DMD # 74138 1 Determination of Incubational Binding in In Vitro Microsomal and Hepatocyte Metabolic Stability Incubations: A Comparison of Methods Sofia Chen, Luna Prieto Garcia, Fredrik Bergström, Pär Nordell and Ken Grime Respiratory, Inflammation and Autoimmunity Department of DMPK, Innovative Medicines and Early Development, AstraZeneca, Gothenburg, Sweden (KG, SC, LPG) Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca, Gothenburg, Sweden (PN) Cardiovascular and Metabolic Diseases Department of DMPK, Innovative Medicines and Early Development, AstraZeneca, Gothenburg, Sweden (FB) This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on January 25, 2017 as DOI: 10.1124/dmd.116.074138 at ASPET Journals on May 21, 2018 dmd.aspetjournals.org Downloaded from
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DMD # 74138
1
Determination of Incubational Binding in In Vitro Microsomal and Hepatocyte Metabolic
Stability Incubations: A Comparison of Methods
Sofia Chen, Luna Prieto Garcia, Fredrik Bergström, Pär Nordell and Ken Grime
Respiratory, Inflammation and Autoimmunity Department of DMPK, Innovative Medicines and Early
Development, AstraZeneca, Gothenburg, Sweden (KG, SC, LPG)
Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca,
Gothenburg, Sweden (PN)
Cardiovascular and Metabolic Diseases Department of DMPK, Innovative Medicines and Early
Development, AstraZeneca, Gothenburg, Sweden (FB)
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fuinc Fraction of unbound drug in incubation, assumed to be at 1 mg/mL microsomal
protein or 1 million hepatocytes/mL unless otherwise stated.
HLM Human liver microsomes
LogD7.4 A distribution-coefficient describing the ratio of compound concentration in octanol
and pH7.4 buffer when the test system is at equilibrium (lipophilicity measure)
RH Rat hepatocytes
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Fraction of drug unbound in an in vitro intrinsic clearance (CLint) incubation, fuinc, is an important
parameter in the pursuit of accurate clearance predictions and is often predicted using algorithms based
on drug lipophilicity measures. However, analysis of an AstraZeneca database suggests that simple
lipophilicity alone is a relatively poor predictor of fuinc measured using equilibrium dialysis. Fuinc can
also be measured directly in CLint assays using multiple concentrations of hepatocytes or microsomal
protein. Since this approach informs of the unbound drug concentration in the assay used to predict in
vivo clearance, it should be considered the gold standard method. As a starting point for building better
predictive algorithms we aimed to determine if equilibrium dialysis really is an appropriate assay for
assessing fuinc. Employing a large number of compounds with a wide range of lipophilicities,
experiments were performed to measure fuinc using rat hepatocytes (RH) and human liver microsomes
(HLM) in both assay formats. A high percentage (94% and 93% for HLM and RH respectively) of the
fuinc values were within 2-fold when compound logD7.4 values were less than 3.5. However, with logD7.4
values greater than these, the agreement was considerably worse. Additional experimental data
generated indicated that this discrepancy was likely due to failings in the direct method when drug
binding is high. Thus we conclude that unbound CLint can be indeed calculated indirectly by
incorporating equilibrium dialysis data with measured CLint but that simple lipophilicity descriptors
alone may be inadequate for predicting fuinc.
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Prediction of in vivo hepatic metabolic clearance from in vitro data uses measured hepatocyte or
microsomal CLint, fuinc and fraction of drug unbound in blood (fub) (Obach, 1996, Ito and Houston, 2005).
