<%BANNER%>

Cytochrome P450 2C8 Reaction Phenotyping

Permanent Link: http://ufdc.ufl.edu/UFE0021832/00001

Material Information

Title: Cytochrome P450 2C8 Reaction Phenotyping Substrate Selection and Inhibition Profile
Physical Description: 1 online resource (64 p.)
Language: english
Creator: Dravid, Prajakta V
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: amodiaquine, cyp2c8, metabolism, pioglitazone
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: About 20% of the adverse drug reactions are related to drug-drug interactions caused by inhibition or induction of a drug metabolizing enzyme determining the elimination of a drug by another concomitantly administered drug. These drug-drug interactions could be predicted by identifying the major drug metabolizing enzymes responsible for the metabolism of a new chemical entity. The reaction phenotyping of Cytochrome P450 2C8 (CYP2C8) did not gain enough attention due to the lack of an enzyme specific probe substrate and inhibitor. Paclitaxel is used as a conventional probe substrate of CYP2C8 in vitro. Recently, amodiaquine (AQ) is reported to be a high affinity and turnover probe substrate of CYP2C8. However, it is not ideal for clinical drug metabolism studies due to its toxicity and very long elimination half-life of its CYP2C8 specific metabolite, N-desethylamodiaquine (DEAQ). Pioglitazone (PIO) hydroxylation to form M-IV is also a CYP2C8 specific reaction. We recognized a need for development of a LC/MS/MS based analytical method for determination of DEAQ and M-IV. The present work involves the development and validation of analytical methods for quantification of these two CYP2C8 specific metabolites. Further, we successfully applied these methods to in vitro drug metabolism studies using pooled human liver microsomes. The potential of M-IV formation as a CYP2C8 specific probe reaction was evaluated by studying the inhibition of montelukast, ketoconazole, terfenadine, beta-estradiol and midazolam and comparing their IC50 values to those obtained by using other CYP2C8 specific probe reactions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Prajakta V Dravid.
Thesis: Thesis (M.S.P.)--University of Florida, 2007.
Local: Adviser: Frye, Reginald F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021832:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021832/00001

Material Information

Title: Cytochrome P450 2C8 Reaction Phenotyping Substrate Selection and Inhibition Profile
Physical Description: 1 online resource (64 p.)
Language: english
Creator: Dravid, Prajakta V
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: amodiaquine, cyp2c8, metabolism, pioglitazone
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: About 20% of the adverse drug reactions are related to drug-drug interactions caused by inhibition or induction of a drug metabolizing enzyme determining the elimination of a drug by another concomitantly administered drug. These drug-drug interactions could be predicted by identifying the major drug metabolizing enzymes responsible for the metabolism of a new chemical entity. The reaction phenotyping of Cytochrome P450 2C8 (CYP2C8) did not gain enough attention due to the lack of an enzyme specific probe substrate and inhibitor. Paclitaxel is used as a conventional probe substrate of CYP2C8 in vitro. Recently, amodiaquine (AQ) is reported to be a high affinity and turnover probe substrate of CYP2C8. However, it is not ideal for clinical drug metabolism studies due to its toxicity and very long elimination half-life of its CYP2C8 specific metabolite, N-desethylamodiaquine (DEAQ). Pioglitazone (PIO) hydroxylation to form M-IV is also a CYP2C8 specific reaction. We recognized a need for development of a LC/MS/MS based analytical method for determination of DEAQ and M-IV. The present work involves the development and validation of analytical methods for quantification of these two CYP2C8 specific metabolites. Further, we successfully applied these methods to in vitro drug metabolism studies using pooled human liver microsomes. The potential of M-IV formation as a CYP2C8 specific probe reaction was evaluated by studying the inhibition of montelukast, ketoconazole, terfenadine, beta-estradiol and midazolam and comparing their IC50 values to those obtained by using other CYP2C8 specific probe reactions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Prajakta V Dravid.
Thesis: Thesis (M.S.P.)--University of Florida, 2007.
Local: Adviser: Frye, Reginald F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021832:00001


This item has the following downloads:


Full Text





CYTOCHROME P450 2C8 REACTION PHENOTYPING: SUBSTRATE SELECTION AND
INHIBITION PROFILE




















By

PRAJAKTA V DRAVID


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN PHARMACY

UNIVERSITY OF FLORIDA




2007

































2007 Prajakta V Dravid

































To the sweet memories of my little heroes, Jayesh and Anay.









ACKNOWLEDGMENTS

Any project no matter how individual will almost certainly require input, assistance and

encouragement from others, my project is no exception!

I take this opportunity to thank my research supervisor, Dr. Reggie Frye, for his invaluable

guidance and abundant help during my work on this project. This research project would not

have been possible without his contribution and support.

I would like to thank Dr. Gunther Hochhaus for being a member of my graduate

committee. Members of Frye lab, Cheryl, Mohamed and Melonie, require a special mention. I

would like to thank them for all the good times and laughter in the lab. I express my gratitude to

departments of Pharmacy Practice and Pharmaceutics for providing me with financial support.

Words fail to express my love and gratitude to my beloved parents and grandparents. I

would like to thank them for their unconditional support and encouragement. Needless to

mention, I would not have seen this day without the emotional support provided by my husband,

Anand. He patiently embraced the lonely time and encouraged me to complete my education. I

thank him for standing by me in every decision I took.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF TA BLES .............. ......... .......................................................... 7

LIST OF FIGURES .................................. .. ..... ..... ................. .8

A B S T R A C T ............ ................... ............................................................ 1 1

CHAPTER

1 IN T R O D U C T IO N ....................................................... ................................................ .. 13

Cytochrome P450 Reaction Phenotyping.................................................... .. .................13
The Centrality of Cytochrome P450 Enzymes in Drug Metabolism............... ..................14
C ytochrom e P450 2C 8..................... ............... ........................ .......... ............. 15
Paclitaxel: A Conventional Probe of CYP2C8 Activity ................................ ................. 16
Amodiaquine: A High Affinity and Turnover Probe Substrate of CYP2C8 ........................17
Pioglitazone as Probe Substrate of CYP2C8 ...............................................19
The Scope of Present W ork ................ ............. ................... ....... ..... ............. 22

