In Vitro - Drug Metabolism and...

DMD#53322

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In Vitro – in Vivo Correlation for Low Clearance Compounds Using Hepatocyte

Relay Method

Li Di, Karen Atkinson, Christine C. Orozco, Carrie Funk, Hui Zhang, Thomas S.

McDonald, Beijing Tan, Jian Lin, Cheng Chang, R. Scott Obach

Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT 06340, USA: LD,

KA, CCO, CF, HZ, TSM, BT, JL, CC, RSO

DMD Fast Forward. Published on July 15, 2013 as doi:10.1124/dmd.113.053322

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 15, 2013 as DOI: 10.1124/dmd.113.053322

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Running Title: IVIVC of Low Clearance

Corresponding Author:

Li Di

Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT 06340, USA

[email protected]

Document Statistics:

Text Pages: 23

Tables: 3

Figures: 2

References: 44

Abstract: 104 words

Introduction: 469 words

Discussion: 702 words

Abbreviations

ADMET = Absorption, Distribution, Metabolism, Excretion, and Toxicity

DMPK = Drug Metabolism and Pharmacokinetics

IVIVC = In Vitro – In Vivo Correlation

PD = Pharmacodynamics

PK = Pharmacokinetics

SAR = Structure-Activity Relationship


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Abstract In vitro-in vivo correlation (IVIVC) of intrinsic clearance in preclinical species of rat and

dog was established using the hepatocyte relay method in order to support high

confidence prediction of human pharmacokinetics for low clearance compounds. Good

IVIVC of intrinsic clearance was observed for most of the compounds, with predicted

values within 2 fold of the observed values. The exceptions involved transporter-

mediated uptake clearance or metabolizing enzymes with extensive extra-hepatic

contribution. This is the first assay available to address low clearance challenges in

preclinical species for IVIVC in drug discovery. It extends the utility of the hepatocyte

relay method in addressing low clearance issues.


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Introduction

An important goal of DMPK scientists in drug discovery is to be able to predict human

pharmacokinetic (PK) and dosing regimen using in vitro data from human-derived

reagents such as hepatocytes and liver microsomes. To increase high confidence in

prediction, it is desirable to accurately predict PK parameters for the same compound(s)

in at least two preclinical species, typically rat and dog using corresponding in vitro

reagents from three species (Wilby et al., 2011). Therefore, developing in vitro – in vivo

correlation (IVIVC) in preclinical species is highly valuable in order to gain confidence

in human translation.

Clearance is one of the most challenging PK parameters to accurately predict due to

species differences in drug metabolizing enzymes, potential for multiple metabolic

pathways, as well as contribution of drug transport. Chemical series that demonstrate

strong IVIVC in preclinical species (e.g., rat, dog) are more likely to give reliable

prediction of human clearance from in vitro data (Wilby et al., 2011). For low clearance

compounds (defined as intrinsic clearance is not measurable using standard liver

microsomal or hepatocyte assays), however, in vitro hepatic clearance values are not

usually available due to lack of appropriate tools in assessing poorly metabolized

compounds in drug discovery. This leads to insufficient supporting evidence of adequate

IVIVC from preclinical species, and therefore low confidence in human translation.

In drug discovery, low intrinsic clearance compounds have increased significantly over

the years due to the effective use of high throughput ADMET screening and early


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metabolite identification, which enable rapid SAR to address high intrinsic clearance as a

liability (Di et al., 2012). In addition, new chemical space of drug discovery is being

explored, such as liver targeting (Oballa et al., 2011; Pfefferkorn et al., 2012) and once

weekly dosing (Rangan et al., 2007), and all these require low clearance. While low

clearance can be a beneficial attribute in a new drug to increase exposure, prolong half-

life and reduce dose, it presents a challenge for DMPK scientists, because tools available

in drug discovery to accurately measure low clearance are limited. As a consequence, it

is often inaccurately assumed that no metabolism is occurring when intrinsic clearance is

below the limit of in vitro microsomal or hepatocyte stability assays. This frequently

leads to inaccurate prediction of human clearance, half-life and dose. To address low

clearance challenges, the hepatocyte relay method was recently developed to predict

human intrinsic clearance for low intrinsic clearance compounds (Di et al., 2012). In this

study, we have expanded the hepatocyte relay method to predict rat and dog intrinsic

clearance in order to establish IVIVC in three preclinical species to support high

confidence translation of human IVIVC for low clearance compounds. This is the first in

vitro tool available to study low clearance in rat and dog when limited to measure

intrinsic clearance by monitoring substrate depletion.


