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Uticaj soli žučnih kiselina na prodor i metabolizam simvastatina u probiotskim bakterijama / The influence of bile salts on simvastatin transport and metabolism in probiotic bacteriaĐanić Maja 15 September 2016 (has links)
<p>Interindividualne razlike u sastavu i aktivnosti crevne mikroflore mogu uticati na metabolizam lekova kao i na njihov konačan terapijski odgovor. Simvastatin je lek iz grupe statina i karakteriše ga izuzetno mala rastvorljivost u vodi, mala bioraspoloživost (<5%) i velike interindividualne razlike u terapijskom odgovoru čiji uzroci nisu u potpunosti objašnjeni. Poslednjih godina velika pažnja se posvećuje ispitivanjima žučnih kiselina u razvoju novih farmaceutskih formulacija zbog svoje uloge u solubilizaciji i modifikaciji prodora lekova kroz biološke membrane. Zbog svega navedenog, u fokusu našeg istraživanja su bile potencijalne interakcije između simvastatina, probiotskih bakterija i žučnih kiselina o kojima se vrlo malo zna, a od izuzetne su važnosti, zbog mogućeg uticaja na farmakokinetske i farmakodinamske osobine simvastatina, pa samim tim i na konačan terapijski odgovor kod pacijenta.Cilj istraživanja je bio da se ispita prodor i metabolizam simvastatina u probiotskim bakterijama kao i uticaj različitih žučnih kiselina na transport ovog leka u bakterijske ćelije. Takođe, cilj je bio da se ispita uticaj soli žučnih kiselina na distribucioni koeficijent simvastatina, kao i interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija kako bi se objasnila priroda očekivanih interakcija.Identifikacija i kvantifikacija uzoraka vršena je metodom tečne hromatografije sa masenom spektrometrijom (LC-MS/MS). Korišćenjem programskih paketa VolSurf+ i Molinspiration, za identifikovane metabolite su izračunati molekulski deskriptori koji opisuju fizičko-hemijske i farmakokinetske osobine molekula. Određivanje distribucionog koeficijenta vršeno je Shake-flask metodom. Interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija ispitane su doking studijama pomoću SwissDock programa. Prilikom dvadesetčetvoročasovne inkubacije sa probiotskim bakterijama uočen je statistički značajan pad koncentracije simvastatina u ekstracelularnom sadržaju. Ukupan sadržaj simvastatina, kao zbir ekstracelulamog i intracelularnog sadržaja, je tokom čitavog ispitivanog perioda bio statistički značajno niži u odnosu na kontrolnu grupu bez probiotika navodeći na zaključak da se deo simvastatina tokom vremena metabolisao pod dejstvom enzima ispitivanih bakterija. Detektovano je i identifikovano 8 metaboličkih produkata simvastatina. Na osnovu izračunatih vrednosti molekulskih deskriptora, očekuje se da će metabolit M-452, koji predstavlja hidroksilovani produkt simvastatinske kiseline, pokazati najbolje rezultate u pogledu fizičko-hemijskih osobina i bioraspoloživosti u biološkom sistemu. Žučne kiseline nisu dovele do statistički značajne modifikacije transporta simvastatina u/iz probiotskih bakterija. Ipak, u nekim vremenskim tačkama primećena je nešto veća koncentracija leka u ekstracelulamom prostoru u grupama sa žučnim kiselinama. Ove razlike se mogu delimično objasniti rezultatima određivanja distribucionog koeficijenta koji su pokazali da ispitivane žučne kiseline dovode do statistički značajnog smanjenja distribucionog koeficijenta simvastatina usled povećanja rastvorljivosti u vodenoj fazi. Rezultatima doking studija procenjeno je da ispitivane žučne kiseline imaju veći afinitet prema čak 80% multidrug transportera ispitivanih bakterija u odnosu na simvastatin što govori o mogućnosti ostvarivanja interakcija žučnih kiselina sa ovim lekom na nivou transportnih proteina probiotskih bakterija. Na osnovu dobijenih rezultata možemo zaključiti da probiotske bakterije imaju ogroman uticaj na sudbinu simvastatina u biološkom sistemu. Uzimajući u obzir činjenicu da probiotske bakterije ulaze u sastav normalne crevne flore i da svaki organizam poseduje specifičan bakterijski sastav, trebalo bi posvetiti više pažnje ispitivanju njegovog uticaja na farmakokinetiku lekova. Neophodna su dalja in vivo ispitivanja kako bi se utvrdila potencijalna farmakološka aktivnost identifikovanih metabolita simvastatina nastalih pod dejstvom enzimske aktivnosti probiotskih bakterija. Povećanje rastvorljivosti simvastatina pomoću žučnih kiselina otvara mogućnost za dalja istraživanja u cilju razvoja novih farmaceutskih formulacija sa poboljšanom bioraspoloživosti i farmakokinetskim osobinama.</p> / <p>Interindividual differences in the composition and activity of the gut microflora may affect the metabolism of drugs as well as their final therapeutic response. Simvastatin is drug from the group of statins and has extremely low water solubility, low bioavailability (<5%) and high interindividual differences in therapeutic response whose causes are not fully understood. In recent years, great attention has been paid to studies of bile acids in the development of new pharmaceutical formulations because of their role in the drug solubilization and modification of drug transport through biological membranes. Accordingly, interactions between simvastatin, probiotic bacteria and bile acids were the focus of our research due to great importance and potential influence on the pharmacokinetic and pharmacodynamic properties of simvastatin, and therefore the final therapeutic response in the patients. The aim of the study was to investigate the simvastatin transport and metabolism in probiotic bacteria as well as the effect of various bile acids on drug transport into the bacterial cell. Additonally, the aim was