Browsing by Subject "MDR1"
Now showing items 1-6 of 6
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2017)Genetiikan tutkimusmenetelmien kehittymisen myötä 2000-luvulla on löydetty useita geneettisiä, lääkeaineiden tehoon ja turvallisuuteen vaikuttavia eroja eläinlajien ja -yksilöiden välillä. Tietoa näistä eroista ei ole aiemmin koottu yhteen suomen kielellä. Kirjallisuuskatsaus keskittyy farmakogeneettisiin eroihin farmakokinetiikassa. Farmakodynaamiset erot ja lääkeaineiden väliset yhteisvaikutukset on rajattu tutkielman aiheen ulkopuolelle. Kuljetinproteiinit kuljettavat aktiivisesti muun muassa lääkeaineita solukalvojen yli soluun sisään tai solusta ulos. Kuljetinproteiineja ilmennetään muun muassa suolistossa, maksassa, munuaisissa, veri-aivoesteessä ja veri-verkkokalvoesteessä ja niillä on merkitys lääkeaineiden jakautumisessa elimistöön. Eläimillä tutkituin kuljetinproteiini on p-glykoproteiini, jonka tehtävä on poistaa lääke- ja vierasaineita esimerkiksi veri-aivoesteessä keskushermostosta. P-glykoproteiinia koodaa ABCB1-geeni (aiemmin MDR1-geeni), jossa useilla koiraroduilla (kuten colliet ja collie-sukuiset rodut) esiintyvä mutaatio aiheuttaa puutteellisen proteiinin muodostumisen ja sitä kautta altistaa tiettyjen lääkeaineiden, kuten ivermektiinin, hermostotoksisille haittavaikutuksille. ABCG2-geeni koodaa ABCG2-kuljetinproteiinia, joka estää lääke- ja vierasaineiden pääsyä esimerkiksi verkkokalvolle veri-verkkokalvoesteessä. Kissalla ABCG2-proteiini on puutteellinen, mikä altistaa kissan esimerkiksi fluorokinolonien aiheuttamalle retinatoksisuudelle ja toisaalta saattaa myötävaikuttaa kissan parasetamoliherkkyyteen. CYP450-entsyymijärjestelmä käsittelee lääkeaineita elimistössä helpommin eritettävään muotoon. CYP-entsyymejä ilmennetään muun muassa maksassa, munuaisissa ja suolistossa ja niiden aktiivisuudessa esiintyy vaihtelua eläinlajien ja -yksilöiden välillä. Vaihtelu entsyymien aktiivisuudessa saattaa johtaa lääkeaineiden tehon puutteeseen, yllättäviin haittavaikutuksiin tai esimerkiksi riittämättömään varoaikaan. Monet rauhoittavina aineina tai anestesiassa käytettävät lääkeaineet metaboloituvat CYP450-entsyymijärjestelmän kautta ja vaihtelu entsyymien aktiivisuudessa saattaa johtaa suurempaan tai pienempään annostarpeeseen eri koiraroduilla. Koiralla ja kissalla esiintyy lajinsisäistä vaihtelua tiopuriinimetyylitransferaasientsyymin (TPMT) aktiivisuudessa. Tämä vaihtelu voi johtaa esimerkiksi atsatiopriinin tehon puutteeseen tai yllättäviin haittavaikutuksiin. Koiralta puuttuvat N-asetyylitransferaasientsyymejä (NAT1 ja NAT2) koodaavat geenit ja kissalta puuttuvat NAT2-entsyymiä koodaavat geenit, millä voi olla vaikutusta esimerkiksi näiden lajien herkkyyteen sulfonamideille ja parasetamolille. Kissalta puuttuu myös UDP-glukuronosyylitransferaasientsyymi (UGT), mikä johtaa puutteelliseen parasetamolin metaboliaan ja aiheuttaa parasetamolitoksisuutta kissalle jo pienillä annoksilla. Kirjallisuuskatsausta voidaan hyödyntää eläinlääkärien käytännön työssä suunniteltaessa lääkehoitoja. Farmakokineettisten erojen tunteminen auttaa arvioimaan sopivaa lääkeannosta esimerkiksi valmisteyhteenvedosta poikkeavassa käytössä. Tutkielman tarkoitus on tuoda eläinlääkärien tietoisuuteen muitakin kuin tutkituimpia geneettisen vaihtelun aiheuttajia. Kirjallisuuskatsaus toimii myös tukena apteekkien farmaseuttisessa työssä valittaessa eläimelle sopivaa itsehoitoon tarkoitettua eläinlääkettä. Lisää tutkimustietoa tarvitaan geneettisten erojen kliinisestä merkityksestä.
