Determination of Unbound Partition Coefficient (K ) and in
Transcript Of Determination of Unbound Partition Coefficient (K ) and in
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Determination of Unbound Partition Coefficient (Kpuu) and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake
Keith Riccardi, Zhenhong Li, Janice A. Brown, Matthew F. Gorgoglione, Mark Niosi, James Gosset, Kim Huard, Derek M. Erion, Li Di
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT (KR, JAB, MN,LD); Cambridge, MA (MFG, JG, KH, DME)
1
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Running Title: Kpuu and IVIVE of SLC13A Transporter-Mediated Uptake
Corresponding Author: Li Di Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. Eastern Point Road, Groton, CT 06345 [email protected]
Text Pages: 25 Tables: 5 Figures: 8 References: 23 Abstract: 238 words Introduction: 726 words Discussion: 714 words
Abbreviations: ABT, 1-aminobenzotriazole; BSA, bovine serum albumin; CES, carboxylesterase; CYP, cytochrome P450; fucell, fraction unbound of cells; IC50, concentration of an inhibitor where the response is reduced by half; I.V., intravenous; IVIVE, in vitro-in vivo extrapolation; JVC, jugular vein cannula; Km, Michaelis-Menten constant; Kpuu, unbound partition coefficient, is defined as the ratio between unbound intracellular free drug concentration and unbound medium concentration (steady state is not assumed) in this paper; Pdiff, clearance via passive diffusion; SBECD, sulfobutyletherβ-cyclodextrin; Vmax, maximum rate
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Abstract Unbound partition coefficient (Kpuu) is important to understand the asymmetric free drug distribution of a compound between cells and media in vitro, as well as tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds that are inhibitors and substrates of transporters from the SLC13A family in hepatocytes and transporter-transfected cell-lines. Enantioselectivity was observed with (R)-enantiomers achieving much higher Kpuu (> 4) than the (S)-enantiomers (< 1) in human hepatocytes and SLC13A5 transfected HEK293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay due to the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid (PF-06649298) directly or via hydrolysis of the ethyl ester prodrug (PF-06757303) resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid vs. 59 for prodrug), which was successfully modeled based on passive diffusion, active uptake and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations potentially due to saturation of transporters. Km of SLC13A5 was estimated to be 24 µM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free BSA showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.
3
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Introduction Intracellular free drug concentration plays a critical role in developing target exposure and pharmacological activity relationships for disease targets located in the intracellular domain. For compounds that reach distribution equilibrium across the cell membrane at steady state, free drug concentration in the media might be considered the same as the intracellular free drug concentration. This is usually the case for most compounds, in which the nominal concentrations can be used to correlate to pharmacological activities (e.g., IC50) of a compound in in vitro assays or after correction of non-specific binding to the proteins/lipids in the assay media. However, when transporters are involved in the distribution process, the intracellular free drug concentration may be different than that in the media due to active processes. In addition, effects of membrane potential, lysosomal trapping, pH gradient and mitochondria accumulation can all impact the intracellular free drug concentration relative to the medium free concentration (Figure 1). Therefore, it is important to understand the intracellular free drug concentration and unbound distribution coefficient (Kpuu) to accurately determine the true potency of a compound, e.g., intrinsic IC50, rather than apparent IC50. Prediction of human in vivo liver-to-plasma Kpuu is of great importance in order to estimate dose, safety margin and drug-drug interaction potentials for compounds with transporter-mediated uptake into the liver. Development of in vitro tools to predict in vivo liver-to-plasma Kpuu will be very useful for human translation in drug discovery and development, since there are no simple tools available to measure human liver concentration.
