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WO2021123851A1 - Dosage à fluorescence add-and-read en temps réel pour tester la fonction et les interactions de médicaments de transporteurs oatp - Google Patents

Dosage à fluorescence add-and-read en temps réel pour tester la fonction et les interactions de médicaments de transporteurs oatp Download PDF

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WO2021123851A1
WO2021123851A1 PCT/HU2020/050062 HU2020050062W WO2021123851A1 WO 2021123851 A1 WO2021123851 A1 WO 2021123851A1 HU 2020050062 W HU2020050062 W HU 2020050062W WO 2021123851 A1 WO2021123851 A1 WO 2021123851A1
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oatp
fluorescence
substrate
membrane
control
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WO2021123851A8 (fr
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Csilla LACZKA
Orsolya UNGVÁRI
Éva TUSNÁDY
Laura KIRÁLY
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Cellpharma Kft
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Cellpharma Kft
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Priority claimed from PCT/HU2020/050014 external-priority patent/WO2021123847A1/fr
Priority claimed from HU2000168A external-priority patent/HUP2000168A1/hu
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Priority to EP20901298.8A priority Critical patent/EP4077707A4/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of assessing transport activity of OATP (Organic anion -transporting polypeptide) transport proteins.
  • OATP Organic anion -transporting polypeptide
  • the invention relates to a real-time, add-and-read fluorescence-based method for determining whether a test compound interferes with substrate transport activity of an OATP, by using a non- fluorescent control substrate which becomes fluorescent within a cell.
  • the method is also applicable to measure the expression of an OATP and to discriminate between a transported substrate and an inhibitor of an OATP.
  • Transmembrane proteins provide a strict control over the transmembrane movement of ions, nutrients, metabolites, hormones and neurotransmitters (Giacomini et al., 2010). Additionally, membrane transporters expressed in various tissue barriers of the human body play important role in the absorption, distribution and elimination of drugs and thus influence their efficacy (Hagenbuch and Stieger, 2013). Cellular entry of drugs is governed by Solute Carriers (SLC) of which Organic anion-transporting polypeptides (OATPs) have renowned role.
  • SLC Solute Carriers
  • OATPs Organic anion-transporting polypeptides
  • OATPs are organic anion exchangers expressed in the cell membrane that mediate 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 (Hagenbuch and Stieger, 2013; Roth et al., 2012).
  • OATP1B1 and OATP1B3 encoded by the SLCOlBl/3 genes are exclusively expressed in the sinusoidal membrane of hepatocytes (Konig et al., 2000; Kullak-Ublick et al., 2001).
  • OATP2B1 is expressed widely in the human body (e.g.
  • OATPs 1B1, 1B3 and 2B1 are multispecific transporters that besides their endogenous substrates also transport numerous clinically applied drugs (Kovacsics et al., 2017). Since these OATPs are renowned participants of pharmacokinetics of their substrates, inhibition of their function due to mutations or by the co-administration of their substrates/inhibitors may lead to altered pharmacokinetics and unexpected side effects (Link et al., 2008; Shitara, 2011; Shitara et al., 2003).
  • Radioactively labeled substrates such as bromosulfophthalein, leukotriene C4, dehydroepiandrosterone sulfate (DHEAS), estrone-3 -sulfate (E1S) or estradiol-17p-D-glucuronide (E217G) have been repeatedly used in such assays (Cui et al., 2001; Hirouchi et al., 2009; Liu et al., 2006; Matsushima et al., 2005).
  • DHEAS dehydroepiandrosterone sulfate
  • E1S estrone-3 -sulfate
  • E217G estradiol-17p-D-glucuronide
  • 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 (De Bruyn et al., 2011; Gui et al., 2010; Izumi et al., 2016; Patik et al., 2018).
  • HTS high-throughput screen
  • CAM is a membrane permeable non-fluorescent compound that rapidly enters the cells by passive diffusion and then it becomes fluorescent when cleaved by esterases within living cells.
  • MRP2 and OATPs 1B1, 1B3 and 2B1 possess numerous common substrates, CAM is not suited for the measurement of OATP function since it is membrane permeable.
  • EP2684963 discloses a method of screening for a compound that enhances or inhibits a transport activity of OATP1B1, using dichlorofluorescein.
  • dichlorofluorescein is not transported by OATP1B3 (Izumi et al., 2016).
  • this method requires the application of adherent cell lines, and, also manual work to perform different washing steps, to remove the extracellular compound at the end of the transport reaction and in the end the lysis of the cells is required. Since drug development, drug- transporter interaction tests or structure-function studies require the screen of a plethora of potential OATP interacting molecules a more robust screening platform is needed.
  • 8-acetoxypyrene-l,3,6-trisulfonate is a non-fluorescent compound that becomes fluorescent after entering a living cell and then cleaved by esterases.
  • 8-acetoxypyrene-l, 3, 6-trisulfonate is cell permeable (e.g. pn ductdis;>lav)/03F.3 ⁇ 47D17C3 ⁇ 46F5E5 80257CA1005BDC6A; all downloaded 17-05-2020; (Wrobel et al, 2011), while other sources identify it as a cell impermeable compound (Langton et al, 2017).
