Browsing by Subject "ABCB1"
Now showing items 1-5 of 5
-
(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.
-
(2023)P-glycoprotein (ABCB1, MDR1) is an efflux transporter expressed widely through the body, but mainly focused on tissues that have protective or excretive function, such as liver and blood-brain-barrier. Many clinically used drugs from variety of therapeutic groups are substrates of P-glycoprotein, and changes in the function of P-glycoprotein may have impact on the drugs pharmacokinetics and -dynamics. The impact of genetic polymorphism on P-glycoprotein activity have been investigated for several years, but due to contradictory results no consensus has been made. The aim of this Master’s thesis was to investigate the effect of five different P-glycoprotein single nucleotide polymorphisms (SNPs) on transport activity. The study was performed by Spodoptera frugiperda (Sf9) membrane vesicles expressing P-glycoprotein variants. Baculovirus-derived expression system was used to introduce the ABCB1 gene to the cells. Vesicle assay was performed with N-methylquinidine (NMQ), and ATP-dependent transport of P-glycoprotein variants was compared to the reference gene. Amino acid change Cys717Tyr led to no transport activity compared to reference gene, and Arg669Cys associated with higher transport activity of NMQ. Arg588Cys, Ser795Cys and Ile836Val indicated no effect on the transport activity. Other aim for this Master’s thesis was to create a new in-house protocol to study P-glycoprotein polymorphism in vitro. Substrate accumulation assay for Rhodamine-123 in Sf9 cells analysed with flow cytometry was established, as flow cytometry is widely used method in other laboratories to study P-glycoprotein polymorphism. The baseline for flow cytometry assay was created successfully by optimizing substrate concentration and incubation time. According to the results, SNPs can impair P-glycoprotein function. New method to study P-glycoprotein function was created, and this method can be used to further study the effects of genetic polymorphism of P-glycoprotein and to compare the result between studies. The results gained from these in vitro studies can be utilized to understand in vivo pharmacogenetic findings.
-
(2023)P-glycoprotein (ABCB1, MDR1) is an efflux transporter expressed widely through the body, but mainly focused on tissues that have protective or excretive function, such as liver and blood-brain-barrier. Many clinically used drugs from variety of therapeutic groups are substrates of P-glycoprotein, and changes in the function of P-glycoprotein may have impact on the drugs pharmacokinetics and -dynamics. The impact of genetic polymorphism on P-glycoprotein activity have been investigated for several years, but due to contradictory results no consensus has been made. The aim of this Master’s thesis was to investigate the effect of five different P-glycoprotein single nucleotide polymorphisms (SNPs) on transport activity. The study was performed by Spodoptera frugiperda (Sf9) membrane vesicles expressing P-glycoprotein variants. Baculovirus-derived expression system was used to introduce the ABCB1 gene to the cells. Vesicle assay was performed with N-methylquinidine (NMQ), and ATP-dependent transport of P-glycoprotein variants was compared to the reference gene. Amino acid change Cys717Tyr led to no transport activity compared to reference gene, and Arg669Cys associated with higher transport activity of NMQ. Arg588Cys, Ser795Cys and Ile836Val indicated no effect on the transport activity. Other aim for this Master’s thesis was to create a new in-house protocol to study P-glycoprotein polymorphism in vitro. Substrate accumulation assay for Rhodamine-123 in Sf9 cells analysed with flow cytometry was established, as flow cytometry is widely used method in other laboratories to study P-glycoprotein polymorphism. The baseline for flow cytometry assay was created successfully by optimizing substrate concentration and incubation time. According to the results, SNPs can impair P-glycoprotein function. New method to study P-glycoprotein function was created, and this method can be used to further study the effects of genetic polymorphism of P-glycoprotein and to compare the result between studies. The results gained from these in vitro studies can be utilized to understand in vivo pharmacogenetic findings.
-
(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.
-
(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 1-5 of 5