to investigate the influence of bile salts on the distribution coefficient of simvastatin, and the interactions of bile acids with simvastatin at the level of probiotic transport proteins in order to elucidate the nature of expected interactions. Identification and quantification of samples were performed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular descriptors that describe the physico-chemical and pharmacokinetic properties of identified metabolites were calculated using the software packages VolSurf+ and Molinspiration. Determination of the distribution coefficient was performed using Shake-flask method. Interaction of bile acids with simvastatin at the level of bacterial transport proteins were studied using docking studies with SwissDock program. During the twenty-four hours of incubation with probiotic bacteria, simvastatin concentrations in the extracellular contet showed a statistically significant decrease. The total amount of simvastatin, as the sum of the extracellular and intracellular amount, during the whole study period, was significantly lower in comparison with control group without probiotics, suggesting that the part of simvastatin was metabolized by the enzymatic activity of studied bacteria. Accordingly, eight metabolic products of simvastatin were detected and identified. Based on the calculated values of molecular descriptors, it is expected that the metabolite M-452, which is the hydroxylated product of simvastatin acid, will show the best results in terms of physico-chemical properties and bioavailability in biological system. Bile acids did not show a significant influence on simvastatin transport into probiotic bacteria. However, in some time points, slightly higher drug concentrations in the extracellular medium in groups with bile acids were observed. These differences can be partly explained by the results of the determination of the distribution coefficients which showed that investigated bile acids lead to a statistically significant decrease in simvastatin distribution coefficient due to increased solubility in the aqueous phase. The results of docking studies estimated that studied bile acids have stronger affinities for the 80% of bacterial multidrug transporters compared to simvastatin indicating the possibility of achieving the interactions of bile acids with simvastatin at the level of transport proteins of probiotic bacteria. Based on the obtained results it could be concluded that probiotic bacteria have great influence on the fate of simvastatin in a biological system. Taking into account the fact that probiotic bacteria are the normal part of gut microflora and that each individual has specific bacterial fingerprint, more attention should be paid on studying its influence on drug pharmakocinetics. Further in vivo studies are required in order to determine potential pharmacological activity of identified simvastatin metabolites. Increased water solubility of simvastatin with bile acids may open the possibility for further investigations with the aim of development of new pharmaceutical formulation with improved bioavailability and pharmacokinetic properties.</p>
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Uticaj žučnih kiselina na prodor u ćelije i tkiva i farmakodinamiku doksorubicina / The influence of bile acids on cell and tissue penetration and pharmacodynamics of doxorubicinStanimirov Bojan 26 March 2018 (has links)
<p>Zahvaljujući amfifilnoj strukturi i mogućnosti građenja konjugata, žučne kiseline - endogeno sintetisani produkti katabolizma holesterola su prepoznate kao potencijalni nosači lekova i promoteri transporta kroz biološke membrane. Otkriće da aktivacijom specifičnih nuklearnih receptora regulišu ekspresiju gena uključenih u plejadu signalnih puteva uključenih u metabolizam, proliferaciju i diferencijaciju ćelija i onkogenezu, proširilo je ulogu žučnih kiselina u odnosu na inicijalno opisanu ulogu intestinalnih emulgatora. Žučne kiseline se danas ne smatraju samo pasivnim nosačima lekova i promoterima transporta kroz biološke membrane već i molekulima sa farmakodinamskom funkcijom, koji regulišu različite aspekte integrativnog ćelijskog metabolizma. Doksorubicin je jedan od najčešće korišćenih antineoplastičkih agenasa i sastavna je komponenta mnogih hemoterapijskih protokola u lečenju solidnih i hematoloških maligniteta. Međutim, hepatotoksični i kardiotoksični efekti značajno ograničavaju upotrebu ovog, inače veoma korisnog antitumorskog agensa. Pojava odložene dozno-zavisne kardiotoksičnosti predstavlja značajan zdravstveni problem onkoloških pacijenata sa uspešno lečenim malignitetom, naročito pacijenata lečenih u pedijatrijskom uzrastu. Budući da je razvoj novih lekova veoma dug i skup proces sa neizvesnim ishodom, poboljšanje farmakodinamskih i farmakokinetskih svojstava već postojećih antitumorskih agenasa sa dokazanom efikasnošću, uz smanjenje toksičnih efekata, predstavlja racionalan istraživački pristup u savremenoj medicini. Osnovni cilj ovog rada je ispitivanje uticaja žučnih kiselina ursodeoksiholne, henodeoksiholne i 12-okso-henodeoksiholne kiseline (12-monoketoholne kiseline) na citotoksičnu aktivnost doksorubicina prema MCF-7 ćelijskoj liniji humanog adenokarcinoma dojke i ispitivanje molekularnih mehanizama odgovornih za farmakodinamske efekte. Takođe su navedene žučne kiseline ispitane kao promoteri transporta koji utiči na prodor i kumulaciju doksorubicina u malignim ćelijama. U ovom radu je ispitan uticaj koadministracije navedenih žučnih kiselina sa doksorubicinom na odložene toksodinamske efekte (hepatotoksičnost i