2 DETERMINATION OF N-DESETHYLAMODIAQUINE BY LC/MS/MS:
APPLICATION TO IN VITRO DRUG METABOLISM STUDIES .............................. 23

In tro du ctio n ................... ...................2...................3..........
E x p e rim e n ta l ..................................................................................................................... 2 5
Chem icals and R agents ............................ ........................... ....... .... ............... 25
Preparation of DEAQ Standards and Quality Control Samples............................... 25
S am p le P rep aration ......... ...... .................................................................. ....... ............... 2 6
L C /M S/M S C onditions.......................................................................... ....................26
V alid atio n ................................................................2 7
Incubation Conditions ............................... ... ..... .. ...... ............... 28
D ata A n aly sis...................................................... ................ 2 9
Results and Discussion ...................................... .. ......... ....... ..... 29
M ethod D evelopm ent .......... ...... ............ ................................. .............. .. 29
M ethod V alidation ..................... ................................ ...... .............. .... ........ .....30
Selectivity, carry over and matrix effect ............. ............. ....... ...... ............ 30
L in e a rity .............................................................................3 2
Precision and accuracy ............................................................. ........... 32
A utosam pler stability ................. ...... ............................. ........... .............. 33
Metabolism of AQ in Pooled Human Liver Microsomes ............................................33
C o n c lu sio n ................... .......................................................... ................ 3 4

3 DETERMINATION OF HYDROXYPIOGLITAZONE (M-IV) BY LC/MS/MS:
APPLICATION TO IN VITRO DRUG METABOLISM STUDIES ....................................36









In tro d u ctio n ................... ...................3...................6..........
E x p e rim e n ta l ..................................................................................................................... 3 9
Chem icals and R eagents................................ ............. ..... ..... ............... ............... 39
Preparation of M-IV Standards and Quality Control samples......................................39
S am p le P rep aration ......... ...... .................................................................. ....... ............... 4 0
L C /M S/M S C onditions.......................................................................... ....................40
V alid atio n ................................................................4 2
Incubation Conditions ............................... .......... ...... ............... 42
Results and Discussion ..................................... ................. ........ ..... 43
Method Development ............. ........ ............ .. ........... 43
M ethod V alidation ..................... ................................ ...... .............. .... ........ .....44
Selectivity, carry over and matrix effect ....... ......................................44
L in e a rity .............................................................................4 6
Precision and accuracy ........ ...... ............ .... ......... ................ .............. 46
A utosam pler stability .................. ....... .......................................... .............. 46
Formation of M-IV in Pooled Human Liver Microsomes.............................................47
C conclusion ...................... ............................ .. .. ......................................... 48

4 DETERMINATION OF IC50 IN POOLED HUMAN LIVER MICROSOMES USING M-
IV FORMATION AS A CYP2C8 SPECIFIC REACTION................................................49

Introduction ............. .......... ................................49
Experim mental ............ ............... .................... ................. 50
C hem icals and R eagents......... ......... ......... .......... .......................... ............... 50
Incubation C conditions ............... ................................................ .......... ... ......... 50
Analysis of Hydroxypioglitazone (M-IV) .............................................. ...............51
D ata A n a ly sis ............................................................................................................. 5 2
Results and Discussion ..................................... ................. ........ .... 52
C o n c lu sio n ........................................................................................................................ 5 4

5 CONCLUSIONS AND FUTURE DIRECTIONS................................... ....................... 56

L IS T O F R E F E R E N C E S .................................................................................... .....................58

B IO G R A PH IC A L SK E T C H .............................................................................. .....................64
















6









LIST OF TABLES


Table page

1-1.Substrates and inhibitors of CYP2C8 for in vitro experiments....................... ................16

2-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for
analysis of DEAQ in 50 mM phosphate buffer, pH 7.4............... ... .............32

2-2.Enzyme kinetic parameters of AQ in pHLM...........................................34

3-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for
analysis of M-IV in 50 mM phosphate buffer, pH 7.4.................................46

3-2.Enzyme kinetic parameters of M-IV formation in pHLM. .....................................................48









LIST OF FIGURES


Figure page

1-1.Proposed structures of pioglitazone metabolites in pLM. ..............................................21

2-1.Representative chromatograms of (A) blank incubation buffer (red) and buffer spiked
with DEAQ at LOQ (black) and (B) incubation sample: blank (red) and after 10 min
incubation of AQ at 0.5 itM (black) (m/z 328 283, overlay offset = 0%). ...................31

2-2.Plot of initial velocity versus amodiaquine concentration for the formation of
desethylam odiaquine in pH LM (n=2)......... ......................................................... .......34

3-1.Chemical structures of (A) pioglitazone and (B) hydroxypioglitazone (M-IV)...................37

3-2.Representative chromatograms of (A) blank buffer (red) and buffer spiked with OH-PIO
at low QC (black) and (B) incubation sample: blank (red) and after 10 min
incubation of PIO at 10tiM (black) (m/z 373 -> 150, overlay offset = 0%)......................45

3-3.Plot of initial velocity versus pioglitazone concentration for the formation of
hydroxypioglitazone (M -IV) in pHLM (n=2)............... ........ ................................. 47

4-1. Structures of the compounds tested for inhibition of in vitro CYP2C8 activity. ....................52

4-2.IC50 plots of pioglitazone hydroxylase inhibition in pHLM by montelukast (circles),
ketoconazole (squares), terfenadine (diamonds), P-estradiol (triangles) and
midazolam (reverse triangles). Data points reflect the average for incubations run in
duplicate + SE ..............................................................................53












P3-NADP:

ADR:

AQ:

AUCo-,:

CYP:

DEAQ:

DME:

DMSO:

ESI:

FDA:

FWHM:

HILIC:

HPLC:

IC5o:


LC/MS/MS:

LOQ:

M-IV

MRM:

NCE:

NIDDM:

PHLM:

PIO:

PPAR:


LIST OF ABBREVIATIONS

P-nicotinamide adenine dinucleotide phosphate

Adverse drug reaction

Amodiaquine

Area under the plasma concentration time curve

Cytochrome P450

N-desethylamodiaquine

Drug metabolizing enzyme

Dimethyl sulfoxide

Electrospray ionization

Food and Drug Administration

Full width at half maximum

Hydrophilic interaction chromatography

High pressure liquid chromatography

Concentration of the inhibitor that reduces the maximum initial velocity to
50%

Liquid chromatography tandem mass spectrometry

Limit of quantification

Hydroxypioglitazone

Multiple reaction monitoring

New chemical entity

Non insulin dependent diabetes mellitus

Pooled human liver microsomes

Pioglitazone

Peroxisome proliferator activated receptor









QC: Quality control

RSD: Relative standard deviation

SRM: Single reaction monitoring

T1/2 Terminal elimination half-life

UV: Ultraviolet

WHO: World Health Organization









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Master of Science in Pharmacy

CYTOCHROME P450 2C8 REACTION PHENOTYPING: SUBSTRATE SELECTION AND
INHIBITION PROFILE


By

Prajakta V Dravid

December 2007

Chair: Reginald Frye
Major: Pharmaceutical Sciences

About 20% of the adverse drug reactions are related to drug-drug interactions caused by

inhibition or induction of a drug metabolizing enzyme determining the elimination of a drug by

another concomitantly administered drug. These drug-drug interactions could be predicted by

identifying the major drug metabolizing enzymes responsible for the metabolism of a new

chemical entity.

The reaction phenotyping of Cytochrome P450 2C8 (CYP2C8) did not gain enough

attention due to the lack of an enzyme specific probe substrate and inhibitor. Paclitaxel is used as

a conventional probe substrate of CYP2C8 in vitro. Recently, amodiaquine (AQ) is reported to

be a high affinity and turnover probe substrate of CYP2C8. However, it is not ideal for clinical

drug metabolism studies due to its toxicity and very long elimination half-life of its CYP2C8

specific metabolite, N-desethylamodiaquine (DEAQ). Pioglitazone (PIO) hydroxylation to form

M-IV is also a CYP2C8 specific reaction.

We recognized a need for development of a LC/MS/MS based analytical method for

determination of DEAQ and M-IV. The present work involves the development and validation of

analytical methods for quantification of these two CYP2C8 specific metabolites. Further, we









successfully applied these methods to in vitro drug metabolism studies using pooled human liver

microsomes. The potential of M-IV formation as a CYP2C8 specific probe reaction was

evaluated by studying the inhibition of montelukast, ketoconazole, terfenadine, P-estradiol and

midazolam and comparing their IC50 values to those obtained by using other CYP2C8 specific

probe reactions.









CHAPTER 1
INTRODUCTION

Cytochrome P450 Reaction Phenotyping

Drug-drug interactions pose a significant health concern causing approximately 20% of

adverse drug reactions (ADRs) (Backmann et al., 2003). Clearly, the probability of ADRs

associated with drug-drug interactions increases as the number of concomitantly administered

drugs increase (Bachmann et al., 2003). Inhibition of one or more drug metabolizing enzymes

(DIMEs) could result in an increased exposure of the parent drug and therefore, an increased

chance of observing drug related toxicity. On the other hand, induction of a DME might result in

subtherapeutic response leading to failure of drug therapy (Venkatakrishnan et al., 2003; Frye,

2004). In order to avoid any unforeseen interactions in the clinic, the Food and Drug

Administration (FDA) requires pharmaceutical companies to identify drug metabolizing

enzymes involved in the elimination of a new chemical entity (NCE) as well as the effect of its

coadministration on the pharmacokinetics of marker substrates of DIMEs that are responsible for

its elimination (http://www.fda.gov/cder/guidance). Common experimental methods to identify

the drug metabolizing enzymes responsible for the metabolism of the NCE are as follows:

(1) Chemical inhibitors: In order to determine the contribution of a particular DME, the

metabolism of the NCE is studied in presence of an enzyme selective chemical inhibitor. In this

case, selectivity could be defined by two factors, the mechanism of inhibition and the relative

affinity of both the inhibitor and the test substrate for the enzyme. The limiting factor in the use

of chemical inhibitors for in vitro studies has historically been the lack of adequate selectivity of

inhibition among cytochrome P450 (CYP) enzymes.

(2) Expressed CYP enzymes: The ability of a panel of expressed CYP enzymes to metabolize a

specific NCE reduces reaction phenotyping to the simplest system of only one enzyme and a









substrate. Although these studies give important qualitative information, the importance of each

CYP enzyme in presence of other enzymatic pathways is difficult to quantify thus providing

incomplete information about overall metabolic fate of the NCE.

(3) Antibodies: The use of anti-CYP antibodies as biological inhibitors of enzyme activity allows

the direct assessment of the role of specific CYP to the metabolism of the NCE in an enzyme

mixture like pooled human liver microsomes (pHLM). A primary limitation of anti-CYP

antibodies for reaction phenotyping is their cross-reactivity with related CYPs. Additionally, the

inhibition of reactions known to be highly specific to a particular CYP isoform is rarely 100%

and a small fraction of the enzyme retains its function.

(4) Correlation analysis: It involves the comparison of the inter-liver variability in the rate of

formation of a specific drug metabolite with the measured activities towards CYP marker

substrates. This approach warrants prior establishment of enzyme kinetic parameters, a thorough

investigation of the correlation between the different CYP enzymes and the use of considerable

number of liver samples with wide range of enzyme activities (Wienkers and Stevens, 2003).

The potential of a NCE to inhibit the metabolism of currently available drugs could be

determined by studying the metabolism of an enzyme specific probe substrate in the presence of

the NCE. The identification of substrates that are selectively metabolized in vitro by specific

CYPs allows for the investigation of the inhibition by the NCE. The results from in vitro

inhibition studies, yielding IC50 or Ki values allows for the prediction of the potential for the

NCE to inhibit specific CYPs in vivo. Information thus obtained is used by the clinician to design

in vivo interaction studies that are relevant to the NCE (Bachmann et al., 2003).