To add confidence to human clearance predictions for any given candidate drug, observed unbound in
vivo CLint should first be predicted to within 2-fold in two preclinical species (Grime et al. 2013). To
meet such stringent criteria, attention to fine detail on experimental conditions and physiological
relevance of the in vitro measurements is paramount. In a Drug Discovery setting, fuinc is typically
measured using equilibrium dialysis in which partitioning of drug between two chambers either side of
the dialysis membrane, one holding inactivated microsomes or hepatocytes and one holding buffer, is
determined. This assay allows a high-throughput of compounds but does not necessarily represent the
conditions experienced by a drug at the site of drug metabolism in a CLint assay and thus assessment of
unbound CLint directly in the metabolic stability assay may be viewed as the ideal. With this in mind, an
approach for determining fuinc using metabolic stability data generated at multiple concentrations of
hepatocytes or microsomal protein can be used, since the rate at which a compound is metabolized is
proportional to the unbound concentration available to enzymes (Giuliano et al., 2005, Grime and Riley,
2006, Nordell et al., 2013). The pharmaceutical industry is under great pressure to reduce costs and as
such it is an appropriate aim to minimise wet screening where possible. As such fuinc in silico prediction
algorithms, based on ion class and lipophilicity (logP or logD7.4 octanol/water partition coefficients,
Austin et al., 2002, Austin et al., 2005, Kilford et al., 2008) as well as other molecular descriptors (Gao
et al., 2008, Gao et al., 2010, Abraham and Austin, 2012; Nair et al., 2016), have become widely used.
On review of an in-house database, we noted that simple lipophilicity based predictions were within
two-fold of equilibrium dialysis measured values for only 64% (HLM) and 62% (RH) of compounds
(Figure 1). As a starting point for building better fuinc predictive algorithms we considered the validity
of equilibrium dialysis measures and compared HLM and RH fuinc values generated by this approach
and directly from the CLint assays. The data sets cover 58 and 74 AstraZeneca proprietary compounds
for HLM and RH, respectively, spanning over five orders of magnitude in measured lipophilicity.
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UltraPool human liver microsomes (mixed gender, lot no 38289) were purchased from BD Gentest
(USA) and cryopreserved Han Wistar rat hepatocytes (mixed gender, lots RJQ and RAL) were
purchased Bioreclamation IVT (Belgium). L-15 Leibovitz medium and phosphate buffers were obtained
from Life technologies (NY, USA). NADPH was obtained from Sigma (USA). Compound stock
solutions DMSO (10 mM) were obtained from the AstraZeneca Compound Management.
Measurement of LogD7.4 and Determination of fuinc by equilibrium dialysis
LogD7.4 was measured as previously described (Austin et al., 2002). Equilibrium dialysis was performed
to determine extent of compound binding to HLM (1mg/mL in 0.1M, pH 7.4 phosphate buffer) and
freshly isolated RH (1 million cells/mL in Leibovitz media). Compound stocks (100 μM in DMSO)
were added at a ratio of 10 L / mL HLM or RH suspensions. Inactivation of drug metabolising enzymes
was performed in accordance with previous studies (Austin et al., 2005) using 1-aminobenzotriazole
(400 mM in DMSO) and salicylamide (300 mM in DMSO), added to the preparations at a ratio of 2.5
L/mL and 5 μL/mL respectively, followed by 1 h of pre-incubation at 37C. Samples were checked for
drug metabolism by analysing parent drug pre- and post- dialysis experiments. Experiments were
performed in triplicate. Dialysis membranes were soaked in ultrapure water for 60 min, in 20% ethanol
for 20 min and finally in phosphate buffer (0.1 M, pH 7.4) for 20 min prior to loading into 96-well
dialysis devices (HTDialysis LLC, Gales Ferry, CT). An aliquot (150 μL) of HLM spiked with test
compound (1 μM) and drug metabolism inhibitors was added to donor wells of the dialysis device with
phosphate buffer (150 μL, 0.1M, pH 7.4) added to acceptor wells. The dialysis device was loaded onto
a rotor housed within an air bath. After 4 hours of incubation (37C, 300 rpm), aliquots (50 μL) from
each donor and acceptor well were transferred to a 96-deep well plate. Samples were quenched with
acetoniltrile (100 µL), diluted, analyzed by LC-MS/MS and quantified from a standard curve. Free
fraction of drug was determined as previously described (Austin et al., 2002). RH were isolated from
adult male Sprague-Dawley rats (250 - 300 g) as described previously (Kenny and Grime, 2006) and
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resuspended in Leibovitz media at 1 million cells/mL. Leibovitz media (150 μL) was added to the
acceptor chamber and hepatocyte suspension (150 μL) containing compound (1 μM) and drug
metabolising enzyme inhibitors was added to the donor chamber. Dialysis procedure, as above. All LC-
MS/MS analyses were performed on a Waters triple quadrupole mass spectrometer, coupled to a liquid
chromatography sample and solvent manager system, as described previously (Nordell et al., 2013). In
order to investigate if non-linear drug binding, with respect to concentration of enzyme, was likely to
influence the estimation of fuinc, equilibrium dialysis experiments were performed as described
above, at HLM concentrations of 0.125, 0.5, 1 and 4 mg/mL for 25 compounds.
