FIELD OF THE INVENTION

The invention relates to the field of assessing transport activity of OATP (Organic anion-transporting polypeptide) and ABC (ATP Binding Cassette) transport proteins. In particular, the invention relates to methods for determining in a single, fluorescence-based assay whether a compound is a modulator of OATP and/or MRP2 (Multidrug resistance associated protein, ABCC) transport activity, using a fluorescent compound that is a substrate of both OATP and MRP2, and is selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II).

BACKGROUND OF THE INVENTION

The liver has a central role in the defense of the body against harmful compounds. Membrane transporters expressed in hepatocytes are key players in the elimination of potentially toxic compounds of endogenous (bile acids, bilirubin) or exogenous (toxins, drugs) origin (Jetter and Kullak-Ublick, 2019). Uptake of harmful substances from the blood into the liver is mediated by Solute Carriers (SLC) of which Organic anion transporting polypeptides OATP1B1, OATP1B3 and OATP2B1 have a renowned role (Hagenbuch and Stieger, 2013). Conversely, following metabolism by hepatic enzymes, toxic compounds are extruded from hepatocytes into the bile or back to the blood stream by the action of efflux transporters of the ABC family, P-glycoprotein (ABCB1), MRPs (ABCC family) and ABCG2 (BCRP) (Kock and Brouwer, 2012). Governed action of hepatic OATPs and ABCs ensures efficient hepatobiliary elimination of their substrates, bilirubin, bile acids and various drugs.

OATP1B1, encoded by the SLC01B1 gene is exclusively expressed in the sinusoidal membrane of hepatocytes (Konig et al., 2000), and is the most abundant OATP of the human liver (Badee et al., 2015; Kimoto et al., 2012; Prasad et al., 2014). OATP1B1 is an organic anion exchanger that mediates the cellular uptake of bile salts, bilirubin, thyroid and sex hormones, and also that of numerous clinically applied drugs, including statins, antivirals, anti-hypertensives and chemotherapeutic agents (Roth et al., 2012). OATP1B1 is a site of drug-drug interactions (DDI), inhibition of its function results e.g. in statin-induced myopathy (Link et al., 2008; Shitara, 2011; Shitara et al., 2003).

MRP2 (ABCC2) is expressed in the canalicular membrane of hepatocytes (Jedlitschky et al., 2006) where it mediates the active efflux of conjugated and unconjugated organic anions (e.g. bilirubin and steroid conjugates), and also the co-transport of uncharged molecules with glutathione into the bile (Konig et al., 1999). Besides its endogenous substrates, MRP2 also recognizes various drugs, including chemotherapeutics, anti HIV drugs, antibiotics and statins (Jedlitschky et al., 2006). Hence, by mediating the extrusion of metabolites from hepatocytes into the bile MRP2 plays a key role in the terminal phase of detoxification (Zhou et al., 2008). Concerted action of hepatic uptake (OATP) and efflux transporters (ABCC2 and ABCG2) is crucial in pharmacokinetics and in the disposition of therapeutic drugs and endogenous substances. Consequently, simultaneous administration of transporter substrates can lead to altered pharmacokinetics and undesired side effects. According to the recommendations of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), interactions with OATP1B1/3 and also potentially with MRP2 should be assayed during drug development (Giacomini et al., 2013). FDA and EMA regulations require the use of sensitive and reliable functional assays for the evaluation of transporter drug interactions. Fluorescent substrates are a good tool to assess OATP and ABC function.

Polarized cell lines (MDCKII, LLC-PK) engineered to overexpress both uptake and efflux transporters are an accepted in vitro model of transepithelial transport measurements. MDCKII cells co-expressing OATP1B and MRP2 or OATP IB and ABCG2 were used in numerous studies for the measurement of vectorial transport of common OATP ABC substrates (Cui et al., 2001; Fahrmayr et al., 2012; Matsushima et al., 2005). These cell lines allow the identification of dual substrates (especially important in cases when the substrate cannot enter the cells without the contribution of an uptake transporter) and also the determination of the involvement of transporters in the transport of a given common substrate (Matsushima, 2005, Sasaki, 2002). OATP and ABC co-expressing MDCKII cells are also used to investigate Drug-Drug Interactions mediated by these transporters. Radioactively labeled substrates, such as bromosulphophthalein, leukotriene C4, dehydroepiandrosterone sulfate (DF1EAS), estrone-3-sulfate (E1S) or estradiol- 17p-D-glucuronide (E217G) have been repeatedly used in such assays (Cui et al., 2001; Flirouchi et al., 2009; Liu et al., 2006; Matsushima et al., 2005). In general, a limitation of the radioligand transport assays is the cost associated with the radiolabeling of the substrates. Fluorescence assays offer a cost effective alternative, and it was shown that fluorescent probe substrates provide an effective and sensitive means to investigate transporter function and drug-transporter interaction. Separate studies have identified common fluorescent substrates of MRP2 and OATPIBs, e.g. fluorescein-methotrexate, cholyl-lysyl- fluorescein, carboxy-dichloro-fluorescein derivatives. Flowever, these were not tested in double transfectants for vectorial transport. One crucial difference between an ideal probe substrate of uptake or efflux transporters is membrane permeability. Uptake transporters require substrates with low membrane permeability, while optimal efflux transporter substrates have high levels of passive uptake (at least in measurements performed on intact cells). EP1392822 reports that a fluorescence compound, Fluo-3, is a substrate of both OATP8 (OATP1B3) and MRP2. Flowever, Fluo-3 is not transported by OATP1B1 or OATP2B1 (Izumi et al., 2016), and therefore its use in testing drug-drug interactions is limited. To date, fluorescent methods allowing the simultaneous investigation of OATP1B1 and ABC transporters have not been identified.

There is thus a need for reliable, safe, selective and easy-to-use methods for the simultaneous investigation of modulators of OATP and MRP mediated transcellular transport.

SUMMARY OF THE INVENTION

The invention provides a method suitable for investigating cellular transfer of compounds by OATP and MRP2. Specifically, a method is provided for investigating hepatic clearance of compounds of interest, by determining the interference of a compound with OATP (Organic anion-transporting polypeptide) transport activity and MRP2 transporter activity in a single, combined, fluorescence-based assay. The method is useful for e.g. screening drug candidates for transport protein mediated drug-drug interactions, such as the inhibition of the transport of an active agent by OATP into a cell (e.g. hepatocyte, enterocyte, blood-brain endothelial cell or kidney cell) and MRP2-mediated efflux of the active agent from the cell. The method is suitable for simultaneously investigating OATP and MRP2 function.

Provided herein is a method for determining whether a test compound interferes with substrate transport by a human OATP and substrate transport by human MRP2, characterized in that substrate transport by the OATP and substrate transport by the MRP2 are assessed in the same polarized cell culture, said cells forming a basolateral and an apical compartment in the culture and expressing the OATP in their basolateral membrane and MRP2 in their apical membrane and a fluorescent compound is used as substrate, wherein the fluorescent compound is a fluorescent substrate of both of the OATP and of MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II) or preferably having any of formula II.2, II.3 and/or II.4.

Preferably the test compound is not taken up into the cells with passive transport.

Preferably the test compound is not taken up into the cells with passive transport or is taken up with a rate significantly lower than the active transport or its transport is negligible.

Preferably, provided herein is a method for determining whether a test compound interferes with substrate transport by a human OATP and substrate transport by human MRP2, characterized by a) providing a polarized cell culture having a polarized layer of cells, said cells forming a basolateral and an apical compartment in the cell culture and expressing the OATP in their basolateral membrane and MRP2 in their apical membrane, b) adding the fluorescent substrate and the test compound to the basolateral compartment of the cell culture, c) measuring the level of fluorescence in the cells and the level of fluorescence in the apical compartment (thereby assessing substrate transport by the OATP and substrate transport by MRP2), d) comparing the levels of fluorescence measured in step c) with the levels of fluorescence in said cells and in the apical compartment, respectively, in the presence of the fluorescent substrate but in the absence of the test compound and therefrom determining whether said test compound interferes with the substrate transport by the OATP, and optionally bl) adding the fluorescent substrate to the basolateral compartment of the cell culture and the test compound to the apical compartment, cl) measuring the level of fluorescence in the apical compartment and the level of fluorescence within the cells, dl) comparing the level of fluorescence measured in step cl) with the level of fluorescence in the apical compartment and the level of fluorescence within the cells, in the presence of the fluorescent substrate but in the absence of the test compound and therefrom determining whether said test compound interferes with the substrate transport by MRP2.

Optionally the levels of fluorescence in said cells and in the apical compartment, respectively, in the presence of the fluorescent substrate but in the absence of the test compound is measured in parallel with steps b) and c). Alternatively the levels of fluorescence in said cells and in the apical compartment are measured separately and the values obtained from the separate measurement are used for comparison. In particular, the levels of fluorescence in the presence of the fluorescent substrate but in the absence of the test compound are measured by carrying out the steps of b’) adding the fluorescent substrate and the test compound to the basolateral compartment of the cell culture, and c’) measuring the level of fluorescence in the cells and the level of fluorescence in the apical compartment (thereby assessing substrate transport by the human OATP and substrate transport by MRP2).

If, in step d), it is determined that the test compound interferes with the substrate transport by human OATP it is identified as a modulator thereof.

If, in step d), it is determined that the test compound interferes with the substrate transport by MRP, it is identified as a modulator thereof; optionally in this embodiment the test compound is added to the apical compartment of the cell culture.

In a preferred embodiment the fluorescent substrate is a sulfopyrene according to general formula (I).

In a further preferred embodiment the fluorescent substrate is a sulforhodamine having general formula (II). Preferably the OATP is selected from OATP1 and OATP2B1. More preferably the OATP is selected from OATP1A2, OATP1B1, OATP1B3 and OATP2B1. Highly preferably the OATP is OATP1B1, OATP1B3, OATP1A2 and OATP2B1.

In a preferred embodiment the fluorescent substrate is selective for OATP1B1, OATP1B3, OATP2B1, OATP1A2 and MRP2. In another preferred embodiment the fluorescent substrate is selective for OATP1B1, OATP1B3, OATP2B1 and MRP2.