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(2013)Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.
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(2012)Elucidation of transporter- and/or metabolic enzyme-mediated drug interactions is important part of early drug development. However the knowledge about clinical consequences of transporter-mediated drug-drug interactions is still limited and more investigation is needed to improve our understanding. MDR1 transporter, widely distributed on the pharmacokinetic barriers in the body (e.g. intestine) and has been shown no limit the bioavailability of drugs. Substrates of MDR1 are exposed to limited intestinal drug absorption and intestinal drug-drug interactions due to inhibition of the transporter. In predicting the clinical significance of an interaction, the principal obstacle has been the limited ability to appropriately scale the preclinical data into in vivo situation. In vitro-in vivo correlations on the extent of MDR1's influence on absorption and standardized predicting methods for drug-drug interactions using the inhibitory constants (IC50 and Ki) would greatly increase the value of in vitro studies. Current in vitro and in silico methods for prediction of the influence of MDR1 on intestinal absorption and related drug-drug interactions are discussed in the literature review. In addition, the latest regulatory draft guidances (FDA, EMA) are reviewed. Aliskiren has been shown to be a sensitive MDR1 substrate in vivo and high affinity substrate for the transporter in vitro. The objective of the experimental work was to study the MDR1-mediated transport of aliskiren and the related drug-drug interactions in vitro and in silico. Vesicular transport assay was used to obtain kinetic parameters for aliskiren (Km and Vmax) and inhibitor potencies (IC50) for ketoconazole, verapamil, itraconazole and its metabolite hydroxyitraconazole. Ki was further calculated for itraconazole and hydroxyitraconazole. Aliskiren showed high affinity to MDR1 transporter with a Km value 5 µM, consistent to what was reported previously in different assay systems. The interactions between aliskiren and the inhibitors in vitro correlated to the observed interactions in vivo in humans. In addition, hydroxyitraconazole was shown to be a potent inhibitor of MDR1-mediated transport of aliskiren in vitro. This suggests that hydroxyitraconazole may contribute to the pronounced interaction observed between aliskiren and itraconazole in a clinical interaction study. A compartmental absorption and transit (CAT) model with added enterocyte compartments and MDR1 efflux was used to describe the influence of MDR1 on intestinal absorption of aliskiren in humans. The integration of kinetic parameters (Km) from in vitro studies requires further optimization on how to describe the intracellular drug concentrations in the model. Aliskiren is however suitable MDR1 probe substrate to be used in in vitro and in vivo trials in humans and therefore gives a good basis for developing vitro-in vivo predictive models.
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(2017)Multidrug resistance protein 1 (MDR1, p-glycoprotein) belongs to the ATP-binding cassette transporter family and it's encoded by ABCB1/MDR1 gene. It is a protein which transports many different kinds of compounds out of cells, for example from endocytes to the lumen with the use of energy from ATP. MDR1 is there for a restrictive factor for several orally administered drugs. It`s important to have knowledge about MDR1-inhibitors, in order to avoid harmful drug-drug and food-drug interactions that might affect medical treatment. The purpose of this master's thesis was to optimize an in vitro MDR1-vesicle uptake method and use it to screen inhibitors from compound libraries. To optimize the method, the effect of cholesterol loading on ATP-dependent transport of test substrate N-methylquinidine (NMQ) was evaluated, transport kinetics of the vesicles and kinetics of known inhibitors were also tested. With the optimized method, screening was done with a library of 25 food additives and a library of 42 synthetic compounds. The chemical structures of the synthetic compounds were analyzed manually in order to find factors that could explain their ability to inhibit MDR1. Only one inhibitor was found among food additives: curcumin. Other additives didn't increase or decrease the ATP-dependent transport of NMQ. Several inhibitors were found from the library of synthetic compounds, also a couple of compounds were found to increase the active transport of NMQ. Results indicate, that the additives used in this study have low risk to cause MDR1 mediated interactions, if curcumin is excluded. The inhibitory effect of curcumin should be investigated in in vivo-situation, because vesicle-based in vitro-results have tendency to overestimate results. Screening results of the synthetic compounds gives more confirmation to the usefulness of the screening method. The MDR1-inhibition screening method described in this Master`s thesis is valid, and it can be used to screen different compound libraries for MDR1-inhibitors. In the future it could be used to screen different kinds of compounds, which might end up inside humans and cause interactions with drugs.