kardiotoksičnost) kod pacova, ali i efekti pretretmana žučnim kiselinama na koncentracije doksorubicina u krvi, bilijarnu ekskreciju leka kao i kumulaciju u jetri i miokardu eksperimentalnih životinja. Žučne kiseline su u netoksičnim koncentracijama potencirale in vitro citotoksične efekte doksorubicina na MCF-7 ćelijskoj liniji pri čemu je henodeoksiholna ispoljila sinergistički efekt, dok su ursodeoksiholna u 12-monoketoholna ispoljile aditivni citotoksični efekt sa doksorubicinom. Ispitivanjem molekularnih mehanizama citotoksičnih efekata utvrđeno je da su žučne kiseline u različitom stepenu potencirale apoptozu ćelija mitohondrijalnim putem uticajem na ekspresiju pro- i antiapoptotskih proteina na transkripcionom nivou i povećale stres endoplazmatskog retikuluma, ali i dovele do alteracija ekspresije gena koji kodiraju sintezu antioksidativnih enzima, transmembranskih efluks proteina i enzima uključenih u metaboličku inaktivaciju leka. Žučne kiseline u netoksičnim koncentracijama su takođe značajno povećale prodor i kumulaciju doksorubicina u MCF-7 ćelijskoj liniji. U in vivo sistemu, koadministracija žučnih kiselina nije rezultovala u poboljšanju odloženih toksodinamskih efekata visokih doza doksorubicina na biohemijskom i molekularnom nivou. Međutim, nakon pretretmana žučnim kiselinama, vrednosti koncetracija doksorubicina u serumu su bile povišene nakon pretretmana urso- i henodeoksiholnom kiselinom i snižene nakon pretretmana 12-monoketoholnom kiselinom uz povećanje bilijarne sekrecije doksorubicina. Pored promena u farmakokinetskom profilu doksorubicina, pretretman žučnim kiselinama je blago redukovao prodor i kumulaciju doksorubicina u hepatocite i kardiomiocite. Na osnovu rezultata ove studije može se zaključiti da primena ispitivanih žučnih kiselina sa doksorubicinom povećava prodor i poboljšava farmakodinamski profil doksorubicina in vitro, na ćelijskom modelu humanog adenokarcinoma dojke. Poboljšanje selektivnog preuzimanja i prodora doksorubicina u maligne ćelije koje nije praćeno povećanom kumulacijom u normalnim tkivima, kao i poboljšanje antitumorskog dejstva doksorubicina sa mogućim smanjenjem doze uz smanjenje pojave dozno-zavisnih neželjenih dejstava doksorubicina čini žučne kiseline molekulima kandidatima za dalja ispitivanja u cilju razvoja novih, poboljšanih antitumorskih terapijskih strategija.</p> / <p>Due to the amphiphilic structure and the significant conjugation potential, bile acids - endogenously synthesized products of cholesterol catabolism have been recognized as potential drug carriers and promoters of transport through biological membranes. The discovery that by activating specific nuclear receptors bile acids regulate the expression of genes involved in various signaling pathways including metabolism, cell proliferation and differentiation as well as carcinogenesis, expanded initially ascribed role of intestinal emulsifiers to the various fields. Bile acids are now considered not to act only as passive carriers of drugs and promoters of transport through biological membranes, but also as the molecules with pharmacodynamic activity, regulating various aspects of integrative cellular metabolism. Doxorubicin is one of the most commonly prescribed antineoplastic agents as an integral component of many chemotherapy protocols in the treatment of both solid and hematologic malignancies. However, hepatotoxic and cardiotoxic effects significantly limit the use of this, otherwise, very useful anti-tumor agent. The development of dose-dependent cardiotoxic side effects represents particular health issue in successfully treated oncological patients, especially among survivors of pediatric malignancies. Since the development of new drugs is very long and expensive process with an uncertain outcome, improving the pharmacodynamic and pharmacokinetic properties of the existing agents with proven efficacy, while reducing toxic side effects, represents a rational approach to research in modern medicine. The main objective of this work is to examine the role of bile acids: ursodeoxycholic, chenodeoxycholic and 12-oxo-chenodeoxycholic acid (12-monoketocholic acid) on the cytotoxic activity of doxorubicin in the MCF-7 human breast adenocarcinoma cell line, and to get insight on molecular mechanisms responsible for underlying pharmacodynamic effects. The capacity of bile acids to promote the transport and accumulation of doxorubicin in malignant cells was also evaluated. In addition, the effect of co-administration of the bile acids with doxorubicin on delayed toxodynamic effects (hepatotoxicity and cardiotoxicity) in rats, as well as the effects of bile acid pretreatment on the doxorubicin serum concentration and pharmacokinetic profile, biliary excretion of the drug as well as accumulation in the liver and myocardial cells of experimental animals were examined. Bile acids applied in non-toxic concentrations potentiated in vitro cytotoxic effects of doxorubicin in MCF-7 cell line. Chenodeoxycholic acid exhibited a synergistic effect, whereas ursodeoxycholic and 12-monoketocholic acid exhibited an additive cytotoxic effect with doxorubicin. By examining the underlying molecular mechanisms of cytotoxic effects, bile acids have been found to potentiate apoptosis of cells by mitochondrial-dependent pathway by modifying the expression of pro- and anti-apoptotic proteins at the transcriptional level and to increase endoplasmic reticulum stress, but also have altered the expression of genes encoding the synthesis of antioxidant enzymes, transmembrane efflux proteins and enzymes involved in metabolic inactivation of the