  • the invention provides a method suitable for investigating the interaction of compounds with OATPs. Specifically, a method is provided for investigating the interaction of compounds of interest with OATPs, by determining the interference of a compound with OATP transport activity or OATP and ABC transport activity in a no- wash, real-time, “add-and-read” fluorescence -based assay. The method is useful for e.g. screening drug candidates for transport protein mediated drug-drug interactions, such as the inhibition or activation of the transport of an active agent by OATP into a cell (e.g. hepatocyte, enterocyte, blood-brain endothelial cell, neuron, muscle or kidney cell). The invention also provides a method for determining whether a test compound which interferes with OATP transport activity is a transported substrate or a non-transported inhibitor of the OATP.
  • a test compound which interferes with OATP transport activity is a transported substrate or a non-transported inhibitor of the OATP.
  • the invention provides a method for determining whether a test compound interferes with substrate transport activity of an organic anion-transporting polypeptide (OATP), wherein the method comprises the steps of a) contacting a membrane -bound vesicle comprising an enzyme and expressing the OATP in the membrane with a control substrate in a test sample in the presence of the test compound and in a control sample in the absence of the test compound, wherein the control substrate is convertible by the enzyme into a fluorescent metabolite, b) measuring the levels of fluorescence produced by the fluorescent metabolite in the vesicles in the test sample and in the control sample, c) comparing the levels of fluorescence measured in b), d) determining that the test compound interferes with the substrate transport activity of the OATP if there is a difference between the levels of fluorescence measured in the test sample and the control sample, wherein the control substrate is a fluorogenic sulfopyrene compound, which is a substrate of the OATP.
  • OATP organic ani
  • test compound If the level of fluorescence measured in b) is higher in the test sample than in the control sample the test compound is identified as an activator of OATP mediated transport.
  • test compound If the level of fluorescence measured in b) is lower in the test sample than in the control sample the test compound is identified as an inhibitor of OATP mediated transport.
  • step d it is determined that the test compound interferes with the substrate transport by the OATP it is identified as a modulator thereof.
  • the method is for determining whether the test compound which interferes with the substrate transport activity of the OATP (i.e. a modulator of the substrate transport activity of the OATP) is a substrate of the OATP, comprising the steps of: a) contacting a membrane -bound vesicle comprising an enzyme and expressing the OATP in the membrane with a control substrate in a test sample in the presence of the test compound and in a control sample in the absence of the test compound, wherein the control substrate is convertible by the enzyme into a fluorescent metabolite, and wherein the test compound is added to the test sample after an appropriate incubation time with the control substrate (when the equilibrium is reached), b) measuring the level of fluorescence in the vesicles produced by the fluorescent metabolite in the test sample and in the control sample, c) comparing the levels of fluorescence measured in b), d) determining that the test compound interferes with the substrate transport activity of the OATP if there is a difference between the levels of fluorescence,
  • step a) of the foregoing embodiment the test compound and additional (equal to the initial concentration) amounts of the control substrate are added to the test sample after an appropriate incubation time and after removing the excess control substrate from the cells.
  • control substrate and the test substrate are removed from the vesicles after an appropriate incubation time, the vesicles are washed and the intracellular pH in the control sample and in the test sample is equalized by addition of a base, and the levels of fluorescence are measured once more.
  • the method is for determining whether a test compound interferes with substrate transport activity of an OATP, and substrate transport of an ABC transporter, wherein
  • a fluorogenic sulfopyrene compound convertible by an enzyme into a fluorescent metabolite is used as the control substrate of the OATP and the fluorescent metabolite is used as the control substrate of the ABC transporter, and
  • the method comprises the steps of a) contacting a membrane -bound vesicle comprising an enzyme and expressing the OATP in the membrane with a control substrate in a test sample in the presence of the test compound and in a control sample in the absence of the test compound, wherein the control substrate is convertible by the enzyme into a fluorescent metabolite, wherein the membrane -bound vesicle is a culture of polarized cells forming a basolateral and an apical compartment in the culture and expressing the OATP in their basolateral membrane and the ABC transporter in their apical membrane and comprising the enzyme, wherein the control substrate and the test compound are added to the basolateral compartment, b) measuring the level of fluorescence produced by the fluorescent metabolite in the cells and the level of fluorescence produced by the fluorescent metabolite in the apical compartment, in the test sample and in the control sample, c) comparing the levels of fluorescence measured in step b) d) determining whether the test compound
  • test compound If the level of fluorescence measured in b) is higher in the vesicles in the test sample than in the vesicles in the control sample, the test compound is identified as an activator of OATP mediated transport.
  • the test compound is identified as an activator of OATP mediated transport. If the level of fluorescence measured in b) is lower in the vesicles in the test sample than in the vesicles in the control sample, the test compound is identified as an inhibitor of OATP mediated transport.
  • the test compound is identified as an inhibitor of the ABC transporter.
  • the test compound is identified as an inhibitor of the ABC transporter.
  • the membrane -bound vesicle is a polarized vesicle.
  • the membrane -bound vesicle is a cell, more preferably a polarized cell.
  • the OATP is selected from OATP1B1, OATP1B3 and OATP2B1, preferably the OATP is OATP1B1 and/or OATP1B3 and/or OATP2B1 and/or OATP1A2. More preferably the OATP is OATP1B1, OATP1B3 and OATP2B1 or the OATP is OATP1B1, OATP1B3, OATP1A2 and OATP2B1.
