Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Proefschrift Universiteit Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

No description available.

Thesis (Ph. D.)--Medical College of Ohio, 2003. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin Sanchez. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 100-124).

Tacrolimus is a lactone macrolide, derived from Streptomyces tsukubaensis. It is an immunosuppressive drug that has been used in the prevention of organs transplant rejection, such as liver, kidney, heart, small intestine, pancreas and bone marron. The drug is also indicated for the treatment of atopic dermatitis, eczema, psoriasis and vitiligo. It is available as capsules, injection and ointment. Few methods have been reported for tacrolimus quantification in the dosage forms. In the present work, spectrophotometric methods were developed and validated for quantification of the drug in capsules. One method was based on the reaction with concentrated sulfuric acid (98 %), with detection at 295 nm. The other method was based in the charge transfer complex with 0.01M Iodine, with detection at 365 nm. The methods showed good linearity (r>0,99), precision (<0,2%) and accuracy (>99%). Preliminary study for in vitro drug release was also performed. Different dissolution medium (phosphate buffer pH 6.8, acetate buffer pH 5.5 and 0.1M HCl) and different stirring rotate (75 and 100 rpm) were evaluated to select the conditions for dissolution test, using basket apparatus. The percents of drug dissolved were evaluated by high performance liquid chromatographic method described in the literature. The percentage of drug dissolved was more than 85 in 60 minutes. / O tacrolimus é uma lactona macrolídea, derivada do Streptomyces tsukubaensis. É um fármaco imunossupressor usado na prevenção da rejeição de transplante de órgãos, tais como fígado, rim, coração, intestino delgado, pâncreas e medula óssea. É também indicado para tratamento de dermatite atópica, lupus eritematoso, psoríase e vitiligo. Encontra-se comercialmente disponível na forma de cápsulas, injetáveis e pomadas. Poucos métodos estão descritos na literatura para quantificação do tacrolimus nas formas disponíveis. Neste trabalho foram desenvolvidos e validados métodos espectrofotométricos para determinação quantitativa do fármaco em cápsulas. Um dos métodos utilizou reação com ácido sulfúrico concentrado (98%), com detecção em 295 nm. O outro se baseou na reação de complexo de transferência de carga com iodo 0,01M, com detecção em 365 nm. Os métodos desenvolvidos apresentaram linearidade (r>0,99), precisão (<0,2%) e exatidão (>99%) adequadas. Realizou-se, igualmente, estudo preliminar para avaliação do perfil de dissolução in vitro do fármaco. Diferentes meios de dissolução (tampão fosfato pH 6,8, tampão acetato pH 5,5 and HCl 0,1M) e diferentes velocidades de rotação (75 e 100 rpm) foram avaliados para definir as condições para o teste de dissolução, empregando o aparato cesta. As porcentagens dissolvidas do fármaco foram determinadas através de método por cromatografia líquida de alta eficiência descrito na literatura. As percentagens dissolvidas foram superiores a 85% em 60 minutos.