drug. Non-toxic concentrations of bile acids also significantly increased the penetration and accumulation of doxorubicin in MCF-7 cell line. In the in vivo system, the co-administration of bile acid did not improved delayed toxodynamic effects of high dose of doxorubicin both at the biochemical and molecular levels. However, pretreatment with bile acids resulted in alterations of serum doxorubicin concentrations. Chenodeoxycholic and ursodeoxycholic acid elevated whereas 12-monoketocholic acid decreased serum doxorubicin concentrations. In addition to changing pharmacokinetic profile of doxorubicin on bile acid species-dependent manner, all bile acids have also increased excretion of drug by the biliary route, and slightly reduced penetration and accumulation of doxorubicin in hepatocytes and cardiomyocytes. Based on the results of this study, the administration of the examined bile acids with doxorubicin increases the penetration and improves the pharmacodynamic profile of doxorubicin in vitro on the cell model of human breast adenocarcinoma. The improvement of selective uptake and penetration of doxorubicin into malignant cells that is not accompanied by increased accumulation in normal tissues, as well as the improvement in the anti-tumor effects of doxorubicin with a possibility to reduce the dose and thereby the occurrence of dose-dependent undesirable effects of doxorubicin, render bile acids as the potential candidate molecules in developing novel antitumor therapeutic strategies.</p>
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Uloga žučnih kiselina u epigenetskoj regulaciji oksidativnog stresa i apoptoze u normalnim i malignim ćelijama / The role of bile acids in epigenetic regulation of oxidative stress and apoptosis in normal and malignant cellsPavlović Nebojša 09 March 2018 (has links)
<p>Žučne kiseline deluju kao signalni molekuli u organizmu i uključene su u regulaciju brojnih metaboličkih, inflamatornih i imunomodulatornih procesa. Ova endogena jedinjenja ostvaruju svoje efekte najvećim delom putem nuklearnih receptora. Farnezoid X receptor (FXR) je glavni regulator homeostaze žučnih kiselina, a pokazano je da je značajno uključen i u procese inflamacije i kancerogeneze, prevashodno u jetri i intestinalnom traktu. Aktivacija FXR receptora predstavlja značajnu farmakološku strategiju za terapiju holestatskih bolesti jetre, inflamatorne bolesti creva i karcinoma kolona. Definisana je uloga žučnih kiselina u signalnim putevima koji regulišu ćelijski ciklus i doprinose razvoju ili regresiji maligniteta, ali je malo poznat uticaj ovih jedinjenja na epigenetske mehanizme regulacije ključnih ćelijskih procesa. Imajući u vidu da su efekti žučnih kiselina determinisani njihovom polarnošću, cilj istraživanja je bio da se ispita uticaj sintetski dobijenog keto derivata holne kiseline, 12-monoketoholne kiseline (MKH), u komparaciji sa prirodnim žučnim kiselinama, hidrofobnom henodeoksiholnom kiselinom (HDH) i hidrofilnom ursodeoksiholnom kiselinom (UDH), na ćelijske procese apoptoze, oksidativnog stresa i inflamacije, koji su od značaja za hemoprevenciju i terapiju karcinoma kolona, u in vitro i in vivo sistemima. Cilj istraživanja je takođe obuhvatao i ispitivanje uloge odabranih žučnih kiselina u epigenetskoj regulaciji ovih procesa u ćelijama karcinoma kolona. Na in vivo modelu intrahepatične holestaze kod eksperimentalnih životinja, pokazano je da UDH i MKH ispoljavaju antiapoptotski, antioksidativni i antiinflamatorni efekat u jetri i intestinumu. Utvrđeno je da UDH i MKH sprečavaju mitohondrijalni put aktivacije apoptoze u jetri, dok UDH ispoljava antiapoptotski efekat i u intestinumu eksperimentalnih životinja sa holestazom. Ove dve žučne kiseline su u značajnoj meri modulirale ekspresiju gena uključenih u antioksidativnu zaštitu, kao i aktivnost antioksidativnih enzima, u jetri i intestinumu eksperimentalnih životinja sa holestazom, ka nivoima ekspresije i aktivnosti kod zdravih, netretiranih životinja. Dok su UDH i MKH u dozi od 4 mg/kg ispoljile antiinflamatorno dejstvo u jetri i intestinumu smanjenjem ekspresije gena za proinflamatorni transkripcioni faktor NF-κB, primena HDH i MKH u dozi od 20 mg/kg je imala suprotan efekat. Na modelu HT-29 ćelijske linije adenokarcinoma kolona, utvrđeno je da polusintetska žučna kiselina MKH ispoljava značajno manju citotoksičnost u odnosu na HDH i nešto veću citotoksičnost u odnosu na UDH. Epigenetski lek vorinostat je ispoljio sinergističko citotoksično dejstvo sa sve tri ispitivane žučne kiseline. Vorinostat je ostvario proapoptotski i antiproliferativni efekat u HT-29 ćelijama, koji je bio najizraženiji u kombinaciji sa MKH, s obzirom da je došlo do značajnog povećanja odnosa ekspresije BAX i BCL2 gena i smanjenja ekspresije gena za marker proliferacije ciklin D1. Vorinostat je, takođe, značajno smanjio antioksidativni kapacitet HT-29 ćelija smanjenjem ekspresije NRF2 gena i sledstvenim smanjenjem ekspresije gena za antioksidativne enzime. HDH je dodatno smanjila, a MKH poboljšala antioksidativni kapacitet HT-29 ćelija modulacijom ekspresije NRF2 gena. U in vitro i in vivo sistemu u okviru ove doktorske disertacije je pokazano da, pored HDH kao poznatog endogenog agoniste FXR receptora, MKH takođe povećava ekspresiju gena za FXR i njegovog ciljnog gena za transkripcioni korepresor SHP, što ukazuje da ova polusintetska žučna kiselina može da aktivira FXR. Osim toga, utvrđeno je da žučne kiseline ispoljavaju različite efekte prema ekspresiji gena za histon deacetilaze HDAC1 i HDAC2 u jetri i intestinumu eksperimentalnih životinja, kao i u HT-29 ćelijama karcinoma kolona, a jedino je UDH značajno smanjila ekspresiju gena za oba ispitivana enzima uključena u epigenetsku regulaciju ćelijskih procesa, i u isptivanim tkivima i HT-29 ćelijama. Rezultati našeg rada ukazuju da bi se UDH i MKH mogle koristiti u hemoprevenciji karcinoma kolona u niskim dozama, s obzirom na utvrđene efekte u modulaciji ekspresije gena uključenih u procese apoptoze, oksidativnog stresa i inflamacije. Takođe, s obzirom na ostvaren sinergistički efekat žučnih kiselina sa epigenetskim antitumorskim agensom vorinostatom, otvara se mogućnost kombinovane farmakološke strategije u terapiji solidnih tumora, koji u najvećem procentu pokazuju rezistenciju prema samom vorinostatu.