Dissertations / Theses on the topic 'Curcumine – Analogues'

La maladie d’Alzheimer, forme la plus commune des démences séniles, est une maladie progressive, chronique, neurodégénérative et irréversible. Les causes physiopathologiques de cette démence sont nombreuses. Parmi elles on peut citer l’agrégation de peptides β-amyloïdes, l’hyperphosphorylation de la protéine Tau, l’inflammation neuronale et le stress oxydatif. Une des approches thérapeutiques récemment développées pour modifier l’évolution de cette maladie multifactorielle est la conception de ligands multicibles. On trouve dans la nature des molécules douées de telles fonctions, dont la curcumine. C’est un polyphénol naturel extrait de la plante Curcuma longa L. Et elle peut être considérée comme un principe actif d’intérêt dans le traitement de la maladie d’Alzheimer, car elle possède des propriétés inhibitrices de l’agrégation β-amyloïde, antioxydantes et anti-inflammatoires. Elle souffre cependant d’une mauvaise biodisponibilité et ne peut être utilisée en thérapeutique. C’est la raison pour laquelle nous avons conçu des analogues de la Curcumine en série pyrrolizinone et aminoindanone en tant que potentiels ligands multicibles d’intérêt dans le traitement de la maladie d’Alzheimer. Dans ce manuscrit, est décrite la démarche de drug design mêlant la pharmacochimie, la modélisation moléculaire et les évaluations biologiques menant à la conception et à la pharmacomodulation de ces analogues. Enfin, une étude préliminaire de la drugabilité des composés néosynthétisés est présentée
Alzheimer's disease, the most common form of elderly dementia in the world, is a progressive, chronic, degenerative and irreversible disease. They are many pathophysiological causes of dementia, among them there may be mentioned β-aggregation of amyloid peptides, tau protein hyperphosphorylation, neuronal inflammation and oxidative stress. A recently developed therapeutic approach to modify the evolution of this multifactorial disease is the design of multi-target directed ligands. In the nature, there are molecules with such functions like curcumin. It’s a natural polyphenol extracted from the plant Curcuma longa L. And it can be considered as an active product useful in the treatment of Alzheimer's disease because of its inhibitory properties of β-amyloid aggregation, antioxidant and inflammatory inhibition activities. However, it suffers from poor bioavailability and can’t be used in therapy. That's why we designed novel curcumin analogs in pyrrolizinone and aminoindanone series as potential multi-target ligands potentially useful in the treatment of Alzheimer's disease. In this manuscript, is described the drug design approach combining medicinal chemistry, molecular modeling and biological assessments leading to these analogues. Finally, a preliminary drugability study of the newly synthesized compounds is presented

Au-delà de la thérapie photodynamique (PDT), il existe plusieurs alternatives thérapeutiques assistées par la lumière parmi lesquelles la thérapie photothermique (PTT) ou encore la photoisomérisation dont l’action mécanique induirait des dommages cellulaires au sein des membranes. De plus, les molécules capables d’absorber simultanément deux photons permettraient de surmonter la nécessité d’une excitation à haute énergie par photon tout en gardant une pénétration suffisante des tissus. Dans ce contexte, nous nous sommes intéressés à la conception et à l’étude d’analogues biomimétiques de la cyclocurcumine (CC), un photoswitch E/Z naturel. Guidé par la modélisation moléculaire, ces nouveaux composés de type donneur-accepteur ont été structurellement conçus pour accroître la section efficace d'absorption à deux photons (σ2). Dans un premier temps, nous avons synthétisé deux classes d’analogues de la CC portant des groupements accepteurs (oxo et malonitrile) et donneurs (hydroxy, alcoxy et amine) ainsi qu’un bras PEG pour augmenter leur hydrophilie. Libres en solution, les dérivés oxo subissent une isomérisation photoinduite réversible, sans aucune isomérisation thermique en retour dans l’obscurité alors que les dérivés malonitrile ne photocommutent plus mais subissent une relaxation vibrationnelle. Elle conduit ainsi à une augmentation de température, d’intérêt en thérapie photothermique. Enfin, confinées dans un environnement lipidique (bicouches de liposomes et monocouches de Langmuir), la cinétique d’isomérisation directe E→Z des composés oxo est fortement ralentie et aucune isomérisation retour Z→E n’est observée, ni thermique ni sous illumination. Ces mêmes composés ont montré une efficacité certaine pour perturber la fluidité membranaire, plus ou moins prononcée selon la forme, Z ou E, la longueur de la chaîne alcoxy ou encore la présence du motif PEG. Les dérivés malonitrile quant à eux se voient diminuer leur effet photothermique au sein des bicouches lipidiques, après plusieurs cycles d’irradiation. L’ensemble de cette étude est une preuve de concept quant au potentiel des réponses photoinduites des analogues de la cyclocurcumine pour la perturbation de la fluidité membranaire vers une potentielle utilisation in vivo
Beyond photodynamic therapy (PDT), there are several light-assisted therapeutic alternatives, including photothermal therapy (PTT) and photoisomerization, whose mechanical action would induce cellular damage within the membranes. Also, molecules able to simultaneously absorb two photons would overcome the need for high energy excitation per photon while maintaining sufficient tissue penetration. In this context, we were interested in designing and studying biomimetic analogues of cyclocurcumin (CC), a natural E/Z photoswitch. Guided by molecular modeling, those novel donor-acceptor compounds were structurally designed to increase the effective two-photon absorption cross section (σ2).First, we synthesized two classes of CC analogues bearing acceptor (oxo and malonitrile) and donor (hydroxy, alkoxy and amine) groups as well as a PEG arm to increase their hydrophilicity. Free in solution, the oxo derivatives show a reversible photoinduced isomerization, without any thermal isomerization in the dark, whereas the malonitrile derivatives do not photoswitch but undergo a vibrational relaxation. This is leading to a temperature increase, of interest in photothermal therapy. Finally, confined in a lipidic environment (liposomes bilayers and Langmuir monolayers), the direct E→Z isomerization kinetics of oxo compounds is strongly slowed down and no Z→E back isomerization is observed. Those compounds also showed a certain efficiency to disrupt membrane fluidity, more or less pronounced depending on the form, Z or E, the length of the alkoxy chain or the presence of the PEG unit. The malonitrile derivatives exhibit a diminished photothermal effect in lipid bilayers after several irradiation cycles.Taken together, this study is a proof of concept for the potential of photoinduced responses of cyclocurcumin analogs for disruption of membrane fluidity towards potential use in vivo