In newborns, unconjugated bilirubin (UCB) is not readily excreted, and when bilirubin levels exceed the serum albumin binding capacity, pathological levels of UCB exist. Hyperbilirubinemia may lead to auditory damage and ultimately cause a hearing disorder called auditory neuropathy/dys-synchrony, characterized by absent or abnormal brainstem auditory evoked potentials (BAEPs) with evidence of normal inner ear function assessed by either otoacoustic emissions or cochlear microphonic responses. Phototherapy and double volume exchange transfusion are used as treatment methods for neonatal hyperbilirubinemia. Spontaneously jaundiced Gunn rat pups given sulfadimethoxine to displace bilirubin from serum albumin develop bilirubin encephalopathy and have abnormal BAEPs comparable to human neonates. BAEPs are a noninvasive electrophysiological measure of neural function of the auditory system. High levels of calcineurin activity are believed to be involved in the mechanism of this bilirubin induced auditory neuropathy. FK506, a calcineurin inhibitor, was administered 3 hours prior to sulfa in concentrations of 0.1mg/kg, 1.0mg/kg, and 10.0mg/kg body weight. Due to the observation that all animals had abnormal BAEPs after treatment with FK506 and sulfa, it can be concluded that none of the treatment doses protected against bilirubin induced auditory impairment.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Class of 2006 Abstract / Objectives: To evaluate the comparative efficacies of Tacrolimus and Pimecrolimus for the treatment of Keratoconjunctivitis Sicca (KCS), Pannus, and other autoimmune related ophthalmological disorders. Methods: A descriptive retrospective study of clinical data obtained through two separate veterinary studies. The improvement in Schirmer Tear Test (STT) between the Tacrolimus group and Pimecrolimus group is compared. Results: There were 27 canines in the Tacrolimus group, with a total of 50 affected eyes. The Pimecrolimus group had 14 total canines, but tear production was measured in only 8, for a total of 16 affected eyes. At baseline, there was no significant difference in tear production between the two groups. At first and second follow up, and in both eyes, Tacrolimus produced a significantly greater increase in tear production than Pimecrolimus (p=0.0093 and p=0.0292 respectively for the right eye first and second follow up; p=0.0425 and p=0.0065 respectively for the left eye first and second follow up). Conclusions: Treatment with Tacrolimus produced a greater increase in tear production that Pimecrolimus in canines with autoimmune keratitis.

Os imunossupressores tacrolimo (Tac) e sirolimo (Srl) são amplamente utilizados no transplante renal. Estes medicamentos apresentam estreita faixa terapêutica e estão associados a uma vasta gama de efeitos colaterais. Polimorfismos de nucleotídeo único (SNP) parecem ter um impacto significativo sobre a farmacocinética dos imunossupressores. Com o objetivo de avaliar a associação de SNP nos genes PPP3CA e MTOR com a resposta farmacológica dos imunossupressores tacrolimo e sirolimo foram selecionados 156 indivíduos indicados para transplante renal entre os pacientes atendidos no Hospital do Rim e Hipertensão da UNIFESP. Esses indivíduos foram tratados com esquema imunossupressor baseado em tacrolimo ou convertido para sirolimo. Amostras de sangue foram coletadas antes do transplante para extração de DNA. As determinações das concentrações sanguíneas de Tac foram determinadas por chemiluminescent microparticle immunoassay (CMIA) e as concentrações sanguíneas de Srl foram obtidas pela técnica de HPLC (High- Performance Liquid Chromatography). Os polimorfismos do MTOR (c.1437T>C, T c.2997C>T e c.4731G>A) e PPP3CA (c.249G>A) foram identificados por PCR em tempo real. O polimorfismo PPP3CA c.246G>A não foi associado à dose diária de tacrolimo ou sirolimo. Já a concentração sanguínea de tacrolimo foi menor nos portadores do alelo A no terceiro dia e terceiro mês de estudo. Os polimorfismos do MTOR foram relacionados à concentração sanguínea corrigida pela dose de tacrolimo. Os portadores dos alelos raros G, T e C dos polimorfismos c.4731G>A, c.14337T>C e c.2997C>T, respectivamente, apresentaram valores de Co/Do de tacrolimo menores em relação aos não portadores destes alelos. As diferenças significativas ocorreram principalmente nos primeiros três meses de estudo. A concentração sanguínea de tacrolimo foi no geral menor nos portadores dos alelos raros, sendo significativamente menor no décimo quarto dia. Doses maiores de tacrolimo foram associadas aos alelos T do c.14337T>C e C do c.2997C>T. No sexto mês de estudo, os portadores dos alelos raros receberam doses de sirolimo significativamente maiores do que os não portadores. O alelo T do polimorfismo c.1437T>C foi associado a menores valores de Co/Do de sirolimo. Os SNPs c.2997C>T e c.1437T>C do MTOR encontram-se em desequilíbrio de ligação (D\'=0,981; r2=0,690). Nos três primeiros meses de estudo, os portadores do haplótipo TC receberam doses menores de tacrolimo e apresentaram a melhor relação Co/Do. Foi possível observar que após a randomização, o haplótipo TC continuou associado a menores doses de tacrolimo e de sirolimo e manteve a tendência de melhores índices de Co/Do de ambos os fármacos. Os polimorfismos c.1437T>C e c.4731G>A foram associados a parâmetros de função