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Material and Methods

Materials

Test compounds were obtained from Pfizer Global Material Management (Groton, CT) or

purchased from Sigma-Aldrich (St. Louis, MO). Other reagents were obtained from

Sigma-Aldrich (St. Louis, MO) unless specified. 96-well polypropylene plates were from

Thermo (Pittsburgh, PA) and 24-well polystyrene plates were obtained from Costar

(Sigma-Aldrich, St. Louis, MO) .

Rat and Dog Hepatocyte Relay Method

The rat and dog hepatocyte relay methods are similar to the human hepatocyte relay assay

previously reported (Di et al., 2012). Pooled cryopreserved male beagle dog hepatocytes

from 5 dogs and male Wistar-Han rat hepatocytes from 12 rats were purchased from

Celsis IVT (Baltimore, MD). Upon thawing, the hepatocytes were re-suspended in

Williams E medium (WEM GIBCO-BRL, cat#C1984, custom formula # 91-5233EC)

supplemented with 50 mM HEPES and 26 mM Na2CO3. The cells were counted using

the Trypan Blue exclusion method, and the 24-well hepatocyte plates containing 0.5

million cells/mL were spiked with a compound at a final concentration of 1 M (final

DMSO of 0.025% and MeOH of 0.125%), in a final incubation volume of 0.50 mL. The

plates were covered with Breathe-EasyTM gas permeable membranes or equivalent

(Diversified Biotech, Dedham, MA) and incubated at 37C with 95% O2/ 5% CO2, 75%

relative humidity for 4 hours at 150 rpm on an orbital shaker in an incubator. At time 0

and 4 hr, 25 µL of hepatocyte suspension was removed from the incubation and added to

50 µL of cold acetonitrile containing internal standard to quench the reaction. The


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samples were centrifuged (Eppendorf, Hauppauge, NY) at 3000 rpm for 10 minutes at

RT and 50 µL supernatant was transferred to a clean plate, dried completely and

reconstituted in 50% methanol / 50% water prior to LC/MS/MS analysis. The remaining

hepatocyte suspension in the incubation plate was centrifuged (3000 rpm , 10 minutes,

RT) and 25 uL supernatant removed and added to 50 µL cold aceontitrile containing

internal standard to determine the concentration of the supernatant. 300 µL of the

remaining supernatant was transferred to a clean 24-well plate and stored at -80C until

the next relay experiment. For the 2nd relay experiment, the supernatant plates were

warmed to 37C for 30 min and hepatocytes were added to the samples to yield a final

cell density 0.5 million cells/mL. The plates were incubated at 37C for 4 hr, sampled

and processed as described above. Five relays were performed to give a total incubation

time of 20 hr. Buffer control plate (without hepatocytes) was run under the same

conditions to monitor any non-metabolic decline. Standard curves were prepared using

the same matrix material.

LC/MS/MS Quantification

The LC mobile phases were: (A) HPLC grade water containing 0.1% formic acid; and

(B) acetonitrile containing 0.1% formic acid or (A) 95% 2 mM ammonium acetate in

water and 5% 50/50 methanol/acetonitrile and (B) 90% 50/50 methanol/acetonitrile and

10% 2 mM ammonium acetate in water. A solvent gradient from 5% (B) to 95% (B)

over 2.0 minutes at the flow rate of 0.5 mL/min was used to elute the compounds from

the column (Kinetex C18, 30x2 mm, 2.6μm, Phenomenex, Torrance, CA). The cycle-

time was 3 minutes / injection. An aliquot of 15 μL sample was injected for analysis