Coordination properties of 4-bromo-N-(diethylcarbamothioyl)benzamide (Hbeb) and 4-bromo-N-(diphenylcarbamothioyl)benzamide (Hbpb) with oxorhenium(V) and rhenium(I) are reported and discussed. Transition metal complexes of these ligands were studied due to the wide range of applications of thiourea derivatives in biological fields. N-[Di(alkyl/aryl)carbamothioyl]benzamide derivatives readily coordinate to metal ions as O,S-donors and the catalytic property of the complexes can be altered by these ligands, due to steric and electronic properties provided by various substituents. The coordination possibilities of curcumin with rhenium(V) are discussed, as well as the difficulties encountered. Analogues of curcumin have been made, which also contains a seven-spacer unit between the phenyl rings, which would be more reactive and more effective in bonding to rhenium and which have greater or a similar biological activity to curcumin. This was done by assessing the coordination properties of 1,5-bis(salicylidene)thiocarbohydrazide (H4salt) and 2,4-bis(vanilidene)thiocarbohydrazide (H4vant) with oxorhenium(V) and rhenium(I) starting materials. Two rhenium(V) complex salts of the core [ReX(PPh3)2]4+ (X = Br, I), containing a coordinated imido nitrogen, are reported. One is a ‘2+1’ complex, coordinating bi- and monodentately, with the other a similar ‘3+0’ complex containing a tridentate imido-coordinated Schiff base. Selected compounds were tested against oesophageal cancer cell lines in order to evaluate and compare their effectiveness in eliminating or reducing the cancer cells in the test medium during biological testing.

Melanoma é o câncer mais agressivo e a mutação BRAF V600E é a mais frequente entre os pacientes. O vemurafenibe foi o primeiro inibidor específico desta mutação aprovado pela Food and Drug Administration. Entretanto, após cerca de seis meses há recidiva e superar os mecanismos de resistência responsáveis por este fenômeno ainda é um desafio. A curcumina é um tumérico com características antitumorais e anti-inflamatórias, entretanto sua baixa biodisponibilidade e estabilidade limitam seu uso e por isso impulsionaram a busca por análogos capazes de serem eficientes e comercializados. O DM-1, é um análogo monocetônico que apresentou efeitos antiumorais in vitro e in vivo em estudos anteriores. O objetivo deste trabalho foi avaliar os efeitos citotóxicos do DM-1 em células de melanoma sensíveis (naive) e resistentes ao vemurafenibe, bem como na modulação de metaloproteinases. As células de melanoma foram tratadas com diferentes concentrações de DM-1, e este composto foi citotóxico para linhagens sensíveis e resistentes ao vemurafenibe, além de induzir parada de ciclo celular em G1/G0 e diminuir o número de colônias, entretanto ele não foi seletivo em ensaios de citotoxicidade realizados com melanócitos e fibroblastos. O tratamento dessas células em doses subtóxicas resultou na modulação de metaloproteinases importantes no processo de invasão celular. O DM-1 reduziu as concentrações das metaloproteinases -1, -2 e -9 (MMP-1, -2 e -9) em um ensaio de quantificação de MMPs e a atividade das MMP-2 e -9 em um ensaio de zimografia de maneira célula dependente. As modulações negativas do inibidor de MMP TIMP-2 e MMP-14 para SKMEL-28 naive foram associadas a diminuição das atividades de MMP-2 e -9, enquanto que as modulações positivas para SKMEL-19 naive foram relacionadas ao aumento de MMP-2. Este composto ainda inibiu a migração das células e a formação de tubos por células endoteliais.
Malignant melanoma is the most aggressive cancer and the BRAF V600E mutation is the most frequent among patients. Vemurafenib was the first specific inhibitor for this mutation approved by Food and Drug Administration. Therefore around six months later there is relapse and overcoming it is still a challenge. Curcumin is a turmeric and it has been deeply researched because of its anti-inflammatory and antitumoral effects. However the low stability limits its use, therefore, encouraged the investigation of analogues capable to be efficient and commercialized. DM-1 is a monoketone curcumin analog and it showed antitumoral effects in vitro and in vivo in previous studies The aim of this project was to evaluate the cytotoxical effects of DM-1 for vemurafenib responsive (naïve) and resistant melanoma cells, as well as metalloproteinases modulation. Melanoma cells were treated with different DM-1 concentrations, and this compound was cytotoxic for responsive and resistant cell lines, besides inducing G1/G0 cell cycle arrest and reducing the number of colonies, nonetheless it was not selective in assays performed with melanocytes and fibroblasts. Subtoxic treatment of those cells modulated important MMPs in the cell invasion process. DM-1 reduced metalloproteinases -1, -2 and -9 (MMP-1,-2 and -9) in a quantification assay, and MMP-2 and -9 activities by zymography in a cell-dependent way. Negative modulations of MMP inhibitor TIMP-2 and MMP-14 for SKMEL-28 naïve were associated with MMP-2 and -9 reduced activities, whereas positive modulations for SKMEL-19 naïve were correlated to MMP-2 increase. Furthermore, this compound reduced migration of those cells and endothelial cell tube formation.

