Lot EAV 10-Donor Pool High AO 5-Donor Pool

Substrate CLin vivo

(mL/min/kg) CLint,in vivo

(mL/min/kg)a fuBb RB

c fu,plasma Suspended In Vitro CLint (mL/min/kg)d

Suspended CLh without bindinge

Suspended CLh with bindinge

Suspended In Vitro CLint (mL/min/kg)d

Suspended CLh without bindinge

Suspended CLh with bindinge

XK-469 0.11 16.0 0.0069 0.52 0.0036 <3.10 <2.70 <0.02 <3.10 <2.70 <0.02 Zaleplon 15.7 98.5 0.66 0.74 0.49 7.10 5.29 3.82 17.5 9.48 7.41

Zoniporide 21.0 9800 0.45 0.75 0.34 20.6 10.3 6.40 57.1 15.2 11.5 O6-Benzylguanine 15.6 452 0.14 0.74 0.10 26.4 11.6 3.14 63.9 15.6 6.25

DACA 17.2 4069 0.025 0.85 0.021 94.8 17.0 2.13 195 18.7 3.95 BIBX1382 17.5f 1132 0.10 1.43 0.15 112 17.5 7.27 202 18.8 10.2

References1. Pryde DC, Dalvie D, Hu Q, et al. (2010) Aldehyde oxidase: an enzyme of emerging importance in drug

discovery. J. Med. Chem. 53: 8441-8460.2. Zientek M, Jiang Y, Youdim K, et al. (2010) In vitro-in vivo correlation for intrinsic clearance for drugs

metabolized by human aldehyde oxidase. Drug Metab. Dispos. 38:1322-1327.3. Hutzler JM, Yang YS, Albaugh D, et al. (2012) Characterization of aldehyde oxidase enzyme activity in

cryopreserved human hepatocytes. Drug Metab. Dispos. 40:267-275.4. Akabane T, Gerst N, Masters JN, et al. (2012) A quantitative approach to hepatic clearance prediction of

metabolism by aldehyde oxidase using custom pooled hepatocytes. Xenobiotica 42:863-871.5. Hutzler JM, Yang YS, Brown C, et al. (2014) Aldehyde oxidase activity in donor-matched fresh and cryopreserved

human hepatocytes and assessment of variability in 75 donors. Drug Metab. Dispos. 42:1090-1097.

OverviewPurpose • Create three separate custom pools of cryopreserved human

hepatocytes (low, moderate, and high activity from 75 donors).• Compare activity using six aldehyde oxidase (AO) substrates that

range in clearance (CL). • Conduct in vitro to in vivo correlation (IVIVC) analysis to assess

scaling approaches.Method • Five individual donors were selected from the low (<30 mL/min/kg),

moderate (30-60 mL/min/kg), and high (>60 mL/min/kg) AO activity ranges and pooled to create custom lots of cryopreserved human hepatocytes, which were compared to a standard commercial 10-donor pool (Lot EAV).

• Analysis was performed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC/MS/MS).

Results • Under-prediction was still noted when comparing in vitro intrinsic

clearance to in vivo intrinsic clearance. • When hepatic clearance was scaled using the well-stirred model and

compared to in vivo total clearance, the predictions were within 2-fold for each of the tested AO substrates, with the exception of XK-469.

• Custom pooling of human hepatocytes for maximal AO activity may help minimize under-prediction of total clearance, but factors involved in why in vivo intrinsic clearance is still drastically under-predicted and require further research.

Introduction• Aldehyde oxidase is a cytosolic drug-metabolizing enzyme that has

emerged in recent years due to the reported negative impact on numerous clinical programs (1).

• Multiple literature reports have pointed towards an under-prediction of clearance bias for substrates of AO (2,3), with multiple potential causes (e.g., extra-hepatic AO contribution, polymorphisms in AOX1, enzyme instability, de-dimerization, variability).

• Custom pools of human hepatocytes prepared with pre-selected donors based on their individual activity may be an approach to maximize activity and, thus, minimize under-prediction of metabolic clearance (4).

• Previously published work in human hepatocytes has identified a high level of variability of AO activity across 75 donors (Figure 1), which translates to measurable but potentially low activity (5).

MethodsHepatocyte Incubations

Five individual donors were selected from the low (<30 mL/min/kg), moderate (30-60 mL/min/kg), and high (>60 mL/min/kg) AO activity ranges and pooled to create custom lots of cryopreserved human hepatocytes. Six AO substrates with a diversity of clearance rates (XK-469, zaleplon, O6-benzylguanine, zoniporide, DACA, and BIBX1382) were then incubated in suspension for up to 4 hours to compare activities across these custom pools, as well as to a standard commercial 10-donor pool (Lot EAV).

