). No. of bitstreams: 1 Marcos_DISSERTACAOCIENCIAANIMAL_29_12.pdf: 1760249 bytes, checksum: 2221031a5e4fc9b1fd527533b56cf561 (MD5) Previous issue date: 2011-12-13 / Keratoconjunctivitis sicca is an ophthalmic disorder that affects the tear. The objective of this study was to evaluate the efficacy of tacrolimus dissolved in almond oil (OA) and linseed (OSL) in the treatment of dry eye syndrome (CCS) induced in rabbits. This study used twenty-five white male rabbits of New Zealand race. The animals were divided into five groups, Group C (control), Group TA (tacrolimus OA), Group TL (OSL tacrolimus), Group A (OA) and Group L (OSL), and were evaluated weekly by Tear Test Schirmer (TLS), Fluorescein Test (TF) and Rose Bengal Test (TRB), monthly by the exfoliative cytology of the ocular surface (CESO), and histopathology at the end of the experiment. The values were significant in TLS with an increase in all treated groups after induction of CCS, featuring improved performance, the group L. In TF, was earlier resolution of corneal ulcers with groups A and L, while in the performance of TRB groups was similar. The TA and TL groups showed less edema and degeneration of the cornea and conjunctiva. Significant increase in the number of goblet cells in all treated groups, especially in groups TA and TL. The evaluation results show that tacrolimus not only with its immunosuppressive effect, but also oily vehicles used for dilution, possibly due to their anti-inflammatory properties, were responsible in improving symptoms of dry eye induced in rabbits / A ceratoconjuntivite seca é um distúrbio oftálmico que afeta a lagrima. O objetivo deste estudo foi avaliar a eficácia do tacrolimus dissolvido em óleo amêndoa (OA) e de semente de linhaça (OSL) no tratamento da ceratoconjuntivite seca (CCS) induzida em coelhos. Neste experimento foram utilizados vinte e cinco coelhos brancos machos da raça Nova Zelândia. Os animais foram alocados em 5 grupos, Grupo C (controle), Grupo TA (tacrolimus em OA), Grupo TL (tacrolimus em OSL), Grupo A (OA) e Grupo L (OSL), sendo avaliados semanalmente pelo Teste de Lágrima de Schirmer (TLS), Teste de Fluoresceína (TF) e Teste de Rosa Bengala (TRB), mensalmente pelo o exame de citologia esfoliativa da superfície ocular (CESO), e análise histopatológica ao final do experimento. Os valores obtidos foram significativo nos TLS com aumento em todos os grupos de tratados após a indução da CCS, apresentando melhor desempenho, o grupo L. No TF, houve resolução mais precoce das úlceras de córnea com os grupos A e L, enquanto no TRB o desempenho dos grupos foi similar. Os grupos TA e TL apresentaram menos edema e degeneração de córnea e conjuntiva. Ocorreu aumento significativo no número de células caliciformes em todos os grupos de tratados, principalmente nos grupos TA e TL. A avaliação dos resultados demonstram que não somente o tacrolimus, com seu efeito imunossupressor, mas também os veículos oleosos utilizados para sua diluição, possivelmente devido às suas propriedades anti-inflamatórias, foram responsáveis na melhora dos sintomas do olho seco induzidos em coelhos.