4
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Several in vitro methods have been developed to measure the intracellular free drug concentration and Kpuu in hepatocytes or transfected cell systems expressed OATP transporters (Figure 2). The binding method measures fucell, medium and total cell concentration at the steady state, from which intracellular free drug concentration and Kpuu are determined (Mateus et al., 2013). The temperature method measures the medium and total cell concentration at both 37°C and 4°C at steady state (Shitara et al., 2013). The total concentration ratio of cell to medium between 37°C and 4°C is considered to be Kpuu. This method does not consider the impact of membrane potential or metabolism. The kinetic method measures the uptake rate at multiple concentrations in the presence of CYP inhibitor, ABT. Kinetic parameters (Vmax, Km, and Pdiff) are obtained by simultaneously fitting all the data (Yabe et al., 2011). Kpuu is calculated based on the kinetic parameters with the extended sequential clearance equation (Yabe et al., 2011). This approach does not consider the impact of efflux transport or metabolism on Kpuu. The permeability method measures the permeability of both neutral and ionized species and determines the Kpuu across a cell membrane based on membrane potential and passive permeability (Ghosh et al., 2014). This method has not incorporated the effects of transporters or metabolism at this point. Other methods reported to determine drug concentration in the subcellular compartments (Pfeifer et al., 2013) are relevant to therapeutic targets localized in the specific subcellular organelles. Validation of the different Kpuu methods with in vitro or in vivo data is minimal in literature. We will discuss our approach for measuring Kpuu and intracellular free drug concentration, as well as validation against in vitro potency, using cell lines expressing different members of the SLC13A family, hepatocytes and in vivo rat liver-to-plasma Kpuu data.
5
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Citrate is a key regulatory metabolic intermediate and is critical in the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake through SLC13A5 has been suggested as a potential therapeutic approach to treat metabolic diseases (Huard et al., 2015; Huard et al., 2016; Li et al., 2016). The SLC13A family contains five family members: SLC13A1 and SLC13A4 mainly transport sulfate, and SLC13A2, SLC13A3 and SLC13A5 transport di- and tri-carboxylates such as citrate (Lee et al., 2005; Bergeron et al., 2013). SLC13A5 is highly expressed in human liver, while SLC13A2 and SLC13A3 are expressed more broadly, including in the intestine and kidney (Pajor, 2014). Therefore, selective SLC13A5 inhibitors have the potential to impact citrate flux in the liver only, while SLC13A2/3 inhibitors can impact citrate flux in the intestine and kidney as well. Development of specific inhibitors for SLC13A5 is proposed to be beneficial in treating metabolic disorders by reducing citrate flux into the liver, and presumably exert the metabolic benefits through modification of intracellular metabolites, while limiting direct effects in other tissues.
6
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Materials and Methods Materials Test compounds were obtained from Pfizer Global Material Management (Groton, CT) or purchased from Sigma-Aldrich (St. Louis, MO). The syntheses of diacids PF-06649298, PF-06649297 and monoacid PF-06757303 were previously reported (Huard et al., 2015). The syntheses of PF-06794266, PF-06793742 and PF-06761281 were also reported (Huard et al., 2016). PF-06761281 was commercially available via Sigma Aldrich (catalog # PZ0318). The syntheses of PF-06746350 and PF-06741415 were similar to the methods described previously (Huard et al., 2016). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless specified. The 96-well equilibrium dialysis (HTD96) device and cellulose membranes with MWCO of 12-14K were purchased from HTDialysis, LLC (Gales Ferry, CT). 1.2 mL deep 96-well plates were from Axygen Scientific Inc. (Union City, CA) and pipette tips were obtained from Apricot Designs (Monrovia, CA).
Measurement of Fraction Unbound Cells (hepatocytes, HEK293 transfected with human SLC13A transporters) at a cell density of 50-60 million cells/mL, rat and human liver tissues (1:5 dilution with phosphate buffered saline (PBS)) were homogenized at room temperature in PBS using an Omni TH tissue homogenizer (Omni International, Kennesaw, GA) with a 7 mm x 110 mm tip at high speed for 30 second pulses. Assay media containing proteins (e.g., BSA) were used directly for binding measurement. The dialysis membranes were prepared prior to experimental setup. The cellulose membranes (MWCO 12-14K) were immersed in de-
7
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