The Centrality of Cytochrome P450 Enzymes in Drug Metabolism

Cytochrome P450 constitutes a superfamily of drug metabolizing enzymes that contributes

to the Phase I metabolism of approximately three-forths of all drugs (Wilkinson, 2001). From the









drug metabolism point of view, the most important CYPs in humans are CYP1A2, CYP2B6,

CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4/5. The CYP2C subfamily

consists of four members namely, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 and is the

second most abundant CYP subfamily after CYP3A, representing about 20% of the total hepatic

P450 (Montellano, Handbook of Drug Metabolism, 1999). Collectively, the CYP2C subfamily is

responsible for the metabolism of about 20% of clinically prescribed drugs. Clinical importance

of CYP2C9 and CYP2C19 is well known and therefore, these two isoforms are extensively

studied and always included in the battery of enzymes used in reaction phenotyping studies

during the development of a new chemical entity. However, until recently, CYP2C8 was often

neglected due to the limited knowledge of its importance as well as the lack of CYP2C8 specific

probe substrate and inhibitor.

Cytochrome P450 2C8

CYP2C8 is a major human hepatic P450, constituting about 7% of total microsomal CYP

content in the liver. It is responsible of at least 5% of drugs cleared by Phase I metabolism

(Rendic and Di Carlo, 1997). Although it is primarily expressed in the liver, CYP2C8 protein is

also found in kidney, intestine, adrenal glands, mammary glands, ovary, heart, aorta as well as in

breast cancer tumors (Klose et al., 1999; Thum and Borlak, 2000; Nishimura et al., 2003;

Knupfer et al., 2004). Ferguson et al. found that the pregnane X receptor, constitutive

androstane receptor and glucocorticoid receptor are involved in the regulation of CYP2C8 gene

expression (Ferguson et al., 2005).

In humans, the CYP2C8 gene is located on chromosome 10q24, spanning 31 kilobases and

consisting of nine exons. Single nucleotide polymorphisms have been identified in exons 3, 5

and 8 (Klose et al., 1999). The most common varient alleles are CYP2C8*2 and CYP2C8*3. The

protein product CYP2C8.2 contains Ile269Phe substitution and is expressed in African American









population with an allle frequency of 18%. CYP2C8*3 has two amino acid susbstitutions,

Argl39Lys and Lys399Arg. It is most commonly found in Caucasians with an allele frequency

of 13% and rarely expressed in African Americans (2%) (Bahadur et al., 2002). Interestingly,

CYP2C8*3 is in incomplete genetic equilibrium with CYP2C9*2.

CYP2C8 plays an important role in the metabolism of various drugs including paclitaxel

(Dai et al., 2001; Bahadur et al., 2002), amodiaquine (AQ) (Li et al., 2002), troglitazone

(Yamazaki et al., 1999), rosiglitazone (Baldwin et al., 1999), pioglitazone (PIO) (Deng et al.,

2005), repaglinide (Deng et al., 2005), cerivastatin (Backman et al., 2002), amiodarone (Soyama

et al., 2002) and verapamil (Busse et al., 1995). It also contributes to the metabolism of various

endogenous substances like all-trans retinoic acid (McSorley and Daly, 2000) and arachidonic

acid (Dai et al., 2001). Common substrates and inhibitors used in in vitro drug metabolism

experiments are listed in Table 1-1.

Table -1. Substrates and inhibitors of CYP2C8 for in vitro experiments.

Substrates Inhibitors
Paclitaxel Gemfibrozil
Torsemide Trimethoprim
Cerivastatin Montelukast
Repaglinide Quercetin
Amodiaquine Thiazolidinediones
Pioglitazone

Paclitaxel: A Conventional Probe of CYP2C8 Activity

Paclitaxel, an antineoplastic agent, was originally isolated from the stem bark of the

western yew, Taxus brevifolia. It is a potent inhibitor of cell replication, blocking the cells in the

late G2 mitotic phase of the cell cycle, presumably by stabilizing the microtubule cytoskeleton.

As a therapeutic antineoplastic agent, paclitaxel is used in patients with breast, lung, esophageal,

head and neck, and advanced platinum-refractory ovarian carcinomas (Walle et al., 1995). In









humans, paclitaxel is primarily metabolized by CYP2C8 to 6-a-hydroxypaclitaxel and to lesser

extent by CYP3A4 to form 3'-p-hydroxypaclitaxel. Further, both metabolites are hydroxylated to

lead to dihydroxypaclitaxel. Paclitaxel and its metabolites are secreted in the bile and feces. The

ratio of 6-a-hydroxypaclitaxel to 3'-p-hydroxypaclitaxel is 6:1 in bile (Cresteil et al., 1994).

Similarly, in pHLM, the concentration of 6-a-hydroxypaclitaxel was about two-fold that of 3'-p-

hydroxypaclitaxel suggesting the major role of CYP2C8 in the elimination of paclitaxel

(Taniguchi et al., 2005). Therefore, formation of 6-a-hydroxypaclitaxel is widely used as a

marker substrate of CYP2C8 activity in reaction phenotyping studies. Dai et al. found that

CYP2C8.2 exhibits two-fold higher Km for paclitaxel 6-hydroxylation (31 tM) compared to that

of CYP2C8.1 (15 tM) whereas CYP2C8.3 had a turnover number of 15% that of CYP2C8.1 for

paclitaxel 6-a-hydroxylation (Dai et al., 2001). Other research groups indicated a moderate

decrease (31 to 84 %) in the enzyme activity by presence of CYP2C8*3 allele (Bahadur et al.,

2002; Soyama et al., 2002).

Amodiaquine: A High Affinity and Turnover Probe Substrate of CYP2C8

Amodiaquine, a 4-aminoquinolone antimalarial drug, is clinically effective against certain

chloroquine resistant strains of Plasmodiumfalciparum. However, it is no longer recommended

for prophylactic antimalarial therapy due to the high risk of agranulocytosis and hepatitis caused

by the reactive quinine-imine metabolite. Although the global use of AQ has declined due to the

intrinsic toxicity, it is still being used as a first-line drug in the treatment of uncomplicated

falciparum malaria, especially in African countries. In 2005, World Health Organization (WHO)

recommended the use of AQ in combination with artemisinin-based antimalarial therapy due to

higher clinical efficacy of the combination, revitalizing the interest in the use of AQ in treating

malaria.