The sulfopyrenes are sulfopyrenes having general formula (I) wherein in the formula

Ri, R ₂ , R3 and R ₄ is selected from H, sulfo, carboxy and OR ₅ , wherein two or three of R _j , R ₂ , R3 and R ₄ is selected from sulfo or carboxy, wherein one or two of R ,, R ₂ , R ₃ and R ₄ is H or OR ₅ , preferably OR ₅ , wherein R ₅ is selected from H (i.e. wherein OR ₅ is hydroxyl); C ₄₄ -C(O)-; _ alkyl; C 1 _ ₄ alkenyl; C 1 _ ₄ alkynyl; a group wherein R ₅ is an at most 30 membered organic moiety comprising one or two four-, five- or six-membered heterocycle(s) each having one or two heteroatoms independently selected from N and O; and comprising one to four groups selected from ester, amide and carboxy groups, preferably 8-[2-(4- { [(2,5-dioxopyrrolidin-l-yl)oxy]carbonyl}piperidin-l-yl)-2-ox oethoxy; a group according to general formula (V) -(CH ₂ ) _n -C(0)-R ₈ , (V) wherein n is an integer between 1 to 3, preferably 1 or 2, more preferably 1, and wherein R ₈ is selected from a moiety comprising a substituted or unsubstituted four-, ftve- or six-membered heterocycle comprising one or two heteroatoms independently selected from N and O, wherein said heterocycle, if substituted, may have a substituent of the formula -C(0)X-Rio wherein X is -O- or -N(H)-, and Ri ₀ is selected from a substituted or unsubstituted Ci_ ₈ alkyl or alkenyl having, if substituted, one to three substituents, preferably selected from carbonyl, carboxy, amide, amine, halogen or ester, and optionally a substituted or unsubstituted four-, five- or six-membered heterocycle;

OR wherein R ₈ is an amine -NR _n Ri ₂ wherein R _n and R _{J 2} are selected from H and substituted or unsubstituted C1.4 alkyl, C1.4 alkenyl and C 1 _ ₄ alkynyl or R _n and R _{J 2} together form a five or six membered heterocycle which may not or may be substituted as defined above; a group according to general formula (VI)

-(CH ₂ ) _n -C(0)-NH-R ₉ , (VI) wherein n is an integer between 1 to 3, preferably 1 or 2, more preferably 1, and wherein R ₉ is selected from H, substituted or unsubstituted C 1 _ ₄ alkyl, C1.4 alkenyl and C1.4 alkynyl and NR13R14 wherein R _J and R _w are selected from H and substituted or unsubstituted C1.4 alkyl, C1.4 alkenyl and C1.4 alkynyl.

Preferably R ₅ is selected from H (i.e. wherein OR ₅ is hydroxyl), C ₄ -C(O)-, C _1.4 alkyl and 8-[2-(4- { [(2,5-dioxopyrrolidin-l-yl)oxy]carbonyl}piperidin-l-yl)-2-ox oethoxy. In a highly preferred embodiment the sulfopyrene compound is Alexa-Fluor-405.

In a preferred embodiment R ₅ is a group according to general formula (V)

-(CH ₂ ) _n -C(0)-R ₈ , (V) wherein n is an integer between 1 to 3, preferably 1 or 2, more preferably 1, and wherein R ₈ is selected from a moiety comprising a substituted or unsubstituted four-, ftve- or six-membered heterocycle comprising one or two heteroatoms independently selected from N and O, wherein said heterocycle, if substituted, may have a substituent of the formula -C(0)X-Rio wherein X is -O- or -N(H)-, and Ri ₀ is selected from a substituted or unsubstituted Ci_ ₈ alkyl or alkenyl having, if substituted, one to three substituents, preferably selected from carbonyl, carboxy, amide, amine, halogen or ester, and optionally a substituted or unsubstituted four-, five- or six-membered heterocycle;

OR wherein R ₈ is an amine -NR _n Ri ₂ wherein R _n and R _{J 2} are selected from H and substituted or unsubstituted C1.4 alkyl, C 1 _ ₄ alkenyl and C 1 _ ₄ alkynyl or R _n and R _{J 2} together form a five or six membered heterocycle which may not or may be substituted as defined above.

In a preferred embodiment R ₅ is a group according to general formula (VI)

-(CH ₂ ) _n -C(0)-NH-R ₉ , (VI) wherein n is an integer between 1 to 3, preferably 1 or 2, more preferably 1, and wherein R ₉ is selected from H, substituted or unsubstituted C i _ ₄ alkyl, C i _ ₄ alkenyl and Ci_ ₄ alkynyl and NR13R14 wherein R _J3 and R _J are selected from H and substituted or unsubstituted C 1 _ ₄ alkyl, C1.4 alkenyl and C1.4 alkynyl _a preferably both R _{J 3} and R _J are H so that NH- Reform a hydrazinyil group. In a highly preferred embodiment the sulfopyrene compound is Cascade Blue hydrazyde.

In a further preferred embodiment R ₅ is H.

In a preferred embodiment in formula I two of R _j , R ₂ , R ₃ and R is selected from sulfo and two of R 1, R ₂ , R ₃ and R is OR5. In a preferred embodiment R ₅ is H. In a preferred embodiment R5 is as defined above.

In a preferred embodiment in formula I three of R _j , R ₂ , R ₃ and R is selected from sulfo and one of R _{| ,} R ₂ , R ₃ and R is OR5. In a preferred embodiment R ₅ is H. In a preferred embodiment R5 is as defined above.

In an embodiment in formula I two of R _j , R ₂ , R ₃ and R is selected from sulfo and one of R _{| ,} R ₂ , R ₃ and R is a carboxy and one of R _j , R ₂ , R ₃ and R is OR5. In a preferred embodiment R ₅ is H. In a preferred embodiment R5 is as defined above.

In a preferred embodiment in formula I R _j , R ₂ and R ₃ are sulfo and R is OR5. In a preferred embodiment R ₅ is H. In a preferred embodiment R5 is as defined above.

In a preferred embodiment in formula I R and R ₂ are sulfo and R ₃ and R are OR5. In a preferred embodiment R ₅ is H. In a preferred embodiment R5 is as defined above.

In a higly preferred embodiment the compound according to formula I is selected from:

8-hydroxypyrene 1,3,6 trisulfonate, (8-hydroxy-l,3,6 pyrene-trisulfonic acid and its trisodium salt),

Cascade Blue hydrazide (CAS name/number: Acetic acid, [(3,6,8-trisulfo-l-pyrenyl)oxy]-, 1-hydrazide, and its trisodium salt),

6, 8-dihydroxypyrene- 1,3-disulfonate (6, 8-dihydroxy- 1,3-pyrene-disulfonic acid and its disodium salt, DHPDS),

Alexa Fluor 405 (tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxo-l-pyrrolidinyl)oxy]carbonyl}-l- piperidinyl)-2-oxoethoxy]-l,3,6-pyrenetrisulfonate),

8-acetoxypyrene-l,3,6 trisulfonic acid (8-acetoxypyrene-l,3,6 trisulfonic acid and its trisodium salt) or any salt, e.g. sodium salt thereof.

The sulforhodamines are sulforhodamines having general formula (II), wherein

R and R ₂ are selected from H, Ci_ alkyl, Ci_ alkenyl and Ci_ alkynyl, preferably H, methyl or ethyl, wherein preferably at least one of R and R ₂ is different from H,

R ₃ and R ₄ are selected from H, _ alkyl, _ alkenyl and _ alkynyl, preferably H, methyl or ethyl, wherein preferably at least one of R ₃ and R ₄ is different from H,

R ₅ and R ₆ are selected from H, _ alkyl, _ alkenyl and Ci_ alkynyl, preferably H, methyl or ethyl,

R ₇ and R ₈ are selected from H, _ alkyl, Ci_ alkenyl and Ci_ alkynyl, preferably H, methyl or ethyl, or wherein any of

R _j and R ₅

R ₂ and R ₆

R ₃ and R ₇ and

R ₄ and Rg together with the N and the adjacent phenyl ring of the anthracene skeleton forms a six membered heterocycle, and

R _n and R ₁₂ are independently selected from sulfo (-S0 H), carboxy (-C(O)OH) and OH with the proviso that at least one of R _n and R ₁₂ is sulfo or any salt (e.g. sodium salt) or hydrate thereof.

Preferably wherein any of

R _j and R ₅ , R ₂ and R ₆ , R ₃ and R ₇ and R ₄ and R ₈ together with the N and the adjacent phenyl ring of the anthracene skeleton forms a six membered heterocycle, R and R ₅ , R ₂ and R ₆ , R ₃ and R ₇ and R ₄ and R ₈ together forms a C _2-4 alkylene, preferably a -(CH ₂ ) ₃ -.

In a preferred embodiment sulforhodamines are sulforhodamines having general formula (II.2), (P 2) wherein

Ri is selected from H, _ ₃ alkyl, _ ₃ alkenyl and C _1.3 alkynyl, preferably H, methyl or ethyl,

R is selected from H, C _1.3 alkyl, C _1.3 alkenyl and C _1.3 alkynyl, preferably H, methyl or ethyl,

R ₆ are selected from H, C _1.3 alkyl, C _1.3 alkenyl and C _1.3 alkynyl, preferably H or methyl,

Rg are selected from H, C _1.3 alkyl, C _1.3 alkenyl and C _1.3 alkynyl, preferably H or methyl,

R _n and R _J2 are independently selected from sulfo (-SO ₃ H), carboxy (-C(O)OH) and OH with the proviso that at least one of R _n and R _J2 is sulfo, preferably both R _n and R _J2 are sulfo or any salt (e.g. sodium salt) or hydrate thereof.

In a preferred embodiment sulforhodamines are sulforhodamines having general formula (II.3), wherein

R ₆ are selected from H, Ci_ ₃ alkyl, Ci_ ₃ alkenyl and Ci_ ₃ alkynyl, preferably H or methyl, Rg are selected from H, Ci_ ₃ alkyl, Ci_ alkenyl and Ci_ alkynyl, preferably H or methyl, R _n and R _{J 2} are independently selected from sulfo (-SO3H), carboxy (-C(O)OH) and OH with the proviso that at least one of R _n and R _{J 2} is sulfo, preferably both R _n and R _{J 2} are sulfo or any salt (e.g. sodium salt) or hydrate thereof.

