Tesi sul tema "Nelfinavir"

Le nelfinavir est un inhibiteur de protéase utilisé dans le traitement du SIDA. Ce médicament est métabolisé par le CYP2C19 en un métabolite actif, le M8. Il existe une grande variabilité interindividuelle des concentrations plasmatiques de nelfinavir et de M8 avec des conséquences pour l’efficacité thérapeutique. Dans quatre publications différentes, nous avons analysé les facteurs de variabilité de ces concentrations grâce à une méthode de pharmacocinétique de population : chez la femme enceinte et au cours de l’accouchement, chez l’enfant et le fœtus et chez les sujets présentant un polymorphisme génétique pour le CYP2C19. Puis à partir des estimations des paramètres pharmacocinétiques individuelles, nous avons calculé la dose nécessaire pour atteindre une concentration efficace. Lorsque nous disposions de mesures d’efficacité et de toxicité, des relations pharmacocinétique – pharmacodynamique ont été étudiées. Ces résultats ont permis des recommandations posologiques adaptées
Nelfinavir is a protease inhibitor used for AIDS treatment. This drug is metabolized by CYP2C19 to an active metabolite, M8. There is a high interindividual variability in nelfinavir and M8 plasma concentrations, with consequences on therapeutic efficacy. In four different publications, we analyzed variability factors of these concentrations thanks to population pharmacokinetics method: in pregnant women and women at delivery, in children and fetus and in adults with genetic polymorphism for the CYP2C19. Then, from the estimated individual pharmacokinetic parameters, we calculated minimal dose necessary to reach an effective concentration. When data concerning efficacy and toxicity were available, pharmacokinetic – pharmacodynamic relationships were studied. The results obtained permitted to give adapted doses recommendations

Ce travail vise à développer des dosages radio-immunologiques de l’IDV et du NFV, deux anti-protéases utilisées en chimiothérapie anti-VIH. Pour cela, nous avons préparé les haptènes et les immunogènes de l’IDV et du NFV en vue d’obtenir les anticorps nécessaires au développement du dosage. Aussi , nous avons développé le premier dosage RIA de l’Indinavir. L’immunisation de lapins par le conjugué IDV-4(S)-HS-KLH et l’utilisation du traceur IDV-4(S)-HS-GT-I* pour la caractérisation des anticorps nous ont permis de sélectionner un immunosérum présentant un titre élevé (1/4. 10 puissance 4) et une affinité importante (IC indice 50 de 1,3. 10 puissance -9 M). Ce dernier possède une spécificité intéressante puisqu’aucune autre anti-PR n’est reconnue par les anticorps anti-IDV. Cette caractéristique nous permettra de doser des échantillons provenant de patients traités par l’IDV. Nous avons appliqué ce dosage à un modèle in vitro mimant les conditions physiologiques permettant d’évaluer les taux intracellulaires de l’IDV. Les premiers résultats montrent des quantités voisines de 2 nmoles. Uns stratégie similaire a été appliquée au NFV. L’immunisation a été réalisée à l’aide de l’immunogène NVF-AcO-BSA. Les premiers anticoprs anti-NFV obtenus ont été caractérisés à l’aide d’un traceur NFV-I*. Dans ces conditions, l’immunosérum sélectionné possède un titre de 1/3000ème et une affinité de 10 puissance -7 M. Cependant, ce dosage nous a semblé trop peu sensible pour la détermination des taux intracellulaires de cette anti-PR. Nous avons alors préféré développer un autre dosage du NFV en changeant la structure chimique de l’immunogène : NFV-AcO-βAla-KLH. La caractérisation des anticorps, à l’aide du traceur NFV-AcO-βAla-GT-I* a permis de sélectionner un immunosérum présentant un titre de 1/1,5. 10 puissance 6. L’affinité des anticorps (4. 10 puissance -9 M) est suffisante pour doser les quantités intracellulaires de NFV. De plus, ce dosage est spécifique puisque les anticorps ne reconnaissent pas les anti-PR utilisées dans le traitement du SIDA.

Made available in DSpace on 2014-06-12T16:31:34Z (GMT). No. of bitstreams: 2 arquivo6106_1.pdf: 2159514 bytes, checksum: 67a8083fdccc3096c53639c1f47b0ac1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005
Os Inibidores de protease (PI) constituem uma potente classe de drogas anti-retrovirais que mudou o tratamento e a evolução da infecção pelo HIV. Em março de 1997, um novo medicamento desta classe, o mesilato de nelfinavir foi aprovado pela Food and Drug Administration (FDA), sendo desde então, extensamente utilizado isoladamente ou em associação a Inibidores da Transcriptase Reversa (RTIs), obtendo-se considerável eficácia clínica no tratamento da AIDS. O mesilato de nelfinavir tem como produto de referência no mercado o Viracept®, fabricado pela indústria Roche. Esta medicação faz parte do coquetel anti-AIDS distribuído no Brasil, sendo usada por 25% dos pacientes. O presente trabalho tem como objetivo apresentar o desenvolvimento farmacotécnico-industrial de comprimidos revestidos de mesilato de nelfinavir 250 mg, baseado em uma planificação qualitativa e quantitativa dos excipientes, além da realização de testes de bancada visando à obtenção de uma forma farmacêutica com qualidade e baixo custo. Para tanto, realizou-se a caracterização da matéria-prima com vistas à qualificação de fornecedores, e executou-se o desenvolvimento da metodologia de dissolução para os comprimidos obtidos, além de estudo comparativo do seu perfil de dissolução frente ao medicamento de referência. O trabalho também contemplou o desenvolvimento e a validação da metodologia para doseamento da matéria-prima e produto acabado, por Cromatografia Líquida de Alta Eficiência (CLAE), obedecendo aos parâmetros estabelecidos na Resolução-RE n° 899, publicada em 02 de junho de 2003 pela Agência Nacional de Vigilância Sanitária (ANVISA). Também foi realizado um estudo comparativo entre o produto de referência Viracept® e o produto desenvolvido mesilato de nelfinavir LAFEPE®. Os comprimidos revestidos de mesilato de nelfinavir desenvolvidos apresentaram boa qualidade e as comparações efetuadas entre estes e o Viracept® não apresentaram diferenças significativas. Encontra-se em curso o estudo de estabilidade nos modelos acelerado e longa duração. Este estudo foi realizado em parceria com o Núcleo de Controle de Qualidade de Medicamentos e Correlatos, o Laboratório de Tecnologia dos Medicamentos, ambos do Departamento de Ciências Farmacêuticas da UFPE e o Laboratório Farmacêutico do Estado de Pernambuco (LAFEPE)