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(2021)P-glycoprotein is an efflux transporter of the ABC family. It is expressed mainly in tissues that have a role in limiting the absorption and distribution of xenobiotics in the body or their elimination. P-glycoprotein is known to have an important role for example in the blood-brain barrier and in protecting the fetus from xenobiotics in the mother’s blood stream. Genetic polymorphisms in transporter proteins can cause individual differences in the pharmacokinetics of drug substances, which can lead to differences in drug efficacy or side effects. In the ABCB1 gene, which codes for p-glycoprotein, several polymorphisms have been discovered. The frequencies of these polymorphisms vary in different ethnic populations. Previous studies have shown that the effects of these polymorphisms are often substrate-dependent. Since there are several confounding factors usually present in clinical association studies, in vitro studies are needed to clarify the effects of individual polymorphisms. Polymorphisms can be studied in vitro by making intentional mutations to the gene sequence and expressing the variant gene in a suitable cell line. In this study four variant p-glycoprotein genes (c.781A>G, c.1199G>T, c.2005C>T and c.3421T>A) were created by site-directed mutagenesis, and expressed in HEK293 cells using a baculovirus recombinant protein expression method. The effects of the polymorphisms were studied by determining the expression level and the transport acitivity of the variant proteins compared to the wild-type. Western blot was used to determine the expression level and a calcein accumulation assay in HEK293 cells was used to compare the transport activities. Also a membrane vesicle transport assay with n-methyl quinidine was set up and optimized, but the variants were not yet studied with this method during this study. In this study no statistically significant differences were found in the transport activities of any of the four variants compared to the wild-type p-glycoprotein. Also the differences in protein expression level between wild-type and variant proteins were small. However, because of the previously reported substrate dependency of polymorphism effects, it would be beneficial to study the variants with at least one other substrate and one other assay method, and thus the membrane vesicle transport assay would be useful to further compare the transport activities of variant proteins to the wild-type p-glycoprotein.
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(2021)P-glycoprotein is an efflux transporter of the ABC family. It is expressed mainly in tissues that have a role in limiting the absorption and distribution of xenobiotics in the body or their elimination. P-glycoprotein is known to have an important role for example in the blood-brain barrier and in protecting the fetus from xenobiotics in the mother’s blood stream. Genetic polymorphisms in transporter proteins can cause individual differences in the pharmacokinetics of drug substances, which can lead to differences in drug efficacy or side effects. In the ABCB1 gene, which codes for p-glycoprotein, several polymorphisms have been discovered. The frequencies of these polymorphisms vary in different ethnic populations. Previous studies have shown that the effects of these polymorphisms are often substrate-dependent. Since there are several confounding factors usually present in clinical association studies, in vitro studies are needed to clarify the effects of individual polymorphisms. Polymorphisms can be studied in vitro by making intentional mutations to the gene sequence and expressing the variant gene in a suitable cell line. In this study four variant p-glycoprotein genes (c.781A>G, c.1199G>T, c.2005C>T and c.3421T>A) were created by site-directed mutagenesis, and expressed in HEK293 cells using a baculovirus recombinant protein expression method. The effects of the polymorphisms were studied by determining the expression level and the transport acitivity of the variant proteins compared to the wild-type. Western blot was used to determine the expression level and a calcein accumulation assay in HEK293 cells was used to compare the transport activities. Also a membrane vesicle transport assay with n-methyl quinidine was set up and optimized, but the variants were not yet studied with this method during this study. In this study no statistically significant differences were found in the transport activities of any of the four variants compared to the wild-type p-glycoprotein. Also the differences in protein expression level between wild-type and variant proteins were small. However, because of the previously reported substrate dependency of polymorphism effects, it would be beneficial to study the variants with at least one other substrate and one other assay method, and thus the membrane vesicle transport assay would be useful to further compare the transport activities of variant proteins to the wild-type p-glycoprotein.
Now showing items 1-6 of 6