Cryopreserved human hepatocytes were thawed in Life Technologies hepatocyte thawing media, centrifuged at 100 x g for 10 minutes and re-suspended in hepatocyte maintenance medium (HMM). The hepatocytes were then counted and percent viability was determined using a Nexcelom K2 Image Cytometer. Cell viability was ≥85% for all studies. A summary of the remaining conditions is in the table below.

Results

Conclusions• Predicting human clearance for AO substrates remains challenging.• In general, standard commercial lots of pooled human cryopreserved

hepatocytes have not been characterized for AO activity by vendors, but reported data (3) has found that while activity is measurable, it is moderate at best.

• Custom pool of cryopreserved hepatocytes from donors with high AO activity demonstrated ~2- to 3-fold higher activity than a commonly used lot of pooled cryopreserved human hepatocytes.

• Pre-selecting donors with high AO activity is suggested for creating a lot of human hepatocytes with high activity.

• A scaling approach using the well-stirred model with no protein binding correction from a high AO activity custom lot provided the most accurate prediction of in vivo clearance.

• In vitro methodology of using custom-pooled human hepatocytes with high AO activity should be integrated with scaling from appropriate pre-clinical species for a higher confidence prediction of clearance.

Acknowledgments• Jeffrey Jones (Washington State University) for providing the AO substrate DACA

Custom-Pooled Human Hepatocytes for Clearance Prediction of Aldehyde Oxidase SubstratesJ. Matthew Hutzler1, Mark VandenBranden1, Olukayode Oluyedun2, Tim Moeller2, Scott Heyward2

1Q2 Solutions, Indianapolis, IN USA; 2BioreclamationIVT, Baltimore, MD USA

Copyright ©2016 Q² Solutions. All rights reserved.

• When comparing in vitro intrinsic clearance to in vivo intrinsic clearance, a 5.6- to 21-fold under-prediction was noted, with zoniporide representing the extreme (171-fold).

• Correcting hepatic clearance estimates with fuB also leads to under-prediction.• When intrinsic clearance was scaled using the well-stirred model to compare to in vivo total clearance, the predictions were within 2-fold for

each of the tested AO substrates, with the exception of XK-469, which did not demonstrate measurable clearance in any of the tested lots of hepatocytes. Hepatic clearance using the well-stirred model without fuB correction from a high AO activity pool of human hepatocytes appears to correlate best with CLin vivo.

Table 1. IVIVC Analysis for Tested AO Substrates

Table 2. Summary of Intrinsic Clearance (CLint) Values for AO Substrates in Standard (Lot EAV) and Custom-Pooled Cryopreserved Human Hepatocytes (Low, Moderate, and High AO)

Calculations

To predict CLint from in vitro data, scaling factors of 120 x 106 hepatocytes/g liver and 25.7 g liver/kg were used (Equation 1), where kdep = slope of time (min) vs. LN% Remaining, Inc Vol = incubation volume, and BWt = body weight.

To calculate in vitro CLh (hepatic clearance) from in vitro data (Equation 2) the well-stirred model was used, with or without the incorporation of the unbound fraction of drug in the blood (fuB = fup/RB where fup = fraction unbound in plasma and RB = red blood cell partitioning) and hepatic blood flow (Qh = 20.7 mL/min/kg).

In addition, to calculate in vivo CLint from observed CL, a back-calculation using the rearranged well-stirred model (Equation 3) was used, where CL = in vivo CLtotal.

Figure 1. Characterization of AO Activity in Cryopreserved Human Hepatocytes from 75 Individual Donors Originally published by Hutzler et al. (5)

Bioanalysis

All substrates were analyzed by LC/MS/MS using a multiple reaction monitoring (MRM) method. A CTC PAL LEAP autosampler was coupled to a Shimadzu HPLC system and an AB SCIEX API 4000 triple quadrupole mass spectrometer operating in positive ion electrospray mode. All analyte methods were validated using an in-batch range of 0.02 to 2.0 µM and labetalol was used as the internal standard. Analytes were separated using a Phenomenex Luna C18 5 µm, 50 x 2 mm column (ambient temperature), with Mobile Phase A (0.05% formic acid in water) and Mobile Phase B (95:5 acetonitrile/0.05% formic acid in water) at a flow rate of 0.5 mL/min under gradient conditions (5% to 95% Mobile Phase B over 2 min). The table below summarizes the ions monitored for each MRM method.