A range of derivatives has previously been synthesized in order to overcome the stability and pharmacokinetic hurdles that hinder the in vivo investigation of the natural product, curcumin. One such derivative, pyrazole 15, has been demonstrated to inhibit the proliferation of a highly metastatic androgen-independent prostate cancer cell line (PC3). In this work, pyrazole analogues, 15 and 32 and the corresponding curcumin analogues, 30 and 31 were synthesized using a microwave-assisted technique. A stability study, and in silico assessment, of curcumin 1 and thirteen pyrazole analogues (15,17, and 32-42) was hindered by the poor solubility of these analogues, as a result of their hydrophobicities. An attempt to improve the solubility of pyrazole 15, through host-guest complexation with three macromolecules, γ-cyclodextrin (γ-CD) 46, p-sulfonatocalix[4]arene sCX[4] 48 and cucurbit[7]uril CB[7] 52, was unsuccessful, presumably due to the lack of sufficient bonding interactions or the incompatibility of the pyrazole with the hydrophobic cavities in these molecules. Eight polymeric micelle (PM) formulations were then fabricated with the aim of generating sufficient hydrophobicity to entrap pyrazole 15; seven formulations of the Pluronic® F127 72 based PMs, via the thin film hydration method, and one formulation incorporating POEGA-b-PS polymer based PMs (POEGA-b-PS-PMs 100), via the dialysis method. All the empty PMs were shown to be stable, with particle sizes below 52 nm and polydispersity indexes (PDI) below 0.34, indicating homogeneity and stability. F127-PMs 93 were not able to accommodate pyrazole 15 (size 253.7 ± 25.4, PDI 0.63 ± 0.13, and lowest %DL 0.29 ± 0.01). Increasing the hydrophobicity of Pluronic® F127 72 by adding TPGS 73 in different ratios, as in the mixed micelles FT41-PMs 94, FT21-PMs 95, FT32-PMs 96 and FT11-PMs 97, resulted in better stability, a higher degree of monodispersity and the encapsulation of the hydrophobic pyrazole 15 (particle sizes 30 – 55 nm, PDIs 0.15 - 0.25, and %DL 1.08 - 1.19). The addition of the targeting moiety [D-Lys6]-LHRH 75, to give the FLHRH-PMs 98, helped improve the adaptability for the drug (particle size 57.6 ± 13.7 nm, PDI 0.41 ± 0.17 and %DL 0.48 ± 0.01), but not to the same levels as the addition of the hydrophobic TPGS 73. Targeted mixed micelles FLHRHT32-PMs 99, reduced the particle size and the PDI, in comparison to targeted FLHRH-PMs 98 (without TPGS 73), as a result of increasing the hydrophobicity increases the stability (particle size 22.0 ± 0.2 nm, PDI 0.28 ± 0.03, and %DL 0.64 ± 0.05). POEGA-b-PS-PMs 100 displayed good stability in comparison to the Pluronic® F127 72 based PMs (particle size of 54.0 ± 0.3 nm and PDI 0.15 ± 0.01). Finally, the resazurin reduction assay and IncuCyte® imaging technique were used to evaluate the biological activity (% cell viability) of pyrazole 15 and four promising formulations in three types of cancer cell lines; (PC-3), (MDA-MB-231) and (MCF-7). The experimental GI50 values for pyrazole 15 using 1% DMSO as a co-solvent were 2.5 ± 0.58 µM in PC-3, 1.26 ± 0.22 µM in MDA-MB-231, and 4.39 ± 0.63 µM in MCF-7. Treatment with FLHRH-PMs 98 containing pyrazole 15 resulted in significant growth inhibition in comparison to the corresponding void in all three tested cell lines, while POEGA-b-PS-PMs 100 showed the same trend but did not produce any significant effect on MCF-7 cells. Treatment with TPGS-containing PMs (FT32-PMs 96 and FLHRHT32-PMs 99), resulted in growth inhibition, both in the presence and absence of pyrazole 15. These results suggest that these formulations can help improve pyrazole 15 solubility and thus enable further biological evaluation.