In vivo pharmacokinetic studies have shown that the primary route of systemic elimination

in humans is via the extensive first-pass metabolism to N-desethylamodiaquine (DEAQ). Li et al.

characterized the metabolism of AQ in various expressed CYP enzymes and pHLM. They found

that AQ is almost exclusively metabolized by CYP2C8 to produce DEAQ (Li et al., 2002).

Therefore, AQ clearance and its metabolism to DEAQ could be used as a measure of CYP2C8

enzyme activity for in vitro reaction phenotyping studies. Recently, Walsky et al. examined 209

frequently prescribed drugs and related xenobiotics for inhibition of CYP2C8 using amodiaquine

N-desethylation as a CYP2C8 specific marker reaction. Forty-eight compounds exhibiting

greater than 50% inhibition were further evaluated for determination of IC50 using expressed

CYP2C8. In pHLM, the leukotriene receptor antagonist, montelukast was found to be the most

potent inhibitor of CYP2C8 with an IC50 of 19 nM (Walsky et al., 2005). In a recent report,

Parikh et al. found that the presence of CYP2C8*2 allele increased the Km of AQ metabolism by

three-fold and decreased intrinsic clearance by six-fold. They also reported a marked decrease in

the AQ metabolism with the presence of CYP2C8*3 allele (Parikh et al., 2007).

Various analytical methods are described for determination of DEAQ (Trenholme et al.,

1974; Pussard et al., 1985; Mount et al., 1986; Pussard et al., 1987; Winstanley et al., 1987;

Laurent et al., 1993; Li et al., 2002; Minzi et al., 2003; Dua et al., 2004; Gitau et al., 2004;

Walsky and Obach, 2004; Bell et al., 2007; Dixit et al., 2007; O'Donnell et al., 2007). Earlier

methods have long run times with high mobile phase flow rates and lack the sensitivity required

for in vitro drug metabolism assays. Most of the recent methods use highly aqueous mobile

phases in order to improve the retention of the polar DEAQ on the column, which is not ideal for

detection by mass spectrometry.









Pioglitazone as Probe Substrate of CYP2C8

Pioglitazone, a thiazolidinedione, is used in the treatment of non-insulin dependent

diabetes mellitus (NIDDM) as monotherapy or in combination with other hypoglycemic agents

(e.g., sulfonylureas, metformin and insulin). Like other thiazolidinediones (e.g., troglitazone and

rosiglitazone), PIO mediates its hypoglycemic effects by activation of peroxisome proliferator

activated receptor (PPAR) y, thereby enhancing the sensitivity of insulin responsive tissues

rather than increasing release of insulin from islet p-cells. In clinical studies, PIO is shown to

produce antihyperglycemic effects by increasing insulin stimulated glucose uptake in peripheral

tissues as well as the ability of insulin to suppress endogenous glucose production in the liver. It

was also shown to decrease plasma levels of insulin, thus reducing the risk of hypoglycemia.

From the toxicity point of view, PIO has a much better safely profile as compared to troglitazone

and rosiglitazone. Additionally, PIO is found to exert hypolipidemic effects by reducing the

serum concentrations of free fatty acids (Mizushige et al., 2002).

After oral administration (30 mg once daily), PIO undergoes minimal first pass metabolism

in the gut and is almost completely absorbed with an absolute bioavailability of 83%. Although it

is distributed to peripheral tissues, plasma concentrations are always higher than tissues

concentrations indicating a small volume of distribution due to high plasma protein binding

(>97%). In humans, PIO undergoes extensive metabolism in the liver to form various

hydroxylated and oxidized metabolites (M-I to M-VII). Oxidative cleavage of aliphatic C-O

bond leads to formation of M-I. M-II and hydroxypioglitazone (M-IV) are formed by the

hydroxylation of aliphatic methylene group whereas terminal ethyl group is hydroxylated to give

M-VII and is oxidized to form M-V. Oxidation of the hydroxyl group in M-IV to a ketone

generates M-III (Yki-Jarvinen, 2004).









In vitro studies in pHLM have shown that PIO undergoes extensive metabolism in the liver

primarily by CYP2C8, with minor contribution from CYP3A4. The metabolites M-II, M-III and

M-IV are pharmacologically active possessing about 40-60% anti-hyperglycemic potency as

compared to PIO. In humans, M-III and M-IV are found to be the major metabolites with

considerably longer terminal half lives (ti/2, 26-28 hours) than that of PIO (mean ti/ 5.8 hours),

presumably contributing to the extended pharmacological activity allowing once-daily

administration of PIO. In another study, a potent and highly selective CYP2C8 inhibitor,

montelukast (1 iM), significantly inhibited depletion ofPIO (IC5o = 0.51 iM) and more strongly

inhibited formation of M-IV (IC5o = 0.18 |iM), clearly indicating the major role of CYP2C8 in

the formation of M-IV (Jaakkola et al., 2006). In a clinical drug interaction study, gemfibrozil

alone raised the mean area under the plasma concentration-time curve (AUCo- ) of PIO 3.2-fold

and prolonged its tl/2 from 8.3 to 22.7 hours. Additionally, it also decreased the AUCO-48 of M-III

and M-IV by 42% and 45%, respectively. However, itraconazole, a potent CYP3A4 inhibitor,

did not have any significant effect on the pharmacokinetics of PIO or either of its metabolites

suggesting a minor role of CYP3A4 in the formation of its major plasma metabolite M-IV

(Jaakkola et al., 2005). In a recent report, Tomio et al., found that trimethoprim, a known

inhibitor of CYP2C8 increased the AUCo-0 of PIO by 42% and reduced the apparent formation

rate of M-IV by 27%, validating the major contribution of CYP2C8 in the formation of M-IV

(Tornio et al., 2007). From the above mentioned in vitro and in vivo drug interaction studies it is

evident that M-IV is almost exclusively formed by CYP2C8. Therefore, formation of M-IV

could be employed as a marker reaction for the quantification of CYP2C8 activity for in vitro

reaction phenotyping studies as well as clinical drug interaction studies. The proposed structures

of PIO metabolites in pHLM are depicted in Figure 1-1. Tornio et al. also investigated the effect










of carrying the CYP2C8*3 allele on the pharmacokinetics of PIO. The weight-adjusted AUCo-,

of PIO was 34% lower in the subjects with the CYP2C8*3/*3 genotype and 26% lower in case of

subjects carrying CYP2C8*1 *3 genotype (Tornio et al., 2007).