BACKGROUND: Highly active antiretroviral therapy(HAART) is used in pregnancy to suppress viral load(pVL) before delivery, reducing risk of vertical HIV-transmission. Nelfinavir(NFV) containing HAART has been highly used in pregnancy, but dosages may be inadequate due to the physiologic changes that occur. Given concerns regarding optimal viral suppression in pregnancy, drug toxicity and resistance development, NFV levels need to be evaluated in this population to guide dosing recommendations. METHODS: As part of a prospective cohort study maternal blood was collected at 18-28wks, 32-37wks and at delivery. Times of last medication dose and blood sampling were recorded and drug levels were measured using HPLC MS-MS. NFV concentration-ratios(NFV-CRs) were calculated by dividing individual levels by a time-adjusted population value. Plasma NFV concentrations and NFV-CRs were compared across gestational age and correlated to variables of interest. Rate and maintenance of viral suppression were analyzed in relation to NFV concentrations and CRs. Statistical tests included ANOVA, χ2, linear regression, and Kaplan Meier estimates. RESULTS: 113 samples were collected from 32 subjects. Samples were eliminated if not in steady state (n=20); 93 samples from 32 subjects were analyzed. Mean NFV-CR at 18-28wks (1.1±0.73) and 32-37wks (0.86±0.73) were not significantly different but were both significantly higher by ANOVA (p=0.049) than the mean NFV-CR at delivery (0.44±0.50). CRs were highly variable. Of 49 antepartum samples, 49%(24) had a CR<0.90 (clinically relevant threshold). Four women reached a pVL <50 copies/mL by 34wks but had a detectable pVL at delivery. One woman never reached an undetectable pVL in pregnancy. Minimum and mean NFV-CRs in these 5 women were not significantly different than those who achieved and maintained virologic suppression. Vertical HIV transmission rate was 0%. CONCLUSIONS: There were no HIV transmissions but 16% (5/32) of women were inadequately suppressed at delivery, which is of concern. Factors associated with inadequate suppression and NFV-CRs need to be explored in conjunction with patient/physician reported adherence and viral resistance profiles. Extreme variability in CRs may limit the potential usefulness of random timed drug levels in all pregnant women.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T15:37:16Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AnaliseConformacionalEnzima.pdf: 2962750 bytes, checksum: a3dc63037d6a89e63bf949b46941a41e (MD5)
Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-14T14:05:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AnaliseConformacionalEnzima.pdf: 2962750 bytes, checksum: a3dc63037d6a89e63bf949b46941a41e (MD5)
Made available in DSpace on 2017-11-14T14:05:55Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AnaliseConformacionalEnzima.pdf: 2962750 bytes, checksum: a3dc63037d6a89e63bf949b46941a41e (MD5) Previous issue date: 2017
CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
O Vírus da imunodeficiência humana (HIV), causador da síndrome da imunodeficiência adquirida (AIDS), é um retrovírus que possui glicoproteínas altamente virulentas que invadem o linfócito TCD4+ através de seus receptores CCR4 e CXCR5. O ciclo biológico do HIV é mediado pelas enzimas protease, transcriptase e integrase. A HIV-1 protease é uma enzima que está presente na fase final do ciclo biológico, onde ocorre a maturação do vírus e é um importante alvo farmacológico. O objetivo principal deste projeto é verificar os efeitos das mutações D30N, I84A e M46I na enzima protease HIV-1 e na formação do complexo com o inibidor nelfinavir através de técnicas de dinâmica molecular e bioinformática. Os resultados baseados nas análises estruturais mostraram diferenças estruturais entre os sistemas estudados. O sistema 1OHR apresentou uma conformação fechada, os sistemas D30N e D30N_I84A_M46I apresentaram conformação semi-aberta e o sistema D30N_I84A apresentou conformação aberta, em que o último apresentou menor valor de energia livre e maior instabilidade nas análises de RMSD, porém a maior flutuação de resíduos de aminoácidos. As análises teóricas mostraram a importância na resistência da dupla mutação D30N_I84A e a capacidade de reestruturação conformacional da mutação M46I e capacidade catalítica.
The Human Immunodeficiency Virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), is a retrovirus that has highly virulent glycoproteins that invade the CD4 + T lymphocyte through its CCR4 and CXCR5 receptors. The biological cycle of HIV is mediated by the protease, transcriptase and integrase enzymes. HIV-1 protease is an enzyme that is present in the final phase of the biological cycle, where virus maturation occurs, and is an important pharmacological target. The main objective of this project is to verify the effects of the D30N, I84A and M46I mutations on the HIV-1 protease enzyme and the complex formation with the nelfinavir inhibitor through molecular dynamics and bioinformatics techniques. The results based on the structural analyzes showed structural differences between the studied systems. The 1OHR system presented a closed conformation, the systems D30N and D30N_I84A_M46I presented semi-open conformation and the D30N_I84A system presented open conformation, in which the latter presented lower free energy value and greater instability in the RMSD analyzes, however the greater flotation of residues Of amino acids. The theoretical analyzes showed the importance in the resistance of the double mutation D30N_I84A and the conformational restructuring capacity of the M46I mutation and catalytic capacity.