Under normal conditions macrophages provide immune surveillance and host defense in tissues to maintain homeostasis. However, upon sensing changes in the microenvironment, macrophages become activated, undergoing a morphological and functional switch. Activation of these cells is not an “all-or-none” process, but rather a continuum characterized by a wide spectrum of molecular and functional phenotypes ranging from the “classical” M1 activated phenotype, with a highly pro-inflammatory profile, to the “alternative” M2 phenotype, associated with a beneficial, less inflammatory, protective profile. The possibility to promote a macrophage protective phenotype has therefore become a therapeutic goal in the treatment of inflammatory conditions, and the identification of factors that control cell activation is currently an area of active research. Most studies in the field so far have been performed using primary mouse macrophages or macrophage cell lines, and a variety of monocyte differentiation protocols and macrophage activation markers are used by different labs. Moreover, the pharmacological control of human macrophage polarized activation has not been extensively explored. A number of natural and synthetic compounds, including chalcones and curcumin, the major active component isolated from the turmeric plant Curcuma longa, have been shown to induce effects on macrophage function (including antioxidant, anti-microbial, anti-carcinogenic and anti-inflammatory action) through multiple pharmacological mechanisms, including interference with TLR4 signaling. On these grounds, the specific aims of the present thesis were: a) to test cell models and differentiation protocols other than spontaneous blood-derived macrophage differentiation, namely the THP-1 cell line and CSF-1-driven differentiation, respectively; b) to profile the cytokine pattern into the culture medium, and c) to determine the modulation of phenotypic markers by pharmacological agents, namely curcumin derivatives known to suppress microglial activation through reduced production and release of pro-inflammatory mediators, as well as the underlying mechanisms of action. Macrophages were differentiated from human PBMCs isolated by density gradient centrifugation, and cultured in RPMI 1640 medium with 10% FBS with CSF-1 for 6 days to obtain resting macrophages (M0). Classical (M1) and alternative (M2) phenotypes were generated using specific cytokines (0.1-1 μg/ml LPS or 20 ng/ml IL-4 plus 5 ng/ml IL-13, respectively) in the presence or absence of curcumin analogues or dexamethasone used a reference compound. Macrophage phenotypes were determined by flow cytometry using fluorocrome-labeled antibodies. Gene expression was analysed using qRT-PCR. The composition of macrophage conditioned media (MCM) was assessed with the Luminex technology. Curcumin analogues were kindly provided by Dr. Federica Belluti (University of Bologna). When M2 polarization was induced with IL-4/IL-13 for 24h, we observed increased expression of M2 markers compared with M0. In terms of gene expression analysis of the CSF-1 driven macrophages, as expected, M1-polarized macrophages after 6 or 48 h showed higher mRNA levels of TNF-α and IL-1β compared with M0. The increase in mRNA was more marked after 48 h for all genes except TNF-α, which rose more sharply after 6 h. The anti-inflammatory cytokine IL-10 mRNA was unexpectedly more abundant in M1- than in M2-polarized macrophages, and peaked after 6 h. Compared with M0, M2 MCM showed higher levels of anti-inflammatory cytokines including CCL22 and IL-4. In contrast, M1 MCM was associated with higher levels of IL-1α, IL-1β, IL-6, IL-8, MCP-1, VEGF and TNF-α. Treatment with the curcumin analogue GG9 as well as CLI095, an inhibitor of TLR4 intracellular domain, reversed the LPS-induced up-regulation of CD80+ (M1) cells. A similar effect was maintained with the double positive CD80+/CCR2+ population. Unlike dexamethasone, which increased the percentage of CD163+ (M2) cells, the curcumin analogue GG9 did not affect M2 markers. Treatment with GG9 significantly blocked IL-1β cytokine production at the cell-bound level, in the protein lysate and in the medium. By contrast, curcumin and GG6 did not affect the levels of intra- and extracellular IL-1β. To investigate intracellular signaling pathways involved in cell activation, we performed Western blot analysis of factors involved in the NF-κB pathway. Activation with LPS significantly decreased the relative expression of IκB-α. By contrast, curcumin and GG6 were able to restore IκB-α amounts as did CLI095, whereas no effect was induced by GG9 treatment. Therefore, M1 and M2 macrophages showed specific profiles of gene expression and surface markers, which were modulated by pharmacological treatment with dexamethasone or a curcumin analogue. Overall, these data suggest that polarized activation protocols may have an impact on the functional status of macrophages and are critical to further investigate pharmacological macrophage targeting.
In condizioni normali i macrofagi sono i responsabili della risposta immunitaria e della difesa dell’ospite per mantenere l’omeostasi tissutale. In seguito a stimoli presenti nel microambiente, i macrofagi possono attivarsi, andando incontro a uno switch morfologico e funzionale. L’attivazione di queste cellule non è un processo “tutto o nulla” ma piuttosto un continuum caratterizzato da un ampio spettro di fenotipi molecolari e funzionali, i cui estremi sono rappresentati dal fenotipo definito “classico” o M1, con un profilo pro-infiammatorio, e da quello “alternativo” o M2, anti-infiammatorio e protettivo. La possibilità di promuovere un fenotipo macrofagico protettivo sta diventando un obiettivo terapeutico nel trattamento delle condizioni infiammatorie e l’identificazione di fattori che regolano l’attivazione cellulare è attualmente un’area di ricerca molto attiva. La maggior parte degli studi in questo ambito sono stati condotti utilizzando culture primarie di macrofagi di topo o linee cellulari; inoltre, i vari laboratori usano protocolli diversi di isolamento/differenziamento dei monociti e marcatori diversi di attivazione. Inoltre, il controllo farmacologico dell’attivazione macrofagica non è ancora stato indagato sufficientemente. Una serie di composti naturali e sintetici, ad esempio il calcone e la curcumina, il principale componente attivo della Curcuma longa (pianta nota per effetti anti-ossidanti, anti-microbici, anti-tumorali e anti-infiammatori) hanno dimostrato un effetto sulla funzione dei macrofagi, agendo attraverso diversi meccanismi farmacologici, ad esempio interferendo con il signaling del TLR4. Sulla base di queste premesse, gli scopi specifici di questo lavoro di tesi erano: a) testare un modello cellulare e un protocollo di differenziamento diverso da quello spontaneo, in particolare la linea cellulare monocitaria THP-1 e il differenziamento in presenza di CSF-1; b) determinare il profilo delle citochine presenti nel terreno di coltura e c) determinare la modulazione dei marcatori dei fenotipi di attivazione da parte di agenti farmacologici, in particolare da derivati di sintesi della curcumina, noti per la loro capacità di modulare l’attivazione di cellule murine di microglia attraverso la riduzione della produzione e rilascio di mediatori pro-infiammatori. I macrofagi sono stati differenziati a partire dai linfo-monociti umani isolati tramite gradiente di densità e coltivati in terreno RPMI + 10% FBS con aggiunta di CSF-1 per 6 giorni, ottenendo così macrofagi in condizioni basali (M0). Il fenotipo classico (M1) e il fenotipo alternativo (M2) sono stati ottenuti incubando i macrofagi M0 rispettivamente con 0.1-1 μg/ml LPS e IL-4 20 ng/ml + IL-13 5 ng/ml, in presenza o assenza di analoghi della curcumina, desametazone o CLI095, un inibitore del dominio intracellulare del TLR4 (questi ultimi utilizzati come composti di riferimento). I fenotipi macrofagici sono stati determinati tramite citofluorimetria utilizzando specifici anticorpi legati a fluorofori. L’espressione genica è stata valutata tramite qRT-PCR. La composizione dei terreni condizionati (macrophage conditioned media, MCM) è stata valutata con la tecnologia Luminex. Gli analoghi della curcumina sono stati gentilmente forniti dalla Prof.ssa Federica Belluti (Università di Bologna). In seguito a polarizzazione per 24 h con IL-4/IL-13 abbiamo osservato un aumento dell’espressione dei marcatori M2 rispetto a M0. Per quanto riguarda l’espressione genica di macrofagi differenziati con CSF-1, come atteso, i macrofagi M1 dopo 6 o 48h presentavano livelli superiori di mRNA per TNF-α e IL-1β rispetto a M0. L’aumento dell’mRNA era più marcato dopo 48 h per tutti i geni tranne TNF-α, che raggiungeva il picco a 6h. L’mRNA della citochina anti-infiammatoria IL-10 inaspettatamente era più espresso nei macrofagi M1 rispetto a M2 e raggiungeva il picco dopo 6 h. Rispetto a M0, l’MCM M2 era caratterizzato da alti livelli di citochine anti-infiammatorie tra le quali CCL22 e IL-4. Al contrario, l’MCM M1 presentava livelli elevati di IL-1α, IL-1β, IL-6, IL-8, MCP-1, VEGF e TNF-α. Il pre-trattamento con l’analogo della curcumina GG9 e il controllo positivo CLI095 ha prevenuto l’aumento indotto da LPS del marcatore M1 CD80. Un effetto simile è mantenuto anche sulla frazione di cellule a doppia marcatura CD80+/CCR2+. Al contrario del desametazone, che aumenta la percentuale di cellule CD163+ (M2), il GG9 non ha modulato i marcatori M2. Il trattamento con GG9 ha bloccato in maniera significativa la produzione di IL-1β valutata come citochina cell-bound, nel lisato cellulare o rilasciata nel terreno di coltura. Al contrario, l’analogo GG6 non ha modificato i livelli di IL-1β intra- ed extra-cellulare. Per indagare più nel dettaglio le vie di segnale coinvolte nell’attivazione di queste cellule, abbiamo effettuato analisi Western Blot di fattori coinvolti nella via di segnale di NF-κB. L’attivazione con LPS ha ridotto significativamente l’espressione relativa di IκB-α. Curcumina, GG6 e CLI095 hanno riportato IκB-α ai livelli di controllo, a differenza del GG9 che non modificava significativamente l’espressione di questa proteina. Pertanto, i macrofagi M1 ed M2 presentano specifici profili di attivazione genica e di marcatori di superficie che possono essere modulati in seguito a trattamento farmacologico con desametazone o analoghi della curcumina. Nel complesso, questi dati suggeriscono che protocolli di attivazione macrofagica possono avere un impatto sullo stato funzionale di queste cellule e sono fondamentali per definire nuove strategie di targeting farmacologico nei macrofagi umani.

