Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 161-195.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.

Angiotensin-converting enzyme (ACE) inhibitors are some of the most commonly prescribed medications for hypertension. They are cited in many papers as the treatment most often recommended by guidelines and favoured over other antihypertensive drugs as first-line agents especially when other high-risk conditions are present, such as diabetic nephropathy. The development of captopril (CPT) was amongst the earliest successes of the revolutionary concept of structure-based drug design. Due to its relatively poor pharmacokinetic profile or short half-life of about 1 hour, the formulation of sustained-release microcapsule dosage form is useful to improve patient compliance and to achieve predictable and optimized therapeutic plasma concentrations. Currently, CPT is mainly administered in tablet form. One of the difficulties of CPT formulation has been reported to be its instability in aqueous solutions. CPT is characterized by a lack of a strong chromophore and, therefore, not able to absorb at the more useful UV–Vis region of the spectrum. For this reason, an accurate, simple, reproducible, and sensitive HPLC-ECD method was developed and validated for the determination of CPT in dosage forms. The method was successfully applied for the determination of CPT in commercial and developed formulations. Possible drug-excipient and excipient-excipient interactions were investigated prior to formulating CPT microcapsules because successful formulation of a stable and effective solid dosage form depends on careful selection of excipients. Nuclear magnetic resonance spectroscopy, Fourier transform infra-red spectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used for the identification and purity testing of CPT and excipients. The studies revealed no thermal changes during stress testing of binary and whole mixtures which indicate absence of solid state interactions. There were no shifts, appearance and disappearance in the endothermic or exothermic peaks and on the change of other associated enthalpy values on thermal curves obtained with DSC method. Characteristic peaks for common functional groups in the FT-IR were present in all the mixtures indicating the absence of incompatibility. The techniques used in this study can be said to have been efficient in the characterization and evaluation of the drug and excipients. The technique of microencapsulation by oil-in-oil was used to prepare CPT microcapsules. The effects of polymer molecular weight, homogenizing speed on the particle size, flow properties, morphology, surface properties and release characteristics of the prepared CPT microcapsules were examined. In order to decrease the complexity of the analysis and reduce cost response surface methodology using best polynomial equations was successfully used to quantify the effect of the formulation variables and develop an optimized formulation thereby minimizing the number of experimental trials. There was a burst effect during the first stage of dissolution. Scanning electron microscopy (SEM) results indicated that the initial burst effect observed in drug release could be attributed to dissolution of CPT crystals present at the surface or embedded in the superficial layer of the matrix. During the preparation of microcapsules, the drug might have been trapped near the surface of the microcapsules and or might have diffused quickly through the porous surface. The release kinetics of CPT from most formulations followed Fickian diffusion mechanism. SEM photographs showed that diffusion took place through pores at the surface of the microcapsules. The Kopcha model diffusion and erosion terms showed predominance of diffusion relative to swelling or erosion throughout the entire test period. Drug release mechanism was also confirmed by Makoid-Banakar and Korsmeyer-Peppas models exponents which further support diffusion release mechanism in most formulations. The models postulate that the total of drug release is a summation of a couple of mechanisms; burst release, relaxation induced controlled-release and diffusional release. Inspection of the 2D contour and 3D response surfaces allowed the determination of the geometrical nature of the surfaces and further providing results about the interaction of the different variables used in central composite design (CCD). The wide variation indicated that the factor combinations resulted in different drug release rates. Lagrange, canonical and mathematical modelling were used to determine the nature of the stationery point of the models. This represented the optimal variables or stationery points where there is interaction in the experimental space. It is difficult to understand the shape of a fitted response by mere inspection of the algebraic polynomial when there are many independent variables in the model. Canonical and Lagrange analyses facilitated the interpretation of the surface plots after a mathematical transformation of the original variables into new variables. In conclusion, these results suggest the potential application of Eudragit® / Methocel® microcapsules as suitable sustained-release drug delivery system for CPT.