ionized water for 15 minutes, followed by 15 minutes in 30% ethanol / de-ionized water, then at least 15 minutes or overnight in PBS. The equilibrium dialysis device was assembled according to manufacturer’s instructions (http://htdialysis.com/page/1puq4/Operating_Instructions.html). DMSO stock solutions of test compounds were prepared at 200 μM, added in 1:100 ratio to matrices, and mixed thoroughly with an 8-channel pipettor (Eppendorf®, VWR, Radnor, PA). The final compound concentration was 2 μM containing 1% DMSO. A 150 μL aliquot of matrix (cell and tissue homogenate, assay media or plasma) spiked with 2 μM compound was added to one side of the membrane (donor) and 150 μL of PBS was added to the other side (receiver). The equilibrium dialysis device was covered with Breathe Easy™ gas permeable membranes (Sigma-Aldrich, St. Louis, MO). Compounds were assessed in quadruplicate. Equilibrium dialysis devices were placed on an orbital shaker (VWR, Radnor, PA) at 200 RPM and incubated for 6 hours in a humidified (75% RH) incubator at 37°C with 5% CO2/95% O2. At the end of the incubation, 15 μL of matrix samples from the donor wells were added to a 96-well plate containing 45 μL of PBS. Aliquots of 45 μL dialyzed PBS from the receiver wells were added to 15 μL of blank matrix. Before and after incubation, 15 μL of matrix spiked with 2 μM of compounds was added to a 96well plate containing 45 μL of PBS. These samples were used for the recovery calculation and stability evaluation. All the samples were quenched with 200 μL of cold acetonitrile containing internal standard (a cocktail of 0.5 ng/mL tolbutamide and 5 ng/mL terfenadine). The plates were sealed and mixed with a vortex mixer (VWR, Radnor, PA) for 3 minutes, then centrifuged at 3000 RPM (Beckman Coulter, Fullerton, CA) at RT for 5 minutes. The supernatant was transferred to a new 96-well plate, dried
8
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
down, reconstituted and subsequently analyzed using LC-MS/MS. Sertraline was used as a quality control sample on every plate. Cell diameter was measured using ViCELL XR (Beckman-Coulter, Inc., Danvers, MA) to calculate cell volume assuming spherical shape and dilution factor.
In Vitro Kpuu Measurement Transfected Cells (SLC13A5- and SLC13A3-HEK293) Kpuu within this paper is defined as the ratio between unbound intracellular free drug concentration and unbound medium concentration (steady state is not assumed). Protocols to generate the stable cell line overexpressing sodium citrate transporters and culture conditions were discussed previously (Huard et al., 2015). Test compounds (1 μM unless otherwise specified) were incubated with the plated cells (1-4 hours unless otherwise specified) at 37 °C in a humidified (75% RH) incubator with 5% CO2/95% O2. At the end of the incubation, 100 μL of the media were removed for concentration determination, and cold acetonitrile was added. The rest of the media was gently removed from the cells by pipette aspiration. The cells were washed three times with cold PBS of the same starting volume. After wash, the cells were lysed by adding 80 μL of MPER buffer (Thermo Scientific, Rockford, IL). An aliquot of the lysed cells was transferred to a new plate, matrix matched, and cold acetonitrile containing IS was added. The solutions were centrifuged at 3000 RPM for 10 min at room temperature. The supernatants were transferred and analyzed by LC-MS/MS against standard curves that were matrix matched for both media and cells.
Human and Rat Hepatocytes 9
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Cryopreserved human hepatocytes (Lot DCM) consisting of 10 donors, both males and females, were custom-pooled and prepared by BioreclamationIVT (Baltimore, MD). Wistar-Han rat hepatocytes (Lot VSU) consisting 35 male donors were also purchased from BioreclamationIVT. Upon thawing, the hepatocytes were re-suspended in Williams E medium (WEM GIBCO-BRL, custom formula supplemented with 50 mM Hepes and 26 mM sodium bicarbonate). The cells were counted and viability was determined using the Trypan Blue exclusion method. Test compounds were dissolved in DMSO at 1 mM and 1 µL was added to 0.5 million cells/mL suspended hepatocytes in 1000 µL (final concentration 1 µM containing 0.1% DMSO). The suspension was incubated in a humidified (75% RH) incubator at 37 °C with 5% CO2/95% O2. For rat hepatocytes, InVitroGRO HI media (Fisher Scientific,Waltham, MA) containing 0.2, 2 and 4% fatty acid free BSA (catalog # A4612, Sigma-Aldrich, St. Louis, MO) were also used in the study for IVIVE development. At appropriate time points, hepatocyte suspension was centrifuged at 500 RPM for 3 minutes and supernatant was collected and removed from the cells to determine the medium concentration. Cells were washed 3 times with cold PBS (1 mL each) and lysed by adding 80 μL of MPER buffer (Thermo Scientific, Rockford, IL). An aliquot of the lysed cells, as well as the medium supernatant, were transferred to a new plate, matrix matched, and cold acetonitrile containing IS was added. The solutions were centrifuged at 3000 RPM for 10 min at room temperature. The supernatants were transferred and analyzed by LC-MS/MS against standard curves that were matrix matched for both media and cells.