renal no grupo TAC. O alelo G do SNP c.4731G>A relacionou-se a valores menores de ureia no pré-Tx, menor redução de ureia e creatinina entre o pré-Tx e o sexto mês de estudo. O alelo T do SNP c.1437T>C também foi relacionado a menores valores de ureia no pré-Tx e menor redução de creatinina. No grupo TAC, o alelo raro do SNP PPP3CA c.249G>A foi relacionado a menores valores de triglicérides no pré-Tx e no grupo SRL uma menor variação de LDL-colesterol. Os portadores do alelo C do SNP c.2997C>T apresentaram menor aumento de colesterol total e LDL colesterol entre o pré-Tx e o sexto mês de estudo, maiores valores de HDL colesterol no pré-Tx e menores valores de triglicérides no sexto mês de estudo. Os portadores do alelo T do SNP c.1437T>C apresentaram menor aumento de colesterol total, LDL colesterol, VLDL colesterol e triglicérides. No sexto mês de estudo apresentaram menores valores de triglicérides em relação aos não portadores deste alelo. Os portadores do alelo G do SNP c.4731G>A tiveram variação menor de colesterol total, VLDL colesterol e triglicérides. Não foi encontrada relação dos polimorfimos estudados e a rejeição aguda comprovada por biópsia ou com a nefropatia crônica do enxerto. Esses resultados são sugestivos de que os polimorfismos do MTOR e PPP3CA estão associados com a dose e concentração sanguínea dos imunossupressores tacrolimo e sirolimo, assim como um perfil lipídico menos aterogênico. / The immunosuppressant tacrolimus (Tac) and Sirolimus (Srl) are widely used in renal transplantation. These drugs have a narrow therapeutic range and are associated with a wide range of side effects. Single nucleotide polymorphisms (SNPs) have a significant impact on the pharmacokinetics of immunosuppressants. In order to evaluate the association of SNPs in genes MTOR and PPP3CA with the pharmacological response of immunosuppressive drugs tacrolimus and sirolimus were selected 156 individuals referred for kidney transplantation among patients treated in the Hospital do Rim e Hipertensão, UNIFESP. These individuals were treated with tacrolimus-based immunosuppressive regimen or converted to sirolimus. Blood samples were collected before transplantation for DNA extraction. Determinations of blood concentrations of Tac were determined by Chemiluminescent microparticle immunoassay (CMIA) and blood concentrations Srl were obtained by the technique of HPLC (High-Performance Liquid Chromatography). Polymorphisms of MTOR (c.1437T>C, T c.2997C>T and c.4731G>A) and PPP3CA (c.249G>A) were identified by real-time PCR. The Polymorphism PPP3CA c.246G>A was not associated with the daily dose of tacrolimus or sirolimus. The blood concentration of tacrolimus was lower in carriers of the allele on the third day and third month of study. The Polymorphisms of MTOR were related to blood concentration corrected by the dose of tacrolimus. The carriers of rare alleles G, T and C polymorphisms c.4731G> A, c.14337T> C and c.2997C> T, respectively, had values of Co/Do tacrolimus lower than the non-carriers of these alleles. Significant differences occurred mainly during the first three months of study. The blood concentration of tacrolimus was generally lower in carriers of the rare alleles being significantly lower on the fourteenth day. Higher doses of tacrolimus were associated with alleles c.14337T T>C and C c.2997C>T. In the sixth month of study, the carriers of rare alleles received doses of sirolimus significantly higher than non-carriers. SNPs c.2997C>T and c.1437T>C MTOR are in linkage disequilibrium (D \'= 0.981; r2 = 0.690). In the first three months of study, carriers of the TC haplotype received lower doses of tacrolimus and presented the best value for Co/Do. It was observed that after randomization, the TC haplotype remained associated with lower doses of tacrolimus and sirolimus and continued the trend of higher rates of Co/Do of both drugs. Polymorphisms c.1437T>C and c.4731G>A were associated with renal function parameters in the TAC group. The G allele of SNP c.4731G> A was related to lower levels of urea in the pre-Tx, a smaller reduction of urea and creatinine between the pre-Tx and sixth months of study. The T allele of SNP c.1437T>C was also related to lower levels of urea in the pre-Tx and a smaller reduction of creatinine. In the TAC group, the rare allele of SNP PPP3CA c.249G>A was related to lower levels of triglycerides in the pre-Tx and the SRL group a smaller variation of LDL-cholesterol. The C allele of the SNP c.2997C>T showed a lower increase in total cholesterol and LDL cholesterol between pre-Tx and sixth months of study, higher HDL cholesterol in pre-Tx and lower levels of triglycerides in the sixth month of study. The T allele of SNP c.1437T>C showed a lower increase in total cholesterol, LDL cholesterol, VLDL cholesterol and triglycerides. In the sixth month of the study, they had lower triglyceride levels compared to non-carriers of this allele. The G allele of SNP c.4731G>A change had lower total cholesterol, VLDL cholesterol and triglycerides. There was no relationship between the studied polymorphisms and biopsy-proven acute rejection or chronic allograft nephropathy. These results suggest that MTOR and PPP3CA polymorphisms are associated with dose and blood concentration of immunosuppressants tacrolimus and sirolimus, as well as a less atherogenic lipid profile.