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using a CTC PAL autosampler (LEAP Technologies, Carrboro, NC). The analysis was

conducted with Shimadzu HPLC AD20 pumps (Columbia, MD) connected to an AB

Sciex (Foster City, CA) API 5500 triple quadrupole mass spectrometer equipped with a

TurboIonSpray source using MRM mode. Analyst™ 1.5.2 software (Applied

Biosystems, Foster City, CA) was applied to data collection. MultiQuant software

(version 2.1) from AB Sciex (Foster City, CA) was utilized for data processing and

analysis. Metoprolol (200 ng/mL, positive mode), indomethacin (250 ng/mL, negative

mode), and terfenadine (40 ng/mL, positive mode) were used as internal standards for

LC/MS/MS quantification in positive and negative ion MRM mode, respectively. Only

one internal standard was used in the final calculation depending on the ionization mode.

All the test compounds had good linearity with R2 > 0.99 and the low limit of

quantitation was 1 nM for all the compounds.

Calculations

The data transformation and calculation for the hepatocyte relay assay has been discussed

previously (Di et al., 2012). The hepatocyte relay assay requires transfer of the

supernatant between plates for the different relay incubations. Drug is lost due to

hepatocyte uptake, nonspecific binding, or other sources, and dilution of the drug occurs

as new hepatocytes and medium are added post transfer. These losses necessitate a

correction before comparison to time zero concentrations. The correction equation

follows (Eq. 1):

,

,

, . 1


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Here, Ccorrected is the total concentration of the well after transfer corrected for loss and

dilution. Recovery accounts for the fraction of the compound irretrievable due to

nonspecific binding to the well or other sources, and should be set to unity unless it is

known that drug is lost after each transfer at a location not captured in the total-

concentration measurement. Recovery can be estimated experimentally by measuring the

change in concentration of a representative solution from before and after addition to a

well in the absence of cells (buffer control). N is the number of transfers. The ratio of the

well volume to the supernatant-transfer volume corrects for the dilution which occurs

after each transfer. The above equation assumes that the ratio is fixed, i.e. the total and

transfer volume remain constant throughout the relay assay. Deviations can be handled

by Eq. 2. The final term, the product of the ratios of the total and supernatant

concentrations in the steps previous to the transfer corrects for loss in the hepatocyte

precipitate. The corrected values can be compared directly to values obtained at early

time points prior to any transfer.

,

,

, . 2

Half-life was calculated using Eq. 3 and intrinsic clearance was obtained using Eq. 4 for

rat and Eq. 5 for dog with the corrected concentration (Ccorrected) discussed above (Hosea

et al., 2009).

T1/2 (min) = ln 2 / -(slope of the ln % remaining of drug vs. time plot) (Eq. 3)


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4) (Eq. kg

liver g 40

liver g

Mcells 135

cells M 0.5

incubation mL

(min) T

12ln )mL/min/KgRat,(CL

1/2

in t

5) (Eq. kg

liver g 32

liver g

Mcells 240

cells M 0.5

incubation mL

(min) T

12ln )mL/min/Kg,Dog(CL

1/2

in t

In vivo intrinsic clearance was calculated using the well-stirred model (Ashforth et al.,

1995) (Eq. 6) based on in vivo IV plasma clearance data from the literature, where QH is

hepatic blood flow (70 and 40 ml/min/kg for rat and dog, respectively); CLb is hepatic

blood clearance; fup is fraction unbound in plasma; RB is blood to plasma concentration

ratio.

6) (Eq.

)Q

CL-(1

R

fuCL

CL

H

b

B

p

bin

t

ACD/Labs version 12.0 (Advanced Chemistry Development, Inc. Toronto, Ontario) was

used to calculate Log D/P for all the compounds.