</p> / <p>Bile acids act as signaling molecules in the organism and they are involved in the regulation of numerous metabolic, inflammatory and immunomodulatory processes. These endogenous compounds exert their effects mostly by binding and activation of nuclear receptors. Farnesoid X receptor (FXR) is the main regulator of bile acid homeostasis, and has been shown to be significantly involved in processes of inflammation and carcinogenesis, primarily in the liver and intestinal tract. Activation of FXR receptor represents a significant pharmacological strategy for the treatment of cholestatic liver disease, inflammatory bowel disease, and colon carcinoma. The role of bile acids in signaling pathways regulating the cell cycle and contributing to the development or regression of malignancies is well determined, but the effects of these compounds on epigenetic mechanisms of key cellular processes regulation is yet to be elucidated. Given that the effects of bile acids are mostly determined by their polarity, the aim of our study was to investigate in vitro and in vivo effects of semi-synthetic keto derivative of cholic acid, 12-monoketocholic acid (MKC), in comparison to natural bile acids, hydrophobic chenodeoxycholic acid (CDC) and hydrophilic ursodeoxycholic acid (UDC), on processes of apoptosis, oxidative stress and inflammation, which are significant for both chemoprevention and therapy of colon cancer. Besides, the aim of our study was to examine the role of selected bile acids in the epigenetic regulation of these processes in colon cancer cells. In in vivo model of intrahepatic cholestasis in experimental animals, it has been demonstrated that UDC and MKC exhibit antiapoptotic, antioxidant, and antiinflammatory effects in the liver and intestine. It was shown that UDC and MKC prevent the mitochondrial pathway of apoptosis activation in the liver, while UDC exhibits an antiapoptotic effect in the intestine of experimental animals with cholestasis as well. These two bile acids significantly modulated the expression of genes involved in antioxidant protection, as well as the activity of antioxidant enzymes, in the liver and intestine of experimental animals with cholestasis, towards levels of expression and activity in healthy, untreated animals. While UDC and MKC at a low dose of 4 mg/kg exhibited an antiinflammatory effect in the liver and intestine by reducing the expression of the gene encoding the proinflammatory transcription factor NF-κB, the application of CDC and MKC at a high dose of 20 mg/kg exerted the opposite effect. In HT-29 human adenocarcinoma cell line, it has been demonstrated that semi-synthetic bile acid MKC exhibits significantly lower cytotoxicity than CDC and slightly higher cytotoxicity than UDC. The epigenetic drug vorinostat has exhibited a synergistic cytotoxic effect with all three investigated bile acids. Vorinostat exerted proapoptotic and antiproliferative effects in HT-29 cells, which were most pronounced in combination with MKC, as there was a significant increase in the ratio of BAX and BCL2 genes expression and a decrease of the proliferation marker cyclin D1 gene expression. Vorinostat also significantly reduced the antioxidant capacity of HT-29 cells by reducing the expression of NRF2 gene and consequently decreasing the expression of genes encoding antioxidant enzymes. CDC further reduced, while MKC improved the antioxidant capacity of HT-29 cells by modulating the expression of NRF2 gene. In both in vitro and in vivo systems, it was demonstrated that, in addition to CDC as a known endogenous FXR agonist, MKC also increased the expression of the gene encoding FXR, and FXR target gene encoding transcriptional co-repressor SHP as well, indicating that this semi-synthetic bile acid can also activate FXR. Besides, bile acids have been shown to exert distinct effects on the expression of the histone deacetylases HDAC1 and HDAC2 gene in the liver and intestine of experimental animals, and in HT-29 colon cancer cells. Only UDC significantly reduced the expression of the genes for both studied enzymes involved in the epigenetic regulation of cell processes, in both tissues and HT-29 cells. The results of our work indicate that UDC and MKC could be used in chemoprevention of colon cancer at low doses, considering determined effects in the modulation of expression of the genes involved in processes of apoptosis, oxidative stress and inflammation. Furthermore, synergistic effects of bile acids with the epigenetic antitumor agent vorinostat open up the possibility of a combined pharmacological strategy in the treatment of solid tumors, which are at the high percentage resistant to the effects of vorinostat alone.</p>
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Studies on the regulatory roles of cholesterol and bile acids /Murphy, Charlotte, January 2007 (has links)
Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.
