Lääkeaineen kulkeutumisen tutkiminen veri-aivoesteessä ja pglykoproteiinin vaikutus lääkkeiden jakautumiseen aivoihin (kirjallisuuskatsaus) : P-glykoproteiinin efluksin mallintaminen veri-aivoesteessä : mahdollisuuksia in vivosta in vitroon (erikoistyö)

The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.Veri-aivoeste säätelee tarkasti sekä endogeenisten aineiden että vierasaineiden pääsyä aivoihin tiiviiden liitosten sekä erikoistuneiden kuljetinproteiinien ja metaboliaentsyymien avulla. Koska lääkeainepitoisuuksien määrittäminen aivoissa on työlästä ja kallista, on pyritty kehittämään menetelmiä, joiden avulla pitoisuuksia voisi ennustaa. Jo lääkekehityksen alkuvaiheessa on tärkeää ennustaa pitoisuuksia, jotta keskushermostoon vaikuttavien lääkkeiden teho ja muualle elimistöön kohdennettujen lääkkeiden turvallisuus voidaan varmistaa. Kirjallisuuskatsaus kuvaa yleisimmät käytössä olevat in vitro, in vivo ja in silico -menetelmät, joiden avulla lääkeaineiden kulkeutumista aivoihin ja erityisesti aktiivista kuljetusta veri-aivoesteessä tutkitaan. Katsauksessa tarkastellaan myös p-glykoproteiinin, veri-aivoesteen tunnetuimman kuljetinproteiinin, mahdollisia vaikutuksia lääkehoitoon. Erikoistyön tavoite oli rakentaa farmakokineettinen (PK) malli kuvaamaan p-glykoproteiinin substraattien pitoisuuksia aivoissa. Mallissa käytettiin yleisesti mitattuja parametreja, jotka kuvaavat yhdisteiden jakautumista aivoihin. Työssä tutkittiin myös mahdollisuutta korvata in vivo -parametriarvoja vastaavilla in vitro -arvoilla. Kaikki tutkimuksessa käytetty data otettiin kirjallisuudesta. Yksinkertainen 2-tilamalli rakennettiin STELLA™-ohjelmalla. Mallilla simuloitiin morfiinin, loperamidin ja kinidiinin pitoisuuksia aivoissa ja tuloksia verrattiin julkaistuihin tutkimuksiin. Eri tutkimuksissa mitattujen in vitro -efluksisuhteiden (efflux ratio, ER) arvoja vertailtiin ja tutkittiin korrelaatiota in vitro ja in vivo mitattujen ER-arvojen välillä. Toinen STELLA™-malli rakennettiin jäljittelemään in vitro -permeabiliteettikoetta ja sitä käytettiin arvioimaan mitatun ER:n herkkyyttä passiivisen permeabiliteetin ja efluksin Michaelis-Menten parametriarvojen vaihtelulle. Lajien välisiä eroja tarkasteltiin lääkeaineiden aivopermeabiliteetin ja aivokudokseen sitoutumisen osalta. PK malli onnistui kuvaamaan kaikkien kolmen lääkeaineen pitoisuus-aikaprofiilien muodon sekä aivoissa että plasmassa. Morfiinin aivopitoisuuksien ennustus oli hyvä, mutta malli yliarvioi loperamidin ja aliarvioi kinidiinin pitoisuudet aivoissa. Koska lääkeaineen pitoisuuksien suhde aivoissa ja veressä on riippuvainen ER-arvosta, epäonnistumisen yksi mahdollinen syy on ER-arvon epätarkkuus, sillä siitä on esitetty vaihtelevia arvioita kirjallisuudessa. Jotta voitaisiin vetää lisää johtopäätöksiä, on mallia validoitava laajemmalla yhdistejoukolla. In vitro ER-arvojen korrelaatio tutkimusten välillä oli vaihtelevaa, mikä viittaa koeolosuhteiden, kuten lääkeaineen pitoisuuden, vaikutukseen. Kaiken kaikkiaan erot olivat kuitenkin pieniä. Korrelaatio in vivo ja pienillä pitoisuuksilla määritetyn in vitro ERarvon välillä oli hyvä, mikä puoltaa in vitro -arvojen mahdollista käyttöä PK mallissa tämän parametrin osalta. In vitro -simulaatio osoitti, että tässä simulaatiossa efluksin vaikutus oli merkittävä vain kun passiivinen permeabiliteetti on pieni. Tämä korostaa sitä, että ER-arvon määrittämiseen käytettävän solumallin tulee olla riittävän tiivis, jotta tulos vastaisi in vivo tilannetta