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Results

A set of low clearance compounds in rat and dog was selected for the study based on

available literature data and low intrinsic hepatic metabolic clearance values. In vivo IV

clearance values were obtained from the literature and hepatic clearance values were

calculated by subtracting renal and biliary clearance from plasma clearance. Variability

of the in vivo data can lead to disconnect of IVIVC. IV data from multiple strains were

used assuming strain difference in clearance to be minimal (Richmond et al., 2010). No

oral PK data was included in order to ensure data quality. In vivo intrinsic clearance was

calculated from in vivo hepatic clearance using the well-stirred model (Hallifax et al.,

2010) by incorporating literature or in-house plasma protein binding and blood-to-plasma

ratio. Intrinsic clearance values from hepatocyte relay were obtained using five relays (20

hour accumulative incubation time) with 0.5 million cell density per mL and were

reported as mean values of two or three replicates. All test compounds had significant

turnover under the assay conditions and demonstrated good linearity. Hepatocyte binding

was not incorporated for correction of unbound intrinsic clearance, since all the

compounds have fu,hep close to 1 based on the calculated values (Kilford et al., 2008)

estimated using Log P/D from ACD software.

Rat IVIVC for Low Clearance Compounds

The intrinsic clearance values of rat from both in vivo and the in vitro hepatocyte relay

method are summarized in Table 1. The in vivo rat intrinsic clearance values ranged

from 4 to 2000 mL/min/Kg. The in vivo literature clearance values were from a variety

of rat strains (e.g., Sprague–Dawley, Wistar, Fischer) and both genders, since not all the


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compounds had data from Wistar Han rat. Strain and gender difference in drug

metabolism was assumed to be minimal (Richmond et al., 2010), though exceptions do

exist for certain compounds. The hepatocytes used in the rat relay assay were Wistar

Han, which was a standard strain for the Pfizer internal rat hepatocyte stability assay. In

general, most compounds showed good IVIVC with the in vitro hepatocyte relay assay

within 2-3 fold of in vivo intrinsic clearance values (Figure 1). The exceptions had a

high hepatic uptake component by transporters (e.g., fexofenadine (Poirier et al., 2009),

fluvastatin (Watanabe et al., 2010a), ketoprofen (Morita et al., 2005), prazosin (Qin et al.,

1994), and bosentan (Huang et al., 2012)) or potential contribution from extra-hepatic

metabolism [e.g., hydrolysis of acebutolol (Andresen and Davis, 1979), UGT metabolism

of ketoprofen (Terrier et al., 1999)].

Dog IVIVC for Low Clearance Compounds

The intrinsic clearance values of dog from both in vivo and the in vitro hepatocyte relay

method are summarized in Table 2 for a set of low clearance compounds (as limited

literature data is available for low clearance compounds in dogs). The in vivo dog

intrinsic clearance values of the test compounds ranged from 7 to 25 mL/min/Kg. The in

vivo clearance values were carefully selected from the literature using only IV beagle dog

PK parameters (Suh et al., 1997; Kimura et al., 1999; Bayliss and Cross, 2000; Balani et

al., 2002; Granero and Amidon, 2008; Neirinckx et al., 2011) (with the exception of one

report for ketoprofen being mongrel dog, but it showed no significant difference from

beagle dog data). All the compounds showed good correlation between dog hepatocyte

relay assay and in vivo intrinsic clearance values (Table 2, Figure 1) with the exception


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of ketoprofen, potentially due to the impact of extra-hepatic contribution (Granero and

Amidon, 2008) and uptake transporter [reported in rat (Morita et al., 2005)].

Human IVIVC for Low Clearance Compounds

Human IVIVC of low clearance compounds using the hepatocyte relay method has been

reported previously (Di et al., 2012). Additional low clearance compounds with rat and

dog data were included in this study for cross-species IVIVC (Table 3). Figure 1 shows

all the IVIVC data from this and previous study (Di et al., 2012). All the compounds

showed good IVIVC between in vivo human intrinsic clearance and the hepatocyte relay

method, with the exception of ketoprofen.


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Discussion

Rat and dog are the most common preclinical species used in drug metabolism,

PK, PD, and safety evaluation studies of new chemical entities. Good IVIVC in rat and

dog usually translates to a high confidence prediction of human PK and clinical dose.