In a preferred embodiment sulforhodamines are sulforhodamines having general formula (II.4), wherein

R _n and R ₁₂ are independently selected from sulfo (-S0 H), carboxy (-C(O)OH) and OH with the proviso that at least one of R _n and R ₁₂ is sulfo, preferably both R _n and R ₁₂ are sulfo or any salt (e.g. sodium salt) or hydrate thereof.

In a higly preferred embodiment the compound of formula II is selected from Sulforhodamine 101 and any salt (e.g. sodium salt) or hydrate thereof.

Preferably the method comprises a) providing a polarized monolayer of cells, said cells forming a basolateral and an apical compartment and expressing the human OATP in their basolateral membrane and the human MRP2 in their apical membrane, b) adding the test compound and the fluorescent substrate to the basolateral compartment, c) measuring the level of fluorescence in the cells and the level of fluorescence in the apical compartment, d) identifying the test compound as a compound that does not interfere with substrate transport by the OATP and MRP2, if the level of fluorescence in said cells and in the apical compartment in the presence of the test compound is unaltered compared to the level of fluorescence in said cells and in the apical compartment, respectively, in the absence of the test compound.

The method preferably comprises the following steps: i) providing a polarized monolayer of cells, said cells forming a basolateral and an apical compartment and expressing the OATP in their basolateral membrane and MRP2 in their apical membrane, ii) adding the fluorescent substrate and the test compound to the basolateral compartment iii) measuring the level of fluorescence in the cell and in the apical compartment and iv) identifying the test compound as

- an inhibitor of substrate transport by the OATP if the level of fluorescence in the cells is lower in the presence of the test compound than in the absence of the test compound, and optionally the level of fluorescence in the apical compartment is lower in the presence of the test compound than in the absence of the test compound,

- an activator of substrate transport by the OATP if the level of fluorescence in the cells is higher in the presence of the test compound than in the absence of the test compound, and preferably the method also comprises the following steps: v) adding the fluorescent substrate to the basolateral compartment and the test compound to the apical compartment and measuring the level of fluorescence in the cells and in the apical compartment, and vi) identifying the test compound as

- an inhibitor of substrate transport by MRP2, if the level of fluorescence in the apical compartment is lower in the presence of the compound than in the absence of the compound or

- an activator of substrate transport by MRP2, if the level of fluorescence in the apical compartment is higher in the presence of the compound than in the absence of the compound.

Preferably the method comprises the following steps: i) providing a polarized monolayer of cells, said cells forming a basolateral and an apical compartment and expressing the OATP in their basolateral membrane and MRP2 in their apical membrane, ii) adding the fluorescent substrate and the test compound to the basolateral compartment iii) measuring the level of fluorescence in the cell and in the apical compartment and iv) identifying the test compound as

- an inhibitor of substrate transport by the OATP if the level of fluorescence in the cells is lower in the presence of the test compound than in the absence of the test compound,

- an activator of substrate transport by the OATP if the level of fluorescence in the cells is higher in the presence of the test compound than in the absence of the test compound,

- an inhibitor of substrate transport by MRP2 if the level of fluorescence in the presence of the test compound in the cells is higher than or unaltered compared to the level of fluorescence in the cells in the absence of the test compound and the level of fluorescence in the apical compartment is lower in the presence of the test compound than in the absence of the test compound,

- an activator of substrate transport by MRP2 if the level of fluorescence in the cells in the presence of the test compound is lower than or unaltered compared to the level of fluorescence in the cells in the absence of the test compound and the level of fluorescence in the apical compartment is higher in the presence of the test compound than in the absence of the test compound and optionally v) adding the fluorescent substrate to the basolateral compartment and the test compound to the apical compartment and measuring the level of fluorescence in the cells and in the apical compartment, and vi) identifying the test compound as - an inhibitor of substrate transport by MRPP2, if the level of fluorescence in the apical compartment is lower in the presence of the compound than in the absence of the compound or

- an activator of substrate transport by MRP2, if the level of fluorescence in the apical compartment is higher in the presence of the compound than in the absence of the compound.

In another aspect of the invention a use of a fluorescent substrate for assessing a human OATP and human MRP2 activity in a single assay is provided. Accordingly the invention relates to the use of a fluorescent substrate for determining in a single, fluorescence-based assay whether a test compound is an activator or inhibitor of substrate transport by the OATP and substrate transport by the MRP2, characterized in that the fluorescent substrate is a substrate of both the OATP and MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II).

In another aspect of the invention a method for measuring expression level of a human OATP and human MRP2 in a cell is provided, the method comprising providing polarized cells expressing the OATP and MRP2, adding a fluorescence compound which is a substrate of both the OATP and MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II) to the part of a cell membrane where expression of the OATP is expected, measuring fluorescence intensity of the fluorescent compound in the cell and at the part of the membrane where expression of MRP2 is expected, wherein the fluorescent compound can only be transported to the part of the membrane where expression of MRP2 is expected by the OATP and the MRP2 and evaluating the expression level of the OATP and MRP2 based on the measured fluorescence.

The invention also provides an assay kit, comprising polarized assay cells expressing the OATP in their basolateral membrane and MRP2 in their apical membrane, and capable of forming a basolateral and an apical compartment in culture and a fluorescent compound which is a substrate of both the OATP and MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II) and optionally polarized control cells expressing the OATP in their basolateral membrane and lacking MRP2 expression, and capable of forming a basolateral and an apical compartment in culture, and/or polarized control cells expressing MRP2 in their apical membrane and lacking the OATP expression, and capable of forming a basolateral and an apical compartment in culture and/or polarized control cells transfected with the same type of expression vector as the polarized control cells expressing the OATP and with the same type of expression vector as the polarized control cells expressing MRP2 but without the nucleic acid encoding the OATP and the nucleic acid encoding MRP2, respectively and/or polarized control cells transfected with the same type of expression vector as the polarized control cells expressing the human OATP but without the nucleic acid encoding the human OATP.

Preferably, the polarized control cells are of the same type as the polarized assay cells. In another aspect of the invention a method for determining whether a cultured human hepatocyte is transport competent is provided, the method comprising adding a fluorescent compound to the sinusoidal membrane of the hepatocyte, detecting fluorescence at the canalicular side of the hepatocyte, and identifying the hepatocyte as a transport competent hepatocyte expressing OATP and MRP2 if transported fluorescence is present at the canalicular side of the hepatocyte, characterized in that the fluorescent compound is a substrate of both OATP and MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II). Preferably the fluorescent compound can only be transported to the part of the membrane where expression of MRP2 is expected by the OATP and MRP2. Preferably, the hepatocyte is in a 3 dimensional culture.

In any one of the foregoing aspects of the invention, the fluorescent compound is a substrate of the OATP and MRP2, thus the OATP and MRP2 are capable of transporting the fluorescent compound through the cell membrane. The fluorescent compound is a compound with low or no membrane permeability to avoid passive transport through the cell membrane. Preferably, the fluorescent compound can only be taken up into and released from the cell by the OATP and MRP2 mediated transport.

In any one of the foregoing aspects of the invention, the cells preferably form a polarized monolayer in culture, wherein tight junctions are formed between the cells, thereby forming a separated apical and basal (basolateral) compartment in the culture. The basolateral cell membrane of the cell is localized in the basal compartment and the apical cell membrane of the cell is localized in the apical compartment. In another embodiment the cells form a 3 dimensional culture, such as spheroids and hepatocyte organoid cultures. In cultured hepatocytes the OATP is expressed in the sinusoidal membrane and MRP2 is expressed in the canalicular membrane.

In any one of the foregoing aspects of the invention, the cells may and preferably are overexpressing the OATP and MRP2. Cells without overexpression may also be used, provided the OATP and MRP2 mediated transport can be detected in them. Suitable cells include both genetically engineered prokaryotic and eukaryotic cells and not engineered cells, such as kidney cells, hepatocytes, enterocytes, endothelial cells, Caco-2, LLC-PK, hepatoma cell lines, HepG2, HepaRG cells. Highly preferred are MDCKII cells overexpressing the OATP and MRP2.

In any one of the foregoing aspects of the invention, in a preferred embodiment the cells do not express other human OATP or MRP2 proteins than those involved in the assay.

In any one of the foregoing aspects of the invention, preferably the OATP is selected from OATP1 and OATP2B1. Highly preferably the OATP is OATP1B1, OATP1B3, OATP1A2 and OATP2B1.

In a preferred embodiment the fluorescent substrate is selective for MRP and OATP1 and OATP2B1. Highly preferably the fluorescent substrate is selective for OATP1B1, OATP1B3, OATP2B1, OATP1A2 and MRP2 or OATP1B1, OATP1B3, OATP2B1 and MRP2.

In any one of the foregoing aspects of the invention, the fluorescent compound is preferably selected from the compounds according to general formula I, wherein at least two of R _j , R ₂ , R and R is selected from sulfo or carboxy, wherein preferably at least two of Ri, R ₂ , R and R is a sulfo, preferably two or three of R , R ₂ , R and R is a sulfo, wherein at most one or two of R _j , R ₂ , R ₃ and R is H or OR ₅ , preferably OR ₅ , wherein R ₅ is selected from H (i.e. wherein OR ₅ is hydroxyl), and C ₁ _ ₄ alkyl, and 8-[2-(4-{ [(2,5- dioxopyrrolidin- 1 -yl)oxy]carbonyl }piperidin-l -yl)-2-oxoethoxy, or wherein R ₅ is a group according to general formula (VI)

-(CH ₂ ) _n -C(0)-NH-R ₉ , (VI) wherein n is an integer between 1 to 3, preferably 1 or 2, more preferably 1, and wherein R ₉ is selected from H, substituted or unsubstituted C ₁ _ ₄ alkyl, C _1.4 alkenyl and C _1.4 alkynyl and NR ₁₃ R ₁₄ wherein R ₁₃ and R _w are selected from H and substituted or unsubstituted C _1.4 alkyl, C _1.4 alkenyl and C _1.4 alkynyl.

In a preferred embodiment R ₅ is H.