Stability in Human Hepatocytes and Media
10
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Determination of Unbound Partition Coefficient (Kpuu) and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake
Keith Riccardi, Zhenhong Li, Janice A. Brown, Matthew F. Gorgoglione, Mark Niosi, James Gosset, Kim Huard, Derek M. Erion, Li Di
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT (KR, JAB, MN,LD); Cambridge, MA (MFG, JG, KH, DME)
1
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 23, 2022
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Running Title: Kpuu and IVIVE of SLC13A Transporter-Mediated Uptake
Corresponding Author: Li Di Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. Eastern Point Road, Groton, CT 06345 [email protected]
Text Pages: 25 Tables: 5 Figures: 8 References: 23 Abstract: 238 words Introduction: 726 words Discussion: 714 words
Abbreviations: ABT, 1-aminobenzotriazole; BSA, bovine serum albumin; CES, carboxylesterase; CYP, cytochrome P450; fucell, fraction unbound of cells; IC50, concentration of an inhibitor where the response is reduced by half; I.V., intravenous; IVIVE, in vitro-in vivo extrapolation; JVC, jugular vein cannula; Km, Michaelis-Menten constant; Kpuu, unbound partition coefficient, is defined as the ratio between unbound intracellular free drug concentration and unbound medium concentration (steady state is not assumed) in this paper; Pdiff, clearance via passive diffusion; SBECD, sulfobutyletherβ-cyclodextrin; Vmax, maximum rate
2
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Abstract Unbound partition coefficient (Kpuu) is important to understand the asymmetric free drug distribution of a compound between cells and media in vitro, as well as tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds that are inhibitors and substrates of transporters from the SLC13A family in hepatocytes and transporter-transfected cell-lines. Enantioselectivity was observed with (R)-enantiomers achieving much higher Kpuu (> 4) than the (S)-enantiomers (< 1) in human hepatocytes and SLC13A5 transfected HEK293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay due to the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid (PF-06649298) directly or via hydrolysis of the ethyl ester prodrug (PF-06757303) resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid vs. 59 for prodrug), which was successfully modeled based on passive diffusion, active uptake and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations potentially due to saturation of transporters. Km of SLC13A5 was estimated to be 24 µM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free BSA showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems.
3
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Introduction Intracellular free drug concentration plays a critical role in developing target exposure and pharmacological activity relationships for disease targets located in the intracellular domain. For compounds that reach distribution equilibrium across the cell membrane at steady state, free drug concentration in the media might be considered the same as the intracellular free drug concentration. This is usually the case for most compounds, in which the nominal concentrations can be used to correlate to pharmacological activities (e.g., IC50) of a compound in in vitro assays or after correction of non-specific binding to the proteins/lipids in the assay media. However, when transporters are involved in the distribution process, the intracellular free drug concentration may be different than that in the media due to active processes. In addition, effects of membrane potential, lysosomal trapping, pH gradient and mitochondria accumulation can all impact the intracellular free drug concentration relative to the medium free concentration (Figure 1). Therefore, it is important to understand the intracellular free drug concentration and unbound distribution coefficient (Kpuu) to accurately determine the true potency of a compound, e.g., intrinsic IC50, rather than apparent IC50. Prediction of human in vivo liver-to-plasma Kpuu is of great importance in order to estimate dose, safety margin and drug-drug interaction potentials for compounds with transporter-mediated uptake into the liver. Development of in vitro tools to predict in vivo liver-to-plasma Kpuu will be very useful for human translation in drug discovery and development, since there are no simple tools available to measure human liver concentration.
4
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Several in vitro methods have been developed to measure the intracellular free drug concentration and Kpuu in hepatocytes or transfected cell systems expressed OATP transporters (Figure 2). The binding method measures fucell, medium and total cell concentration at the steady state, from which intracellular free drug concentration and Kpuu are determined (Mateus et al., 2013). The temperature method measures the medium and total cell concentration at both 37°C and 4°C at steady state (Shitara et al., 2013). The total concentration ratio of cell to medium between 37°C and 4°C is considered to be Kpuu. This method does not consider the impact of membrane potential or metabolism. The kinetic method measures the uptake rate at multiple concentrations in the presence of CYP inhibitor, ABT. Kinetic parameters (Vmax, Km, and Pdiff) are obtained by simultaneously fitting all the data (Yabe et al., 2011). Kpuu is calculated based on the kinetic parameters with the extended sequential clearance equation (Yabe et al., 2011). This approach does not consider the impact of efflux transport or metabolism on Kpuu. The permeability method measures the permeability of both neutral and ionized species and determines the Kpuu across a cell membrane based on membrane potential and passive permeability (Ghosh et al., 2014). This method has not incorporated the effects of transporters or metabolism at this point. Other methods reported to determine drug concentration in the subcellular compartments (Pfeifer et al., 2013) are relevant to therapeutic targets localized in the specific subcellular organelles. Validation of the different Kpuu methods with in vitro or in vivo data is minimal in literature. We will discuss our approach for measuring Kpuu and intracellular free drug concentration, as well as validation against in vitro potency, using cell lines expressing different members of the SLC13A family, hepatocytes and in vivo rat liver-to-plasma Kpuu data.