  • the ABC transporter is MRP2.
  • the enzyme is an esterase and the control substrate comprises an acetate moiety which is cleavable by the esterase.
  • control substrate is a sulfopyrene compound having the following general formula (I) wherein in the formula
  • Ri, R 2 , R 3 and R 4 is selected from H, sulfo, carboxy and OR 5 , preferably from sulfo and OR 5 , wherein at least one, preferably two or three of Ri, R 2 , R 3 and R 4 is sulfo, wherein one, two or three preferably one or two of Ri, R 2 , R 3 and R 4 is OR 5 , wherein R 5 is Ci- 4 -C(0)-, preferably R 5 is selected from acetyl and propanoyl and butanoyl and the control substrate is a fluorogenic, being convertible into a fluorescent metabolite by an enzyme.
  • Ri, R 2 , R 3 and R 4 is selected from H, sulfo, carboxy and OR 5 , preferably from sulfo and OR 5 , wherein two or three of Ri, R 2 , R 3 and R 4 is sulfo, wherein one or two of Ri, R 2 , R 3 and R 4 is OR 5 , wherein R 5 is Ci- 4 -C(0)-, preferably R 5 is selected from acetyl and propanoyl.
  • Ri, R2, R3 and R4 is selected from sulfo and OR5.
  • OATP is selected from OATP1B1, OATP1B3 and OATP2B1, and in formula (I)
  • Ri, R 2 , R 3 and R 4 is selected from H, sulfo, carboxy and OR 5 , preferably from sulfo and OR 5 , wherein two or three of Ri, R 2 , R 3 and R 4 is sulfo, wherein one or two of Ri, R 2 , R3 and R 4 is OR 5 , wherein R5 is Ci- 4 -C(0)-, preferably R5 is selected from acetyl and propanoyl, preferably R5 is acetyl.
  • Ri, R 2 , R 3 and R 4 is selected from sulfo and OR 5 .
  • the OATP is selected from OATP1B1, OATP1B3 and OATP2B1.
  • the OATP is OATP1B1.
  • OATP is OATP1 A2, and in formula (I)
  • Ri, R2, R3 and R4 is selected from H, sulfo, carboxy and OR5, preferably from H, sulfo and OR5, wherein any one, preferably only one of Ri, R 2 , R 3 and R 4 is sulfo, wherein one, two or three, preferably two or three of Ri, R2, R3 and R4 is OR5, wherein R5 is Ci-4-C(0)-, preferably R5 is selected from acetyl and propanoyl, preferably R5 is acetyl.
  • Ri, R2, R3 and R4 is selected from H, sulfo and OR5.
  • control substrate is 1 -pyrenesulfonic acid or a salt thereof.
  • the control substrate is selected from the group consisting of 8-acetoxypyrene-l,3,6-trisulfonic acid and a salt thereof, 6,8-diacetoxypyrene-l,3-disulfonic acid sodium salt and a salt thereof. More preferably, the control substrate is 8-acetoxypyrene-l,3,6-trisulfonic acid or a salt thereof. Particularly preferably the control substrate is 8-acetoxypyrene-l,3,6-trisulfonic acid trisodium salt.
  • control substrate is selected from the group consisting of 8-acetoxypyrene- 1,3,6-trisulfonic acid and a salt thereof, 6,8-diacetoxypyrene-l,3-disulfonic acid sodium salt and a salt thereof.
  • the fluorescent metabolite is 8-hydroxypyrene-l,3,6- trisulfonic acid or a salt thereof or 6,8-dihydroxy-l,3-pyrenedisulfonic acid or a salt thereof.
  • FIG. 1 Dye uptake in A431 cells expressing human OATPs.
  • A431 cells overexpressing human OATPs, 1A2, 1B1, 1B3 or 2B1, and their mock transfected controls seeded on 96 well plates were incubated with 20 mM dye (pyrene, 1 -pyrenesulfonic acid, 6, 8-dihydroxy-l,3-pyrenedisulfonate, 8-hydroxypyrene-l,3,6-trisulfonic acid, 8- acetoxypyrene-l,3,6-trisulfonic acid or 1,3,6,8-pyrenetetrasulfonic acid) for 30 minutes at pH 5.5 or pH 7.4.
  • Cellular fluorescence was determined in an Enspire fluorescence plate reader. Fluorescence was determined in A431 -OATP and A431 mock transfected cells, and the fluorescence ratio (OATP/mock) is shown. Experiments were performed in 3 biological replicates, average +/- SD are shown.
  • FIG. 3 Effect of known OATP -interacting molecules on dye uptake.
  • A431 cells seeded on 96 well plates were incubated with 5 mM 8-acetoxypyrene-l, 3, 6-trisulfonate, 8-hydroxy-l, 3, 6-trisulfonate (10 mM for OATP1B1, 20 mM for OATP2Bl) or 6,8-dihydroxy- 1 ,3-pyrenedisul Ionic acid (5 pM forOATPIBl, 10 mM for OATP2B1) in the absence or presence of increasing concentrations of BSP or CsA for 5, 10 or 15 minutes. After the incubation time, the excess dye was removed from the cells and after repeated washing, fluorescence was determined in an Enspire fluorescence plate reader.