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Termodinamička stabilnost odabranih micelarnih sistema žučnih soli značajnih za nove farmaceutske formulacije / Thermodynamic stability of selected bile salt micellar systems relevant for new pharmaceutical formulationsPopović Kosta 27 April 2017 (has links)
<p>Da bi se dobio sistem surfaktanata željenih osobina moguće je hemijski modifikovati već postojeće molekule površinski aktivnih supstanci, a druga mogućnost je konstrukcija binarnih smeša surfaktanata. U farmaceutskoj i prehrambenoj industriji uveliko se primenjuju binarne smeše površinski aktivnih molekula. Ukoliko je binarna mešovita micela termodinamički stabilnija od hipotetičke idealne binarne mešovite micele, onda je kritična micelarna koncentracija binarne smeše surfaktanata niža čak i od hidrofobnije gradivne jedinice mešovite micele, što znači da je za isti efekat površinske aktivnosti potrebna manja količina binarne smeše nego čistog surfaktanta. Različite gradivne jedinice binarne micele u micelarnoj pseudofazi mogu formirati specifične regije koje mogu vezivati lekove određenih strukturnih karakteristika. Pogodno je da jedna gradivna jedinica bude krute konformacije, npr. soli žučnih kiselina, dok je druga gradivna jedinica konformaciono pokretljiva (ugljovodonični nizovi iznad C10). Na taj način se povećava zapremina hidrofobne micelarne faze u odnosu na zapreminu hidrofobne micelarne faze monokomponentne micelle konformaciono krutog surfaktanta, što povećava solubilizacioni kapacitet mešovite micele u odnosu na monokomponentnu micelu krutog surfaktanta. Povećanjem dužine ugljovodoničnog niza konformaciono pokretnog surfaktanta povećava se stepen unutrašnje pokretljivosti u hidrofobnom domenu mešovite micele, što takođe povećava verovatnoću prihvatanja molekula gosta. Micelarni sistemi, kako monokomponentnih micela tako i binarnih mešovitih micela dodatno se mogu termodinamički stabilizovati povećanjem jonske jačine rastvora. Za hidrataciju katjona troše se molekuli vode iz sistema, što povećava efekat desolvatizacije hidrofobne površine surfaktanata, pa se zbog toga pospešuje samoasocijacija.</p> / <p>To obtain the surfactant system with the desired properties it is possible to chemically modify existing molecules of surface active agents. The other possibility is the construction of binary mixtures of surfactants. Binary mixtures of surface active molecules are widely used In the pharmaceutical and food industry. If the binary mixture micelle is more thermodynamically stable than the hypothetical ideal binary mixed micelle, then the critical micellar concentration (CMC) of the binary mixture of surfactants is even lower than the CMC of the more hydrophobic building block of the binary mixture. That means that for the same effect of surface activity less the amount of the binary mixture than the pure surfactants is required. The different building blocks of binary micelles in micelar pseudophase can form specific regions that can bind drugs of certain structural characteristics. It is suitable that one building block is of a rigid conformation, i.e. bile acid salts, while the second building block is of a flexible conformation (above C10 hydrocarbon arrays). In this way the volume of the hydrophobic micellar phase is increased in relation to the volume of the hydrophobic micellar phase of the monocomponent micelles of conformationally rigid surfactant, which increases the capacity of solubilisation of the mixed micelles, compared to the mono-component surfactant micelle of the rigid conformation. By increasing the length of the hydrocarbon array of the the conformational flexible surfactant, the degree of internal mobility in the hydrophobic domain of mixed micelles is also increased, which also increases the likelihood of acceptance of guest molecules. Micellar systems, of both monocomponent micelles and mixed micelles can be additionally thermodynamically stabilized by increasing the ionic strength of the solution. The hydration of cations uses the molecules of water from the system, which increases the effect of desolvatisation of the hydrophobic surface of the surfactants, and therefore promotes self-association.</p>
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Uticaj sintetske i prirodne žučne kiseline na oksidativni stres i apoptozu hepatocita / Influence of synthetic and natural bile acid on oxidative stress and apoptosis in hepatocytesAndrejić Višnjić Bojana 03 March 2016 (has links)
<p>Žučne kiseline (ŽK) su strukturno raznoliki molekuli, koji pored uloge koju ostvaruju putem žuči, deluju i kao signalni molekuli i ostvaruju kako endokrina tako i parakrina dejstva. Činjenica da je do sada u terapijske svrhe primenjivana samo ursodeoksiholna kiselina (UDK), posledica je brojnih ograničenja u mogućnosti primene ostalih prirodnih ŽK, i ističe potrebu za otkrivanjem novih sintetskih ŽK i liganda. Cilj istraživanja bio je ispitivanje sintetske 12-monoketoholne kiseline (MK) i prirodne UDK u modelu holestaze i aloksanom izazvanog dijabetesa. Ispitivanja su vršena na pacovima soja Wistar. Analizirana je telesna masa, glikemija, pokazatelji jetrene funkcije (AST; ALT, γ-GT, ukupni i direktni bilirubin), a iz homogenate jetre određen je intenzitet lipidne peroksidacije i aktivnost antioksidativnih enzima (CAT, GSH-Px, GSH-R, GSH-ST). Isečci tkiva jetre su histološki obrađeni i bojeni hematoksilin-eozin metodom i histohemijskim metodama (retikulin, Mallory, Periodic Acid Schiff- Alcian Blue (PAS/AB)). Imunohistohemijski je ispitana proliferacija hepatocita (Ki-67), markeri apoptoze (p53, Bcl-2, Bcl-X, Bax) i ekspresija nuklearnog farnesoid X receptora (FXR). Rezultati istraživanja pokazuju da ispitivane ŽK pomažu očuvanje telesne mase u holestazi i dijabetesu, i značajno snižavaju glikemiju kod dijabetičnih jedinki. Parametri jetrene funkcije u holestazi i dijabetesu su regulisani primenom MK i UDK. Obe ŽK u značajnoj meri smanjuju intenzitet lipidne peroksidacije i pojačavaju enzimsku antioksidativnu odbranu hepatocita u holestazi i dijabetesu. Ekspresija markera apoptoze nije značajno promenjena izazvanjem modela holestaze i dijabetesa, kao ni primenom ispitivanih ŽK. Nasuprot tome, izazivanje holestaze i dijabetesa značajno smanjuje proliferaciju hepatocita, dok primena MK i UDK poništava ovaj efekat i značajno povećava proliferaciju hepatocita. Hiperglikemija u aloksanskom dijabetesu nije dovela do pojačane ekspresije FXR. Izazivanje holestaze kod zdravih i dijabetičnih životinja dovelo je do porasta ekspresije FXR, koja je redukovana primenom MK i UDK. Na osnovu dobijenih rezultata može se zaključiti da sintetska 12-monoketoholna kiselina pokazuje slična hipoglikemijska, hepatoprotektivna i antioksidativna dejstva kao i prirodna ursodeoksiholna kiselina.