La miastenia gravis es una enfermedad autoinmune mediada por anticuerpos anti-receptor de acetilcolina del músculo esquelético. Su tratamiento incluye: La timectomia y la inmunosupresión con los corticoides, la azatioprina y la ciclosporina. Estos inmunosupresores son efectivos, pero el manejo de los pacientes con miastenia gravis generalizada refractaria y con efectos adversos secundarios al tratamiento prolongado con corticoides continúa sin solución. Tacrolimus (FK506) es un macrólido parecido a la ciclosporina y con potente efecto inmunosupresor. Basados en prometedores resultados iniciales con este fármaco, hemos iniciado dos trabajos prospectivos tratando con tacrolimus a un grupo de pacientes afectos de miastenia gravis refractaria, en que no es posible reducir la dosis de prednisona y/o presentan importantes efectos secundarios a la prednisona y/o ciclosporina. El primer estudio corresponde a un grupo de 15 pacientes, 2 fueron excluidos, uno por no cumplir los criterios de inclusión y el otro por decisión propia, los 13 incluidos completaron el estudio, todos timectomizados y afectos de miastenia gravis refractaria al tratamiento con prednisona y ciclosporina, que han sido tratados con tacrolimus (FK506) durante 12 meses a dosis de 0.1 mg/Kg de peso/día repartidos en dos tomas y ajustando la dosis en función de los niveles plasmáticos de este fármaco, para alcanzar concentraciones entre 7 y 8 ng/mL. La dosis de prednisona se ha reducido progresivamente hasta retirarla. Los títulos de los anticuerpos anti-receptor de acetilcolina y la escala del grado de severidad de la miastenia gravis se han reducido de manera significativa y la fuerza muscular ha aumentado en un 37%. Todos los pacientes presentaron remisión farmacológica, 11 asintomáticos y 2 con discreta ptosis palpebral. Tacrolimus fue bien tolerado y puede ser una alternativa en enfermos miastenicos que no responden a los inmunosupresores convencionales. El segundo trabajo corresponde a un grupo de 86 pacientes con 7 excluidos, 3 por no acudir a las visitas y 4 por decisión propia, de los 79 pacientes incluidos, timectomizados y con dependencia de la prednisona y ciclosporina que han recibido tacrolimus durante una media de 2.5 ± 0.8 años. La prednisona se ha retirado en todos los enfermos excepto dos. Los títulos de anticuerpos anti-receptor de acetilcolina y la escala de severidad de la enfermedad se han reducido de manera significativa y la fuerza muscular ha mejorado en un 39%. Estos dos estudios representan el núcleo de esta tesis doctoral y las conclusiones son: 1) El tratamiento con tacrolimus, ha permitido suspender la ciclosporina y una reducción progresiva de la prednisona hasta poder retirarla de forma total en el 98% de los casos, con la consiguiente reducción del 63.2% de los efectos secundarios a estos dos fármacos.2) Se ha observado un aumento de la fuerza muscular del 39% con el uso del tacrolimus. Esto ha permitido mejorar la calidad de vida de estos pacientes a nivel social, laboral y familiar.3) Los niveles de anticuerpos anti-receptor de acetilcolina han experimentado una reducción del 86.14% con la utilización del FK506. 5) Con el tacrolimus, a dosis de 0.1mg/kg./día y durante más de tres años, no hemos observado la aparición de recidivas en los casos de timomas invasivos.6) El tratamiento con tacrolimus mejora los resultados, con remisión completa del 5.1% y remisión farmacológica del 87.3 % sin provocar efectos secundarios importantes y con buena tolerancia en la mayoría de los pacientes.7) No se ha evidenciado diferencias en la respuesta terapéutica al tacrolimus en los pacientes con o sin timoma.8) Los resultados nos permiten apuntar al tacrolimus como un inmunosupresor a tener muy en cuenta