  • Figure 4 A) Uptake of 8-acetoxypyrene-l, 3, 6-trisulfonate in A431 or MDCKII cells expressing human
  • OATP1B1 A431 or MDCKII cells overexpressing human OATP1B1 or their mock transfected controls (ctr) were incubated with 20 mM 8-acetoxy-l, 3-6-trisulfonate diluted in buffer pH 5.5 for 30 minutes. After washing, cellular fluorescence was measured in an Enspire fluorescent plate reader at Ex/Em: 460/510 nm. Experiments were repeated in 3 biological replicates. Average +/- SD values are shown. B) Transcellular basolateral to apical transport of 8- acetoxypyrene-1, 3, 6-trisulfonate in MDCKII cells.
  • A431 cells mock-transfected (ctr) or overexpressing OATP1B1, OATP1B3 or OATP2B1 were incubated with 20 mM of 8- acetoxypyrene-1, 3, 6-trisulfonate in buffer pH 5.5 at 37°C for 0-60 minutes, and fluorescence was monitored at Ex/Em: 460/510 nm without removing the dye.
  • B) A431 mock-transfected (ctr) or overexpressing OATP1B1, OATP1B3 or OATP2B1 were incubated with 1, 5 or 20 mM of 8-acetoxypyrene-l, 3, 6-trisulfonate in buffer pH 5.5 at 37°C for 10 minutes.
  • A431 cells mock-transfected (ctr) or overexpressing OATP1B1, OATP1B3 or OATP2B1 were incubated with 20 mM of 8-acetoxypyrene-l, 3, 6-trisulfonate in buffer pH 5.5 at 37°C without or with the inhibitors
  • CsA cyclosporin A (10 mM final concentration, OATP1B1 and OATP1B3)
  • BSP bromosulfophthalein (100 mM final concentration, OATP1B1, OATP1B3 and OATP2B1)
  • E1S estrone-3 -sulfate (10 mM final concentration, OATP2B1).
  • Cellular fluorescence without removing the dye was detected in every 5 minutes using an Enspire plate reader. Experiments were repeated in at least 3 biological replicates, average +/- SD values are shown.
  • FIG. 7 Comparison of the effect of test compounds in the uptake, competitive counterflow and efflux assay using 8-acetoxypyrene-l, 3, 6-trisulfonate. Uptake was performed as described at Figure 3 with preincubation with increasing concentrations of the tested compounds. In the case of counterflow and efflux A431 cells overexpressing OATP1B1 or mock transfected controls were preloaded with 5 mM 8-acetoxypyrene-l, 3, 6-trisulfonate by incubation at 37°C for 15 minutes.
  • Figure 8 The non-fluorescent non-cell permeable 8-acetoxypyrene-l, 3, 6-trisulfonate is transported into the cells by the function of OATPs, and cleaved inside the cells by esterases and transformed to fluorescent pyranine (8- hydroxypyrene- 1 , 3 ,6-trisulfonate) .
  • Figure 9 The counterflow assay distinguishes between a competitive substrate and a non-transported inhibitor.
  • the invention provides a “no wash” method for measuring OATP substrate/inhibitor interactions, i.e. whether a test compound interferes with the transport activity of the OATP, using a control substrate that becomes fluorescent upon transportation by the OATP.
  • Substrate transport by an OATP and/or an ABC transporter may be activated or inhibited by the test compound if the test compound interacts with the OATP or ABC. 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 a control substrate, whereas an “inhibitor” substance decreases or inhibits substrate transport.
  • An inhibitor may be a competitive inhibitor.
  • a “competitive inhibitor” may be a substrate that is transported by the OATP while an “inhibitor” may be a compound that is not transported by the OATP (or the ABC transporter).
  • substrate refers to a compound that can be transported by the OATP or the ABC transporter.
  • the test compound and the control 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 or membrane-bound vesicle.
  • the test compound may be added to either the section of the membrane where the OATP is expressed (e.g. to the basolateral compartment of a polarized cell culture) or to the section of the membrane where the ABC transporter is expressed (e.g. to the apical compartment of a polarized cell culture) in case when transport activity of ABC transporter is also tested, while the control substrate is added to the section of the membrane where the OATP is expressed.
  • the amount of the test compound and the amount of the control substrate, as well as the timing of addition of the test compound and the control substrate may vary depending on a number of factors, such as the affinity of the test compound and/or the control substrate to the transport protein, the kinetics of the transport by the OATP (and/or the transport by the ABC transporter, where appropriate) (e.g. it may be necessary to calculate Km, Vmax at different concentrations of a test compound, and IC50 values 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 ABC transporter, where appropriate). Examples are also described herebelow. An exemplary method is adding first the test compound and 5 minutes later the control substrate.
  • Control experiments The skilled person is able to select appropriate control conditions (such as cells, compounds, buffers) most suited for the aims of the study. An example is provided herebelow.