Preferably, the fluorescent compound having general formula I is selected from pyranine (trisodium 8- hydroxypyrene-1, 3, 6-trisulfonate, 8-hydroxypyrene-l,3,6-trisulfonic acid), Cascade Blue (pyrenyloxytrisulfonic acid), Cascade Blue hydrazide (CAS name/number: Acetic acid, [(3,6,8-trisulfo-l-pyrenyl)oxy]-, 1-hydrazide, trisodium salt 137182-38-8), 6, 8-Dihydroxy- 1,3-pyrenedisulfonic acid disodium salt (DHPDS), Alexa Fluor 405, 8-acetoxypyrene-l,3,6 trisulfonic acid trisodium salt. Highly preferably the fluorescent compound is selected from pyranine, DHPDS, Alexa Fluor 405, 8-acetoxypyrene-l,3,6 trisulfonic acid trisodium salt and Cascade Blue hydrazide.

In any one of the foregoing aspects of the invention, the fluorescent compound is preferably selected from compounds of general formula II.2, general formula II.3, and general formula II.4 as defined above.

More preferably, the fluorescent compound having general formula II is Sulforhodamine 101.

In any one of the foregoing aspects of the invention, the compound to be tested and the fluorescent compound are added to the basal compartment, thus preferably the cell is incubated with the compound and the fluorescent compound in a (transport) buffer at the basolateral compartment. The compound to be tested is a compound with low or no membrane permeability to avoid passive transport through the cell membrane and interaction with the transporter in the opposite membrane.

In any one of the foregoing aspects of the invention, in an embodiment of the invention the compound to be tested is added to the apical compartment and the fluorescent compound is added to the basal compartment.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Pyranine is a novel substrate of OATP1B1, OATP1B3 and OATP2B1. Concentration dependent uptake of pyranine was measured in A431 cells overexpressing OATP1B1, OATP1B3 or OATP2B1, or mock transfected controls (Ctr.) seeded onto 96-well plates at 37°C in the linear phase of transport, at 10 (OATP1B1) or 15 (OATP1B3 and OATP2B1) minutes at pH 5.5. Fluorescence was determined using an Enspire plate reader (Ex/Em: 403/517 nm). Transport kinetics was determined by subtracting fluorescence measured in mock transfected cells. Average +/- SD values of at least 3 independent measurements are shown. Figure 2. Dye uptake in A431 cells expressing human OATPs. A431 cells seeded on 96 well plates were incubated with 20 mM dye for 30 minutes at pH 5.5. Cellular fluorescence was determined in an Enspire fluorescent plate reader. Trisulfo=pyranine, monosulfo= 1-pyrenesulfonic acid, disulfo= 6, 8-dihydroxy- 1,3- pyrenedisulfonic acid, tetrasulfo=l,3,6,8-pyrenetetrasulfonic acid, acteoxy=8-acetoxy-l, 3, 6-pyrenetrisulfonic acid. Experiments were performed in 2 biological replicates, average +/- SD are shown.

Figure 3: Uptake of the fluorescent dyes, ZV, LDV and LDG (0.2 mΐ/sample), 5 mM CB, 5mM pyranine, 1 mM SR101, 1 mM FMTX or IOmM LY in MRP2- (panels A, B) or ABCG2- (panels C, D) containing or control membrane vesicles (50 pg) was measured for 30 minutes (CB), 20 minutes (pyranine) or 10 minutes (ZV, LDV, LDG, SR101, FMTX, LY) in the presence of 4 mM Mg ATP or 4 mM Mg AMP at 37°C. Experiments were repeated 3-times. Average of at least 3 independent replicates +/- SD are shown. Statistical significance was calculated between ATP-dependent signals. Delta values generated by subtracting signals with MgAMP from that measured with MgATP were compared for statistical significance by Student’s t-test, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 4: Expression and function of OATP1B1, MRP2 or ABCG2 in the MDCKII cell lines. 3A: 10 or 20 pg of total cell lysates were analyzed for transporter expression by Western blot. Signals of OATP 1 B l/MRP2/ABCG2/l. -actin were visualized by the antibodies raised against these proteins. Transporter expression is equally present for OATP1B1/MRP2/ABCG2 among the appropriate cell lines. B-actin signals verified the equal amount of proteins in the samples tested. Experiments were repeated at least 3-times. The result of one representative experiment is shown. B: Transporter function of OATP IB 1, MRP2 or ABCG2 in MDCKII cell lines. Functionality of the transporters OATP1B1, MRP2 or ABCG2 was determined based on the transport of CB, CaAM (Calcein AM) or DCV (DyeCycle Violet) substrates, respectively. 5 x 10 ⁵ MDCKII cells were incubated at 37°C for 10 min (CaAM)/30 min (CB, DCV) in 100 mΐ fluorescent dye (final concentrations: 0.5 mM CaAM (Calcein-AM), 1-1 mM CB or DCV) diluted in the appropriate buffer (pH 5.5/7.4 for OATP/ABC function). Cellular fluorescence was determined of at least 20000 living cells from each sample using an Attune Acoustic Focusing Cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA, US). Transport measurements were repeated at least 3-times. One representative experiment is shown. C: Transcellular transport of fluorescein-methotrexate: Basolateral to apical transcellular transport of FMTX (ImM) in MDCKII- OATP1B1, MDCKII-MRP2, MDCKII-MRP2-OATP1B1 or control (Ctr., mock transfected) cells grown on transwell inserts for 4 days prior to the experiment was followed for 30 minutes at 37°C. Average of 3 independent measurements +/- SD are shown.

Figure 5: Transcellular transport of pyranine. A: Basolateral to apical transport of pyranine (5 mM) in MDCKII- OATP1B1, MDCKII-MRP2, MDCKII-MRP2-OATP1B1 or control (Ctr., mock transfected) cells grown on transwell inserts for 4 days prior to the experiment was followed for 30 minutes at 37°C. Average of 5 independent measurements +/- SD are shown. B: Intracellular levels of pyranine were determined in MDCKII cells after 30 minutes of incubation with 5 mM pyranine administered from the basolateral side of the transwells. Average of 3 independent measurements +/- SD are shown. Statistical analysis for multi comparison was evaluated by Tukey-Kramer HSD (Honest Significant Differences) procedure, as a post-hoc test, after rejecting HO in One-Way ANOVA (a=0,05). Means +/- SD marked with the same letter (“a” for Ctr. and MRP2) were not significantly different (p > 0.05, Tukey-Kramer HSD test) from each other unlike “b” and “c” which mean significant difference. C: Lack of apical to basolateral transport of pyranine. Experiments were performed with pyranine (5 mM) added either to the apical or the basolateral compartment and samples were taken from the basolateral (A-B) or the apical compartment (B-A), respectively until 25 minutes. Average of 3 independent measurements +/- SD are shown. D: Inhibition of pyranine transcellular transport by cyclosporin A (CsA) or benzbromarone (Benz). Transcellular (B-A) transport of 5 mM pyranine can be inhibited by known OATP1B1 and MRP2 inhibitors, cyclosporin A (CsA) and benzbromarone (Benz). Vectorial transport was measured as described at Fig.4A, except that 10 mM CsA or 40 mM benzbromarone was added to the basolateral or apical compartment, respectively prior to the addition of pyranine (5 mM). Fluorescence of pyranine was measured using an Enspire plate reader at Ex/Em 403/517 nm. Data obtained from 3 independent experiments +/- SD values are presented as a percent of transport measured in MDCKII-MRP2-OATP1B1 cells without any inhibitor (NT).

Figure 6: Transcellular transport of Cascade Blue hydrazide or sulforhodamine 101. Basolateral to apical transport of CB (5 mM, panel A) or SR101 (1 mM, panel B) in MDCKII-OATP1B1, MDCKII-MRP2, MDCKII- MRP2-OATP1B1, MDCKII-ABCG2-OATP1B1 (panel A) or control (Ctr., mock transfected) cells grown on transwell inserts for 4 days prior to the measurement was followed for 30 minutes at 37°C. Average of 4 independent measurements +/- SD are shown for MDCKII-OATP1B1, MDCKII-MRP2, MDCKII-MRP2- OATP1B1 and Ctr. cells on panel A, and average of 3 independent measurements +/- SD are shown for MDCKII-ABCG2-OATP1B1 cells (panel A) and on panel B. C: Lack of apical to basolateral (A-B) transport of the fluorescent dyes. Experiment was performed on MDCKII cells grown on transwell inserts for 4 days prior to the measurement. CB (5 mM) or SR101 (1 mM) were added to the apical (A-B transport) or basolateral (B-A transport) compartment and after 25 minutes of incubation at 37°C samples were taken from the basolateral (A-B transport) or apical (B-A transport) compartment, and fluorescence was determined. Data are shown as a percent of B-A transport measured in MDCKII-MRP2-OATP1B1 cells. Average of 3 independent measurements +/- SD are shown. D: Inhibition of CB or SR101 transcellular transport. Transcellular (B-A) transport of 5 mM CB or 1 mM SR101 can be inhibited by known OATP1B1 and MRP2 inhibitors, cyclosporin A (CsA) and benzbromarone (Benz). Vectorial transport was measured as described at Fig.4A, except that 10 mM CsA or 40 mM benzbromarone was added to the basolateral or apical compartment, respectively prior to the addition of the dyes 5 mM CB or 1 mM SR101. Data represent average +/- SD values of 3 independent experiments and are presented as a percent compared to the B-A transport measured in MDCKII-MRP2-OATP1B1 cells (NT).

Figure 7: Transcellular transport of sulfopyrene compounds. Average +/- SD values obtained in 2 independent experiments are shown. CsA stands for cyclosporin A (10 mM final concentration), Benz is benzbromarone (40 mM final concentration).

Figure 8: Schematic presentation of the fluorescent assay.

DETAILED DESCRIPTION OF THE INVENTION

Hepatic clearance of drugs by OATP and ABC proteins alters their bioavailability. Interaction of drugs or compounds naturally present in the human body with OATP and ABC transporters may cause undesired effects including drug resistance, altered pharmacokinetics or even toxicity. The invention provides a method for determining in a single assay whether a test compound, e.g. a drug candidate is an inhibitor or activator of an OATP and/or an ABC transporter, specifically of MRP2 and OATP1 and/or OATP2B1. The method comprises detecting the inhibition or augmentation of the transport of a fluorescent compound by the OATP and MRP2, wherein the fluorescent compound is a substrate of both the OATP and MRP2. Although cell lines engineered to overexpress pairs of uptake and efflux transporters or even metabolic enzymes are an accepted model of in vitro drug interaction screens, to recapitulate in vivo conditions more complex models, e.g. human derived hepatocytes are needed. The dual OATP-MRP2 substrates identified in our study are good candidates to monitor the function and drug interactions of these transporters in hepatocytes.