Les études pharmacocinétiques en pédiatrie sont importantes en raison des transformations liées à la maturation dont il faudra tenir compte dans la prescription des médicaments. Dans la première partie de notre thèse nous exposerons les données bibliographiques concernant les modifications des paramètres pharmacocinétiques chez l'enfant ainsi que l'intérêt de l'utilisation de la pharmacocinétique de population en pédiatrie. Nous présenterons ensuite notre travail personnel concernant la pharmacocinétique de population de la ciprofloxacine, du nelfinavir et du mycophénolate mofétil. Le logiciel NONMEM a été utilisé pour ces analyses. Après un bref rappel sur la pharmacologie des fluoroquinolones, les relations structure activité et structure toxicité, nous présenterons les résultats d'une étude pharmacocinétique réalisée chez 55 patients âgés de 1 jour à 24 ans. Le modèle structurel prend en compte une relation entre clairance totale de la ciprofloxacine et âge et état clinique du patient (fibrose cystique ou non). Une stratégie de prélèvement limité est proposée. Ce travail constitue la deuxième partie de notre thèse. Dans une troisième partie, après un rappel sur la pharmacologie des antirétroviraux, nous présenterons l'étude pharmacocinétique du nelfinavir et de son métabolite actif (M8) chez des enfants âgés de moins de 3 mois. Les paramètres pharmacocinétiques individuels ont été estimés par approche Bayésienne empirique. Le modèle structurel permet l'analyse simultanée des données plasmatiques du nelfinavir et du M8. L'influence de l'âge et du poids sur les paramètres pharmacocinétiques estimés a été évaluée. Enfin, la dernière partie de notre travail concerne la détermination des paramètres pharmacocinétiques du mycophénolate mofétil chez des enfants et adolescents transplantés rénaux. Une stratégie avec deux prélèvements permet l'estimation de l'aire sous la courbe des concentrations plasmatiques d'acide mycophénolique sans biais et avec une bonne précision

The human immunodeficiency virus (HIV) can be divided into HIV-1 and HIV-2. The former can be divided into groups: M, N and O. Group M, which represents 90% of infections, is divided into several subtypes (A, B, C, D, F, G, H, J and K). It is known today that the most prevalent subtype in the world (and in Africa) is the subtype C, although the most studied is B (prevalent in the U.S. and Western Europe). Several stages the HIV-1 replicating cycle have been identified as a target for pharmacologic intervention. One of the main targets is the enzyme aspartyl protease (PR), which processes the viral polyproteins Gag and Gag-Pol. Its inhibition results in the formation of non-infectious virus particles. Currently 10 PR inhibitors are used in clinic. However, the emergence of resistance to these inhibitors leads to a therapeutic failure. Several mutated amino acid residues that are present in resistant isolates have been identified. One of such resistance mutations is the D30N, which confers primary resistance exclusively to nelfinavir, has been described in patients infected with subtype B. However, clinical and laboratory studies showed that virus of subtype C with the mutation D30N (CD30N) has low incidence in clinical and reduced adaptability in vitro. To try to understand these differences caused by mutation D30N in subtypes B and C, we studied the interaction of these PRs with the peptide KARVLAEAM (analogous to the natural substrate of cleavage between the protein the capsid (CA) and p2 of HIV-1) and with the inhibitor nelfinavir. We have also studied the PR CD30N with the compensatory mutations N83T or N88D, found in vitro and in vivo, respectively, which occur when the subtype C acquires the mutation D30N. This work aimed to study the molecular and atomic mechanisms of mutation D30N in the PR of subtypes B and C. The results showed that the inhibitor and backbone of models BD30N and CD30N/N83T possessed the greatest variation, with respect to the initial structure. Although the mutants CD30N and CD30N/N88T have not suffered similar variations, they showed, as well as the other two mutants, a reduction in the intensity of the h-bonds that occur between PR and inhibitor which are located near the catalytic and the flaps regions. Also, all mutants had reduced hydrophobic contacts between the receptor and the ligand. Some data indicated that the flap of one of the chains is highly immobile in a model CD30N suggesting the mutation D30N impairs the contact of flap with the substrate in subtype C. Also, the analysis of the PR structure interacting with the substrate, indicated that the CD30N mutant has one of its α-helix regions unstructured, which can be directly associated with substrate cleavage. Our work provides important insights in to the effect of D30N mutation in the PR structure of the subtype C, and on its interaction with the substrate and the inhibitor. These data confirm and explain, at least in part, the smaller incidence of the studied mutation in that genetic subtype of HIV-1.
O HIV pode ser dividido em HIV-1 e HIV-2. Aquele, por sua vez, pode ser divido nos grupos: M, N e O. O grupo M, que representa 90% das infecções, foi dividido em vários subtipos (A, B, C, D, F, G, H, J e K). Sabe-se hoje que o subtipo mais circulante no mundo (a maior parte na África) é o C, entretanto o mais estudado é o B (prevalente nos EUA e Europa). Diversas etapas do ciclo replicativo do HIV-1 têm sido identificadas como alvos para intervenção farmacológica. Um dos principais alvos é a enzima aspartil protease (PR); é ela que processa as poliproteínas virais Gag e Gag-Pol e sua inibição resulta na formação de partículas virais não infecciosas, sendo atualmente 10 inibidores utilizados em clínica. No entanto, o aparecimento de resistência a esses inibidores leva à falha terapêutica, tendo sido identificados e estudados vários resíduos que se apresentam mutados em isolados resistentes. Uma dessas mutações de resistência é a D30N, que consiste numa mutação primária de resistência exclusiva ao nelfinavir descrita em pacientes soropositivos infectados pelo subtipo B. Entretanto, observações clínicas e laboratoriais mostraram que vírus do subtipo C com a mutação D30N (CD30N) têm baixíssima ocorrência clínica e adaptabilidade reduzida in vitro. Para tentar entender as diferenças causadas pela mutação D30N nos subtipos B e C, foi estudada a interação da PR destes vírus com o peptídeo KARVLAEAM (análogo ao substrato natural de clivagem entre a proteína do capsídeo (CA) e a proteína p2 do HIV-1) e com o inibidor nelfinavir. Também foi estudada a PR CD30N com as mutações compensatórias N83T e N88D, encontradas in vitro e in vivo respectivamente, que se manifestam quando o subtipo C sofre a mutação D30N. Este trabalho teve como objetivo estudar os mecanismos moleculares e atômicos dos efeitos da mutação D30N na PR dos subtipos B e C. Os resultados mostram que o inibidor e o esqueleto peptídico dos modelos BD30N e CD30N/N83T sofreram as maiores variações, em relação à estrutura inicial. Embora os mutantes CD30N e CD30N/N88D não tenham sofrido variação semelhante, eles apresentaram, assim como os outros dois mutantes, uma redução na intensidade das ligações de hidrogênio que ocorrem entre a PR e o inibidor que estão localizadas próximas à região catalítica e aos flaps. Além disso, todos os mutantes apresentaram redução em seus contatos hidrofóbicos ocorridos na interação receptor/ligante. Alguns dados obtidos indicam que a alça de uma das cadeias é altamente imóvel no modelo CD30N sugerindo que a mutação D30N prejudica o contato do flap com o substrato no subtipo C. Além disso, a análise da estrutura das PRs, interagindo com o substrato, indicou que o mutante CD30N tem uma de suas regiões de α-hélice desestruturada, o que pode estar diretamente associado a não clivagem do substrato. O nosso trabalho provê importantes insights sobre o efeito da mutação D30N na estrutura da PR do subtipo C, bem como na sua interação com o substrato e com o inibidor. Tais dados corroboram e explicam, ao menos em parte, a menor ocorrência da mutação estudada naquele variante genético do HIV-1.