Metabolic stability at multiple microsomal protein or hepatocyte concentrations
CLint was determined in duplicate for each of the HLM or RH concentrations used. HLM stock solutions
were thawed and diluted in phosphate buffer (0.1M, pH 7.4) containing NADPH (1 mM) to the
following protein concentrations: 0.14, 0.21, 0.28, 0.41, 0.56, 0.83, 1.11, 2.22, 4.44 mg protein/mL.
Suspensions were mixed with compound stock solutions (50 M in 50% acetonitrile) to a final
concentration of 0.5 M and incubated in a 96-well plate at 37°C with plate shaking. Reactions were
terminated at 0.5, 5, 10, 15, 20 and 30 minutes by quenching with cold acetronitrile containing 0.1%
formic acid and subsequently analyzed by LC-MS/MS. RH were suspended in Leibovitz media to the
following cell concentrations: 0.13, 0.19, 0.25, 0.38, 0.50, 0.75, 1.0, 2.0 and 4.0 million cells/mL. Cell
suspensions were mixed with compound solutions (50 M in 50% acetonitrile) to a final concentration
of 0.5 M and incubated in 96-well plate at 37°C with plate shaking. Reactions were terminated (0.5, 5,
15, 20, 30, 45, 60, 80, 100 and 120 minutes) and analyzed by as described above.
Model of incubational binding
Incubational binding, treated as an equilibrium between drug in aqueous and the membrane phases, is
described by a partitioning constant Kp which is related to the fraction unbound (fuinc): Kp = drug bound
to the membrane phase/unbound in the aqueous phase = (1 – fuinc / fuinc) (Austin et al., 2002). Since only
unbound drug is available to metabolizing enzymes, measured intrinsic metabolic clearance (CLint)
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becomes a function of the microsomal or hepatocyte concentration, related to the unbound intrinsic
CLint,u: CLint = fuinc x CLint,u. The fraction unbound at standard conditions (microsomal protein or
hepatocyte concentration of 1 mg/mL or 1 million cells/mL, respectively), fuinc,0, is related to the fraction
unbound and CLint at a second enzyme concentration, allowing definition of the true unbound intrinsic
clearance, CLint,u (where CLint represents observed intrinsic clearance at any enzyme concentration C).
𝐶𝐿𝑖𝑛𝑡 =𝐶𝐿𝑖𝑛𝑡,𝑈
1 +𝐶𝐶0
×1 − 𝑓𝑢𝑖𝑛𝑐,0𝑓𝑢𝑖𝑛𝑐,0
Eq. 1
Direct determination of fuinc from in vitro depletion curves
Analytical peak areas of samples, withdrawn from drug incubations at the various HLM or RH
concentrations, were loge-transformed (as exemplified in Figure 2a) and elimination rate constants k
(min-1) were derived from the slopes of the loge[substrate]-time plots. Since CLint,= k x V, where V
represents the incubation volume (mL/mg protein or mL/million cells), the entire dataset from multiple
microsomal protein or hepatocyte concentrations was used to define fuinc and CLint,U (from Eq. 1) as
follows: Using nonlinear least squares solver lsqnonlin in Matlab 2014b (The MathWorks Inc., Natick,
MA), the sum-of-squares of the vertical distances between loge-transformed percentage substrate
remaining and the linear best fit, at all HLM or RH concentrations, was minimized and the intercept
(from the family of CLint values analyzed with their associated microsomal protein or hepatocyte
concentrations, Figure 2b) was allowed to vary freely for optimal fit. Prior to summation, squared
residuals were weighted to account for number of data points included at each enzyme concentration.