Chronic inflammation is closely related to the emergence of a number of cancers, including Breast cancer. Inflammation causes damage to the cell’s DNA which leads to its abnormal growth and formation of tumor mass. One of the most commonly known receptor responsible for inflammatory reactions is Toll-like receptor 4 (TLR4). It is activated majorly by bacterial LPS. Its activation further activates Cyclooxygenase enzyme that catalyzes the conversion of arachidonic acid into prostaglandins that lead to inflammation-like conditions. COX2 has also been correlated to the promotion of tumor growth. It enhances metastasis, neoplasia, lymphangiogenesis, etc., and is also related to poor prognosis in the breast cancer patients. Curcumin derived from turmeric is a proven inhibitor of COX2. In my project I have aimed to analyse and compare the inhibitory properties of other analogues of curcumin that have previously been known to inhibit COX2. The experimental layout began with screening the molecules on the basis of drug-likeness using Lipinski rule of five. The suitable ligand molecules were further subjected to other experiments, i.e., ligand docking and drug potential assessment. After all the experiments, out of the five selected Curcumin analogues, Isoeugenol (extracted from clove) was determined as the best fit molecule. The druglikeliness and drug potential assessment results further validate its use as a potential inhibitor and can further be tested for in-vivo efficacy. This drug can further be used in the 1st line therapy of locally advanced and metastatic breast cancer patients as it will inhibit COX2 that promotes metastasis of cancer cells. Isoeugenol extracted from Eugenia caryophyllus (Cloves) can further be proven as a better COX2 inhibitor than its chemical counterparts, as it is a natural compound and will therefore have significantly less side effects.

Curcumin analogues are novel antineoplastic agents designed by structural modifications of the natural product curcumin to enhance its therapeutic effects. Various curcumin analogues displayed a significant cytotoxic effect towards different cancer cell lines including leukemia, melanoma, and colon cancer. In order to evaluate the safety, efficiency and metabolism of the new anticancer candidates, sensitive and high throughput analytical methods are needed. Thirteen curcumin analogues with the backbone structure of 3,5-bis(benzylidene)-4-piperidone were tested. The ionization behavior of curcumin analogues was investigated to reveal the possible mechanisms for the unusual formation of the positively charged [M-H]+ ions during single stage positive ion mode MALDI-MS analysis. Different ionization techniques (i.e., ESI, APCI, APPI, and MALDI) were used to evaluate this phenomenon. The results showed that curcumin analogues ionize into [M-H]+ along with the expected [M+H]+ species during MALDI and dopant free APPI-MS. In contrast, ESI, APCI and the dopant mediated APPI showed only the expected [M+H]+ peak. Our experiments revealed that photon energy triggers the ionization of the curcumin analogues even in the absence of any ionization enhancer such as matrix, solvent or dopant. Three proposed mechanisms for the formation of [M-H]+ were evaluated, two of them are probably involved in the [M-H]+ formation: (i) hydrogen transfer from the analyte radical cation and (ii) hydride abstraction. In addition to the ionization behavior, the collision induced dissociation-tandem mass spectrometric (CID-MS/MS) fragmentation behavior of curcumin analogues was evaluated showing similar dissociation pathways that centered on the piperidone ring of the 3,5-bis(benzylidene)-4-piperidone moiety. The presence of different substitutes on that moiety resulted in specific product ions for each curcumin analogue. The fragmentation patterns were established to confirm the chemical structure of the tested compounds and identify the diagnostic product ions of each compound. Twelve common product ions were identified resulting from the breakage of various bonds within the piperidone moiety. There was a tendency for the formation of highly conjugated product ions that are stabilized via resonance. Common product ions were identified allowing for the establishment of a general MS/MS behavior for any curcumin analogue that belongs to the 3,5-bis(benzylidene)-4-piperidone structural family. The fragmentation routes and the genesis of the product ions were confirmed via MS3 and neutral loss analysis. In summary, the ionization of curcumin analogues provided insights into the formation of unique [M-H]+ ions which were linked to photo ionization of such compounds without the need for additives, such as matrix, dopant or solvent. As such, curcumin analogues should be evaluated as MALDI matrices in the future. The CID-MS/MS analysis of curcumin analogues revealed a common fragmentation behavior of the tested compounds. It will be applied, in the future to determine metabolic by-products of the tested compounds as well as to develop targeted liquid chromatography (LC)-MS/MS methods.