There were no adequate in vitro tools to determine low clearance in drug discovery. If a

compound has no measurable turnover in liver microsomes or hepatocytes under the

standard assay conditions, compounds would be reported as having no metabolism or

below a certain cutoff clearance value. The development of the hepatocyte relay method

enables determination of clearance values for low clearance compounds early in drug

discovery to support SAR, prioritization of chemical series, and for human PK and dose

projection (Di et al., 2012). The hepatocyte relay method has been shown to correlate

well with in vivo human intrinsic clearance. Here, the IVIVC in rat and dog was

evaluated for low clearance compounds to assess application of the method in preclinical

species.

Typically, the hepatocyte relay method used an incubation time of 20 hours, which

allowed an estimation of half-life in hepatocytes up to 40 hours (Di et al., 2004). In

theory, the limit of the low clearance value that can be measured decreases with

increasing number of relays. However, based on the theoretical calculation, intrinsic

clearance values drop most significantly for the first few relays. As incubation time goes

beyond 20 hrs, there is little gain in clearance values from each additional relay.

Therefore, in practice, five relays are the standard assay conditions used to support drug

discovery programs. To achieve lower limit of intrinsic clearance, one approach is the


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increase the cell density in the incubation when hepatocyte binding is low. For example,

the intrinsic clearance of antipyrine in human was determined using 2.0 million cell/mL

in this study due to the very low clearance.

As shown in Tables 1-3 and Figure 1, the rat, dog and human hepatocyte relay method

gave good prediction of in vivo intrinsic clearance [additional human data was collected

during this study and added to the previously published set (Di et al., 2012)]. For most

compounds, the in vivo and in vitro intrinsic clearance ratios were within 2-fold,

suggesting good IVIVC. The exceptions involved significant active uptake from

transporters or contribution of extra-hepatic metabolism. Figure 2 shows the IVIVC of

five compounds with rat, dog and human data. Good human IVIVC was observed when

both rat and dog demonstrated strong IVIVC (i.e., ranitidine, tolbutamide, antipyrine, and

naproxen). For ketoprofen, the rank-order of the species is correct, the in vivo values are

under-predicted. About 5-fold under-prediction of human in vivo intrinsic clearance was

observed for ketoprofen. Correction factor derived from rat and dog can be applied to

enhance human prediction accuracy of intrinsic clearance. Weak or no IVIVC in

preclinical species often translated to low confident prediction of human in vivo

clearance from in vitro data.

In rat, significant contribution of transporter mediated clearance was observed for

ketoprofen (Poirier et al., 2009), leading to under-prediction of in vivo clearance based on

metabolic clearance from hepatocytes since transporter uptake also contributes the

systemic clearance (Yamazaki et al., 1996; Shitara et al., 2006; Watanabe et al., 2010b).


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In addition, potential extra-hepatic contribution of UGT metabolism in rat can lead to

under-prediction of in vivo clearance (Terrier et al., 1999). In dog, the major elimination

pathway for ketoprofen was through UGT metabolism and kidney had a significant

contribution to the metabolism (Granero and Amidon, 2008). In human, ketoprofen was

mainly metabolized by CYP2C9 and UGT2B7 (Kilford et al., 2009). The extra-hepatic

contribution of UGT metabolism could potentially contribute to the under-prediction of

intrinsic clearance using hepatocytes for ketoprofen.

This study indicated that the rat and dog hepatocyte relay method gave good prediction of

in vivo hepatic clearance for low clearance compounds. The hepatocyte relay method

extended the low limit for intrinsic clearance measurement by about 10-fold, which

enhanced our ability to address the challenges of low intrinsic clearance compounds. The

good in vivo prediction of the rat and dog hepatocyte relay method on clearance

highlights the wide applications of the relay approach not only in human clearance

prediction (Di et al., 2012), but also in preclinical species.