</p> / <p>Bile acids (BAs) are structurally diverse molecules, which have theroles in the digestive system, which are exercised through the bile. Beside those, BAs act as a signaling molecules and achieve endocrine and paracrine effects. In addition to its own metabolism, bile acids modulate the metabolism of lipids and glucose. The fact that so far only ursodeoxycholic acid (UDC) is used for therapeutic purposes, speak clearly about of numerous limitations on the application of other natural BAs, and highlights the need to develop new synthetic Bas and ligands. The aim of this study was to investigate the influence of synthetic 12-monoketocholic acid (MC) and natural bila acid UDC in the model of cholestasis and alloxan-induced diabetes. Tests were performed on male Wistar rats. We analyzed the body mass, glucose, liver function tests (AST, ALT, γ-GT, total and direct bilirubin). Using liver tissue homogenates we determined intensity of lipid peroxidation (by concentration of malondilaldehyde) and the activity of antioxidant enzymes (CAT, GSH-Px, GSH -R, GSH-ST). Liver tissue were histologically processed and stained with hematoxylin-eosin method and histochemical methods (reticulin, Mallory, Periodic Acid Schiff- Alcian Blue (PAS / AB)). Imunohistochemical examination included hepatocyte proliferation (Ki-67), markers of apoptosis (p53, Bcl-2, Bcl-X, Bax), and expression of the nuclear farnesoid X receptor (FXR). Results of the research show that MC prevented decrease in body mass during cholestasis and diabetes, and significantly reduced glycemia in diabetic animals. The liver function tests in cholestasis and diabetes are normalised by MC and UDC aplication. Both BAs significantly reduce lipid peroxidation and enhance enzymatic antioxidant defense of hepatocytes in cholestasis and diabetes. The expression of markers of apoptosis was not significantly changed in models of cholestasis and diabetes, as well as the application of the tested BAs. In contrast, in cholestasis and diabetes model, the proliferation of hepatocytes was significantly reduced, while the use of MC and UDC reversed this effect and significantly increased the proliferation of hepatocytes. Hyperglycemia in alloxan-induced diabetes did not lead to overexpression of FXR. Induction of cholestasis in healthy and diabetic animals resulted in an increase in the expression of FXR, which is reduced by using the MK and the UDC. Based on these results we can conclude that a synthetic 12-monoketocholic acid shows similar hypoglycemic, hepatoprotective and antioxidant effects as natural ursodeoxycholic acid.</p>
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Uticaj žučnih kiselina na bioraspoloživost makrolidnih antibiotika / The effects of bile acids on macrolide antibiotics bioavailabilityTrifunović Jovana 13 May 2016 (has links)
<p>Uvod: U prošlosti žučne kiseline su uglavnom razmatrane sa stanovišta njihove funkcije koju obavljaju u crevima jer posreduju u varenju masti i apsorpciji liposolubilnih vitamina. Nedavne studije potvrđuju da žučne kiseline ne igraju samo ulogu u varenju masti, nego se ponašaju i kao signalni molekuli koji stupaju u interakciju sa raznim receptorima uključujući nuklearne receptore i receptore vezane za G-proteine. Kao amfipatični molekuli one su sposobne da reaguju sa fosfolipidima ćelijskih membrana i da poboljšavaju prolazak lekova kroz njih. Stoga se žučne kiseline razmatraju kao promoteri u bukalnim, okularnim i nazalnim farmaceutskim formulacijama. Cilj: Svrha ovog istraživanja je bila da se ispitaju žučne kiseline i njihovi okso derivati kao jedinjenja koja utiču na propustljivost ćelijskih membrana i prolazak lekova do ciljnih tkiva. Materijal i metod: Interakcije makrolidnih antibiotika i žučnih kiselina su ispitivane uz pomoć NMR difuzionih merenja i relaksacije paramagnetičnim jonima. Retencioni parametri odabranih žučnih kiselina su dobijeni korišćenjem hromatografije na normalnim fazama i evaluisani su primenom pet različitih softvera. In vivo ekaperimenti su sprovedeni na 126 eksperimentalnih životinja koje su bile podeljene u 21 grupu. Rezultati: Vezivanje žučnih kiselina za micele je indikovano razlikama u hemijskom pomeranju makrolida i proširenju signala kao posledica redukovane mobilnosti unutar micela. Dodatak micela žučnih kiselina povećava solubilizaciju makrolida za faktor približno 2-3. Sprovedena korelaciona analiza pokazala je značajnu zavisnost između faktora retencije i intestinalne apsorpcije, prodora u MDCK epitelne ćelije, permeabilnost kroz kožu, logBB i PPB%. Putem implementacije in vivo eksperimentalnog dela pokazano je da žučne kiseline utiču na prolazak makrolida u tkivo mozga, bubrega i jetre. Zaključak: Ispitivane žučne kiseline pokazuju dobre farmakokinetske karakteristike i olakšavaju prolazak makrolida kroz različite ćelijske membrane.</p> / <p>Introduction: In the past, bile acids were mostly considered to function in the intestine where they play a role in digestion of fats and mediate absorption of fat-soluble vitamins. Recent studies confirm that bile acids not only facilitate solubilization of fats but behave as signal molecules that interact with various receptors including nuclear receptors and G protein-coupled receptors. As amphipathic molecules they are able to interact with phospholipids of cells membranes and enhance drugs permeation. Thus, bile acids are considered as drug promoters in buccal, ocular, nasal, and transdermal dosage forms. Purpose: The purpose of this research was to investigate bile acids and its oxo derivatives as enhancers in drug permeability. Three research methods to evaluate the characteristics of bile acids and its properties were used. Material and method: The interaction between macrolide antibiotics and bile acids was investigated by NMR chemical-shift titration, self-diffusion measurements and paramagnetic relaxation enhancements. Retention parameters of selected bile acids are acquired by normal-phase thin layer chromatography and evaluated using five different softwares. In vivo experiments were conducted on 126 animals which were divided in 21 groups. Results: Binding bile acids to the micelles is indicated by differences in the chemical shift of the macrolides and line broadening as a consequence of reduced mobility in the micelle. Addition of bile micelles increases the solubility of macrolide antibiotics by a factor of approximately 2–3. Examined correlation analysis confirmed significant dependence between retention factor and intestinal absorption, MDCK epithelial cells, skin permeability, logBB and PPB%. Through the implementation of in vivo experiments it is shown that bile acids promote penetration of macrolides in brain tissue, kidney and liver. Conclusion: Investigated bile acids showed good pharmacokinetic properties and facilitate in macrolides permeation through various membranes.</p>
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Uticaj farmaceutsko-tehnološke formulacije u obliku mikrovezikula sa alginatom na resorpciju gliklazida iz digestivnog trakta pacova / The effect of alginate microcapsules pharmaceutical formulation on gliclazide absorption in rat gastrointestinal tractĆalasan Jelena 24 April 2019 (has links)