Made available in DSpace on 2015-03-04T18:51:06Z (GMT). No. of bitstreams: 1 Tese.pdf: 24640886 bytes, checksum: 4120e048629aa78cd493eb576212d8ca (MD5) Previous issue date: 2008-11-28
The human immunodeficiency virus (HIV) can be divided into HIV-1 and HIV-2. The former can be divided into groups: M, N and O. Group M, which represents 90% of infections, is divided into several subtypes (A, B, C, D, F, G, H, J and K). It is known today that the most prevalent subtype in the world (and in Africa) is the subtype C, although the most studied is B (prevalent in the U.S. and Western Europe). Several stages the HIV-1 replicating cycle have been identified as a target for pharmacologic intervention. One of the main targets is the enzyme aspartyl protease (PR), which processes the viral polyproteins Gag and Gag-Pol. Its inhibition results in the formation of non-infectious virus particles. Currently 10 PR inhibitors are used in clinic. However, the emergence of resistance to these inhibitors leads to a therapeutic failure. Several mutated amino acid residues that are present in resistant isolates have been identified. One of such resistance mutations is the D30N, which confers primary resistance exclusively to nelfinavir, has been described in patients infected with subtype B. However, clinical and laboratory studies showed that virus of subtype C with the mutation D30N (CD30N) has low incidence in clinical and reduced adaptability in vitro. To try to understand these differences caused by mutation D30N in subtypes B and C, we studied the interaction of these PRs with the peptide KARVLAEAM (analogous to the natural substrate of cleavage between the protein the capsid (CA) and p2 of HIV-1) and with the inhibitor nelfinavir. We have also studied the PR CD30N with the compensatory mutations N83T or N88D, found in vitro and in vivo, respectively, which occur when the subtype C acquires the mutation D30N. This work aimed to study the molecular and atomic mechanisms of mutation D30N in the PR of subtypes B and C. The results showed that the inhibitor and backbone of models BD30N and CD30N/N83T possessed the greatest variation, with respect to the initial structure. Although the mutants CD30N and CD30N/N88T have not suffered similar variations, they showed, as well as the other two mutants, a reduction in the intensity of the h-bonds that occur between PR and inhibitor which are located near the catalytic and the flaps regions. Also, all mutants had reduced hydrophobic contacts between the receptor and the ligand. Some data indicated that the flap of one of the chains is highly immobile in a model CD30N suggesting the mutation D30N impairs the contact of flap with the substrate in subtype C. Also, the analysis of the PR structure interacting with the substrate, indicated that the CD30N mutant has one of its α-helix regions unstructured, which can be directly associated with substrate cleavage. Our work provides important insights in to the effect of D30N mutation in the PR structure of the subtype C, and on its interaction with the substrate and the inhibitor. These data confirm and explain, at least in part, the smaller incidence of the studied mutation in that genetic subtype of HIV-1.
O HIV pode ser dividido em HIV-1 e HIV-2. Aquele, por sua vez, pode ser divido nos grupos: M, N e O. O grupo M, que representa 90% das infecções, foi dividido em vários subtipos (A, B, C, D, F, G, H, J e K). Sabe-se hoje que o subtipo mais circulante no mundo (a maior parte na África) é o C, entretanto o mais estudado é o B (prevalente nos EUA e Europa). Diversas etapas do ciclo replicativo do HIV-1 têm sido identificadas como alvos para intervenção farmacológica. Um dos principais alvos é a enzima aspartil protease (PR); é ela que processa as poliproteínas virais Gag e Gag-Pol e sua inibição resulta na formação de partículas virais não infecciosas, sendo atualmente 10 inibidores utilizados em clínica. No entanto, o aparecimento de resistência a esses inibidores leva à falha terapêutica, tendo sido identificados e estudados vários resíduos que se apresentam mutados em isolados resistentes. Uma dessas mutações de resistência é a D30N, que consiste numa mutação primária de resistência exclusiva ao nelfinavir descrita em pacientes soropositivos infectados pelo subtipo B. Entretanto, observações clínicas e laboratoriais mostraram que vírus do subtipo C com a mutação D30N (CD30N) têm baixíssima ocorrência clínica e adaptabilidade reduzida in vitro. Para tentar entender as diferenças causadas pela mutação D30N nos subtipos B e C, foi estudada a interação da PR destes vírus com o peptídeo KARVLAEAM (análogo ao substrato natural de clivagem entre a proteína do capsídeo (CA) e a proteína p2 do HIV-1) e com o inibidor nelfinavir. Também foi estudada a PR CD30N com as mutações compensatórias N83T e N88D, encontradas in vitro e in vivo respectivamente, que se manifestam quando o subtipo C sofre a mutação D30N. Este trabalho teve como objetivo estudar os mecanismos moleculares e atômicos dos efeitos da mutação D30N na PR dos subtipos B e C. Os resultados mostram que o inibidor e o esqueleto peptídico dos modelos BD30N e CD30N/N83T sofreram as maiores variações, em relação à estrutura inicial. Embora os mutantes CD30N e CD30N/N88D não tenham sofrido variação semelhante, eles apresentaram, assim como os outros dois mutantes, uma redução na intensidade das ligações de hidrogênio que ocorrem entre a PR e o inibidor que estão localizadas próximas à região catalítica e aos flaps. Além disso, todos os mutantes apresentaram redução em seus contatos hidrofóbicos ocorridos na interação receptor/ligante. Alguns dados obtidos indicam que a alça de uma das cadeias é altamente imóvel no modelo CD30N sugerindo que a mutação D30N prejudica o contato do flap com o substrato no subtipo C. Além disso, a análise da estrutura das PRs, interagindo com o substrato, indicou que o mutante CD30N tem uma de suas regiões de α-hélice desestruturada, o que pode estar diretamente associado a não clivagem do substrato. O nosso trabalho provê importantes insights sobre o efeito da mutação D30N na estrutura da PR do subtipo C, bem como na sua interação com o substrato e com o inibidor. Tais dados corroboram e explicam, ao menos em parte, a menor ocorrência da mutação estudada naquele variante genético do HIV-1.