碩士
輔仁大學
食品科學系碩士班
103
Curcumin is a constituent from root and stem of family Zingiberaceae and Araceae. It has been used as a natural colorant in food industry. Curcumin has a great value in food and medicine, and it has been reported that curcumin could inhibit the activities of xanthine oxidase (XO) and α-glucosidase that make curcumin can be used for treatment of gout and diabetes. Chemical synthesis, which can change the structure of component, can enhance the activities, even create a new one. Therefore, the objective of this research is to evaluate the characterization of curcumin and its analogs as XO and α-glucosidase inhibitors. In this study, after screening XO and α-glucosidase inhibition, the components which have high inhibitory activities were calculated their half-maximal inhibitory concentration (IC50) and enzyme inhibitory kinetics. In order to calculating the inhibitory reaction constant, a docking algorithm simulates binding position between enzyme and inhibitors. The results showed that among all the curcumin and its analogs, CM-F had the strongest anti-oxidant activity with a half-maximal effective concentration (EC50) of 9.39 ± 0.16 μM, which was better than vitamin E (EC50=17.03 ± 0.09 μM). It also had a good XO inhibitory activity, and its IC50 value against XO was 6.14 ± 0.38 μM. The enzyme kinetic result showed it was competitive inhibition. As for α-glucosidase, CM-1 and CM-2 have good α-glucosidase inhibitory activities with the IC50 value of 21.06 ± 0.92 μM and 5.95 ± 0.09 μM, of which kinetic study indicates that both CM-1 and CM-2 are mix-competitive inhibitors on α-glucosidase. Furthermore, docking simulation showed there are 5 hydrogen bonds between XO and CM-F. However, only 1 and 2 hydrogen bonds involved in CM-1 and CM-2 binding to α-glucosidase, respectively. Accordingly, analogs of curcumin have the potentials using in the gout or diabetes patients.

碩士
國立中興大學
微生物暨公共衛生學研究所
101
Curcumin is a commonly used colouring agent and spice in food. Accumulated evidence indicates that curcumin is associated with a great variety of pharmacological activities, including an antimicrobial effect. Previously, we reported curcumin inhibits the infection of type A influenza virus (IAV) and proved one of the mechanisms is through interfering the viral HA activity. To investigate the structure contributing to its anti-IAV activity, structural and functional analogues of Curcumin (Cur), such as Tetrahydrocurcumin (THC), Monoacetylcurcumin (MAC) and Petasiphenol (Pet) were comparatively analyzed in the current study. The result of time-of-drug addition tests revealed that all the analogues analyzed were able to inhibit IAV production in cell cultures; MAC has a similar strength to curcumin, whereas Pet and THC inhibit IAV to a much less extent than curcumin. Comparative analysis of curcumin analogues described herein demonstrated that Thc and Pet have much less effect on suppression of plaque formation ability; the EC50 of pet are 14.65 μM, whereas curcumin and MAC is 0.17 and 0.2 μM, respectively. Surprisingly, none of the analogues harbors HA inhibition effect. Considering that as with curcumin, the structure of MAC contains two double bonds in the central seven-carbon chain, whereas Pet and THC has one or none, respectively, it indicates the presence of double bond is crucial for the anti-IAV activity. The unsaturated carbonyl group acts as the acceptor of Michael addition reaction that involves in the intermolecular conjugation by formation of covalent Michael adducts with certain proteins. It is very possible that via Michael addition reaction, curcumin conjugated with viral protein that in turns alters or interferes with the function of viral surface proteins and inactivate virus infectivity. In conclusion, THC, one of the stable curcumin metabolites exhibits anti-IAV activity and MAC appears to be an effective agent for inhibition of IAV infection. Moreover, comparative analysis of curcuminoids indicated that the two double bonds in the central seven-carbon chain contribute to the curcumin -mediated anti-IAV activity.

碩士
中國醫藥大學
藥物化學研究所碩士班
102
Curcumin is an orange-yellow crystalline powder obtained from rhizome of Curcuma longa Linn (Zingiberaceae). Curcumin has a variety of biological activities. Within the past 30 years, cancer is a leading cause of death in Taiwan. Therefore, developing new anticancer drugs is in an urgent need. In the recent years, there are many studies reported that curcumin displayed anticancer activity. In this study, several curcumin analogs were synthesized and evaluated for anticancer activities. We expected to find a better anticancer agent than curcumin. This study is focus on synthesis of two types of curcumin analogs, which are represented as WC and S series. WC series was used the catecol as a starting material, which was first cyclized with various ketones under P2O5 catalyst to obtain benzodioxole derivatives. The benzodioxoles were reacted with NBS, and then went forward the Grignard reaction to yield the corresponding aldehydes. Finally, the aldehydes were condensed with acetylacetone to give the corresponding curcumin analogs WC4a~4h. As synthetic curcumin analogs S series, bezaldehyde or piperonal was first reacted with acetone under strong base, and then reacted again with the substituted aldehydes to obtain the corresponding curcumin analogs S2a~2h . The anticancer activities of these curcumin analogs WC4a~4h and S2a~2h were evaluated on Hep3B, H460, A498 and COLO-205 human cancer cell lines. The results showed that the S2a exhibited the most potent cytotoxicity against A498 cells with IC50 value 16.8 μM. Moreover, the preliminary mechanism studies indicated that S2a could induce apoptosis in A498 cells by both the mitochondrial pathway and death receptor pathway.