Predicted in vivo rat clearance
Clearance predictions were made using a ‘regression line approach’, whereby an existing in vitro-in vivo
unbound CLint dataset (for which in vivo CLint values represent metabolic clearance only) is used as a
framework for predicting the in vivo clearance for novel compounds (Grime and Riley, 2006; Sohlenius-
Sternbeck et al., 2012).
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0.87, N: 0.001 - 0.95) defined. For RH, 74 (A: 21, B: 20, N: 20) compounds spanning logD7.4 values of
-0.3 - 5.3 (A: -0.4 - 4.5, B: -0.3 - 4.1, N: 0.9 - 5.3) had fuinc (at 1 million cells/mL), values of 0.004 -
0.996 (A: 0.009 - 0.71, B: 0.03 - 0.91, N: 0.004 - 0.995) defined. Fuinc was also determined directly from
the CLint assays as described. In the RH equilibrium dialysis experiments, 1-aminobenzotriazole was
used as a CYP inhibitor. Although a very effective CYP3A4 inhibitor, other enzymes may be less
effectively inhibited including CYP2C9, for which over 60% of activity may remain using 1 mM and a
30 minute pre-incubation (Linder et al., 2009). This was a compromise to avoid the use of several
individual CYP inhibitors designed to remove all CYP activity, but at a cost of greater organic solvent
addition. The pre-incubation was 60 minutes and since the HLM equilibrium dialysis incubations
contained no NADPH and the results were in line with the RH experimental data, it is apparent that the
use of 1-aminobenzotriazole as a CYP inhibitor did not compromise the results. In confirmation of this,
biotransformation assessment of all equilibrium dialysis incubations revealed no drug metabolism in the
RH incubates. The DMSO concentration of 1% v/v used in all metabolic stability incubations may give
rise to sub-optimal rate of metabolism for some drug metabolizing enzymes. However, since the relative
contribution of individual CYPs to the overall CLint is not relevant in determining fuinc by equation 1,
this compromise was made to ensure the solubility of all compounds since many were relatively
lipophilic.
Figure 3 shows a log-scale representation of the agreement between incubational binding estimates
given by the two assays for acids, bases and neutrals in RH and HLM incubations. Only 65% (HLM)
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and 77% (RH) of the fuinc values from the direct assay fell within 2-fold of the values provided by the
equilibrium dialysis assay. However, 94 and 93% (HLM and RH respectively) of the fuinc values were
within 2-fold when compound logD7.4 values were less than 3.5. Although a somewhat arbitrary cut-off,
it allows some quantification of the lipophilicity effect. When considering compounds with logD7.4 >
3.5 only 25% of HLM and 27% of RH fuinc values were within 2-fold. For these compounds (logD7.4 >
3.5), there was significant bias (p = 110-4 and 710-4 for HLM and RH, respectively), with fuinc values
determined by equilibrium dialysis consistently being lower than estimates from direct assessment using
substrate depletion data. Compounds with logD7.4 < 3.5 showed no indication of bias. Using HLM
concentrations of 0.125, 0.5, 1 and 4 mg protein/mL in equilibrium dialysis experiments, we confirmed
that non-linear drug binding, with respect to HLM concentration, was not an influencing factor in the
difference in fuinc values from the two assay formats: For 25 compounds, equilibrium dialysis
incubational binding increased proportionally with increasing HLM concentration (data not shown).