Substrate transport by the OATP and MRP2 may be activated or inhibited by the test compound (test substance). If a test compound interferes with the substrate transport of a transporter it can be identified as a modulator thereof. A modulator can be an “activator” or an “inhibitor”. An “activator” compound increases substrate transport, e.g. the transport of the fluorescent substrate, whereas an “inhibitor” substance decreases or inhibits substrate transport. An inhibitor may be a competitive inhibitor.

The test compound and the fluorescent substrate may be added to the cells simultaneously or sequentially in an amount suitable for detecting changes in fluorescence in the cell, at the side where the compounds were added or at the opposite membrane (side) of the cell. The test compound may be added to either the basolateral compartment or the apical compartment, while the fluorescent substrate is added to the basolateral membrane. Those skilled in the art will understand that the amount of the test compound and the amount of the fluorescent substrate, as well as the timing of addition of the test compound and the fluorescent substrate may vary depending on a number of factors, such as the affinity of the test compound and/or the fluorescent substrate to the transport protein, the kinetics of the transport by the OATP and/or the transport by MRP2 (e.g. it may be necessary to calculate Km and Vmax at different concentrations of a test compound to determine inhibition). It is within the knowledge of the skilled person to select the appropriate test conditions for determining whether a test compound is an activator or inhibitor of the transport activity of the OATP and MRP2. Examples are also described herebelow. An exemplary method is adding first the test compound and 5 minutes later the fluorescent substrate.

Control experiments and calibration curves. The skilled person is able to select appropriate control conditions (such as cells, compounds) most suited for the aims of the study. An example is provided herebelow.

For example - cells which do not express the OATP and/or MRP2, cells which express the OATP and/or MRP2 under a pre-determined threshold value, cells in which the expression of the OATP and/or MRP2 is silenced, cells which express a mutant OATP and/or MRP2 which is not capable of transporting the fluorescent substrate or cells in which the activity of the OATP and/or MRP2 is inhibited, cells which overexpress the OATP alone, and cells which overexpress MRP2 alone or cells transfected with the control vector may be used as control cells.

Calibration curves may be used to assess fluorescence, e.g. to convert measured intensity into the amount of fluorescent substrate. If necessary, background fluorescence may be tested by appropriate controls, e.g. in the absence of the fluorescent compound and only transported fluorescence is used.

A “single assay” as used herein refers to an assay wherein double expressing cells (i.e. expressing both the OATP and MRP2) are used to test compounds for transport by the OATP and MRP2. This setting eliminates the need of using separate cell lines for the OATP and inside out vesicles for MRP2, while provides a model better representing the biological environment of polarized cells, e.g. the liver.

The double expressing cells used in the method of the invention are polarized cells expressing the OATP on a fraction of their cell membrane (the basolateral membrane, as used herein) and MRP2 on another fraction of the cell membrane (the apical membrane, as used herein). The polarized cells preferably form a monolayer when cultured.

The polarized cells form a basolateral compartment and an apical compartment in the culture. The basolateral compartment comprises the portion of the cell comprising the basolateral membrane and the apical compartment comprises the portion of the cell comprising the apical membrane. Such polarized cells forming a basolateral and an apical compartment are well-known in the art.

It is also possible to use artificial membranes and microfluidic constructions to model the cell membrane and the cytosol.

“Unaltered compared to” as used herein may refer to the lack of a statistically significant difference. “Higher” and “lower” as used herein preferably refer to a statistically significant difference.

Several fluorescent dyes were studied, for example: pyranine (8-hydroxypyrene 1, 3, 6 trisulfonate, PYR); Cascade Blue hydrazide (CAS name/number: Acetic acid, [(3,6,8-trisulfo-l-pyrenyl)oxy]-, 1-hydrazide, trisodium salt 137182-38-8CB; CB); Live/Dead Violet (LDV); Live/Dead Green (LDG); Lucifer yellow (LY); sulforhodamine 101 (SR101); pyrene; 1 -pyrenesulfonic acid; 6,8-Dihydroxy-l,3-pyrenedisulfonic acid disodium salt (DHPDS); 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt; Alexa Fluor 405.

Pyranine was tested for transport by hepatic OATPs in A431 cells expressing OATP1B1, OATP1B3 and OATP2B1. We found that although pyranine is a lower affinity substrate compared to CB (pyranine Km values for OATP1B1, OATP1B3 and OATP2B1 were 27.8, 92 and 65.6 mM, respectively, vs. CB Km values of 2.6, 21 and 21 mM (Patik, 2018)), its transport ratio by all three OATPs is about 3-times higher than that of CB, hence resulting in an improved signal to noise ratio. In addition, we found that OATP1A2 can also transport pyranineTherefore we conclude that pyranine can be an excellent tool to investigate hepatic OATP function. More fluorescent dyes were tested in OATP1B1 MRP2 double transfected MDCKII cells for basolateral to apical transport, in which experiments we confirmed pyranine, CB and SR101 as dual probes transported by both OATP1B1 and MRP2 (Figure 5 and 6). Moreover, the vectorial transport of these dyes could be inhibited by known OATP MRP2 inhibitors, indicating that these fluorescent probes can be used for assessing OATP IB MRP2 drug interactions. Additional pyrene derivatives were also tested in OATP1B1 MRP2 double transfected MDCKII cells for basolateral to apical transport and we confirmed that 6,8-dihydroxy-l,3-pyrenedisulfonic acid, 8-acetoxy-l, 3, 6 pyrenetrisulfonic acid and Alexa-Fluor 405 are novel dual fluorescent probes of MRP2 and OATP IB 1 (Figure 7).

Although CB transport was very low in MRP2 and ABCG2 containing IOVs, we still detected significant transport of this dye in double transfected MDCKII-MRP2-OATP1B1 cells. However, although expression and function of both OATP IB 1 and ABCG2 was confirmed in the double transfected MDCKII cell line, we could not detect any transcellular transport of CB. These discrepancies can be explained by the conversion of CB in the cells (but not in IOVs) into a metabolite that is a higher affinity substrate of MRP2 than CB, but not recognized anymore by ABCG2. These results underline the relevance of cell-based assays, which are influenced by the intracellular metabolism of compounds that are also relevant in drug transporter and drug-drug interactions.

The fluorescent substrate compounds of the invention are any of those defined in the SUMMARY OF THE

INVENTION (above), in particular, a fluorescent compound selected from compounds according to any compounds of general formula I or a preferred or more preferred subset thereof as defined above, any compounds of general formula II or a preferred or more preferred subset thereof as defined above, any compounds of general formula II.2 or a preferred or more preferred subset thereof as defined above, any compounds of general formula II.3 or a preferred or more preferred subset thereof as defined above, and/or any compounds of general formula II.4 or a preferred or more preferred subset thereof as defined above.

In the formulae the following definitions apply.

A “sulfonic acid” refers to a group of acidic organosulfur compounds having the general formula R-S(=0) ₂ -0H (i.e. R-S(=0) ₂ -0H or RS(0) ₂ 0H, which are considered as equivalent descriptions herein), wherein R is an organic group, e.g. an alkyl, alkenyl or alkynil or aryl group with a carbon atom adjacent to the sulfur of the sulfo group of the sulfonic acid. The term sulfonate anion (R-S(0) ₂ 0- or R-S(=0) ₂ -0H -) of a sulfonic acid is usually named with the suffix -ate, in keeping with the general pattern of -ic acid and -ate for a conjugate acid and its conjugate base, respectively. The term “sulfonic acid” includes all these forms as well as salts or hydrates thereof.

A „sulfo group” or “sulfo” as a prefix or within a formula definition, as used herein is a group having the formula of -SO ₃ H (i.e. -S(=0) ₂ -0H or S(0) ₂ 0H which are equivalents herein) or -SO ₃ - i.e. its deprotonated, anionic form is included in the definition; moreover, any of its salts or its form in an ion pair with a positively charged ion is also included. The sulfo group is the functional group of a sulfonic acid and can be thought of as a sulfuric acid with one hydroxyl group removed wherein the sulfo group may be covalently bound to a carbon (forming a sulfonic acid) or an oxygen (forming a sulfate). The term „sulfo group” or “sulfo” includes all possible forms as well as salts or hydrates thereof.

A “carboxylic acid” is an organic compound having the general formula R-C(=0)0H (i.e. R-COOH or R- C(0)0H ) which are considered as equivalent descriptions of carboxylic acids herein) that contains a carboxyl group wherein R refers to the rest of the molecule, i.e. an organic group with a carbon atom adjacent to the sulfur of the sulfo group of the sulfonic acid. The carboxylate anion (R-COO- or RCO2-) of a carboxylic acid is usually named with the suffix -ate, in keeping with the general pattern of -ic acid and -ate for a conjugate acid and its conjugate base, respectively. The term “carboxylic acid” includes all these forms as well as salts or hydrates thereof.

A “carboxyl group” or “carboxy” as a prefix or within a formula definition as used herein is a group having the formula of -C(=0)0H (i.e. -C(0)0H or -COOH which are equivalents), or -COO- i.e. its deprotonated, anionic form (also used as carboxylate) is included in the definition; moreover, any of its salts or its form in an ion pair with a positively charged ion is also included. The carboxyl group is the functional group of a carboxylic acid and can be thought of as a carbonic acid with one hydroxyl group removed wherein the carboxyl group may be covalently bound to a carbon (forming a carboxylic acid) or an oxygen (forming a carbonate). The term “carboxyl group” or “carboxy” includes all possible forms as well as salts or hydrates thereof.

The term “ester” refers to an ester of a carboxylic acid unless indicated differently herein having the formula R _r C(=0)0R ₂ (see equivalants above mutatis mutandis) wherein R and R ₂ refers to organic moieties or groups with a respective carbon atom covalently bound to the carbon and to the oxygen of the ester group.

A heterocycle is an organic cyclic compound that has atoms of at least two different elements as members of its ring(s) wherein one of the elements is carbon.