  • cells which do not express the OATP (and/or the ABC transporter, where appropriate) For example cells which do not express the OATP (and/or the ABC transporter, where appropriate), cells which express the OATP (and/or ABC transporter, where appropriate) under a pre-determined threshold value, cells in which the expression of the OATP (and/or the ABC transporter, where appropriate) is silenced, cells which express a mutant OATP (and/or ABC transporter, where appropriate) which is not capable of transporting the control substrate or cells in which the activity of the OATP (and/or the ABC transporter, where appropriate) is inhibited, cells which overexpress the OATP (alone) (and cells which overexpress the ABC transporter alone, where appropriate) 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 the fluorescent metabolite of the control substrate (determination of the amount of the control substrate therefrom is straightforward). If necessary, background fluorescence may be tested by appropriate controls, e.g. in the absence of the control substrate and the fluorescent metabolite of the control substrate with or without the addition of the tested compound, and only transported fluorescence is used.
  • the membrane -bound vesicle is a polarized vesicle.
  • the membrane -bound vesicle is a cell, more preferably a polarized cell.
  • the term “polarized” refers to membrane -bound vesicles comprising the OATP in one part of the membrane of the vesicle and the ABC transporter on the opposite part of the membrane.
  • 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.
  • 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 the ABC transporter is expressed in the canalicular membrane
  • the cells may and preferably are overexpressing the OATP and ABC transporter, where appropriate.
  • Cells without overexpression may also be used, provided the OATP and (the ABC transporter, where appropriate) 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 the ABC transporter, where appropriate).
  • A431 cells may be used as cells overexpressing the OATP.
  • a “single assay” as used herein refers to an assay wherein double expressing cells are used to test compounds for interaction with the OATP and the ABC transporter. This setting eliminates the need of using separate cell lines for the OATP and inside out vesicles for the ABC transporter, while provides a model better representing the biological environment of polarized cells, e.g. hepatocytes.
  • 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 the ABC transporter 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.
  • 8-acetoxypyrene-l, 3, 6-trisulfonate refers specifically to the trisodium salt of 8- acetoxypyrene-1,3,6 trisulfonic acid.
  • 8-acetoxypyrene-l, 3, 6-trisulfonate as used herein may refer to 8-acetoxypyrene- 1,3,6 trisulfonic acid and any salt thereof in general.
  • vesicle and “membrane-bound vesicle” as used herein refers to a structure surrounded by a lipid bilayer (membrane). Any cell falls within the meaning of “vesicle”.
  • an “enzyme” as used herein refers to a protein capable of converting the control substrate into the fluorescent metabolite in a cell.
  • an “esterase” as used herein may refer to any esterase capable of cleaving the control substrate, e.g. intracellular nonspecific esterases.
  • the fluorescent metabolite is produced by the intravesicular or intracellular activity of an esterase.
  • fluorogenic refers to a compound that becomes fluorescent upon crossing a membrane, e.g. a cell membrane, preferably by the activity of an enzyme, preferably by the action of an esterase.
  • the non- fluorescent fluorogenic compound used in the method according to the invention is converted into a fluorescent metabolite inside the vesicle (e.g. cell).
  • Fluorogenic compounds which may be used according to the invention are commercially available (see e.g. Examples) and/or are disclosed in e.g. Austrian Patent AT 385 755 B, EP2707454B1, Wolfbeis et al. Fluorimetric Assay of Hydrolases at Longwave Excitation and Emission Wavelengths with new Substrates Possessing Unique Water Solubility. Analytical Biochemistry, 129, 365-370 (1983), US4585598A.
  • control substrate is a fluorescent sulfopyrene compound, which is a substrate of the OATP.
  • the fluorescent metabolite of the control substrate is a substrate of the ABC transporter, thus the ABC transporter is capable of transporting the fluorescent metabolite through the cell membrane.
  • the term “equilibrium” as used herein in relation with a counterflow assay refers to a state of a sample where the uptake rate (into a vesicle) of a substance and the efflux rate (from the vesicle) of the substance or the metabolite thereof are equal, more accurately, the velocity of the uptake of the substrate (e.g. the control substrate) by OATP is equal with the velocity of the efflux of the metabolite of the substrate (e.g. the fluorescent metabolite of the control substrate) or non-converted substrate (e.g. the control substrate) by OATP.
  • loading refers to adding the compound to a sample in excess, e.g. in an amount that is sufficient to achieve a steady state (equilibrium) between the concentration of the compound (or a metabolite thereof) in the vesicle and outside of the vesicle (e.g. wherein no net flux of the compound is detected or the net flow is negligible) or in an amount that is sufficient to achieve an influx of the compound into the cells.
  • the invention provides a “no wash” method for measuring OATP substrate/inhibitor interactions, i.e. whether a test compound interferes with the transport activity of the OATP, using a control substrate that becomes fluorescent upon transportation by the OATP.
  • the method is suitable for measuring OATP and ABC transporter substrate/inhibitor interactions in a single assay.
  • the method is suitable for distinguishing between a substrate and inhibitor of an OATP.
  • the method can be used to monitor expression and function of an OATP in cells endogenously expressing the OATP, e.g. OATPs 1B1, 1B3, 1A2 or 2B1.
  • the method can be automatized and developed to a high throughput method using 386 (or more) well plates.
  • the invention provides a method for measuring expression level of an OATP in a cell, the method comprising
  • control substrate is a fluorogenic sulfopyrene compound, which is a substrate of the OATP.
  • a method for determining whether a cultured hepatocyte is OATP mediated transport competent comprising
  • the hepatocyte as an OATP mediated transport competent hepatocyte expressing the OATP if fluorescence is present in the hepatocyte or at the canalicular side.