Hepatocyte Incubation Conditions Pooled Cryopreserved Human Hepatocytes

1) Lot EAV, a 10-donor, mixed-gender commercial pool 2) Three custom prepared pools consisting of 5 donors

each with pre-determined AO activity Cell Density 1 x 106 hepatocytes/mL Plate Design 96-well plate Incubation Volume 200 µL (200,000 hepatocytes/well) Incubation Medium HMM AO Substrate Concentration 0.3 µM Shaker (speed) LiCONic (600 rpm) Time Points 0, 15, 30, 60, 120, 240 minutes (duplicate) Incubator Settings 37 °C, 5% CO2

a CLint back-calculated from CLin vivo using Equation 3 b Literature or calculated from reported fu and RB (fuB = fu/RB) c Literature reference (2)d Experimental data from Q² Solutions e Calculated using the well-stirred model with and without fuB f Corrected for blood partitioning (RB); clearance range 17.5 to 38.5

LN%

Rem

aini

ngLN

% R

emai

ning

Figure 2. Substrate Depletion Plots for AO Substrates Tested in Separate Lots of Cryopreserved Human Hepatocytes

0 30 60 90 120 150 180 210 2401

2

3

4

5

0 30 60 90 120 150 180 210 2401

2

3

4

5

0 30 60 90 120 150 180 210 2401

2

3

4

5

0 30 60 90 120 150 180 210 2401

2

3

4

5

0 30 60 90 120 150 180 210 240

1

2

3

4

5

0 30 60 90 120 150 180 210 2401

2

3

4

5

Time (minutes)Time (minutes)LN

% R

emai

ning

■ (AO Low), ● (Lot EAV), ▲ (AO Moderate), ▼ (AO High)Activities for all substrates tested trended in a similar fashion, with intrinsic clearance values rank-ordering as: high AO custom pool > moderate AO custom pool > commercial pool EAV > low AO custom pool. The activity in the high AO custom pool was roughly 2- to 3-fold higher than the commercial pool EAV.

XK-469 O6-Benzylguanine

Zoniporide Zaleplon

DACA BIBX1382

N

N

O

O COOH

Cl

N

HNO

N

N

N N

N

H2N

O

N

NN

NNH H

N

N

Cl

F

CH3

N

N N

CN

N

O

N

NN

ONH

NH2

HN

Individual Lots

CL in

trin

sic (

mL/

min

/kg)

O6 -B

enzy

lgua

nine

Oxi

datio

n

0

10

20

30

40

50

60

70

80

90

100

Individual Donor Cryopreserved Hepatocytes(Mean ± SD, n = 70 donors)39.9 ± 19 mL/min/kg

Pooled Cryopreserved Hepatocytes(n = 19 donors, mixed gender)34.2 ± 1.4 mL/min/kg

Equation 1: In Vitro Intrinsic Clearance:

BWt kgLiver g 25.7

Liver gsHepatocyte 10120

10 sHepatocyte(mL) Inc VolkCL

6

6depint ××

××

×=

Custom pool of cryopreserved hepatocytes from donors with high activity demonstrated ~2- to 3-fold higher activity than commonly used lot of pooled cryopreserved human hepatocytes.

Substrate Hepatocyte Lot CLint (mL/min/kg) Custom/EAV Ratio

Zaleplon EAV (10 donor) 7.1 1.0

AO Low No clearance —

AO Moderate 16.0 2.3

AO High 17.5 2.5

Zoniporide EAV (10 donor) 20.6 1.0

AO Low 6.2 0.3

AO Moderate 46.8 2.3

AO High 57.1 2.8

O6-Benzylguanine EAV (10 donor) 26.4 1.0

AO Low 4.9 0.2

AO Moderate 31.7 1.2

AO High 63.9 2.4

DACA EAV (10 donor) 94.8 1.0

AO Low 34.8 0.4

AO Moderate 140.7 1.5

AO High 194.6 2.1

BIBX1382 EAV (10 donor) 112.4 1.0

AO Low 39.1 0.3

AO Moderate 146.9 1.3

AO High 201.6 1.8

Ions Monitored (MRM) XK-469 Zaleplon O6-Benzylguanine Zoniporide DACA BIBX1382

345.0 → 299.0 306.0 → 236.0 242.2 → 91.0 321.1 → 262.0 295.2 → 250.0 388.0 → 98.0

Equation 2: In Vitro Hepatic Clearance: )]CL ( [CL

CLintuBH

intuBHh ×+

××=

fQfQ

Equation 3: In Vivo Intrinsic Clearance:

−×

HuB

intCL1

CL= CL

Qf

Figure 3. Substrate Depletion Plots of AO Substrates for IVIVC Analysis Comparison

LN%

Rem

aini

ng

Time (minutes) Time (minutes)

Standard Pool: Lot EAV Custom Pool: High Activity

0 30 60 90 120 150 180 210 240

1

2

3

4

5

0 30 60 90 120 150 180 210 240

1

2

3

4

5

XK-469Zaleplon

ZoniporideO6-Benzylguanine

DACABIBX1382