An “alkylene” is a a bivalent saturated aliphatic radical (such as ethylene) regarded as derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from different carbon atoms.

A “member” of an organic compound or group as used herein is any atom covalently bound in the compound which is different from hydrogen. Preferably, members of organic compounds of the present invention are selected from C, N, O and S.

The size of an organic compound or group may be given herein by a number of its members, said number being a positive integer, i.e. atoms different from H and is expressed in giving this number with the term membered added. Thus, an n-membered compound or group is a compound or group having n atoms covalently bound ad being different from H wherein said n-membered compound or group may comprise cyclic parts and branched or unbranched aliphatic parts as well as substituents the member atom(s) of which also included in the number n whereas H atoms are not; or an n-membered ring is a cyclic compound having n ring atom, said cyclic compound being either an aromatic or not aromatic compound and being either a heterocycle or a carbohydrate.

It is to be understood that, as well known by a person skilled in the art, the compounds according to general formulae II can be described by variant formulae which are considered as being covered by any of formulae II (including formulae II.2, II.3 and II.4) as follows: wherein actually the positive charge is distributed in the delocalized electron system of the molecule and/or can also be allocated to other atoms, e.g. carbon atoms, and/or can be illustrated as a partial positive charge on atoms wherein it may be localized, as known by a person skilled in the art.

Other aspects of the invention are given in the following paragraphs.

Pyranine is a novel substrate of hepatic OATPs, 1B1, 1B3 and 2B1 and MRP2 Pyranine did not accumulate in control, mock transfected A431 cells. A typical OATP-mediated uptake was found in A431 cells expressing one of the hepatic OATPs, 1B1, 1B3, 2B1, or OATP1A2 (Figure 2) revealing that pyranine is a common substrate of these uptake transporters.

In order to screen, which other pyrenes are OATP substrates, OATP-mediated uptake of pyranine analogs was tested in A431 cell lines overexpressing human OATP1A2, OATP1B1, OATP1B3 or OATP2B1.

Pyrene and 1,3,6,8-pyrenetetrasulfonic acid were not transported by OATP1B1, OATP1B3, OATP2B1 and OATP1A2. 1 -pyrenesulfonic acid was exclusively transported by OATP1A2. 6, 8-dihydroxy- 1,3- pyrenedisulfonic acid and pyranine proved to be general substrates of all the tested OATPs. 8-acetoxy-l, 3, 6- pyrenetrisulfonic acid was transported by OATPs, 1B1, 1B3 and 2B1, but not by OATP1A2.

To characterize the susceptibility of fluorescent OATP probes to MRP2 mediated transport, we used inside-out membrane vesicles (IOV) prepared from Sfi> ( Spodoptera fmgiperda ) cells overexpressing MRP2 (Figure3). IOVs allow the investigation of the transport of membrane impermeable substrates by efflux transporters that otherwise, in lack of passive uptake, could not be investigated in single transfectants. IOVs prepared from mock transfected Sfi> cells, as well as transport in the presence of MgAMP served as negative controls for transport experiments. As shown on Figure3A and 3B, ATP-dependent transport by MRP2 was observed for ZV, LDG, CB, SR101, FMTX and pyranine, while LDV was not transported.

The IOV-based transport screen identified hitherto undescribed transport of ZV, LDG, pyranine, SR101 and CB by MRP2 (Figure3).

The invention provides a use of a fluorescent substrate for identifying a test compound which is capable of activating or inhibiting the transport of a substrate by human MRP2, wherein the fluorescent substrate is selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II).

Preferably, the fluorescent substrate is selected from sulfopyrenes having general formula (I). Preferably, the fluorescent compound having general formula I is selected from pyranine (trisodium 8-hydroxypyrene-l, 3,6- trisulfonate, 8-hydroxypyrene-l,3,6-trisulfonic acid), Cascade Blue (pyrenyloxytrisulfonic acid), Cascade Blue hydrazide (CAS name/number: Acetic acid, [(3,6,8-trisulfo-l-pyrenyl)oxy]-, 1-hydrazide, trisodium salt 137182- 38-8), 6,8-Dihydroxy-l,3-pyrenedisulfonic acid disodium salt (DF1PDS), Alexa Fluor 405, 8-acetoxypyrene- 1,3,6 trisulfonic acid trisodium salt. Flighly preferably the fluorescent compound is selected from pyranine, DF1PDS, Alexa Fluor 405, 8-acetoxypyrene-l,3,6 trisulfonic acid trisodium salt and Cascade Blue hydrazide. Flighly preferably the fluorescent substrate is selected from pyranine, DF1PDS and Cascade Blue hydrazide. Preferably, the fluorescent substrate is selected from sulforhodamines having general formula (II). Flighly preferably the fluorescent compound is Sulforhodamine 101.

In a preferred embodiment of the use according to the invention, the use comprises a) incubating inside out (IOV) vesicles expressing human MRP2 with the fluorescent compound and the test compound under conditions allowing transport of the fluorescent compound across the vesicle membrane by the human MRP2 and b) measuring the level of fluorescence taken up in the vesicles, wherein the test compound is an inhibitor of the transport of a substrate by the human MRP2 if the level of fluorescence taken up in the vesicles in the presence of the test compound is lower than in the absence of the test compound, the test compound is an activator of the transport of a substrate by the human MRP2 if the level of fluorescence taken up in the vesicles in the presence of the test compound is higher than in the absence of the test compound or the test compound is not a modulator of the transport of a substrate by the MRP2 if the level of fluorescence taken up in the vesicles in the presence of the test compound is unaltered compared to the level of fluorescence taken up in the vesicles in the absence of the test compound.

Use of pyranine is provided for identifying a test compound which is capable of activating or inhibiting the transport of a substrate by a human OATP, preferably human OATP1B1, OATP1B3, OATP1A2 and OATP2B1.

EXAMPLES

Materials and Methods Materials

Zombie Violet was purchased from BioLegend® (San Diego, CA, US). LIVE/DEAD® Fixable Cell Stain Dyes (Violet and Green), fluorescein-methotrexate and Cascade Blue hydrazide were purchased from Thermo Fisher Scientific (Waltham, MA, US). All other materials, if not indicated otherwise, were from Sigma Aldrich, Merck (Budapest, HU).

Generation of cell lines and cell culturing

A431 cells expressing OATPs 1B1, 1B3 or 2B1 and their mock transfected counterparts were generated previously as described in (Patik et al., 2018). The MDCKII-MRP2 cell line was generated previously (Bakos et al., 2000). In order to generate MDCKII cells expressing ABCG2, MDCKII parental cells were transfected with 1 pg plasmid DNA (pSB-CMV-ABCG2, allowing transposon mediated genomic insertion of ABCG2 cDNA (Saranko et al., 2013)) and 100 ng plasmid containing the lOOx Sleeping Beauty transposase using Lipofectamine 2000® reagent (Thermo Fisher Scientific) according to the recommendation of the manufacturer. After 48 hours the transfection medium was removed and the cells were selected in puromycin (1 pg/ml) for two weeks. Transfected cells were sorted based on labeling by the anti-ABCG2 monoclonal antibody 5D3 (Bioscience), which binds to a surface epitope (Ozvegy et al., 2002). Cells showing 5D3 positivity were sorted using a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA, US).

OATP1B1 expression in MDCKII, MDCKII-MRP2 or MDCKII-ABCG2 cells was achieved by recombinant lenti viruses as described earlier (Patik et al., 2018). Briefly, MDCKII parental, MDCKII -MRP2 and MDCKII- ABCG2 cells were transfected with the pRRL-CMV-OATPlBl-MCS-IRES-ACD4 vector. In order to generate mock transfected control cells for transport experiments, MDCKII cells were transfected with the pRRL-EFl- ACD4 vector. Transduced cells were sorted based on their CD4 positivity using a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA, US). OATP1B1 overexpressing cells were sorted based on their increased Live/Dead Green uptake, as described earlier (Patik et al., 2018).

A431 and MDCKII cells were grown in DMEM (Gibco, Thermo Fisher Scientific (Waltham, MA, US)) completed with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin at 37 °C, 5% C02, under sterile conditions.

Western Blot MDCKII cell lysates were separated on 7.5 % Laemmli SDS-PAGE gels and transferred onto PVDF membranes. Immunoblotting was performed as described (Sarkadi et al., 1992). Membranes were incubated overnight with anti-OATPIBl, anti-MRP2 (M2-I-4 / M2-III-6 monoclonal antibody) (Bakos et al., 2000) or anti-ABCG2 (BXP- 21, (Maliepaard et al., 2001)) antibodies or anti-(i-actin antibody (A1978, Sigma). The antibody used for the detection of OATP1B1 was a kind gift from Dr. Bruno Stieger (Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland) (Kullak-Ublick et al., 2001). Secondary antibodies were HRP-conjugated anti-rabbit (OATP1B1) or anti-mouse (MRP2, ABCG2, I.S-actin) antibodies (Jackson ImmunoResearch, Suffolk, UK) in a dilution of 20,000x. Luminescence was detected using the Luminor Enhancer Solution kit by Thermo Scientific (Waltham, MA, US).

MRP2 and ABCG2 expression in insect cells and inside-out membrane vesicle preparation

Recombinant baculovirus containing the ABCC2/MRP2 cDNA (Bakos et al., 2000), ABCG2 cDNA (Ozvegy et al., 2002) or the cDNA of an unrelated protein (Patik et al., 2015) were used to achieve transient expression in Sf9 insect cells. Culturing and infection of Sf9 cells was performed as described earlier (Sarkadi et al., 1992). Virus-infected Sf9 cells were harvested after 72 hours. Following washing with Tris-mannitol buffer (50 mM Tris, pH 7.0, with HC1, 300 mM mannitol and 0.5 mM phenylmethylsulfonyl fluoride), cells were lysed and homogenized in TMEP (50 mM Tris, pH 7.0, with HC1, 50 mM mannitol, 2 mM EGTA, 10 pg/ml leupeptin, 8 pg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mM dithiotreitol) using glass tissue grinder tubes. Undisrupted cells were removed by centrifugation for 10 min at 500 g. Finally, the supernatant containing the membranes was centrifuged for 1 h at 100 000 g, and the pellet was resuspended in TMEP (with freshly appended 0.5 mM phenylmethylsulfonyl fluoride) at concentration of 5-10 mg/ml. Membranes were stored at - 80°C in aliquots. (Bakos et al., 2000; Sarkadi et al., 1992). In the case of ABCG2 cholesterol enriched membranes (and their controls) IOVs were prepared in order to achieve maximal ABCG2 activity (Telbisz et al., 2007). Cholesterol loading was performed by incubation of the membranes with TMEP containing 2 mM Cholesterol-RAMEB (Cyclolab, Hungary) on ice for 30 minutes, prior to the final centrifugation step (Telbisz et al., 2007).