  • an assay kit comprising a membrane-bound vesicle comprising an enzyme and expressing an OATP in the membrane and a control substrate, which is convertible by the enzyme into a fluorescent metabolite, and optionally membrane -bound control vesicles not expressing the OATP in the membrane or not comprising the enzyme, wherein the control substrate is a fluorogenic sulfopyrene compound, which is a substrate of the OATP.
  • a method for determining whether a test compound interferes with substrate transport by an OATP and substrate transport by an ABC transporter in a single assay is also provided.
  • the method can be used to monitor expression and function of an OATP and an ABC transporter in cells endogenously expressing the OATP and the ABC transporter.
  • the method can be automatized and developed to a high throughput method using 386 (or more) well plates.
  • a method for measuring expression level of an OATP and an ABC transporter in a cell comprising
  • control substrate is a fluorogenic sulfopyrene compound, which is a substrate of the OATP, and wherein the fluorescent metabolite is a substrate of the ABC transporter.
  • a method for determining whether a cultured hepatocyte is OATP and the ABC transporter mediated transport competent comprising adding a control substrate to the sinusoidal membrane of the hepatocyte, detecting fluorescence of a metabolite of the control substrate at the canalicular side of the hepatocyte, and identifying the hepatocyte as an OATP and the ABC transporter mediated transport competent hepatocyte if fluorescence is present at the canalicular side of the hepatocyte, wherein the control substrate is a fluorogenic sulfopyrene compound, which is a substrate of the OATP, and wherein the control substrate is convertible by an enzyme present in the hepatocyte into a fluorescent metabolite, and wherein the fluorescent metabolite is a substrate of the ABC transporter.
  • the hepatocyte is in a 3 dimensional culture.
  • an assay kit comprising a control substrate, which is convertible by an enzyme into a fluorescent metabolite, which is a substrate of the ABC transporter and polarized assay cells expressing the OATP in their basolateral membrane and the ABC transporter in their apical membrane, and capable of forming a basolateral and an apical compartment in culture and optionally polarized control cells expressing the OATP in their basolateral membrane and lacking the ABC transporter expression, and capable of forming a basolateral and an apical compartment in culture, and/or polarized control cells expressing the ABC transporter 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 the ABC transporter but without the nucleic acid encoding the OATP and the nucleic
  • the polarized control cells are of the same type as the polarized assay cells.
  • a competitive counterflow assay based on the method of the invention.
  • Indirect assays based on the uptake of radioactively labeled OATP substrates are routinely used by researchers and by pharmaceutical industry to determine the interaction of a potential OATP interacting molecule.
  • one of the drawbacks of these fluorescence or radioligand based assays is that they cannot distinguish between a transported substrate and a non-transported inhibitor - or only in a time and cost consuming way. Therefore when e.g. the potential liver toxicity of an OATP substrate is to be determined additional measurements are required.
  • a competitive counterflow (CCF) assay based on the method of the invention has different readouts in the case of an OATP inhibitor and substrate.
  • CCF is based on the activity of OATPs as exchangers, i.e.
  • control substrate it is not necessary for the control substrate to be fluorogenic in a competitive counterflow assay, i.e. a fluorescent compound may be used in embodiments where the cells are washed before measuring fluorescence.
  • fluoerescent control substrate also must be a substrate of the OATP.
  • a fluorescent compound the compound is not necessary to be converted by the enzyme to practice the method of the invention, and therefore the presence of the enzyme in the vesicle is optional.
  • a preferred embodiment of this aspect is a method comprising the following steps: a) contacting a membrane -bound vesicle comprising the OATP in the membrane and optionally comprising an enzyme with a control substrate in a test sample in the presence of the test compound and in a control sample in the absence of the test compound, wherein the test compound is added to the test sample after an appropriate incubation time of the control substrate, b) measuring the levels of fluorescence in the vesicles produced by the fluorescent metabolite in the test sample and in the vesicles in the control sample, c) comparing the levels of fluorescence measured in b), d) identifying the test compound as a substrate of the OATP if the fluorescence in the vesicles in the test sample is lower than the fluorescence in the vesicles in the control sample, wherein the control substrate is a fluorogenic or fluorescent compound, which is a substrate of the OATP and wherein if the control substrate is a fluorogenic compound,
  • step a) a) the test compound is added to the test sample after an appropriate incubation time and after removing the excess control substrate from the samples.
  • step b) the control substrate and the test substrate are removed from the vesicles after an appropriate incubation time, the vesicles are washed and the intracellular pH in the control sample and in the test sample is equalized by addition of a base, and the levels of fluorescence are measured.
  • the method comprises the steps of: i) loading the vesicles with the control substrate until equilibrium is reached, using a suitable amount of the control substrate and an appropriate incubation time, in a test sample and in a control sample, ii) removing the excess control substrate from the test sample and from the control sample, iii) adding the control substrate and the test compound to the test sample, and adding the control substrate without the test compound to the control sample, (using a suitable amount of the control substrate and the test compound and an appropriate incubation time to allow transport to take place), iv) measuring the levels of fluorescence by the control substrate (or the fluorescent metabolite of the control substrate) in the test sample and in the control sample, and identifying the test compound as a substrate of the OATP if the fluorescence measured in iv) in the test sample is lower than the fluorescence in the control sample.