Orientador: Prof. Dr. Luciano Puzer / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017. / As calicreinas 5 (KLK5) e 7 (KLK7) sao serino proteases que pertencem a um grupo de 15 calicrei.nas teciduais humanas (KLKs) encontradas em diversos tecidos do corpo. A KLK5 e encontrada principalmente no tecido mamario, sistema nervoso central, testiculos, prostata e traqueia, enquanto a KLK7 e encontrada no esofago, figado, rins e glandulas salivares. Alem disso, ambas as enzimas sao mais abundantemente expressas na pele humana, onde acredita-se que exercam importante papel no processo de descamacao epidermal, a partir da hidrolise de proteinas que compoem as estruturas de adesao intercelular dos corneodesmossomos. Estudos tambem demonstram, que a atividade dessas duas enzimas aparece aumentada em patologias relacionadas ao processo de descamacao da pele, como psoriase e dermatite atopica. Desta forma, o desenvolvimento de inibidores para KLK5 e KLK7 podera contribuir nao apenas para elucidar seus mecanismos fisiologicos e patologicos, mas como um arquetipo de novas drogas, visando ao desenvolvimento de novos procedimentos terapeuticos para patologias relacionadas ao processo de descamacao epidermal. Neste projeto, foram testados 17 pseudopeptideos e 10 compostos derivados do acido tetramico frente as calicrei.nas teciduais humanas 5 e 7, tripsina e quimotripsina. Dos 17 pseudopeptideos, oito compostos nao foram soluveis na fase de teste em meio aquoso e nove compostos foram soluveis em meio aquoso e puderam ser testados como inibidores das KLK 5 e KLK 7, sendo que quatro destes apresentaram inibicao utilizando concentracoes da ordem micromolar perante a KLK5. No entanto, apenas um composto obteve acao inibitoria frente a KLK7, porem com potencial inibitorio menor, comparado com resultados anteriores, relatados pelo nosso grupo de pesquisa. Os dez compostos aciltetramicos foram soluveis em tampao aquoso e apenas tres nao apresentaram atividade inibitoria contra as enzimas. Os resultados referentes as KLKs e tripsina foram significativos com uma acao inibitoria da ordem de micromolar. Os compostos derivados do acido tetramico nao inibiram a quimotripsina. Esses resultados abrem uma discussao sobre a especificidade da KLK7, que e classificada como uma enzima do tipo quimotripsina-like, no entanto, neste trabalho, apresentou um padrao de inibicao semelhante a tripsina. / The kallikreins 5 and 7 are serine proteases which belong to a group of fifteen human tissue kallikreins (KLKs) found in a diverse of body tissues. The KLK5 is mainly found on mammary tissue, central nervous system, testicles, prostate and trachea, while the KLK7 is found in the esophagus, liver, kidneys and salivary glands. Moreover, both enzymes are abundantly expressed on the human skin, and its believed that they have an important role on the epidermal desquamation process, from the hydrolysis of proteins that make up the corneodesmosomes intercellular adhesion structures. Recently it was shown that both kallikreins activity appears to be increased in diseases relates to the desquamation process, such as psoriasis and atopic dermatitis. Thus, the development of inhibitors for KLK5 and KLK7 would not only contribute to the elucidation of their physiological and pathological mechanisms, but also serve as an archetype for new drugs, aiming the development of new therapeutic procedures for diseases related to epidermal desquamation. In this project, 17 pseudo-peptides and 10 tetramic acid derived compounds were tested against the human tissue kallikreins 5 and 7, trypsin and chymotrypsin. From the 17 pseudo-peptides, nine were soluble in aqueous medium and could be tested; four of them showed inhibition in the micro molar range against the KLK5. Just one compound was effective against the KLK7, but with low inhibitory action. The 10 aciltetramic compounds were soluble in aqueous buffer, and only three of them did not show any inhibitory activity against the kallikreins. The results referring to the kallikreins and trypsin were significant, with an inhibitory action in the micro molar range. The compounds did not inhibit the chymotrypsin. These results open a discussion about the KLK7 specificity, which despite of being classified as a chymotrypsin-like enzyme, showed, in this work, an inhibition pattern similar to trypsin.

Made available in DSpace on 2015-08-20T17:09:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2015-08-20T17:26:16Z : No. of bitstreams: 1 000841508_20160426.pdf: 1577360 bytes, checksum: f910ad7dfc28e389bbc2cb39d7f2de0f (MD5) Bitstreams deleted on 2016-05-04T13:08:26Z: 000841508_20160426.pdf,. Added 1 bitstream(s) on 2016-05-04T13:09:30Z : No. of bitstreams: 1 000841508.pdf: 3356205 bytes, checksum: 3b5f5a833cae5f299c4dce5b2edae0de (MD5) / YacG é uma pequena proteína membro da família dos zinc-fingers, descoberta em E. coli. Sua função biológica está ligada a inibição da atividade de DNA girase e ao metabolismo do stress em procariotos. YacG atua na inibição da atividade de DNA girase por um mecanismo bipartido, a região do domínio zinc-finger atua impedindo a ligação do DNA à subunidade B e a região c-terminal liga-se a subunidade A. Embora a literatura cite YacG como sendo um inibidor de DNA girase, nada foi observado a respeito da atividade de inibição de YacG de E.coli e de outras