<p>Gliklazid je jedan od najčešće korišćenih lekova u terapiji dijabetes melitusa tip 2. U poslednje vreme, utvrđeno je da gliklazid ispoljava i druge pozitivne farmakološke efekte kao što su imunomodulatorni i anti-koagulacioni efekti, ukazujući na njegovu potencijalnu primenu u terapiji dijabetes melitusa tip 1. Gliklazid se odlikuje varijabilnim stepenom apsorpcije nakon peroralne primene i iz tog razloga pretpostavlja se da bi tehnike njegove ciljane isporuke, kao što je mikroinkapsulacija, mogle da dovedu do poboljšanja njegove apsorpcije i njegove potencijalne primene u terapiji T1DM. Pokazano je da različite žučne kiseline, uključujući i holnu, imaju stabilizacione efekte u domenu primene mikrovezikula i kontrolisanog osobađanja lekova, te je moguće da bi njihov dodatak u mikrovezikularnu formulaciju gliklazida mogao dodatno da poboljša oslobađanje gliklazida, njegovu apsorpciju i antidijabetičke efekte. S tim u vezi, cilj ovog istraživanja je da se ispita hipoglikemijski efekat gliklazida primenjenog u obliku alginatnih mikrovezikula, sa ili bez dodatka holne kiseline na T1DM modelu pacova. Trideset šest pacova obolelih od T1DM indukovanog aloksanom i odgovarajuće zdrave kontrolne životinje su nasumično raspoređene u šest grupa (n=6) i tretirane jednokratnom dozom fiziološkog rastvora, suspenzijom gliklazida, gliklazidom u obliku alginatnih mikrovezikula, samo holnom kiselinom, i mikrovezikulama gliklazida sa ili baz dodatka holne kiseline. Uzorkovana je krv tokom 10 h nakon unete doze i merena je koncentracija glukoze u krvi I koncentracija gliklazida u serumu korišćenjem HPLC metode. Mikrovezikule gliklazida su ispoljile hipo-glikemijski efekat kod pacova obolelih od dijabetesa, uprkos njegovim smanjenim koncentracijama u serumu, dok je dodatak holne kiseline u mikrovezikularnu formulaciju smanjio hipoglikemijski efekat gliklazida. Ovo potvrđuje izostanak sinergističkog efekta između gliklazida i holne kiseline. Takođe, ni proces mikroinkapsulacije niti dodatak holne kiseline nisu doprineli poboljšanju apsorpcije gliklazida, što ukazuje na činjenicu da su njegovi hipoglikemijski efekti nezavisni od njegove apsorpcije i koncentracije u serumu. Stoga se može pretpostaviti da su hipoglikemijski efekti gliklazida pre pod uticajem crevno-metaboličke aktivacije nego ciljanog oslobađanja u digestivnom traktu sistemske apsorpcije. Mikrovezikule gliklazida ispoljavaju hipoglikemijski efekat kod pacova obolelih od T1DM nezavisno od insulina, te mogu imati potencijalnu primenu u terapiji T1DM. Ovaj rad su podržali: HORIZON 2020 MEDLEM projekat broj 690876; Projekat Sekretarijata naučnog i tehnološkog razvoja Vojvodine broj . 114-451-2072-/2016-02; Projekat Ministarstva obrazovanja, nauke i tehnološkog razvoja Republike Srbije broja 41012.</p> / <p><!--[if gte mso 9]><xml> <o:DocumentProperties> <o:Author>mladen</o:Author> <o:Version>16.00</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings></xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF/> <w:LidThemeOther>EN-US</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> 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Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Salutation"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Date"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text First Indent"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text First Indent 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Note Heading"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text Indent 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Body Text Indent 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Block Text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Hyperlink"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="FollowedHyperlink"/> <w:LsdException Locked="false" Priority="22" QFormat="true" Name="Strong"/> <w:LsdException Locked="false" Priority="20" QFormat="true" Name="Emphasis"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Document Map"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Plain Text"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="E-mail Signature"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Top of Form"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Bottom of Form"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Normal (Web)"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Acronym"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Address"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Cite"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Code"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Definition"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Keyboard"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Preformatted"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Sample"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Typewriter"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="HTML Variable"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Normal Table"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="annotation subject"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="No List"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Outline List 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Simple 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Classic 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Colorful 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Columns 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 6"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 7"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Grid 8"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 4"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 5"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 6"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 7"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table List 8"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table 3D effects 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Contemporary"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Elegant"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Professional"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Subtle 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Subtle 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 1"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 2"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Web 3"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Balloon Text"/> <w:LsdException Locked="false" Priority="39" Name="Table Grid"/> <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true" Name="Table Theme"/> <w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/> <w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading"/> <w:LsdException Locked="false" Priority="61" Name="Light List"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/> <w:LsdException Locked="false" Priority="70" Name="Dark List"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/> <w:LsdException Locked="false" SemiHidden="true" Name="Revision"/> <w:LsdException Locked="false" Priority="34" QFormat="true" Name="List Paragraph"/> <w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/> <w:LsdException Locked="false" Priority="30" QFormat="true" Name="Intense Quote"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/>
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Chemoprevention for Colorectal CancerKrishnan, K, Ruffin, M T., Brenner, D E. 01 March 2000 (has links)
No description available.
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