L’apoptose est un événèment clef dans le maintien de l’intégrité de l’organisme et peut être impliqué dans des pathologies humaines. La compréhension des mécanismes de contrôle et l’identification de nouveaux régulateurs peut être essentiel au developpemment de nouvelle statégies thérapeutiques. L’objectif de cette thèse a été d’identifier de nouveaux régulateurs de l’étape mitochondriale de l’apoptose. Trois axes de recherche ont été explorés :(i) L’etude de la cytotoxicité de Bax chez la levure. La surexpression de la protéine pro-apoptotique Bax induit la mort des cellules et une perméabilisation de la membrane externe mitochondriale (PMM). Une approche génétique chez la levure a permis de mettre en évidence l’implication du processus de l’autophagie dans la cytotoxicité induite par Bax. (ii) L’identification de la cible cellulaire du nelfinavir (NFV). Cet inhibiteur de la protéase du virus de l’immunodéficience humaine (VIH) présente également un effet anti-apoptotique. L’approche in cellulo et biochimique a permis d’identifier la cible du NFV comme étant le translocateur à adénine, un des constituants du pore de transition de perméabilité. (iii) L’étude de la fonction pro-poptotique de la glycéraldéhyde 3 phosphate deshydrogenase (GAPDH). La GAPDH est une protéine glycolitique également décrite pour intervenir dans l’apoptose. Dans ce contexte, nous avons montré qu’une augmentation de la GAPDH dans les mitochondries pouvait induire une activation de la PMM en intéragissant avec la porine VDAC. L’utilisation de ces différentes approches a permis de caractériser des régulateurs exogènes et endogènes de la phase mitochondriale de l’apoptose
Apoptosis is a key event for the organism intergrity throughout life. This process is frequently implied in human diseases. Understanding of the mechanisms involved in apoptosis control and characterization of new regulators are essential for development of therapeutic strategies. The objectives of my thesis deal with identification of regulators of the mitochondrial step of apoptosis. Three research orientations have been explored: (i) Study of Bax cytotoxicity in yeast model. Surexpression of the pro-apoptotic protein Bax induces cell death and itochondrial membranes permeabilization. This genetic approach in yeast highlighted the involvment of autophagy pathway in Bax’s death. (ii) Identification of nelfinavir (NFV) cellular target. This protease inhibitor of the Human Immunodeficiency Virus also presents an antiapoptotic effect, whose regulation can be a key role in apoptosis control in HIV syndrome. The biochemical and in cellulo approach we developed lead to identification of the Adenine Nucleotide Translocator (ANT), one of the components of permeability transition pore, as a target of NFV. (iii) investigation of proapoptotic function of glyceraldehyde 3 phosphate deshydrogenase (GAPDH). GAPDH is a well known protein, involved into glycolysis, but can also play a role into apoptosis. In this context, we have shown that an increase in the mitochondrial GAPDH induces the activation of the mitochondrial permeability transition, through its interaction with the porine VDAC. Using different but complementary approaches during this work allowed us to characterize one exogenous (NFV) and two endogenous (Bax, GAPDH) regulators of mitochondrial step of cell death

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-09T19:23:03Z No. of bitstreams: 1 carinadealmeidabastos.pdf: 1640331 bytes, checksum: 96c6f61dba624b337759b27baecc7a34 (MD5)
Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T14:34:40Z (GMT) No. of bitstreams: 1 carinadealmeidabastos.pdf: 1640331 bytes, checksum: 96c6f61dba624b337759b27baecc7a34 (MD5)
Made available in DSpace on 2017-05-17T14:34:40Z (GMT). No. of bitstreams: 1 carinadealmeidabastos.pdf: 1640331 bytes, checksum: 96c6f61dba624b337759b27baecc7a34 (MD5) Previous issue date: 2015-03-20
CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Um método cromatográfico eletrocinético micelar para a determinação simultânea do mesilato de nelfinavir e das impurezas ácido 3-hidroxi-2-metilbenzóico e benzoato de (2R,3R)-4-((3S,4aS,8aS)-3-(terc-butilcarbamoil)octahidroisoquinolina-2(1H)-il)-3-hidroxi-1-(feniltio)butano-2-amônio, com tempo de análise de 25 minutos, foi proposto. O eletrólito composto por tampão tetraborato de sódio (pH 9,24; 25 mmol L−1), dodecil sulfato de sódio (9 mmol L−1) e metanol (10%, v/v) foi otimizado utilizando planejamento fatorial misto, com detecção direta em 200 nm. Após avaliação das figuras de mérito seletividade, linearidade, precisão, limite de detecção, limite de quantificação, exatidão e robustez (Teste de Youden), o método foi aplicado na análise do mesilato de nelfinavir e suas impurezas em uma formulação farmacêutica (comprimidos). O método otimizado pode ser útil na determinação desses analitos em processos de monitoramento de síntese, matérias-primas e formulações farmacêuticas, oferecendo como vantagens baixo consumo de solventes, pequena demanda de amostra e uso de colunas não específicas.
A methodology for the simultaneous determination of nelfinavir mesylate and the impurities 3-hydroxy-2-methylbenzoic acid and (2R,3R)-4-((3S,4aS,8aS)-3-(tert-butylcarbamoyl) octahydroisoquinolin-2(1H)-yl)-3-hydroxy-1-(phenylthio)butan-2-aminium benzoate by micellar electrokinetic chromatography, with an analysis time of 25 min, was proposed. An electrolyte composed of sodium tetraborate buffer (pH 9.24; 25 mmol L−1), sodium dodecyl sulphate (9 mmol L−1) and methanol (10%, v/v) was optimized using a mixed-level factorial design, with direct detection at 200 nm. After evaluating some figures of merit, such as selectivity, linearity, precision, limit of detection, limit of quantification, accuracy and robustness (Youden’s test), the method was successfully applied to the analysis of nelfinavir mesylate and its impurities in a pharmaceutical formulation (tablets). The optimized methodology is demonstrated to be useful in the determination of these analytes in a synthesis monitoring process, in raw materials and in pharmaceutical formulations, while offering low solvent consumption, requiring a small sample and using non-specific columns as advantages.

The genetic differences and polymorphisms between HIV-1 subtype B and non-B subtypes have been well documented. Classically, however, antiretrovirals (ARVs), including protease inhibitors (PIs), have been designed based on structural and functional information obtained utilizing subtype B HIV-1. With the advent of antiretroviral therapy (ART) in developing countries and the emergence of non-B infections in developed countries, the impact of these polymorphisms must be evaluated in terms of ART efficacy.
The 36th amino acid in the viral protease (PR) of B subtypes is Methionine (M), while non-B subtypes code for Isoleucine (I). I at position 36 is associated with PI resistance in subtype B HIV-1; therefore, we sought to investigate the effect of this single nucleotide polymorphism on emergence of resistance mutations and PI susceptibility in various HIV-1 subtypes in vitro. Our results indicate that the effect of this single nucleotide polymorphism appears to be subtype specific and PI specific.
Les différences génétiques (polymorphismes) entre les différents sous-types du VIH-1, soit B et non-B, sont bien documentées. Toutefois, les antirétroviraux incluant les inhibiteurs de la protéase, ont été conçus de façon structurelle et fonctionnelle en utilisant les informations obtenues à partir du sous-type B. L'impact des polymorphismes des différents sous-types du VIH-1 sur l'efficacité des antirétroviraux doit être évalué dû à l'émergence des infections de sous-types non-B dans les pays développés ainsi qu'avec l'arrivée de la thérapie antirétrovirale dans les pays en voie de développement.
Le 36e acide aminé de la protéase virale de sous-type B du VIH-1 est une méthionine. Cependant, cet acide aminé est remplacé par une isoleucine dans les sous-types non-B. La présence d'une isoleucine à la position 36 entraîne une résistance du VIH-1 sous-type B pour les inhibiteurs de la protéase. Nous avons examiné l'effet de ce polymorphisme sur les mutations de résistance dans les différents sous-types de VIH in vitro. Nos résultats indiquent que l'effet de ce polymorphisme serait différent selon le sous-type.