5
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 23, 2022
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837 Citrate is a key regulatory metabolic intermediate and is critical in the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake through SLC13A5 has been suggested as a potential therapeutic approach to treat metabolic diseases (Huard et al., 2015; Huard et al., 2016; Li et al., 2016). The SLC13A family contains five family members: SLC13A1 and SLC13A4 mainly transport sulfate, and SLC13A2, SLC13A3 and SLC13A5 transport di- and tri-carboxylates such as citrate (Lee et al., 2005; Bergeron et al., 2013). SLC13A5 is highly expressed in human liver, while SLC13A2 and SLC13A3 are expressed more broadly, including in the intestine and kidney (Pajor, 2014). Therefore, selective SLC13A5 inhibitors have the potential to impact citrate flux in the liver only, while SLC13A2/3 inhibitors can impact citrate flux in the intestine and kidney as well. Development of specific inhibitors for SLC13A5 is proposed to be beneficial in treating metabolic disorders by reducing citrate flux into the liver, and presumably exert the metabolic benefits through modification of intracellular metabolites, while limiting direct effects in other tissues.
6
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 23, 2022
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Materials and Methods Materials Test compounds were obtained from Pfizer Global Material Management (Groton, CT) or purchased from Sigma-Aldrich (St. Louis, MO). The syntheses of diacids PF-06649298, PF-06649297 and monoacid PF-06757303 were previously reported (Huard et al., 2015). The syntheses of PF-06794266, PF-06793742 and PF-06761281 were also reported (Huard et al., 2016). PF-06761281 was commercially available via Sigma Aldrich (catalog # PZ0318). The syntheses of PF-06746350 and PF-06741415 were similar to the methods described previously (Huard et al., 2016). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless specified. The 96-well equilibrium dialysis (HTD96) device and cellulose membranes with MWCO of 12-14K were purchased from HTDialysis, LLC (Gales Ferry, CT). 1.2 mL deep 96-well plates were from Axygen Scientific Inc. (Union City, CA) and pipette tips were obtained from Apricot Designs (Monrovia, CA).
Measurement of Fraction Unbound Cells (hepatocytes, HEK293 transfected with human SLC13A transporters) at a cell density of 50-60 million cells/mL, rat and human liver tissues (1:5 dilution with phosphate buffered saline (PBS)) were homogenized at room temperature in PBS using an Omni TH tissue homogenizer (Omni International, Kennesaw, GA) with a 7 mm x 110 mm tip at high speed for 30 second pulses. Assay media containing proteins (e.g., BSA) were used directly for binding measurement. The dialysis membranes were prepared prior to experimental setup. The cellulose membranes (MWCO 12-14K) were immersed in de-
7
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 23, 2022
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
ionized water for 15 minutes, followed by 15 minutes in 30% ethanol / de-ionized water, then at least 15 minutes or overnight in PBS. The equilibrium dialysis device was assembled according to manufacturer’s instructions (http://htdialysis.com/page/1puq4/Operating_Instructions.html). DMSO stock solutions of test compounds were prepared at 200 μM, added in 1:100 ratio to matrices, and mixed thoroughly with an 8-channel pipettor (Eppendorf®, VWR, Radnor, PA). The final compound concentration was 2 μM containing 1% DMSO. A 150 μL aliquot of matrix (cell and tissue homogenate, assay media or plasma) spiked with 2 μM compound was added to one side of the membrane (donor) and 150 μL of PBS was added to the other side (receiver). The equilibrium dialysis device was covered with Breathe Easy™ gas permeable membranes (Sigma-Aldrich, St. Louis, MO). Compounds were assessed in quadruplicate. Equilibrium dialysis devices were placed on an orbital shaker (VWR, Radnor, PA) at 200 RPM and incubated for 6 hours in a humidified (75% RH) incubator at 37°C with 5% CO2/95% O2. At the end of the incubation, 15 μL of matrix samples from the donor wells were added to a 96-well plate containing 45 μL of PBS. Aliquots of 45 μL dialyzed PBS from the receiver wells were added to 15 μL of blank matrix. Before and after incubation, 15 μL of matrix spiked with 2 μM of compounds was added to a 96well plate containing 45 μL of PBS. These samples were used for the recovery calculation and stability evaluation. All the samples were quenched with 200 μL of cold acetonitrile containing internal standard (a cocktail of 0.5 ng/mL tolbutamide and 5 ng/mL terfenadine). The plates were sealed and mixed with a vortex mixer (VWR, Radnor, PA) for 3 minutes, then centrifuged at 3000 RPM (Beckman Coulter, Fullerton, CA) at RT for 5 minutes. The supernatant was transferred to a new 96-well plate, dried
8
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 23, 2022
DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
down, reconstituted and subsequently analyzed using LC-MS/MS. Sertraline was used as a quality control sample on every plate. Cell diameter was measured using ViCELL XR (Beckman-Coulter, Inc., Danvers, MA) to calculate cell volume assuming spherical shape and dilution factor.