O O OO/H O OH
H OH
OH OH 0 O N 0 S








Pioghtazone C H3
M-MI CH3 HO CO










O OH N O O
N N H
SC-OH
M-IV H CH3 M-V

ON
0H H 0 H 0

HO0



Figure 1-1.Proposed structures of pioglitazone metabolites in pHLM.




Several analytical methods are available for the quantification of M-IV in various

biological fluids (e.g., plasma and urine) (Kiyota et al., 1997; Lin et al., 2003; Deng et al., 2005;

Jaakkola et al., 2005; Tornio et al., 2007) and subcellular fractions (e.g., recombinant CYP

enzymes and pHLM) (Shen et al., 2003; Baughman et al., 2005; Jaakkola et al., 2006; Tornio et

al., 2007). Most of these methods involve slow mobile phase gradients with long run times as

they were developed for characterization of all of the metabolites. Many of the recent methods









are either not sensitive enough for the detection of metabolites or assign arbitrary units to M-IV

due to the lack of commercially available metabolite standard at the time of development.

The Scope of Present Work

Considering the limitations of the current analytical methods for determination of DEAQ

and potential application of DEAQ formation as a CYP2C8 specific reaction for in vitro reaction

phenotyping studies, there is a need for development of a simple, sensitive and robust mass

spectrometric method that could be easily applied to drug metabolism studies. Therefore, the

current work involves development and validation of a liquid chromatography tandem mass

spectrometry (LC/MS/MS) based method for determination of DEAQ and its application to drug

metabolism studies using pHLM.

Despite the number of drug interaction studies involving PIO, until recently, very little was

known about the affinity of PIO towards CYP2C8. Therefore, one of the aims of the present

work is to characterize the formation of M-IV with respect to Michaelis-Menten kinetics in

pHLM. In order to facilitate the detection of M-IV, we developed and validated a LC/MS/MS

based method that can be applied to drug metabolism studies. Additionally, the focus of the

current work was to evaluate the potential of PIO as a CYP2C8 specific substrate by comparing

the IC5o values of montelukast, ketoconazole, P-estradiol, midazolam and terfenadine with those

obtained using AQ.









CHAPTER 2
DETERMINATION OF N-DESETHYLAMODIAQUINE BY LC/MS/MS: APPLICATION TO
IN VITRO DRUG METABOLISM STUDIES

Introduction

Amodiaquine, a 4-aminoquinolone antimalarial drug, is clinically effective against certain

chloroquine resistant strains of Plasmodiumfalciparum. Although the global use of AQ has

declined due to the the high risk of agranulocytosis and hepatitis caused by the reactive quinine-

imine metabolite (Jewell et al., 1995), it is still being used as a first-line drug in the treatment of

uncomplicated falciparum malaria, especially in African countries (Basco et al., 2002; Cavaco et

al., 2005; Hombhanje et al., 2005; Rower et al., 2005). After oral administration, AQ undergoes

rapid and extensive metabolism in the liver to form the pharmacologically active metabolite

DEAQ, which is primarily responsible for the antimalarial effects (Winstanley et al., 1987). In

humans, desethylation of AQ is the major pathway of elimination with other minor metabolites

being 2-hydroxyl DEAQ and N-bisdesethylAQ (Pussard et al., 1985a; Pussard et al., 1987;

Laurent et al., 1993; Jewell et al., 1995). Studies in pHLM and recombinant enzymes show that

AQ desethylation is almost exclusively catalyzed by Cytochrome P450 2C8 (Li et al., 2002).

Therefore, AQ is used as an enzyme-selective probe substrate to quantify CYP2C8 enzyme

activity in vitro (Li et al., 2002).

Several analytical methods are available for quantification of DEAQ in various biological

fluids (e.g., blood, plasma and urine) and subcellular fractions (e.g., pHLM) (Trenholme et al.,

1974; Pussard et al., 1985b; Mount et al., 1986; Pussard et al., 1987; Winstanley et al., 1987;

Laurent et al., 1993; Li et al., 2002; Minzi et al., 2003; Dua et al., 2004; Gitau et al., 2004;

Walsky and Obach, 2004; Bell et al., 2007). Early reverse phase chromatographic methods

suffered from poor retention of AQ and DEAQ, long run times and high mobile phase flow rates

(Pussard et al., 1985b; Gitau et al., 2004). Analytical methods based on UV detection did not









permit the accuracy and sensitivity required for the quantification of the analytes due to

endogenous interference from the biological matrices as a result of poor baseline resolution

between AQ and its metabolites. Additionally, all of these methods involved tedious multiple

extraction steps and large volumes of organic solvents (Pussard et al., 1987; Winstanley et al.,

1987; Laurent et al., 1993; Minzi et al., 2003; Dua et al., 2004; Gitau et al., 2004; Bell et al.,

2007). Higher sensitivity was achieved by Trenholme and coworkers through conversion of AQ

to a fluorescent product by refluxing it with borate buffer. Although this normal phase

chromatographic method improved sensitivity and retention, it was found to be non-specific

because the concentration of AQ was confounded by its metabolites (Trenholme et al., 1974).

Mount and coworkers developed the most sensitive method for assaying DEAQ in human blood

and urine (LOQ 1 ng/ml) by employing electrochemical detection (Mount et al., 1986).