Curcumin is one of the major curcuminoids produced by the ginger family Zingiberaceae. These curcuminoids possess pharmacological properties that include anticancer activities. We have evaluated some synthetic curcumin analogues that have shown potential as anticancer drugs. These antineoplastic agents bearing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore are electrophiles which are designed to preferentially react with sulfhydryl groups present in proteins as opposed to amino and hydroxyl groups present in DNA. In previous pilot studies, three derivatives examined in this thesis showed inhibition towards human cancer cell lines such as Molt 4/C8 and CEM T-lymphocytes. In this thesis work, I determined the cytotoxicity of these derivatives and curcumin towards human colon cancer (HCT-116) cells and also normal colon epithelial (CRL-1790) cells, and examined the possible mechanism(s) involved. I hypothesized that they act via induction of reactive oxygen species (ROS) which elicit a transient surge of mitochondrial ROS generation and a phenomenon known as ROS-induced ROS release (RIRR), along with the mitochondrial permeability transition (MPT) and mitochondrion –dependent apoptosis. I asked whether these agents react with some of the key protein thiols in the mitochondria whose oxidation/alkylation results in mitochondrion - dependent apoptosis. NC-2109 and NC-2346 were found to be potent cytotoxic agents based on their GI50 values of 0.87 ± 0.38 μM and 0.90 ± 0.22 μM, respectively, and were more potent than the anticancer drug 5-fluorouracil (GI50 = 5.47 ± 0.55 μM) and curcumin (GI50 = 3.50 ± 0.36 μM). However NC-2109 was found to have a better selectivity towards cancer cells over normal cells (a selectivity index of 18.81 versus 5-FU, curcumin and NC-2346 which had selectivity indices of 1.87, 16.75 and 4.61, respectively). In the investigations of the mechanisms involved, both curcumin and curcumin analogues were able to induce mitochondrial ROS production. Moreover, curcumin and its synthetic counterparts showed a biphasic ROS profile which is most characteristic of RIRR. Treatment with these agents also led to the disruption of the mitochondrial membrane potential, suggesting oxidation of protein thiols and the opening of the mitochondrial permeability transition pore which is an important step to initiate mitochondria-directed apoptosis. This possibility was confirmed based on GSSG/GSH ratios, since curcumin, NC-2346 and NC-2109 all produced a higher GSSG/GSH ratio than the controls. In addition to their ability to depolarize the mitochondrial membrane in HCT-116 cells, that these molecules acted via the mitochondrial pathway were further authenticated based on their ability to induce mitochondrial swelling in rat liver mitochondria. In another part of this thesis I evaluated the involvement of the critical thiol protein adenine nucleotide translocase (ANT), a bifunctional protein that plays a central role in mitochondrial apoptosis. ANT has four different isoforms; ANT1 and ANT3 are proapoptotic, while ANT2 and ANT4 are antiapoptotic and are overexpressed in cancer states. A combination approach using ANT2 siRNA however did not conclusively show whether these agents acted synergistically with ANT2 knockdown to potentiate mitochondria-mediated cell death. An alternative combination approach was the use of carboxyatractyloside (CAT) which binds to and retains ANT in its ‘c’ conformation, exposing thiols and potentially driving a cell towards programmed cell death. The presence of CAT enhanced the ability of curcumin and its synthetic analogs to collapse the mitochondrial membrane potential, an important step in mitochondrial-mediated apoptosis. In conclusion, curcumin and the curcumin analogue NC-2109 were found to be cytotoxic in vitro, towards HCT-116 cells and also showed good selectivity. In addition, these two molecules were found to be ROS inducers, and coincidentally oxidized cellular thiols and caused depolarization of the mitochondrial membrane potential. The results support a mechanism of mitochondrial-mediated cell death upon MPT pore formation (mitochondrial swelling), perhaps involving ANT2. This conclusion was further supported by the potentiation of cell death in the presence of the ANT2 inhibitor, CAT.

碩士
輔仁大學
食品科學系碩士班
107
Curcumin (CM) is a natural lipophilic polyphenol with a variety of pharmacological properties, that provide antioxidizing, antiinflammatory, antimicrobial, antiviral, antirheumatic, anticancer, and neuroprotective effects. However, curcumin is limited in terms of water-solubility, and curcumin analogues as well. Researchers have recently begun using biopolymers to encapsulate hydrophobic compounds in order to improve their bioavailability. The objective of this study was to evaluate the feasibility of using zein nanoparticles as an oral delivery vehicle for curcumin and its analogues. The investigation centered on the radical scavenging activities of ABTS and DPPH as well as the inhibition of α-glucosidase and xanthine oxidase by curcumin and its analogues, including CM-1, CM-2, CM-A, CM-F, and tetrahydrocurcumin. Fourier transform infrared spectroscopy was used to identify and characterize zein-curcumin analogues nanoparticles. All of the analogues except CM-1 presented ABTS radical scavenging activity on par with or superior to that of vitamin E. The α-glucosidase inhibitory activity of the compounds in this study was as follows: CM-F > CM > CM-A > CM-1. The α-glucosidase inhibitory effects of curcumin and its analogues were superior to those of quercetin. Furthermore, only CM-F had a good xanthine oxidase inhibitory activity (IC50 = 2.05 ± 0.15 μM), suggesting a better inhibition than that of allopurinol (IC50 = 12.04 ± 1.54 μM). Amide I, amide II and amide III of zein were observed in the nanoparticles with the peak positions either existed or slightly shifted. The main peaks of all compounds themselves were also occurred or shifted in nanoparticles, indicating that partial hydrogen bonds occurred between the hydrophobic region of zein and compounds. The resulting nanoparticles without surfactant were spherical, small (mean particle size ≈ 125-150 nm), and had a narrow size distribution (polydispersity index < 0.4). Additionally, the encapsulated compounds were in an amorphous as detected by differential scanning calorimetry. These results demonstrate that zein can indeed be used as a carrier of curcumin and its analogues. Especially, zein-CM-F nanoparticle is the most potential inhibitors using in the treatment of gout and diabetes.