In Vitro Kpuu Measurement Transfected Cells (SLC13A5- and SLC13A3-HEK293) Kpuu within this paper is defined as the ratio between unbound intracellular free drug concentration and unbound medium concentration (steady state is not assumed). Protocols to generate the stable cell line overexpressing sodium citrate transporters and culture conditions were discussed previously (Huard et al., 2015). Test compounds (1 μM unless otherwise specified) were incubated with the plated cells (1-4 hours unless otherwise specified) at 37 °C in a humidified (75% RH) incubator with 5% CO2/95% O2. At the end of the incubation, 100 μL of the media were removed for concentration determination, and cold acetonitrile was added. The rest of the media was gently removed from the cells by pipette aspiration. The cells were washed three times with cold PBS of the same starting volume. After wash, the cells were lysed by adding 80 μL of MPER buffer (Thermo Scientific, Rockford, IL). An aliquot of the lysed cells was transferred to a new plate, matrix matched, and cold acetonitrile containing IS was added. The solutions were centrifuged at 3000 RPM for 10 min at room temperature. The supernatants were transferred and analyzed by LC-MS/MS against standard curves that were matrix matched for both media and cells.
Human and Rat Hepatocytes 9
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DMD Fast Forward. Published on July 14, 2016 as DOI: 10.1124/dmd.116.071837 This article has not been copyedited and formatted. The final version may differ from this version.
DMD#71837
Cryopreserved human hepatocytes (Lot DCM) consisting of 10 donors, both males and females, were custom-pooled and prepared by BioreclamationIVT (Baltimore, MD). Wistar-Han rat hepatocytes (Lot VSU) consisting 35 male donors were also purchased from BioreclamationIVT. Upon thawing, the hepatocytes were re-suspended in Williams E medium (WEM GIBCO-BRL, custom formula supplemented with 50 mM Hepes and 26 mM sodium bicarbonate). The cells were counted and viability was determined using the Trypan Blue exclusion method. Test compounds were dissolved in DMSO at 1 mM and 1 µL was added to 0.5 million cells/mL suspended hepatocytes in 1000 µL (final concentration 1 µM containing 0.1% DMSO). The suspension was incubated in a humidified (75% RH) incubator at 37 °C with 5% CO2/95% O2. For rat hepatocytes, InVitroGRO HI media (Fisher Scientific,Waltham, MA) containing 0.2, 2 and 4% fatty acid free BSA (catalog # A4612, Sigma-Aldrich, St. Louis, MO) were also used in the study for IVIVE development. At appropriate time points, hepatocyte suspension was centrifuged at 500 RPM for 3 minutes and supernatant was collected and removed from the cells to determine the medium concentration. Cells were washed 3 times with cold PBS (1 mL each) and lysed by adding 80 μL of MPER buffer (Thermo Scientific, Rockford, IL). An aliquot of the lysed cells, as well as the medium supernatant, were transferred to a new plate, matrix matched, and cold acetonitrile containing IS was added. The solutions were centrifuged at 3000 RPM for 10 min at room temperature. The supernatants were transferred and analyzed by LC-MS/MS against standard curves that were matrix matched for both media and cells.
Stability in Human Hepatocytes and Media
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