In vitro evaluation of the pharmacokinetic effects of BCRP interactions

Transport proteins embedded in the cell membranes of many organs can affect the absorption, distribution and elimination of numerous drugs. This can lead to the enhanced or restricted uptake or distribution of the drugs, nonlinear pharmacokinetics, transporter-mediated drug-drug interactions (DDIs) and inter-individual variability. Transporters may therefore alter the safety and efficacy of drugs, thus it is important to study drug-transporter interactions in drug development. The breast cancer resistance protein (BCRP, ABCG2) is one of the transporters involved in drug disposition. It belongs to the ATP-binding cassette (ABC) transporter family and uses ATP to expel drugs and other substrates out from cells. BCRP was initially found to cause drug resistance in cancer cell lines, but it is also expressed in healthy tissues such as the intestine, liver and blood-brain barrier, where it is one of the transporters limiting the uptake of many structurally diverse compounds. Despite interest in BCRP and other ABC transporters, it remains poorly understood how they recognize their substrates and which chemical structures are liable to interaction. In this thesis, a vesicle-based in vitro method was used to study the ligand preferences of BCRP. The results were compared to those obtained for the multidrug resistance associated protein 2 (MRP2, ABCC2), which is also implicated in drug transport. The results show that a range of natural compounds and their derivatives are able to inhibit BCRP transport and among these, flavonoids were identified as the most important group for inhibition. Conversely, MRP2 transport was affected by only few of the tested compounds. However, a more similar pattern of inhibition was seen for the two transporters when selected food additives were studied, where several food colourants were identified as inhibitors. In addition, the effect of one assay component (bovine serum albumin, BSA) on the in vitro transport kinetics of BCRP and MRP2 was evaluated. The inclusion of BSA in the vesicle assay lead to moderate changes (up to 2-fold) in transport activity, but the effects on in vitro − in vivo extrapolation are expected to be minor, at least based on the tested compounds. Finally, the vesicle assay was used to study the functionality of selected BCRP variants with polymorphisms in the transmembrane helices and they were found to have significantly impaired transport activity and expression compared to wild type BCRP. In summary, the vesicle-based transport assay was successfully applied to identify and evaluate the effects that BCRP interactions may have on the pharmacokinetics of BCRP substrates.Lääkkeiden kulkuun elimistössä voivat vaikuttaa kuljetinproteiinit, joita ilmenee monien kudosten solukalvoilla. Nämä proteiinit voivat edistää tai estää lääkkeiden imeytymistä ja jakautumista kudoksiin, aiheuttaa yksilöiden välistä vaihtelua tai välittää lääkeinteraktiota. Näin ollen kuljetinproteiinit voivat vaikuttaa lääkeaineiden tehoon ja turvallisuuteen minkä vuoksi on tärkeää tutkia kuljetinproteiinien toimintaa sekä vuorovaikutusta lääkkeiden kanssa. Rintasyöpäresistenssiproteiini (engl. breast cancer resistance protein, BCRP) on yksi lääkeaineiden kulkuun vaikuttava ABC-perheen kuljetinproteiini. ATP:sta saamansa energian avulla se poistaa lääkkeitä ja muita yhdisteitä soluista suojaten elimistöä. BCRP tunnistettiin alun perin syöpälääkkeille vastustuskykyisistä soluista, mutta sitä ilmentyy myös terveissä kudoksissa kuten suolessa, maksassa ja veri-aivoesteessä, joissa se rajoittaa monenlaisten yhdisteiden kulkua. On epäselvää, miten BCRP on vuorovaikutuksessa kuljettamiensa yhdisteiden kanssa ja mitkä rakenteelliset ominaisuudet ovat tärkeitä tälle vuorovaikutukselle. Tässä väitöskirjassa tutkittiin BCRP:n vuorovaikutusta erilaisten yhdisteiden kanssa, mukaan lukien jotkin luonnonaineet, luonnonainejohdannaiset ja elintarvikkeiden lisäaineet. Näitä tuloksia verrattiin monilääkeresistenssiin liittyvän proteiinin 2 (engl. multidrug resistance associated protein 2, MRP2) tutkimuksissa saatuihin tuloksiin. Myös MRP2 osallistuu joidenkin lääkeaineiden kuljetukseen elimistössä. Solukalvovesikkeleihin perustuvan in vitro – koeasetelman tulosten perustella useat luonnonaineet, erityisesti flavonoidit, voivat estää BCRP:n toimintaa. Vain harva näistä yhdisteistä vaikutti MRP2:n toiminaan, mutta lisäainetutkimuksissa löydettiin useita väriaineita, jotka estivät molempia kuljetinproteiineja. Näin ollen on mahdollista, että ravinnosta saadut yhdisteet voivat estää BCRP:n toimintaa, mutta riski MRP2-vuorovaikutukseen on pienempi. Väitöskirjassa tutkittiin myös, miten albumiinin lisääminen muuttaa näiden proteiinien toimintaa käytetyssä vesikkelikokeessa. Albumiini tehosti kuljetusta hieman (enintään kaksinkertaiseksi), mutta oletettu vaikutus in vivo ennusteisiin on ainakin tässä tutkimuksessa tutkittujen aineiden perusteella vähäinen. Viimeisenä vesikkelikokeen avulla tutkittiin valikoitujen BCRP:n perinnöllisten varianttien toimintaa ja todettiin, että BCRP:n solukalvoja läpäisevissä osissa olevat muutokset voivat vaikuttaa haitallisesti sen ilmentymiseen ja kuljetuskykyyn

Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ± 1.11 μM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda-derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,' 7'-dichlorofluorescein (CDCF), estradiol-17 beta-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 +/- 1.11 mu M. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.Peer reviewe

The Effect of Albumin on MRP2 and BCRP in the Vesicular Transport Assay

The ABC transporters multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are of interest in drug development, since they affect the pharmacokinetics of several drugs. Membrane vesicle transport assays are widely used to study interactions with these proteins. Since albumin has been found to affect the kinetics of metabolic enzymes in similar membrane preparations, we investigated whether albumin affects the kinetic parameters of efflux transport. We found that albumin increased the V-max of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) and estradiol-17-beta-D-glucuronide uptake into MRP2 vesicles in the presence of 0.1% bovine serum albumin (BSA) by 2 and 1.5-fold, respectively, while BSA increased Lucifer yellow uptake by 30% in BCRP vesicles. K-m values increased slightly, but the change was not statistically significant. The effect of BSA on substrate uptake was dependent on the vesicle amount, while increasing BSA concentration did not significantly improve substrate uptake. These results indicate a minor effect of albumin on MRP2 and BCRP, but it should be considered if albumin is added to transporter assays for example as a solubilizer, since the effect may be substrate or transporter specific.Peer reviewe

Efflux transport of nicotine, cotinine and trans-3 '-hydroxycotinine glucuronides by human hepatic transporters

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3 '-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K-m value of 895 mu mol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 mu mol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.Peer reviewe

The Role of Uptake and Efflux Transporters in the Disposition of Glucuronide and Sulfate Conjugates

Glucuronidation and sulfation are the most typical phase II metabolic reactions of drugs. The resulting glucuronide and sulfate conjugates are generally considered inactive and safe. They may, however, be the most prominent drug-related material in the circulation and excreta of humans. The glucuronide and sulfate metabolites of drugs typically have limited cell membrane permeability and subsequently, their distribution and excretion from the human body requires transport proteins. Uptake transporters, such as organic anion transporters (OATs and OATPs), mediate the uptake of conjugates into the liver and kidney, while efflux transporters, such as multidrug resistance proteins (MRPs) and breast cancer resistance protein (BCRP), mediate expulsion of conjugates into bile, urine and the intestinal lumen. Understanding the active transport of conjugated drug metabolites is important for predicting the fate of a drug in the body and its safety and efficacy. The aim of this review is to compile the understanding of transporter-mediated disposition of phase II conjugates. We review the literature on hepatic, intestinal and renal uptake transporters participating in the transport of glucuronide and sulfate metabolites of drugs, other xenobiotics and endobiotics. In addition, we provide an update on the involvement of efflux transporters in the disposition of glucuronide and sulfate metabolites. Finally, we discuss the interplay between uptake and efflux transport in the intestine, liver and kidneys as well as the role of transporters in glucuronide and sulfate conjugate toxicity, drug interactions, pharmacogenetics and species differences.Peer reviewe

Novel inhibitors of breast cancer resistance protein (BCRP, ABCG2) among marketed drugs

Drug-drug interactions (DDIs) are a major concern for the safe use of medications. Breast cancer resistance protein (BCRP) is a clinically relevant ATP-binding cassette (ABC) transporter for drug disposition. Inhibition of BCRP increases the plasma concentrations of BCRP substrate drugs, which potentially could lead to adverse drug reactions. The aim of the present study was to identify BCRP inhibitors amongst a library of 232 commonly used drugs and anticancer drugs approved by the United States Food and Drug Administration (FDA). BCRP inhibition studies were carried out using the vesicular transport assay. We found 75 drugs that reduced the relative transport activity of BCRP to less than 25% of the vehicle control and were categorized as strong inhibitors. The concentration required for 50% inhibition (IC50) was determined for 13 strong inhibitors that were previously poorly characterized for BCRP inhibition. The IC50 ranged from 1.1 to 11 mu M, with vemurafenib, dabigatran etexilate and everolimus being the strongest inhibitors. According to the drug interaction guidance documents from the FDA and the European Medicines Agency (EMA), in vivo DDI studies are warranted if the theoretical intestinal luminal concentration of a drug exceeds its IC50 by tenfold. Here, the IC50 values for eight of the drugs were 100-fold lower than their theoretical intestinal luminal concentration. Moreover, a mechanistic static model suggested that vemurafenib, bexarotene, dabigatran etexilate, rifapentine, aprepitant, and ivacaftor could almost fully inhibit intestinal BCRP, increasing the exposure of concomitantly administered rosuvastatin over 90%. Therefore, clinical studies are warranted to investigate whether these drugs cause BCRP-mediated DDIs in humans.Peer reviewe

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2

To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.Peer reviewe

Substrate binding to 1.0% BSA as measured using equilibrium dialysis.

<p>The f_u is calculated as the ratio of binding in the presence and absence of BSA and is shown with standard deviation (SD) (n = 3).</p