However, it involved lengthy extraction steps and consumed high amounts of organic solvents

making it unsuitable for analysis of large number of samples. Considering the prospects of the

use of amodiaquine as a CYP2C8 probe substrate in drug metabolism studies, high throughput

LC/MS/MS based methods were developed for analysis of DEAQ (Li et al., 2002; Walsky and

Obach, 2004; Turpeinen et al., 2005; Walsky et al., 2005; Dixit et al., 2007; O'Donnell et al.,

2007). All of these methods use simple processing methods and are sensitive enough for

determination of DEAQ concentration in in vitro assays as well as clinical studies. However,

DEAQ was separated on reverse phase columns resulting in the use of highly aqueous mobile

phase gradients to prolong retention of DEAQ, which is not ideal for mass spectrometric

detection. Additionally, they involved separation of DEAQ by gradient elution with long run

times. Thus, a simple, sensitive and robust mass spectrometric method that could be easily

applied to drug metabolism studies is needed.









The purpose of the present work was to develop a LC/MS/MS method using hydrophilic

interaction chromatography (HILIC) that involved minimal sample preparation. HILIC

chromatography yielded excellent separation of AQ from DEAQ by prolonging DEAQ retention

time while using high proportions of organic solvent in the mobile phase. The method was used

to determine enzyme kinetic parameters for DEAQ formation in pHLM.

Experimental

Chemicals and Reagents

Amodiaquine, P-nicotinamide adenine dinucleotide phosphate (3-NADP), glucose-6-

phosphate, glucose-6-phosphate dehydrogenase, magnesium chloride and ammonium acetate

were purchased from Sigma (St. Louis, MO, USA). The DEAQ metabolite standard and

deuterated internal standard, DEAQ-d3, were obtained from BD-Gentest (San Jose, CA, USA).

Potassium phosphate, sodium citrate and dimethyl sulfoxide (DMSO) were purchased from

Fisher Scientific (Fair Lawn, NJ, USA). Glucose 6-phosphate dehydrogenase solution was

prepared by dissolving lyophilized enzyme in 5 mM sodium citrate to produce 40 U/ml solution

and was stored at -20'C until use. All chemicals used in the study were of analytical grade.

HPLC grade acetonitrile was obtained from EMD Chemicals (Gibbstown, NJ, USA). Deionized

water was prepared by using a Barnstead Nanopure Diamond UV Ultrapure Water System

(Dubuque, IA, USA). Pooled HLM were purchased from BD Biosciences (San Jose, CA, USA).

Preparation of DEAQ Standards and Quality Control Samples

Two sets of stock solutions were prepared in acetonitrile and water (50:50 v/v) at

concentrations of 0.1 and 0.5 mM. One set of stock solutions was used to spike standards and the

other set was used to spike quality control (QC) samples. Standards were prepared by spiking

phosphate buffer (50 mM, pH 7.4) at seven concentrations ranging from 10 nM to 1500 nM. For

validation, QC samples were prepared by spiking phosphate buffer (50 mM, pH 7.4) at three









concentration levels (50, 500 and 1200 nM). The standards and QC samples were stored at -20C

until analysis. The internal standard solution was prepared by dissolving DEAQ-d3 in

acetonitrile to produce a final concentration of 200 nM and stored at 4 C.

Sample Preparation

The internal standard solution in acetonitrile (200 nM, 1400 1l) was added to DEAQ

standard or QC sample (250 [l). After shaking for 2 minutes on a vortex shaker, samples were

centrifuged at 20,817 g for 8 minutes. An aliquot of clear supernatant was transferred to a 96

well plate and 10 [l was injected on the column. All samples were protected from light exposure

during processing in order to avoid photodecomposition (Motten et al., 1999).

LC/MS/MS Conditions

The LC system was comprised of a ThermoFinnigan Surveyor HPLC autosampler and

ThermoFinnigan Surveyor MS quaternary pump. Chromatographic separation was achieved on a

BETASIL Silica-100 (50 x 2.1 mm, 5[i, ThermoElectron Corporation) analytical column.

Isocratic elution was performed at a flow rate of 220 pl/min for 4.7 minutes using a mobile phase

consisting of 5 mM ammonium acetate and 0.1% (v/v) formic acid in water and 5 mM

ammonium acetate and 0.1% (v/v) formic acid in acetonitrile (15:85 v/v). The autosampler was

maintained at 10C and 10 [l of sample was injected on the column. The mobile phase flow was

diverted from the mass spectrometer to waste for the first 1.5 minutes of run time to remove

nonvolatile salts. After each injection, the needle was washed and flushed with 1000 Il of

solution containing: acetonitrile:2-propanol:water (35:35:30 v/v) and 0.1% (v/v) formic acid.

The mass spectrometer was a TSQ Quantum Discovery triple quadrupole mass

spectrometer equipped with electrospray ionization (ESI) source. The mass spectrometer was

calibrated with a solution of polytyrosine-1, 3, 6 per manufacturer's instructions. The operating









conditions were optimized by infusing DEAQ in the mobile phase in order to maximize the

detector signal. ESI source was operated in positive mode and was set orthogonal to the ion

transfer capillary tube.

For quantification, the TSQ quantum was operated in multiple reaction monitoring (MRM) mode

and the precursor-product ion pair was 328 -- 283 m/z for DEAQ and 331 -* 283 m/z for

DEAQ-d3. The acquisition parameters were: spray voltage 4.0 kV, source CID -10 V, heated

capillary temperature 325"C and capillary offset 35V. Nitrogen was used as a sheath and

auxiliary gas set to 35 and 10 (arbitrary units), respectively. The argon collision gas pressure was

set to 1.5 mTorr. The collision energy was 24 eV for the analyte as well as the internal standard.

The peak full width at half maximum (FWHM) was set at 0.2 Th and 0.7 Th for Q1 and Q3,

respectively. Scan width was fixed to 0.1 Th for both SRM channels and scan time was set to

250 ms.

Calibration curves were constructed by linear regression of the peak area ratio of analyte to

that of the internal standard (Y-axis) and the nominal standard concentration (X-axis) with a

weighting factor of 1/y2. Concentrations of QCs and incubation samples were calculated by using

the regression equation of the calibration curve. Chromatographic peaks were quantified by

using XcaliburTM software (version 1.4) and the peak area ratio of DEAQ to DEAQ-d3 was

plotted against the nominal DEAQ concentrations.