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Conclusions

Rat and dog hepatocyte relay method has been successfully developed and applied to

predict in vivo clearance for low clearance compound. Good IVIVC was observed for

most of the compounds with in vivo to in vitro intrinsic clearance ratio within 2-fold. The

outliers were due to the contribution of transporter mediated clearance or extra-hepatic

metabolism. This is the first method available to study low clearance compounds in rat

and dog in drug discovery. It highlights the broad applications of the hepatocyte relay

method in preclinical species as well as human for developing IVIVC.


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Acknowledgements

The authors thank Larry Tremaine, Tess Wilson and Charlotte Allerton for their

leadership and support.


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Authorship Contributions

Participated in research design: Li Di, Karen Atkinson, Christine C. Orozco, Carrie Funk,

Hui Zhang, Thomas S. McDonald, Beijing Tan, R. Scott Obach

Conducted experiments: Karen Atkinson, Christine C. Orozco, Carrie Funk, Thomas S.

McDonald, Beijing Tan

Performed data analysis: Li Di, Karen Atkinson, Christine C. Orozco, Carrie Funk, Hui

Zhang, Thomas S. McDonald, Beijing Tan, Jian Lin, Cheng Chang, R. Scott Obach


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Legends for Figures

Figure 1. IVIVC of Intrinsic Clearance within Species for Rat, Dog and Human Using

Hepatocyte Relay Method. Lines indicate lines of unity. Open circles represent

compounds with significant transporter-mediated and/or extra-hepatic contribution to

clearance.

Figure 2. IVIVC of Intrinsic Clearance within Compounds for Rat, Dog and Human

Using Hepatocyte Relay Method. Lines indicate lines of unity.


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Table 1. IVIVC of Intrinsic Clearance Using Rat Hepatocyte Relay Method

Compounds In Vivo Rat

Intrinsic Clearance

(mL/min/Kg)

In Vitro Intrinsic Clearance

from Rat Hepatocyte Relay

Method (mL/min/Kg) ± SD

Fold Difference

In Vivo / Relay

Method

Caffeine 4.3 (Nakazawa et

al., 1985)

3.0 ± 0.76 1.4

Tolbutamide 9.9 (Ashforth et

al., 1995; Mandula

et al., 2006)

9.8 ±0.71 0.61

Antipyrine 6.5 (Belpaire et al.,

1990)

2.4 ± 1.35 2.7

Famotidine 9.4 (Matsuzaki et

al., 2008)

6.6 ± 2.31 1.4

(±)-Warfarin 19 (Yacobi and

Levy, 1977; Hirate

et al., 1990)

12 ± 0.01 1.6

Ranitidine 20 (Eddershaw et

al., 1996)

18 ± 0.39 1.1

Cimetidine 27 (McPherson

and Lee, 1977;

Kurata et al.,

2010)

16 ± 1.6 1.7

Naproxen 38 (Huntjens et al., 37 ± 5.04 1.0


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2006)

Fexofenadine 47 (Tahara et al., 2005)

21 ± 1.41 2.3

(±)-Acebutolol 88 (Piquette-Miller and Jamali, 1997)

7.9 ± 0.89 11

Fluvastatin 85-154 (Watanabe

et al., 2010a)

54 ± 2.77 1.6-2.9

(±)-Ketoprofen 155 (Satterwhite and Boudinot, 1992)

70 ± 12 2.2

Prazosin 196 (Ohkura et al.,

1999)

45 ± 0.76 4.3

Bosentan 1916 (Huang et al., 2012)

79 ± 0.05 24


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Table 2. IVIVC of Intrinsic Clearance Using Dog Hepatocyte Relay Method

Compounds In Vivo Dog Intrinsic

Clearance

(mL/min/Kg) a


from Dog Hepatocyte

Relay Method

(mL/min/Kg) ± SD

Fold Difference

In Vivo / Relay

Method

Ranitidine 7.7 (Bayliss and Cross,

2000)

7.4 ± 0.6 1.0

Tolbutamide 11 c (Kimura et al.,

1999)