  • the method further comprises v) removing the excess control substrate (or the fluorescent metabolite of the control substrate) and the excess test compound from the test sample and from the control sample, vi) measuring the levels of fluorescence by the control substrate (or the fluorescent metabolite of the control substrate) in the test sample and in the control sample, and identifying the test compound as a substrate of the OATP if the fluorescence measured in vi) in the test sample is lower than the fluorescence in the control sample and optionally vii) adding a base to the test sample and to the control sample to equalize intracellular pH and measuring fluorescence by the control substrate or the fluorescent metabolite of the control substrate in the test sample and optionally in the control sample, viii) identifying the test compound as a substrate of the OATP if the fluorescence measured in vii) in the test sample is lower than the fluorescence in the control sample.
  • the fluorescent metabolite has low or no membrane permeability to avoid passive transport through the cell membrane.
  • the fluorogenic compound control substrate
  • the fluorescent metabolite can only be taken up into the vesicle (or cell) and the fluorescent metabolite can only be released from the cell by the OATP and ABC mediated transport, respectively.
  • the vesicle does not comprise or express any other transporter than the OATP or the OATP and the ABC.
  • the fluorescent metabolite may be detected using an excitation wavelength of about 300 to 500 nm and an emission wavelength of about 400 to 600 nm.
  • the OTP and the ABC transporter are human OTP and ABC transporter
  • the cell or hepatocyte is a human cell or hepatocyte.
  • Pyranine, pyrene, 1 -pyrenesulfonic acid, 6,8-dihydroxy-l,3-pyrenedisulfonic acid, 1,3,6,8-pyrenetetrasulfonic acid were purchased from Sigma, Merck (St. Louis, MO, US). 8-acetoxy-l, 3, 6 pyrenetrisulfonic acid was bought from Santa Cruz Biotechnology (VWR International, Hungary).
  • A431 cells expressing OATPs 1A2, 1B1, 1B3 or 2B1 and their mock transfected counterparts were generated previously as described in (Bakos et al., 2019; Patik et al., 2018).
  • the MDCKII-MRP2 cell line was generated previously (Bakos et al., 2000).
  • OATP1B1 expression in MDCKII and MDCKII-MRP2 cells was achieved by recombinant lentiviruses as described earlier (Patik et al., 2018). Briefly, MDCKII parental and MDCKII-MRP2 cells were transfected with the pRRL-CMV- OATP1B1-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 cells 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% CO2, under sterile conditions.
  • A431 cells (8 x 10 4 /well) were seeded on 96- well plates in 200 m ⁇ 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 m ⁇ uptake buffer (125 mM NaCl, 4.8 mM KC1, 1.2 mM CaC 1.2 mM KH2PO4, 12 mM MgS04, 25 mM MES, and 5.6 mM glucose, with the pH adjusted to 5.5 using 1 M HEPES and 10 N NaOH, pH 5.5 or pH 7.4) at 37°C.
  • 50 m ⁇ uptake buffer 125 mM NaCl, 4.8 mM KC1, 1.2 mM CaC 1.2 mM KH2PO4, 12 mM MgS04, 25 mM MES, and 5.6 mM glucose
  • the reaction was started by the addition of 50 m ⁇ uptake buffer containing pyranine (Sigma), pyrene (Sigma), 1 -pyrenesulfonic acid (Sigma), 6, 8-dihydroxy- 1,3-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 m ⁇ 1 x PBS was added to each well and fluorescence was determined using an Enspire plate reader. Ex/Em wavelengths were the following:
  • Transport activity was determined as a ratio of fluorescence in A431-OATP cells/ mock transfected controls. Experiments were repeated in 3 biological replicates.
  • the reaction was started as described above, with the exception that the supernatant was not removed and fluorescence in the cells was continuously monitored by incubating the cells in an Enspire fluorescence plate reader by setting the temperature to 37°C, Ex/Em: 460/510 nm.
  • the cells were preincubated with 50 m ⁇ uptake buffer (pH 5.5) containing bromosulfophtalein (100 mM final concentration), cyclosporin A (10 mM final concentration), or estrone-3-sulfate (10 mM final concentration), for 5 minutes at 37°C.
  • the reaction and real-time measurement of fluorescence was started by the addition of 50 m ⁇ buffer containing 40 mM 8-acetoxypyrene-l, 3,6- trisulfonate. Experiments were repeated in 3 biological replicates.
  • the transcellular transport of 8-acetoxypyrene-l, 3, 6-trisulfonate was tested in polarized MDCKII cells co-expressing OATP1B1 and MRP2, or their single (MDCKII-OATP1B1, MDCKII-MRP2) or mock transfected controls (ctr.) ⁇
  • OATP1B1 and/or MRP2 overexpressing MDCKII cells (9 x 10 4 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 m ⁇ 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 m ⁇ uptake buffer (see above) with the pH adjusted to pH 7.4
  • the wells were washed three times with 1 ml pH 5.5 buffer (see above).
  • 300 m ⁇ 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 buffer pH 5.5 containing 1 mM 8-acetoxypyrene-l, 3, 6 -trisulfonate to the wells at 37°C.
  • 20 mM cyclosporin A (CsA) or 40 mM benzbromarone was added to the lower or upper compartment, respectively was added to the lower or upper compartment, respectively.