linhagens de micro-organismos frente a outras DNA topoisomerases e não foi realizado nenhum trabalho envolvendo fragmentos peptídicos desta proteína, e tampouco ensaios de inibição do crescimento celular por estes. Sendo assim YacG de Klebsiella pneumoniae foi obtida por expressão heteróloga e uma série de peptídeos derivados desta foram sintetizados pelo método de fase sólida. Os peptídeo sintéticos foram projetados de forma a manter a região zinc-finger nativa (YacGAG1), substituídos os resíduos de cisteína por serina (YacGAG4), alanina (YacGAG5), histidina (YacGAG6 e YacGAG8) e também sintetizada a sua região c-terminal (YacGAG7). Os ensaios de inibição da atividade de DNA girase pelos peptídeos sintéticos e proteína, mostraram que YacG, YacGAG4, YacGAG5 e YacGAG7 conseguem inibir a atividade desta enzima, ao contrario do fragmento nativo YacGAG1. Por outro lado YacGAG1 conseguiu inibir a atividade de Topoisomerase IIα humana juntamente com os demais peptídeos YacGAG4, YacGAG5 e YacGAG7 e proteína YacG. Os ensaios aqui apresentados corroboram com os dados apresentados na literatura onde a região c-terminal por si só é capaz de inibir a atividade de DNA girase. Os ensaios de anisotropia de fluorescência demonstraram que a interação dos peptídicos sintéticos com DNA girase se dá pela interação destes com... / YacG belongs to zinc-finger's protein family discovered in E. coli. Its biological function is connected to the catalytic inhibition of DNA gyrase and stress metabolism in prokaryotes. This protein inhibits DNA gyrase (gyrase) through a bipartite mechanism where the zinc-finger domain prevents the DNA ligation to the B subunit (GyrB) and the C-terminal bindings to the A subunit (GyrA). Although it is classified as a gyrase inhibitor neither the inhibition activity of YacG from E. coli and other microorganisms against other DNA topoisomerases nor the biological activity of fragments in cellular assays has been evaluated until present. In this study, YacG from Klebsiella pneumoniae was obtained by heterologous expression and derivative peptides from this protein were synthesized by the solid-phase method. The synthetic peptides were designed to keep the native region of zinc-finger (YacGAG1), to substitute cysteines to serines (YacGAG4), to alanine (YacGAG5), to histidine (YacGAG6 and YacGAG8) and also having only the C-terminal region (YacGAG7). The inhibition assays of gyrase by the peptides and the native protein revealed that YacG, YacGAG4, YacGAG4 e YacGAG7 inhibited this enzyme and the human topoisomerase IIα (htopoIIα). However, the same was not observed for the native fragment YacGAG1, which inhibited only the htopoIIα. The results are in agreement with literature showing that solely the C-terminal region is able to inhibit the gyrase. The assays of fluorescence anisotropy indicated that these synthetic peptides interact with GyrA. Besides inhibiting the gyrase and the htopoIIα, these peptides also revealed bacteriostatic activity against gram-negative and gram-positive bacteria. A computational study by applying a molecular dynamics protocol highlighted the importance of residues I47, R46 and W40 from YacG in the process of molecular recognition and interaction with GyrB. The results...

Les analogues de nucléosides représentent une famille d’agents thérapeutiques très largement utilisée en chimiothérapie anticancéreuse. Cependant des phénomènes de résistance d’origine multifactorielle apparaissent chez les patients atteints de leucémie et il semble que deux protéines appartenant à la famille des 5’-nucléotidases (la nucléotidase cytosolique de type II, cN-II et l’ectonucléotidase, CD73) puissent être impliquées. Nous nous sommes donc intéressés à la synthèse et à l’étude d’inhibiteurs potentiels de cN-II ou de CD73 du type analogue de nucléosides phosphonates. Ce manuscrit rapporte dans un premier temps le contexte biologique des travaux de thèse, et les découvertes ayant supposées l’implication des protéines cibles dans les mécanismes de résistance. Le deuxième chapitre décrit la synthèse d’une série d’analogues beta-hydroxyphosphonates incluant une nucléobase modifiée de type 1,2,3-triazolo, ainsi que leur évaluation biologique vis-à-vis de la cN-II purifiée. Enfin, des résultats préliminaires ont été obtenus concernant de nouvelles structures capables d’inhiber modestement l’activité enzymatique de CD73. / Nucleosidic analogs are widely used as therapeutic agents in antitumoral chemotherapy. However, cellular resistance appears in a multifactorial manner in leukemic patients and it seems that two proteins belonging to the 5’-nucleotidase family (the 5’-cytosolic nucleotidase, cN-II and the ectonucleotidase, CD73) may be involved in these phenomenon. We focused our interest on the synthesis and the study of potential inhibitors belonging to the family of phosphonate nucleoside analogs. First, we reviewed literature data about the biological context of our research, especially concerning the involvement of these two targeted proteins in resistance phenomenon. Then, we described the synthesis of a series of beta-hydroxyphosphonate nucleosides incorporating 1,2,3-triazolo moiety as nucleobase, as well as their evaluation as inhibitors against the purified enzyme. Finally, preliminary results concerning the design of novel inhibitors of CD73 were reported.