en el tratamiento de la miastenia gravis. / Myasthenia gravis is an autoimmune disease caused usually by antibodies against the acetylcholine receptor of skeletal muscle. Its current treatment includes thymectomy as an early consideration, and several immunosuppressive agents, such as corticosteroids, azathioprine and cyclosporine. Although immunosuppressive regimens are very effective, important issues related to the management of patients with generalized uncontrollable myasthenic symptoms or serious side effects of long-term steroid treatment remained unanswered. Tacrolimus (FK506) is a macrolide molecule of the same immunosuppressant class as cyclosporine and has a potent immunosuppressive. Successful treatment of myasthenia gravis with tacrolimus has been recently reported in patients with intractable myasthenia. Based on these encouraging initial results we started two prospective studies to evaluate the efficacy of Tacrolimus in myasthenic patients with residual myasthenia gravis in whom it was not possible to reduce the doses of prednisone and/or due to the occurrence of important side effects of cyclosporine and/or side effects of prednisone.A first study consisted of 15 patients, 2 were excluded. In one patient, inclusion criteria were not met at baseline assessment and another patient through his own decision. Therefore, 13 patients completed the study. All with myasthenia gravis, unresponsive to prednisone and cyclosporine after thymectomy, received FK506 (tacrolimus) for 12 months, at starting doses of 0.1 mg/kg per day b.i.d. and then adjusted to achieve plasma concentrations between 7 and 8 ng/mL. The doses of prednisone were progressively reduced and finally discontinued. Anti-acetylcholine antibodies and myasthenia gravis score for disease severity decreased significantly and muscular strength increased by 37%. All patients achieved pharmacological remission, 11 were asymptomatic and two had minimal weakness of eyelid closure. Tacrolimus was well tolerated and appears a suitable approach after unsuccessful treatment with conventional immunosuppressants in patients with disabling myasthenia.A second prospective study consisted of 86 patients, seven were excluded due to failure to attend control visits (n=4) and voluntary decision to quit (n=3). Of the seventy-nine patients with cyclosporine- and prednisone dependent myasthenia gravis (MG) after thymectomy received tacrolimus for a mean of 2.5 ± 0.8 years. Prednisone was withdrawn in all but two patients. Anti-acetylcholine antibodies and MG score for disease severity decreased significantly and muscular strength increased by 39%. Those two studies represent the nucleus of this Doctoral Thesis and the conclusions are: 1) The treatment with tacrolimus permitted withdrawn cyclosporine and the dose of prednisone were reduced and finally withdrawn in 98% of patients and the adverse effects related to both drugs was decreased in 63.2%.2) We observed an improvement of muscular strength with an increase of 39% using tacrolimus. All patients resumed full activities of daily living. 3) Anti-acetylcholine antibodies also decreased significantly in 86.14% using FK506.4) Tacrolimus for more than three years, at starting doses of 0.1 mg/kg per day, we don't observe relapse en cases with invasive thymoma.5) Tacrolimus improve results; complete stable remission was achieved in 5.1% of patients and pharmacologic remission in 87.3% without an important side effect and was well tolerated by all patients.6) The clinical response of patient with thymic hyperplasia and patients with thymoma was similar.7) The present results add evidence to the benefits of including tacrolimus in the therapeutic armamentarium of myasthenia gravis.