  • 30 m ⁇ samples from the upper compartment were collected every 5 minutes and pipetted into 70 m ⁇ 1 x PBS for fluorescence measurements. The fluorescence intensity of the samples was determined using an Enspire plate reader (Perkin Elmer) at the wavelengths indicated above. Experiments were repeated in 2 biological replicates. Counterflow assay
  • A431 cells expressing OATP1B1 and mock transfected control were seeded on 96-well plates in 200 m ⁇ DMEM 16-24 h prior to the 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 loaded with 5 mM 8-acetoxypyrene-l, 3,6- trisulfonate in a final volume of 100 m ⁇ pH 5.5 uptake transport buffer by incubation at 37°C for 15 minutes. According to our previous measurements, dye uptake reaches its equilibrium (influx and efflux rate of the dye and its metabolite are equal) at 15 minutes and stays stabile for further 20 minutes. In order to control loading of the cells, fluorescence was measured after 15 minutes of incubation at Ex/Em 460/510 nm in an Enspire fluorescence plate reader.
  • the supernatant was removed and 5 mM 8-acetoxypyrene-l, 3, 6-trisulfonate in 100 m ⁇ pH 5.5 with or without the tested compound, BSP, benzbromarone, CsA, E1S or 0.5 % PFA was added.
  • the plates were incubated for 20 minutes at 37°, then the fluorescence was measured at 460/510 nm.
  • the supernatant was removed, and the cells were washed with 200 m ⁇ ice-cold PBS three times. The fluorescence was measured again at 460/510 nm.
  • Patik, L Szekely, V., Nemet, O., Szepesi, A., Kucsma, N., Varady, G., Szakacs, G., Bakos, E., Ozvegy-Laczka, C., 2018. Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1. Sci Rep 8, 2630.

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Abstract

L'invention concerne le domaine de l'évaluation de l'activité de transport de protéines de transport OATP (polypeptides de transport d'anions organiques). En particulier, l'invention concerne un procédé par fluorescence add-and-read en temps réel pour déterminer si un composé d'essai interfère avec l'activité de transport de substrat d'un OATP, en utilisant un substrat de commande non fluorescent qui devient fluorescent à l'intérieur d'une cellule. Le procédé est également applicable pour mesurer l'expression d'un OATP et pour discriminer un substrat transporté et un inhibiteur d'un OATP.
PCT/HU2020/050062 2019-12-20 2020-12-19 Dosage à fluorescence add-and-read en temps réel pour tester la fonction et les interactions de médicaments de transporteurs oatp Ceased WO2021123851A1 (fr)

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HUPCT/HU2020/050014 2020-04-27
PCT/HU2020/050014 WO2021123847A1 (fr) 2019-12-20 2020-04-27 Procédé de fluorescence permettant l'évaluation de l'activité de transport d'oatp et de mrp2
IBPCT/IB2020/050014 2020-04-27
HUP2000168 2020-05-22
HU2000168A HUP2000168A1 (hu) 2020-05-22 2020-05-22 Valós idejû, fluoreszcencia alapú, "add-és-mérd" eljárás OATP fehérjék funkciójának és drog interakcióinak vizsgálatára

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EP1645627A1 (fr) * 2004-10-07 2006-04-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Lignée cellulaire quadruplement transfectée pour l'identification d'inhibiteurs de transport

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* Cited by examiner, † Cited by third party
Title
"Fluorogenic Substrates", SCBT - SANTA CRUZ BIOTECHNOLOGY, 8 May 2013 (2013-05-08), XP055834571, Retrieved from the Internet <URL:https://www.scbt.com/browse/chemicals-Stains-Dyes-Probes-Labels-Fluorogenic-Substrates/_/N-z80cs8> [retrieved on 20210326] *
LANGTON MATTHEW J., KEYMEULEN FLORE, CIACCIA MARIA, WILLIAMS NICHOLAS H., HUNTER CHRISTOPHER A.: "Controlled membrane translocation provides a mechanism for signal transduction and amplification", NATURE CHEMISTRY, vol. 9, no. 5, May 2017 (2017-05-01), pages 426 - 430, XP055834564, DOI: 10.1038/nchem.2678 *
MATSUSHIMA SOICHIRO, MAEDA KAZUYA, KONDO CHIHIRO, HIRANO MASARU, SASAKI MAKOTO, SUZUKI HIROSHI, SUGIYAMA YUICHI: "Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby Canine Kidney II cells expressing human organic anion- transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidtug resistance 1, and OATP1B1/breast cancer ", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS : JPET, vol. 314, no. 3, 18 May 2005 (2005-05-18), US , pages 1059 - 1067, XP009538029, ISSN: 0022-3565, DOI: 10.1124/jpet.105.085589 *
MUSTAFA DANA, MA DONGPING, ZHOU WENHUI, MEISENHEIMER PONCHO, CALI JAMES J.: "Novel no-wash luminogenic probes for the detection of transporter uptake activity", BIOCONJUGATE CHEM. 2016, vol. 27, no. 1, 20 January 2016 (2016-01-20), pages 87 - 101, XP055834563, DOI: 10.1021/acs.bioconjchem.5b00495 *
See also references of EP4077707A4 *

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