Transport measurements in inside-out vesicles

Membrane vesicles (50 pg/tube) were incubated in transport buffer (Bakos et al., 2000) with 4 mM MgATP or 4 mM MgAMP and with the fluorescent dyes 1 mM sulforhodamine 101 (SR101)/fluorescein-methotrexate (FMTX), 5 mM pyranine/Cascade Blue (CB), 10 mM Lucifer Yellow (LY) or 0.2 mΐ/tube ZV/LDV/LDG for 10 (ZV/LDV/LDG/FMTX/SR101/LY) / 20 (pyranine) / 30 (CB) minutes in 150 mΐ final volume at 37°C. These experimental conditions were evaluated by measuring time- and concentration dependent transport of the fluorescent dyes in preliminary experiments. For each compound, time and concentration values yielding the highest signal/noise ratio measured were chosen. When inhibition was investigated, the vesicles were pre incubated with 1 mM Kol43, a known ABCG2 inhibitor (Allen et al., 2002) prior to the addition of the fluorescent dyes. Reaction was stopped by the addition of 550 mΐ ice cold transport buffer and by placing the samples on ice. Eppendorf tubes were centrifuged for 5 min at 22000 g. Supernatant was eliminated and the pellet was suspended in 200 mΐ RT (room temperature) 1 x Phosphate Buffered Saline (PBS). The suspensions were pipetted onto 96 well plates and fluorescence intensity was measured in an Enspire Fluorescent plate reader (Perkin Elmer) at the following wavelengths: 405/423 nm (ZV), 416/451 nm (LDV), 495/520 nm (LDG), 403/517 nm (pyranine), 400/419 nm (CB), 586/605 nm (SR101), 428/540 nm (LY), 497/516 nm (FMTX). Transport activity in the case of CB, pyranine, LY, SR101 and FMTX was determined based on a calibration curve.

Transport measurements in MDCKII cells by flow cytometry

MDCKII cells were collected following trypsinization (0.2% trypsin) in complete DMEM. After washing in 1 ml uptake buffer (125 mM NaCl, 4.8 mM KC1, 1.2 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 12 mM MgSCF, 25 mM MES, and 5.6 mM glucose, with the pH adjusted to 5.5/7.4 using 1 M HEPES and 10 N NaOH for OATP/ABC function respectively), 5 x 10 ⁵ cells were incubated at 37°C for 10 min (calcein-AM (CaAM)) / 30 min (CB, DCV) in 100 pi fluorescent dye (final concentrations: 0.5 mM CaAM, 1-1 mM CB or DCV) diluted in the appropriate buffer. CB/CaAM/DCV were used as substrates of OATP1B1/MRP2/ABCG2 for validating functionality. The reaction was stopped by the addition of 700 mΐ ice-cold 1 x PBS, and the samples were kept on ice until the flow cytometry analysis. Cellular fluorescence was determined of at least 20000 living cells from each sample using an Attune Acoustic Focusing Cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA, US). OATP-mediated transport of sulfopyrenes using a microplate based transport assay

Uptake of pyranine in A431 cells overexpressing OATPs 1B1, 1B3 or 2B1 was measured as described previously (Patik et al., 2018). Briefly, A431 cells (8 x 10 ⁴ /well) were seeded on 96-well plates in 200 mΐ DMEM one day prior to the transport measurement. Next day the medium was removed, and the cells were washed three times at room temperature with 1 x Phosphate Buffered Saline (PBS). The cells were pre-incubated with 50 mΐ uptake buffer (see above) at 37°C. The reaction was started by the addition of 50 mΐ uptake buffer containing increasing concentrations of pyranine. Cells were then incubated at 37°C for 10 minutes (OATP1B1) or 15 minutes (OATP1B3 and OATP2B1). The applied incubation time was evaluated during previous unpublished data. The reaction was stopped by removing the supernatant and washing the cells three times with ice-cold 1 x PBS. Wells were loaded with 200 mΐ ice-cold 1 x PBS and fluorescence was determined using an Enspire plate reader Ex/Em: 403/517 nm.

In case of other pyrene derivatives, The reaction was started by the addition of 50 mΐ uptake buffer containing pyranine (Sigma), pyrene (Sigma), 1-pyrenesulfonic acid (Sigma), 6, 8-dihydro xy-l,3-pyrenedisulfonic acid (Sigma), 1,3,6,8-pyrenetetrasulfonic acid (Sigma) or 8-acetoxy-l, 3, 6 pyrenetrisulfonic acid (VWR) in a final concentration of 20 mM. After 30 min of incubation at 37°C the reaction was stopped by removing the supernatant and washing the cells three times with ice-cold 1 x PBS. Finally, 200 mΐ 1 x PBS was added to each well and fluorescence was determined using an Enspire plate reader. Ex/Em wavelengths were the following:

Ex/Em nm:

Pyranine (8-hydroxy-l, 3, 6-pyrenetrisulfonic acid): 403/517

Pyrene: 365/476

1-pyrenesulfonic acid: 347/378

6.8-dihydroxy-l,3-pyrenedisulfonic acid: 460/510

1.3.6.8-pyrenetetrasulfonic acid: 353/406

8-acetoxy-l, 3, 6 pyrenetrisulfonic acid: 460/510. OATP-dependent transport was determined by extracting fluorescence measured in mock transfected cells. Transport activity was calculated based on a calibration curve. Experiments were repeated in 3 biological replicates.

Transcellular transport measurements

For transcellular transport experiments, OATP1B1 and/or MRP2 or ABCG2 overexpressing MDCKII cells (9 x 104 cells/insert) were grown on Tissue culture plate inserts (6.5 mm diameter, 0.4 mM pore size, VWR Ltd., Hungary) for four days. The cells were seeded in 300 mΐ complete DMEM onto the insert membranes and 1 ml media was added to the wells around the inserts in 24 well plates. The transport measurement was started by the removal of the medium from the transwell inserts and by washing the cells two times with 300 mΐ pH 7.4 uptake buffer (see above). The wells were washed three times with 1 ml pH 5.5 uptake buffer (see above). After washing, 300 mΐ pH 7.4 buffer was pipetted into the inserts containing the cells and 1 ml pH 5.5 buffer into the wells and a 10 min pre-incubation period at 37°C was applied. The reaction was started by the addition of 1 ml pyranine, CB, SR101, or FMTX (final concentrations 5-5 mM and 1-1 mM respectively) to the wells at 37°C. When inhibitors were tested, 10 mM cyclosporin A (CsA) or 40 mM benzbromarone was added to the lower or upper compartment, respectively. To determine transport, 30 mΐ samples from the upper compartment were collected every 5 minutes and pipetted into 70 mΐ 1 x PBS for fluorescence measurements. In the case of MDCKII-ABCG2-OATP1B1 cells, CB transport was determined at the following time points: 0, 15 and 30 minutes. The fluorescence intensity of the samples was determined using an Enspire plate reader (Perkin Elmer) at the following wavelengths: 403/517 nm (pyranine), 400/419 nm (CB), 586/605 nm (SR101), or 497/516 nm (FMTX).

In order to evaluate the transport in the opposite direction (A-B), transport reaction was started from the apical side by adding the substrates in the same concentration as applied before but in 300 mΐ pH 7.4 buffer. Samples were collected from the wells (B side) to a 96 well plate, 100 mΐ at each time points until 25 minutes and fluorescence intensity was determined as described before.

For evaluating intracellular accumulation of pyranine, transport reaction in B-A direction was stopped after 30 minutes by removing the solutions from the wells and inserts and washing the cells three times with cold 1 x PBS on ice. Transwell inserts were then cut out and placed into 200 mΐ 1% Triton-PBS in Eppendorf tubes. Inserts were incubated at RT for 75 minutes, and then cell lysates were pipetted onto 96 well plates. Fluorescence intensity was determined using an Enspire plate reader as described above, Ex/Em: 403/517. In order to define the amount of the dyes in the samples, a calibration curve was generated by determining the fluorescence of increasing amounts of the given dye dissolved in 200 mΐ 1 x PBS. We found that fluoresce of the dyes diluted in PBS or in cells remained stable even after 90 minutes of incubation at room temperature (data not shown).

Data analysis and statistics

Kinetic parameters shown in Figure 1 of dye uptake were analyzed by Hilll fit using the OriginPro 8 software (GraphPad, La Jolla, CA, USA). Statistical significance was calculated by Student’s t-test between ATP- dependent signals. Delta values were generated by subtraction of MgAMP or MgAMP+Ko signals from MgATP or MgATP+Ko signals, respectively. These delta values were compared for statistical significance by Student’s t-test, *: p < 0.05, **: p < 0.01, ***: p < 0.001 (Figure 3).

Statistical analysis of the samples shown in Figure 5B for multi comparison was evaluated by Tukey-Kramer HSD (Honest Significant Differences) procedure, as a post-hoc test, after rejecting HO in One-Way ANOVA (a=0.05). Samples marked with the same letter (“a” for MRP2 and Ctr.) were not significantly different from each other.

Results

Pyranine is a novel substrate of hepatic OATPs, 1B1, 1B3 and 2B1

While pyranine did not accumulate in control, mock transfected A431 cells, a typical OATP-mediated uptake was found in A431 cells expressing one of the hepatic OATPs, 1B1, 1B3 or 2B1 (Figure 1), or OATP1A2 (Figure 2) revealing that pyranine is a common substrate of these uptake transporters.