Overall the analysis indicates that more lipophilic compounds may have a strong bias towards
equilibrium dialysis yielding lower fuinc estimates than the direct approach, independent of whether
whole cells or microsomes are studied. In line with this, Guiliano et al. (2005) identified two outliers,
both extensively bound, deviating from the otherwise good correlation between equilibrium dialysis and
metabolic stability based fuinc when investigating HLM-drug binding behaviour. A possible explanation
for these observations is that when incubational binding is high, the rapidly changing CLint-enzyme
concentration curve (Figure 2b) is not sufficiently well-described by the input data at low hepatocyte or
microsomal protein concentrations to allow appropriate back extrapolation to the true CLint,u value (Y-
axis intercept), making fuinc direct from CLint approach less reliable for more lipophilic compounds.
In order to substantiate the idea that, for molecules with higher hepatocyte binding, equilibrium dialysis
gives a more accurate description of binding, we investigated the in vivo rat clearance of two of the
compounds with a big discrepancy in fuinc from the two assays. Using the previously described method
for predicting clearance at AstraZeneca with input rat hepatocyte CLint, fuinc and plasma protein binding
data (Sohlenius-Sternbeck et al., 2012), the clearance predictions were 37 & 1 mL/min/kg when using
fuinc determined from the equilibrium dialysis method and the direct from CLint approach respectively
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for the first compound. The observed rat plasma clearance was 65 mL/min/kg. Similarly, for the second
compound, the predicted clearance values were 48 and 10 mL/min/kg using equilibrium dialysis and
direct method fuinc respectively, whilst the observed clearance was 51 mL/min/kg. Although such an
assessment is not completely robust since there can be other explanations for poor clearance prediction,
this evidence does point to the greater general utility of equilibrium binding data for making accurate
clearance predictions. This study supports the long-held assumption that unbound CLint, required for
predicting in vivo hepatic metabolic clearance, can indeed be calculated indirectly using equilibrium
dialysis data and CLint taken under standard conditions. Additionally, our analysis of an in-house data
base indicates that for prediction of incubational binding requires more sophisticated prediction
approaches than simple lipophilicty algorithms, as discussed elsewhere (Gao et al., 2010; Nair et al.,
2016).
Authorship contributions
Participated in research design: Grime, Nordell, Bergström, Chen, Prieto Garcia.
Conducted experiments: Chen and Prieto Garcia
Performed data analysis: Chen, Prieto Garcia, Bergström and Nordell
Wrote the manuscript: Grime and Nordell
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Figure 1. Human liver microsomal (A) and rat hepatocyte (B) incubational binding: Comparison of
lipophilicity based predicted (Austin et al 2002; Austin et al., 2005) and measured (equilibrium
dialysis) for acidic compounds (upward red triangle), bases (downward blue triangle), neutrals (green
circles) and zwitterions (orange squares). Lines depict unity (solid) and two-fold of unity (dashed).
The number of compounds represented: 512 (RH) and 544 (HLM) of which 319 and 347 respectively
are within 2-fold (observed/predicted).
Figure 2. Principle of data analysis for fuinc determined directly from the CLint assay (RH data shown
for compound 1 in Figure 3). A: loge-transformed percentage of drug remaining for incubations run
between 4 (top-left) and 0.125 (bottom-right) million cells/mL. B: effect of hepatocyte concentration on
CLint. While fuinc reflects the curvature, CLint,U is given by the intercept with the Y-axis according to
equation 1 (Methods). Circles indicate hepatocyte concentration at which incubations were performed.
Figure 3. Figure 3. Comparison between fuinc determined by equilibrium dialysis and directly from
substrate depletion CLint data for HLM (A) and RH (B) incubations. Acidic compounds (upward red
triagles), bases (downward blue triangles), neutrals (green circles). In 3B the solid green circle is
compound 1 (see Figure 2). Comparison between observed and predicted plasma clearance were made
for two compounds indicated by solid red upward triangles. C: Comparison of the fuinc methods
(substrate depletion fuinc / equilibrium dialysis fuinc) with increasing logD7.4 for HLM (upward red
triangles) and RH (downward blue triangles) data. The horizontal lines indicate unity and two-fold of
unity (dashed), the vertical line indicates logD7.4 > 3.5 threshold.
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