Hintergrund Die Strahlentherapie ist neben der radikalen Prostatektomie eine Standardtherapie zur Behandlung von Prostatatumoren und führt zu sehr guten Ergebnissen für die lokale Tumorkontrolle und für das Überleben. Allerdings ist, wie bei der Operation auch, dabei das Risiko eines Rezidivs für fortgeschrittene Tumoren im Gegensatz zu Tumoren in früheren Stadien relativ hoch. Daher besteht eine hohe Dringlichkeit zur Verbesserung der Strahlentherapie vor allem bei fortgeschrittenen Tumoren. Ein Ansatz hierfür ist die Kombination der Bestrahlung mit molekularen Therapien. Ziel dabei ist es, bestimmte Zielproteine zu blockieren, um die Strahlensensibilität der Prostatakarzinomzellen zu erhöhen. Ein potentielles Target könnte hierbei die Blockade des VEGF-C/NRP-2/Akt-Signalwegs (VEGF-C – vascular endothelial growth factor C; NRP-2 – Neuropilin 2; Akt – Proteinkinase B) sein. Im Prostatakarzinom sind die Konzentrationen von VEGF-C und NRP-2 im Vergleich zu normalen Prostatazellen erhöht. Aus Untersuchungen ist bekannt, dass beide Proteine eine progressive Wirkung auf die Tumorgenese haben. In Vorarbeiten zeigen Muders et al. (2009) zudem eine Aktivierung von Akt über die VEGF-C/NRP-2-Achse und eine darüber vermittelte Resistenz gegenüber oxidativem Stress durch H2O2. Akt wirkt in verschiedenen Tumorentitäten außerdem protektiv gegenüber Bestrahlung. Es besteht die Annahme, dass dies auch für Prostatakarzinomzellen gilt. Zielstellung Im Rahmen dieser Arbeit wurde untersucht, ob und über welchen Mechanismus VEGF-C, NRP-2 und Akt die Strahlenresistenz in Prostatakarzinomzelllinien beeinflussen. Methoden Es wurden in vitro- und in vivo-Experimente in den humanen Prostatakarzinomzelllinen PC-3, DU145, LNCaP sowie in PC-3-Xenografts durchgeführt. Der Einfluss von VEGF-C und NRP-2 auf die Strahlenresistenz wurde in vitro nach Herunterregulierung der entsprechenden Gene mittels siRNA beziehungsweise nach Supplementierung mit humanem rekombinanten VEGF-C in Koloniebildungsassays untersucht. Zur Ermittlung des Einflusses von VEGF-C und von NRP-2 auf mögliche Zellüberlebensmechanismen wurden der autophagische Flux nach Blockade der Autophagie mit Bafilomycin A1 mittels Western Blot, die DNA-Doppelstrangbruch-Reparatur mittels Quantifizierung der γH2AX Foci sowie die Zellzyklusverteilung mittels Durchflusszytometrie untersucht. Die Signalweiterleitung von VEGF-C über Akt sowie, als weitere Möglichkeit, die Signalweiterleitung über ERK1/2 wurden nach siRNA-Transfektion mit und ohne Bestrahlung mittels Western Blot geprüft. Weitere Versuche zu Akt erfolgten in vitro und in vivo mit dem PI3K/Akt-Inhibitor Nelfinavir in PC-3-Zellen. Der in vitro Effekt von Nelfinavir auf die Strahlenresistenz wurde dabei mithilfe eines Koloniebildungsassays nach Behandlung der Zellen mit 10 µM Nelfinavir getestet. In vivo wurde die Wirkung von Nelfinavir ohne sowie in Kombination mit Bestrahlung in PC-3-Xenografts in Nacktmäusen untersucht. Für die Bestimmung der Tumorwachstumszeit wurden die Mäuse mit 80 mg Nelfinavir/kg Körpergewicht 30 mal innerhalb von 6 Wochen behandelt. In einem weiteren Versuch wurde die lokale Tumorkontrolle bei gleichzeitiger fraktionierter Bestrahlung mit Gesamtdosen von 30 bis 120 Gy und einer Nachbeobachtungszeit von 180 Tagen bestimmt. Ergebnisse Die Untersuchungen zur Strahlenresistenz über den VEGF-C/NRP-2/Akt-Signalweg haben ergeben, dass in den drei Prostatakarzinomzelllinien PC-3, DU145 und LNCaP VEGF-C signifikant Strahlenresistenz vermittelt. Für NRP-2 hingegen wurde festgestellt, dass es in Abhängigkeit von der Zelllinie entweder zur Strahlenresistenz (DU145) oder zur Strahlensensibilisierung (PC-3) führt. Weiterhin wurde nachgewiesen, dass durch VEGF-C in PC-3 und DU145 weder über Akt noch über ERK1/2 Strahlenresistenz vermittelt wird. Die Versuche zu Strahlenresistenz vermittelnden Mechanismen ergaben, dass VEGF-C in unbestrahlten PC-3-Zellen die Autophagie fördert, NRP-2 jedoch nicht. Unter Bestrahlung war ein Effekt von VEGF-C und NRP-2 auf die Autophagie nicht reproduzierbar nachweisbar. Ein weiterer Versuch hat gezeigt, dass in PC-3 Autophagie keinen Einfluss auf das klonogene Überleben nach Bestrahlung hat. Außerdem wurde festgestellt, dass VEGF-C in PC-3 die DNA-Doppelstrangbruch-Reparatur nicht beeinflusst. Darüber hinaus wurde nachgewiesen, dass eine Verminderung des VEGF-C-Gehalts in PC-3 zum G2/M-Arrest führt. In DU145 konnte jedoch kein Effekt beobachtet werden. In den Untersuchungen zum Einfluss von Akt auf die Strahlenresistenz unabhängig von VEGF-C und NRP-2 wirkte Nelfinavir inhibierend auf die Akt-Phosphorylierung am Ser473 und beeinflusste das klonogene Überleben von PC-3-Zellen minimal. In PC-3-Xenografts führte Nelfinavir zu keiner Tumorwachstumsverzögerung und wirkte in vitro und in vivo nicht strahlensensibilisierend. Schlussfolgerung In den Versuchen konnte gezeigt werden, dass VEGF-C in Prostatakarzinomzellen Strahlenresistenz vermittelt. Diese Erkenntnis könnte als ein Forschungsansatz zur Entwicklung einer kombinierten Therapie aus VEGF-C-Blockade und Bestrahlung dienen. Ein potentieller Mechanismus, über den VEGF-C die Strahlenresistenz vermittelt, ist, in Abhängigkeit von der Zelllinie, die Aufhebung des G2/M-Arrests. NRP-2 wirkt in der Vermittlung von Strahlenresistenz beziehungsweise sensibilität je nach Zelllinie unterschiedlich. Hierzu sollten weitere Untersuchungen bezüglich möglicher Interaktionen innerhalb anderer Signalwege mit strahlensensibilisierendem Einfluss erfolgen. Innerhalb des untersuchten Signalwegs konnte weiterhin festgestellt werden, dass VEGF-C Strahlenresistenz nicht über Akt vermittelt. Die vorliegende Arbeit enthält die erste Studie sowohl zur Untersuchung des Einflusses von Nelfinavir in Kombination mit Bestrahlung auf das Überleben von Prostatakarzinomzellen in vitro als auch auf die Tumorwachstumszeit und die lokale Tumorkontrolle in vivo. Hierin konnte keine strahlensensibilisierende Wirkung von Nelfinavir nachgewiesen werden. Da Nelfinavir in Zellen anderer Tumorentitäten strahlensensibilisierend wirkt und außerdem bekannt ist, dass es in eine Reihe von Signalwegen eingreift, die das Zellüberleben fördern oder hemmen, sollte weiter geklärt werden, ob Tumorzellen mit einem bestimmten genetischen Profil besser auf die Behandlung mit Nelfinavir ansprechen