碩士
臺北醫學大學
生藥學研究所
97
Curcumin, a yellowish pigment existed in turmeric plant has been shown to exhibit numerous biological properties, such as anti-inflammation, antioxidation, anti-invasion, and antimetastatic activity. Chemical modifications of curcumin have been studied intensively in an attempt to find an analog with similar but enhanced biological activities of curcumin. In literatures, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) played important roles in cancer cell invasion by hydrolysing extracellular matrix (ECM). In this study, curcumin and 23 novel analogs were used to screen for inhibition of matrix metalloproteinase-9 (MMP-9) activities, protein and gene expression of highly metastatic HT-1080 human fibrosarcoma cells to explore the mechanisms of action. All tested compounds, except analog 24 and analog 31 at concentration of 2.5 μM showed no cytotoxic activities at 5 μM. The analog 2 (six methoxy substitutes in phenyl groups) and analog 7 (three hydroxyl and one methoxyl substitutes in phenyl groups) showed higher MMP-9 inhibitory activities than curcumin did at 5 μM in gelatin zymography analysis. We comparatively examined the influence of analog 2, 7, 24 (three hydroxyl substitutes in phenyl groups), and 31(two hydroxyl and two methoxyl substitutes in phenyl groups) on the expression of MMP-9 of HT-1080 cells at concentration of 2.5 μM. Analog 2, 7, 24 and 31 suppressed the gene expression of MMP-9 as the results of RT-PCR assay and Western blot assay, and decreased their corresponding activities in HT-1080 cells revealed by gelatin zymography assay, MMP-9 activity ELISA and fibrin zymography assay which resulted in inhibition of HT-1080 cell invasion and migration differentially. All in all, analog 2 and 7 showed higher anti-metastatic activities than analog 24 and analog 31 did. Heme oxygenase 1(HO-1), a stress-responsive enzyme, which was found to correlate with production of reactive oxygen species (ROS), suggesting a causative relationship on MMP inhibition. Therefore, the effect of curcumin and analog 2, 7, 24 and 31 on HO-1 protein expression were also studied. Interestingly, exposure to curcumin, analog 2 and 31 treatment maximally induce HO-1 protein expression in HT-1080 cells, however, analog 7 and 24 were less apparently. These data suggested that the inhibitory effects of curcumin and analog 2, 7, 24 and 31 on MMP-9 gene expression and uPA activity was closely related to tumor invasion and migration in vitro. Furthermore, analog 2 showed highly MMP-9 inhibitory activity which possibly involved mechanisms related to its ability to induce HO-1 to suppress PMA-induced ROS, which can activate MMP-9 gene expression. In summary, these data demonstrated that analog 2, 7, 24 and 31 show higher anti-metastasis potency than curcumin by the differentially down-regulation of MMP-9 and uPA.

碩士
臺北醫學大學
生藥學研究所
98
Curcumin is the primary bioactive constituent of turmeric, which is an herbal medicine used in Asia for a long time. It has been shown to possess anti-inflammatory, antioxidant, anti-metastatic, and apoptosis- induced properties. In this study, curcumin and 23 different kinds of curcumin analogs were evaluated for cytotoxicity effects on two breast cancer cell lines—MCF-7 and MDA-MB-231. According to the results of cell viability assay, analog 2, 6 and 85 showed stronger growth inhibition activities in both MCF-7 and MDA-MB-231 cell lines than curcumin under concentration of 5 μM and were selected as test candidates. The analog 2, 6, and 85 together with negative control of analog 9 with four to six methoxyl substituents in the phenyl groups were chosen to compare structure-activity relationships. Apoptosis activity was examined by Annexin-V-FITC fluorescence stain by a confocal microscopy under 5 μM treatments. Among them, analog 6 was demonstrated to effectively induce apoptosis activity. By flow cytometry analysis for Annexin-V-FITC/PI fluorescence under concentration of 15 μM treatment for 24 hours, analog 6 showed almost 100% apoptosis in both MCF-7 and MDA-MB-231 cell lines; analog 2 induced 80% apoptosis in MDA-MB-231 cell line; analog 85 exhibited 80% apoptosis in MCF-7 cell line. Cell cycle distribution was arrested by curcumin, analog 2, 6, and 85 in G2/M phase in MCF-7 cells. In MDA-MB-231 cells, curcumin, analog 2, and 85 caused cell cycle arrest in G2/M phase; analog 6 caused cell cycle arrest in G0/G1 phase. Analog 9 showed no influence on cell cycle distribution in both cell lines. To study the mechanism of apoptosis induction by analog 6 and curcumin, cellular reactive oxygen species (ROS) produced levels were detected. It was found that curcumin and analog 6 elevated ROS levels in MDA-MB-231 cells after being treated with 30 minutes. Furthermore, curcumin and analog 6 for 24-h treatments induced heme oxygenase-1 (HO-1) protein expressions in both cell lines. Analog 6 increased MDA-MB-231 cells caspase-9 and caspase-3 actived form expression. We concluded that analog 6 induce MDA-MB-231 cells undergo apoptosis through intrinsic pathway. In addition, according to gelatin zymograhy and wound healing assay, analog 6 inhibits MDA-MB-231 cells migration through suppressing MMP-9 activity.

碩士
臺北醫學大學
生藥學研究所
102
Hydrogen peroxide can produce large amounts of intracellular reactive oxygen species (reactive oxygen species, ROS) which can cause oxidative stress in cells. Many diseases and skin aging are associated with oxidative stress in cells. To find out which drug can reduce intracellular oxidative stress, hydrogen peroxide is used to treat cells for drug screenings. Curcumin, a polyphenol in turmeric, it has been reported to have various biological activities. In this study, we used curcumin and its analogs as materials to find out the protective activities against hydrogen peroxide-induced oxidative stress in HaCaT cells and the possible mechanisms. In the cell viability tests, it was found that the pretreatment of samples 7 and 24 at 5 μM for 2.5-h have significant protection against 150 μM hydrogen peroxide-induced apoptosis in human keratinocyte cell line (HaCaT cells). Using the flow cytometric analysis, it was found that the pretrement of samples 7 and 24 at 5 μM for 2.5-h could reduce the levels of early stage apoptosis in HaCaT cells. The pretreatment of samples 7 and 24 also reduce the expressions of p53 tumor suppressor protein, and reverse the loss of mitochondrial membrane potentials in HaCaT cells. The c-jun, c-fos, and iNOS mRNA expressions in HaCaT cells were also significantly decreased after being pretreated with samples 7 or 24. It was also found that the pretreatments of samples 7 and 24 at 5 μM for 2.5-h could significantly reduce intracellular ROS levels elevated by 150 μM hydrogen peroxide treatments. Using western blotting and enhanced chemiluminescence (ECL) system, it was found that the pretreatments of samples 7 and 24 could significantly induce heme oxygenase-1 (HO-1) expressions and glutathione peroxidase (GPx) activity in HaCaT cells. These results indicate that curcumin analogs, samples 7 and 24, show significantly protective effects against hydrogen peroxide-induced oxidative stress in HaCaT cells which may have protentials in cosmetics to develop anti-aging for skin cares.