9.0 ± 3.9 1.2

Antipyrine 14 (Balani et al., 2002) 14 ± 1.4 1.0

Naproxen 22d (Suh et al., 1997) 20 ± 3.0 1.1

(±)-Ketoprofen 48 – 114e (Granero

and Amidon, 2008;

Neirinckx et al., 2011)

24 ± 1.2 2.0 - 4.7 b

a Fu and Rb values were from literature (Berry et al., 2011) or in-house data.

b The in vitro – in vivo differences may be due to extra-hepatic contribution of UGT (e.g.,

kidney metabolism (Granero and Amidon, 2008)).

c Equation, CLiv = Dosepo/AUCpo x F, was used to derive IV clearance.

d PK profile(Suh et al., 1997) was digitized and IV clearance value was calculated using

WinNonlin®.

e Two IV studies were reported in the literature (Granero and Amidon, 2008; Neirinckx et

al., 2011). One used racemic ketoprofen in mongrel dogs with three different doses

(Granero and Amidon, 2008) and the other was a beagle dog study with individual


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enantiomers (Neirinckx et al., 2011). The clearance values of the individual enantiomers

were converted to that of the racemate using dose (sum of the two enantiomers) divided

by AUC (sum of the two enantiomers) (Neirinckx et al., 2011). It gave a similar

clearance value (CLint 113 mL/min/Kg) as that in the racemate study with mongrel dog

(CLint 48-114 mL/min/Kg). Therefore, data from both reports were included for IVIVC

determination.


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Table 3. IVIVC of Intrinsic Clearance Using Human Hepatocyte Relay Method

Compounds In Vivo Human

Intrinsic Clearance

(mL/min/Kg)


from Human Hepatocyte Relay

Method (mL/min/Kg) ± SD

Fold Difference

In Vivo / Relay

Method

Ranitidine 4.4 (Hallifax et al.,

2010) 3.1 ± 0.48 1.4

Tolbutamide 4.9 (Brown et al.,

2007) 7.4 ± 0.40 (Di et al., 2012) 0.66

Antipyrine 0.6 (Hallifax et al.,

2010) 1.3 ± 0.25 0.46

Naproxen

25 (Huntjens et al.,

2006; Hallifax et al.,

2010)

18 ± 3.6 1.4

(±)-Ketoprofen 78 (Hallifax et al.,

2010) 17 ± 6.6 4.6

Human hepatocyte relay method was the same as reported previously (Di et al., 2012), with

the exception of antipyrine. A cell density of 2 million/mL was used for antipyrine due to

the very low clearance.


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Figure 1

1

10

100

1000

10000

1 10 100 1000

In V

ivo

Intr

insi

c C

lear

ance

(mL/

min

/Kg)

In Vitro Intrinsic Clearance (mL/min/Kg)

Rat IVIVC

1

10

100

1 10 100

In V

ivo

Intr

insi

c C

lear

ance

(mL/

min

/Kg)


Dog IVIVC

0.1

1

10

100

0.1 1 10 100

In V

ivo

Intr

insi

c C

lear

an

ce (m

L/m

in/K

g)


Human IVIVC


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Figure 2

0

5

10

15

20

0 5 10 15 20

In V

ivo

CL i

nt(m

L/m

in/K

g)

In Vitro Hepatocyte Relay CLint (mL/min/Kg)

Human

Dog

Rat

Antipyrine

10

15

20

25

30

35

40

45

10 15 20 25 30 35 40 45

In V

ivo

CL i

nt(m

L/m

in/K

g)


Human Dog

Rat

Naproxen

2

4

6

8

10

12

14

2 4 6 8 10 12 14

In V

ivo

CL i

nt(m

L/m

in/K

g)


Human

Dog

Rat

Tolbutamide

0

5

10

15

20

25

0 5 10 15 20 25

In V

ivo

CL i

nt(m

L/m

in/K

g)


Human

Dog

RatRanitidine

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

In V

ivo

CL i

nt(m

L/m

in/K

g)


Human

Dog

RatKetoprofen


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