Background In addition to radical prostatectomy, radiotherapy is a standard therapy for the treatment of prostate tumours and leads to good results for local tumour control and survival. However, as with the resection, the risk of recurrence for advanced tumours is relatively high compared to tumours in earlier stages. Therefore, there is a high urgency to improve radiotherapy especially for advanced stages. One approach is the combination of irradiation with molecular therapies. The aim is to block certain target proteins to increase the radiosensitivity of the prostate carcinoma cells. A potential target could be the blockade of the VEGF-C/NRP-2/Akt signalling pathway (VEGF-C – vascular endothelial growth factor C; NRP-2 – neuropilin 2; Akt – protein kinase B). In prostate cancer the concentrations of VEGF-C and NRP-2 are increased compared to normal prostate cells. Studies have shown that both proteins have a progressive effect on tumourigenesis. In preliminary work Muders et al. (2009) also showed the activation of Akt via the VEGF-C/NRP-2 axis and a resistance to H2O2 induced oxidative stress. Akt also has a protective effect against irradiation in various tumour entities. It is assumed that this also applies to prostate carcinoma cells. Aim of the study Within the framework of this thesis, it was investigated whether and via which mechanism VEGF-C, NRP-2, and Akt affect the radioresistance in prostate carcinoma cell lines. Methods In vitro and in vivo experiments were performed in the human prostate carcinoma cell lines PC-3, DU145, LNCaP, as well as in PC-3 xenografts. The influence of VEGF-C and NRP-2 on the radioresistance was examined in vitro after knock down of the corresponding genes using siRNA or after supplementation with human recombinant VEGF-C in colony formation assays. In order to determine the influence of VEGF-C and NRP-2 on possible cell survival mechanisms, the autophagic flux was examined after the blockade of autophagy with bafilomycin A1 using western blot, the DNA double strand break repair by quantification of the γH2AX foci, and the cell cycle distribution by flow cytometry. The signal transduction of VEGF-C via Akt as well as, as a further possibility, the signal transduction via ERK1/2 were tested after siRNA transfection with and without irradiation using western blot. Further experiments on Akt were performed in vitro and in vivo with the PI3K/Akt inhibitor nelfinavir in PC-3 cells. The in vitro effect of nelfinavir on radioresistance was tested using a colony formation assay after treatment of the cells with 10 μM nelfinavir. In vivo, the effect of nelfinavir without and in combination with irradiation in PC-3 xenografts was investigated in nude mice. For the determination of the tumour growth time, the mice were treated with 80 mg nelfinavir/kg body weight 30 times within 6 weeks. In a further experiment, the local tumour control was determined with simultaneous fractionated irradiation with total doses of 30 to 120 Gy and a follow-up time of 180 days. Results The investigations on radioresistance via the VEGF-C/NRP-2/Akt signalling pathway showed that in the three prostate carcinoma cell lines PC-3, DU145, and LNCaP VEGF-C significantly mediates radioresistance. For NRP-2 however, it was found that, depending on the cell line, it either leads to radioresistance (DU145) or radiosensitization (PC-3). Further, it was shown that in PC-3 and DU145 VEGF-C does not mediate radioresistance via Akt or ERK1/2. The experiments on radioresistance mediating mechanisms revealed that VEGF-C promotes autophagy in untreated PC-3 cells, but NRP-2 does not. Under irradiation, an effect of VEGF-C and NRP-2 on autophagy could not be detected reproducibly. A further experiment has shown that in PC-3 autophagy has no influence on the clonogenic survival after irradiation. In addition, it was found that VEGF-C does not affect the DNA double strand break repair in PC-3. Furthermore, it was shown that a reduction in the VEGF-C content leads to a G2/M arrest in PC-3. However, no effect could be observed in DU145. In studies regarding the influence of Akt on radioresistance independent of VEGF-C and NRP-2, nelfinavir inhibited Akt phosphorylation at Ser473 and minimally affected the clonogenic survival of PC-3 cells. In PC-3 xenografts, nelfinavir did not lead to any tumour growth delay and did not have a radiosensitizing effect in vitro or in vivo. Conclusion In the experiments, it was shown that VEGF-C mediates radioresistance in prostate cancer cells. This finding could serve as a research approach for the development of a combined therapy of a VEGF-C blockade and irradiation. A potential mechanism by which VEGF-C mediates radioresistance is the reverse of the G2/M arrest, depending on the cell line. NRP-2 acts differently in the mediation of radioresistance or radiosensitivity, depending on the cell line. On this, further investigations should be carried out with regard to possible interactions within other signalling pathways with a radiosensitizing influence. Within the investigated signalling pathway, it was further shown that VEGF-C does not mediate radioresistance via Akt. The present work contains the first study examining the effect of nelfinavir in combination with irradiation on prostate cancer cell survival in vitro as well as on growth time and local tumour control in vivo. Herein no radiosensitizing effects of nelfinavir could be detected. Since nelfinavir radiosensitizes cells of other tumour entities and is also known to interfere with a series of signalling pathways that promote or inhibit cell survival, it should be clarified whether tumour cells with a particular genetic profile are more responsive to treatment with nelfinavir

En cas d’infection à VIH, le traitement doit être poursuivi en cours de grossesse pour la santé de la patiente et éviter la transmission mère-enfant du virus, mais il apparaît nécessaire d’évaluer son impact potentiel sur le fœtus. L’utilisation du modèle du cotylédon perfusé a permis d’évaluer le passage transplacentaire de plusieurs antirétroviraux et déterminé l’intérêt et les limites de la méthode. Certains transporteurs actifs placentaires sont capables d’effluer certains médicaments, limitant ainsi l’exposition fœtale à des molécules potentiellement toxiques. Nous avons montré qu’il existait une variabilité interethnique des polymorphismes génétiques des gènes ABCB1 et ABCG2, et de l’expression placentaire de la P-glycoprotéine (ABCB1). L’étude de l’impact des antirétroviraux seuls ou en association sur l’expression et la fonctionnalité du transporteur d’efflux BCRP (ABCG2) sur un modèle de cellules BeWo a permis d’évaluer leurs conséquences placentaires potentielles
