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

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.

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.

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. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

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.

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 &#945;-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 &#945;-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.

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.

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.

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