Test of additional pyranine analogs for transport by OATPs 1A2, 1B1, 1B3 and 2B1

In order to screen, which other pyrenes are OATP substrates, OATP-mediated uptake of pyranine analogs was tested in A431 cell lines overexpressing human OATP1A2, OATP1B1, OATP1B3 or OATP2B1, or their mock transfected controls (Bakos et al., 2019, Patik et al., 2018). Pyrene and 1,3,6,8-pyrenetetrasulfonic acid was not transported by OATP1B1, OATP1B3, OATP2B1 and OATP1A2 (Figure 2). 1-pyrenesulfonic acid was exclusively transported by OATP1A2 (Figure 2). 6, 8-dihydroxy- 1,3-pyrenedisulfonic acid and pyranine proved to be general substrates of all the tested OATPs (Figure 2). 8-acetoxy-l, 3, 6-pyrenetrisulfonic acid was transported by OATPs, 1B1, 1B3 and 2B1, but not by OATP1A2.

Identification of novel common fluorescent OATP1B1, MRP2 and ABCG2 substrates To characterize the susceptibility of fluorescent OATP probes to MRP2 or ABCG2 mediated transport, we used inside-out membrane vesicles prepared from Sj9 ( Spodoptera fmgiperda ) cells overexpressing either MRP2 or ABCG2 (Figure3). IOVs allow the investigation of the transport of membrane impermeable substrates by efflux transporters that otherwise, in lack of passive uptake, could not be investigated in single transfectants. IOVs prepared from mock transfected Sj9 cells, as well as transport in the presence of MgAMP served as negative controls for transport experiments. On the other hand, Lucifer yellow (LY) and fluorescein-methotrexate (FMTX), documented substrates of ABCG2 or MRP2, respectively, were used as positive controls. As shown on Figure 3A and 3B, ATP-dependent transport by MRP2 was observed for ZV, LDG, CB, SR101 and pyranine, while LDV was not transported. In the case of ABCG2, although uptake of the known substrate Lucifer yellow (LY) indicated functionality, there was no transport of ZV, LDG, pyranine or SR101. On the other hand, we observed significant transport of LDV and a weak transport of CB by ABCG2. ATP-dependent transport of SR101 was also present in control vesicles (Figure3B and 3D). To reveal the nature of the SR101 uptake observed in control vesicles, transport was measured using EDTA as a Mg2+ chelator or Na-ortho vanadate as a general ATP-ase inhibitor. These experiments confirmed the involvement of a yet undefined insect transporter involved in SR101 transport.

Transcellular transport of pyranine, a novel common substrate of OATP1B1 and MRP2

In order to test whether the newly identified dual fluorescent substrates can indeed be applied for simultaneous investigation of OATP1B1 and MRP2 function, we examined their transport in double transfected polarized MDCKII cells. Since the structure of Z V, LDG and LDV is unknown, these dyes were excluded from further experiments. First, stable MDCKII cell lines co-expressing these transporters, termed as MDCKII-MRP2- OATP1B1 or MDCKII-ABCG2-OATP1B1 were generated. Cell lines containing solely OATP1B1, MRP2, ABCG2 or mock transfected cells (MDCKII-1B1, MDCKII-MRP2, MDCKII- ABCG2 and MDCKII-Ctr.) served as controls. Expression was confirmed by Western blot analysis (Figure 4A), and functionality of the transporters was verified by transport assays (using CB, CaAM and DCV as OATP1B1, MRP2 or ABCG2 substrates, respectively (Figure 4B).

For transcellular transport measurements, MDCKII cells were grown on transwell inserts for 4 days to reach a polarized state, when OATP1B1 is localized to the basolateral membrane, and MRP2 or ABCG2 are found apically. First, FMTX, a previously identified substrate of OATP1B1 (Gui et al., 2010) and MRP2 (Notenboom et ah, 2005; Prevoo et ah, 2011) was used for the setup of the transcellular transport measurement. As shown on Figure 4C a time-dependent transcellular basolateral to apical (B-A) transport could be observed in MRP2- OATP1B1 double transfectants that was not present in the control, single transfected MDCKII-1B1, MDCKII- MRP2 or MDCKII-Ctr. (mock) cells. Next, transcellular transport of pyranine was determined. As shown on Figure 5 A, a time-dependent B-A transport of pyranine could be observed in MRP2-OATP1B1 double transfectants, and there was no B-A transport in single or mock transfected cells. When intracellular accumulation of pyranine was measured, we found that pyraninePyranine cannot enter the cells without the function of OATP1B1, hence it cannot be detected in control or MRP2 single transfected cells (Fig 5 B). To exclude leakage of the cell monolayer, concurrent pyranine transport in the apical to basolateral (A-B) direction was investigated. Transport of pyranine in both directions was measured on the MRP2-OATP1B1 double and OATP1B1 single transfectant cells (Fig.5C). We found negligible transcellular transport from the apical to the basal compartment in both cell lines. These results indicate that the B to A directed transport is indeed derived from the interaction of the dye with the OATP1B1 and MRP2 transporters. Finally, in order to assure that the increasing fluorescent pyranine signal at the A side of double transfected cells is a result of the concerted action of OATP1B1 and MRP2, inhibitory measurements were performed for both transporters (Fig. 5D). Double transfected cells treated with either cyclosporin A at the basal side or benzbromarone at the apical side showed no detectable transcellular transport. To control these experiments, OATP1B1 and MRP2 single transfected cells were also treated with cyclosporin A or benzbromarone, at the basal or apical side, respectively. All these results were consistent with the transcellular transport of pyranine in double transfectants, mediated by OATP1B1- mediated uptake and MRP2-mediated efflux.

Identification of Cascade Blue hydrazide and sulforhodamine 101 as dual OATP1B1 and MRP2 probes in transwell measurements

Based on the experiments evaluated in the Sf9 IOV assay, SR101 is another potential common substrate of OATP1B1 and MRP2, and a weak accumulation of CB in IO Vs with MRP2 or ABCG2 was also detected. Therefore, in order to test their applicability as fluorescent probes in vectorial transport measurements their transcellular transport was investigated in transwells (Figure 6). When following time dependent accumulation of CB or SR101 in the apical compartment of the transwells, vectorial transport in MRP2-OATP1B1 double transfected cells was detected for both substrates (Figure 6A-B). Interestingly, although we detected ABCG2- mediated transport of CB in IOVs, transcellular transport activity in ABCG2-OATP1B1 cells could not be observed (not shown). Transcellular transport of CB and SR101 in the A-B direction was negligible (Fig. 6C), and inhibitory measurements performed with these dyes also confirmed OATP1B1- and MRP2-mediated transport of CB and SR101 (Fig. 6D).

Transcellular transport of pyranine analogs by the function of OATP1B1 and MRP2

After the selection of fluorescent dyes that are transported into the cells by the action of OATP1B1 Figure 2, we tested the transcellular transport of these dyes in polarized MDCKII cells co-expressing OATP1B1 and MRP2, or their single (MDCKII-OATP1B1, MDCKII-MRP2) or mock transfected controls (ctr.). Additionally, Alexa Fluor 405, a previously identified OATP1B and OATP2B1 substrate (Patik et al., 2018) was also tested for transcellular transport.6, 8-dihydroxy- 1,3-pyrenedisulfonic acid, 8-acetoxy-l, 3, 6 pyrenetrisulfonic acid and Alexa-Fluor 405 are novel dual fluorescent probes of MRP2 and OATP1B1 (Figure 7).

Illustrative example of a selective fluorescent assay for complex drug interaction screening of the

OATP1B1 and MRP2 transporters

The assay (kit) contains the following elements:

• double overexpressing polarized cells (e.g. MDCKII cells): MRP2-OATP1B1 overexpressing cells

• fluorescent compound which is a substrate of both O ATP IB and MRP2, selected from sulfopyrenes having general formula (I) or sulforhodamines having general formula (II) (e.g. pyranine)

• polarized cells (of the same type as the double overexpressing polarized cells, e.g. MDCKII cells) mock transfected, OATP1B1 overexpressing, MRP2 overexpressing cells

The assay is based on the measurement of transcellular (basolateral to apical) transport of the fluorescent dye in MRP2-OATP1B1 overexpressing polarized MDCKII cells grown on transwell inserts.

Potential readouts:

A: validation of the assay. In the absence of added inhibitors, the basolaterally administered dye is transported from the basolateral compartment to the apical compartment by the function of OATP1B1 and MRP2. This results in an increased fluorescent signal in the intracellular and apical compartments (Figure 8A and 8B, left panel). In MDCKII mock, MDCKII-OATP1B1, MDCKII-MRP2 cells there is no signal in the apical compartment. In MDCKII-OATP1B1 cells an intracellular dye accumulation can be observed.

B: Effect of inhibition of OATP1B1 function: if the tested compound inhibits the function of OATP1B1 than there is no transcellular transport, no signal can be detected either intracellularly or apically (Figure 8A, middle panel).

C: Effect of inhibition of MRP2 function: if the tested compound inhibits the function of MRP2 than there is no transcellular transport. No signal can be detected apically, but an increased intracellular signal can be measured (Figure 8A, right panel).

D: Effect of activation of OATP1B1 function: if the tested compound activates the function of OATP1B1 than increased fluorescence signal can be detected in the cells with unaltered fluorescence in the apical compartment, or increased fluorescence signal can be detected both within the cells and in the apical compartment (Figure 8B, middle panel). E: Effect of activation of MRP2 function: if the tested compound activates the function of MRP2 than increased fluorescence signal can be detected in the apical compartment with unaltered/lower signal in the cells (Figure 8B, right panel).

In cell-free experiments the fluorescence of the test compound alone and in the presence of the fluorescent probe should be measured in order to exclude indirect (not related to transporter function) effect of the test compound on fluorescence (quenching, etc.)

Selective fluorescent assay for monitoring transporter expression/function in hepatocytes

Cultured hepatocytes and differentiated hepatocytes are needed for modeling transport processes of the liver. However, it is often difficult to achieve proper expression levels of uptake and efflux transporters in these cultured cells. The fluorescent substrate(s) described herein (i.e. fluorescent compound which is a substrate of both O ATP IB and MRP2, selected from sulfopyrenes having general formula (I) (e.g. pyranine) or sulforhodamines having general formula (II)) can be applied to measure the function of the hepatic OATP uptake transporters, OATP1B1, OATP1B3 and OATP2B1, and also that of the ABC efflux transporter MRP2. Canalicular transport of the novel dyes can be expected if these transporters are expressed in these cells.

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