In case of HIV infection, treatment must be continued during pregnancy for mother’s health or prevention of mother-to-child transmission, but it is necessary to evaluate the potential impact on the foetus. The model of perfused cotyledon has been used to evaluate the placental of several antiretrovirals and we determined the interest and the limits of the method. Some placental ATP-dependent drug efflux pumps can limit fetal exposition to potential toxic drugs. We have showed a interethnic variability for genetic polymorphisms of ABCB1 and ABCG2, and for placental expression of P-glycoprotein (ABCB1). The study of impact of antiretrovirals, alone or in association, on expression and functionality of the transporter BCRP (ABCG2) with BeWo model has allowed to evaluate their potential placental consequences

Background: Systemic chemotherapy and radiotherapy play an important role in the treatment of colorectal cancer. Tumour perfusion and oxygenation is known to influence radiosensitivity and chemosensitivity. In this thesis, I propose that the evaluation of changes in tumour perfusion using perfusion CT (pCT) and dynamic contrast-enhanced (Dce) MRI can guide the rational sequencing of drugs and radiation. Methods: Dce-MRI and pCT scans were incorporated into a clinical trial of hypofractionated pelvic radiotherapy and nelfinavir in 10 patients with rectal cancer. Toxicity and tissue biomarkers (tumour cell density, microvessel density, CAIX, HIF1-alpha, phospho-Akt and phospho-PRAS40) were evaluated. pCT liver scans were incorporated into an imaging study in patients with colorectal liver metastases randomised to receive either oxaliplatin/ 5FU chemotherapy or oxaliplatin/ 5FU chemotherapy plus selective internal radiotherapy. Results: After 7 days of nelfinavir concurrent with hypo-fractionated pelvic radiotherapy, there was a mean 42% increase in median K^trans (P=0.03, paired t test) on Dce-MRI and a median 30% increase in mean blood flow on pCT (P=0.028, Wilcoxon Rank Sum), although no statistically significant changes in perfusion parameters were demonstrated after 7 days of nelfinavir prior to radiotherapy. The feasibility of evaluating tumour cell density in rectal biopsies before and after radiotherapy and a radiosensitising drug as an early endpoint of response was demonstrated. In patients with colorectal liver metastases who received oxaliplatin and modified de Gramont chemotherapy alone, after 4 cycles of chemotherapy, a 28% decrease in the mean hepatic arterial fraction was observed (P=0.018, paired t test). Between pCT scans 2 days before SIRT and 39-47 days following SIRT and continued 2-weekly chemotherapy, there was a mean 62% (P=0.009) reduction in Blood Flow and 61% (P=0.006) reduction in Blood Volume (paired t test). Conclusions This research does not support the hypothesis that nelfinavir before radiotherapy improves blood flow to human rectal cancer. Increases in rectal tumour perfusion during radiotherapy and concurrent nelfinavir are likely to be primarily explained by the acute biological effects of radiation. Four or more cycles of oxaliplatin and modified de Gramont chemotherapy may result in changes in tumour perfusion of colorectal liver metastases which would be detrimental to subsequent radiotherapy. Selective internal radiotherapy resulted in substantial reductions in tumour perfusion 39-47 days after the treatment. Perfusion imaging can be used to detect changes in tumour perfusion in response to radiotherapy and systemic therapy which have implications for the sequencing of therapies.

acase@tulane.edu
Objectives Human Immunodeficiency Virus (HIV) infects immune cells and lowers cell-mediated immunity leading to acquired immune deficiency syndrome or AIDS. The virus causes damage/dysfunction of helper T cells, macrophages, and dendritic cells. One of the long-term complications of untreated AIDS is severe cognitive impairment caused by HIV-associated dementia (HAD). Highly Active Antiretroviral therapy, the HAART regimen, which inhibits virus replication has been shown to reduce the incidence of HAD. HAART includes several mechanistically diverse classes of drugs such as protease inhibitors, nucleoside inhibitors of reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors. The protease inhibitor, nelfinavir, is used in HIV therapy in order to mitigate the effects of the HIV virus by breaking down HIV-1 and HIV-2 proteases, which are essential to the replication of the virus within the host cell. HAART has decreased the incidence of HAD yet milder cognitive dysfunction, considered to be a consequence of anti-HIV drug toxicity, often manifests. Previous studies have shown that protease inhibitors may play a role in causing oxidative stress in endothelial cells. The present study involves understanding the effect of nelfinavir on mitochondrial oxidative stress and its role in the injury to human brain microvascular endothelial cells that form the blood-brain barrier. Methods and Results Our studies utilized primary human brain microvascular endothelial cells (hBMECs). We performed measurements of mitochondrial superoxide levels (ESR Spectrscopy), oxygen consumption rates or OCR (Seahorse XFe Extracellular Flux Analyzer and MitoStress Test Assay), cell viability/proliferation (CCK8 based Cellular Viability Assay). Sub-therapeutic doses of nelfinavir (1 µmol/L) increased the cell proliferation whereas therapeutic (3-5 µmol/L) and supra-therapeutic (10 µmol/L) doses of nelfinavir reduced the cell viability. In addition, treatment with sub-therapeutic levels of nelfinavir has no effect on the levels of mitochondrial superoxide in hBMECs but therapeutic and supra-therapeutic levels of nelfinavir increased mitochondrial superoxide levels. Measurements of OCR showed that sub-therapeutic doses of nelfinavir enhanced the basal and maximal respiration in hBMECs. In contrast, therapeutic concentration of nelfinavir reduced ATP production and spare respiratory capacity although basal respiration, proton leak, and non-mitochondrial respiration were unchanged. However, supra-therapeutic dose of nelfinavir significantly reduced basal respiration, ATP production, and spare respiratory capacity accompanied by reduced non-mitochondrial respiration and proton leak. Conclusions We identified that nelfinavir treatment was associated with a decrease in cellular proliferation at therapeutic and supra-therapeutic levels. Furthermore, we identified an increase in mitochondrial superoxide species in cells treated with nelfinavir in concentrations beyond therapeutic levels which was accompanied by a decrease in basal respiration, ATP production, and mitochondrial spare capacity. These results are indicative of nelfinavir causing cellular cytotoxicity in BMECs that are likely mediated by mitochondrial oxidative stress and impaired mitochondrial respiration.
1
Gowthamram Rajaprabhakaran