Validation

The newly developed analytical method was validated with respect to selectivity, carry

over, linearity, precision, accuracy and autosampler stability.

For selectivity, samples of blank incubation matrix were analyzed to check the lack of

interference in the quantification of DEAQ. Carry-over was evaluated by placing vials of blank

mobile phase at several locations in the analysis set.









Standards at all concentrations were analyzed in duplicate except the LOQ, which was run in

triplicate. To assess linearity, the maximum allowable deviation of the back calculated

concentration was set at 15% for all standards and at 20% for the LOQ.

The accuracy and precision of the assay was determined by the analysis of QC samples of

DEAQ at concentrations of 50.0, 500.0 and 1200.0 nM. Six of each QC sample were analyzed on

the same day to determine intra-day precision and accuracy, and on three different occasions to

assess inter-day precision and accuracy.

Reanalysis of standards and QC samples tested the stability of the analyses. The stability of

the samples in the autosampler was tested after the samples were left in the autosampler for up to

36 hours by reanalyzing the standards and QC samples. Stability was defined as less than 10%

deviation in concentration from that on the day samples were processed.

Incubation Conditions

Preliminary experiments were conducted to optimize the microsomal protein concentration

(0.01-0.2 mg/ml) and incubation time (5-20 minutes) in order to assure the linearity of DEAQ

formation Amodiaquine and pHLM (0.1 mg/ml) were mixed with phosphate buffer (50 mM,

pH 7.4) and warmed at 37"C for five minutes. Incubations were commenced by addition of the

NADPH regenerating system, which consisted ofMgCl2 (assay concentration, 3.3 mM), NADP+

(1.25 mM), glucose 6-phosphate (3.3 mM) and glucose 6-phosphate dehydrogenase (0.32 U/ml)

in 5 mM sodium citrate solution. Final incubation volume was 250 l. After incubating for 10

minutes at 37"C, the reaction was terminated by addition of 1400 il of ice-cold acetonitrile

containing DEAQ-d3 (0.28 nmol). Samples were processed as described above.

Enzyme kinetic parameters were obtained by performing incubations at nine different

concentrations of AQ ranging from 0.5 iM to 80 riM. AQ was dissolved in acetonitrile and

water (50:50 v/v, final acetonitrile concentration of 0.4% v/v). Microsomes were stored at -80C









and thawed immediately before use. Polypropylene microcentrifuge tubes were used to store AQ

stocks as well as to conduct the microsomal incubations. All incubations were performed in

duplicate and were protected from light to avoid photodecomposition of AQ and the metabolite.

Data Analysis

Enzyme kinetic parameters were obtained by nonlinear regression using GraphPad Prism

(San Diego, CA, USA). Data were typically fit to the following Michaelis-Menten equation:

Vm xS
V = ax Equation 2-1
Km+S

in which V is the initial velocity, Vmax is the maximal velocity, S is the substrate concentration

and Km is the substrate concentration at half-maximal velocity.

Results and Discussion

Method Development

In order to improve the retention of DEAQ and to avoid the use of highly aqueous mobile

phases, we selected BETASIL Silica-100 (50 x 2.1 mm, 5 i) column, which separates analytes

based on the principles of HILIC. It elutes analytes by passing a hydrophobic or mostly organic

mobile phase across a neutral hydrophilic stationary phase causing solutes to elute in order of

increasing hydrophilicity resulting in better separation of highly polar compounds. Alternative to

reverse phase chromatography, HILIC worked best for separation of DEAQ (retention time, 2.9

min) from AQ (retention time, 1.2 min) while still allowing use of 85% acetonitrile in the mobile

phase.

Ammonium acetate buffer was used to volatilize the mobile phase and aid ionization of

DEAQ. Addition of 0.1% (v/v) formic acid in the mobile phase also enhanced ionization and

improved the peak shape of DEAQ. ESI was chosen as the mode of ionization because it gave

high signal intensity for DEAQ. A full scan mass spectrum of DEAQ was obtained in the









positive and negative mode. The most abundant parent ion of DEAQ (328 m/z) was obtained in

the negative mode, which was selected for SRM scanning. Further, the fragmentation pattern of

the precursor ion was obtained and a highly specific ion pair (328 283 m/z) was selected

based on the intensities of three most abundant product ions. Thus, for quantification purpose,

TSQ quantum was operated in MRM mode and the precursor-product ion pair of 328 283 m/z

and 331 283 m/z was followed for DEAQ and DEAQ-d3, respectively. In order to minimize

the sample preparation time, a one step protein precipitation method was utilized by addition of a

solution of internal standard in acetonitrile followed by a short mixing and centrifugation step.

Considering the simplicity of sample processing, the present method could potentially be applied

to a high throughput drug metabolism assay.

Method Validation

Validation of the assay method was conducted according to the FDA guidelines with

respect to selectivity, carry over, linearity, precision, accuracy and autosampler stability

(http://www.fda.gov/cder/guidance). For validation purposes, QC samples at low, medium and

high concentrations were prepared independently and six of each QC sample was analyzed on

three occasions.

Selectivity, carry over and matrix effect

To determine the selectivity of the method, blank microsomal incubation samples were

used to investigate the potential interference due to the endogenous compounds in the matrix. A

clear baseline was observed without any significant interference at the retention times of DEAQ

and DEAQ-d3. Representative chromatograms of (A) blank buffer and buffer spiked with DEAQ

at LOQ (10 nM) and (B) blank incubation sample and a sample after 10 minute incubation of AQ

at 0.5 [tM are depicted in Figure 2-1. No carry over was observed in any of the blank samples.

The potential of ion suppression or enhancement due to the matrix components was evaluated for















DEAQ and DEAQ-d3. No significant matrix effect was observed in microsomal incubations, as



the use of a deuterated internal standard compensated for any variation in matrix effect.


100
95
90
85-
80-
75
70-
65
60
S55
50-
45
40
35
30
25-
20


10


00
00o


35 40 45


n-v ---~~p /


05 10 15 20 25
Time (mm)


35 40


Figure 2-1.Representative chromatograms of (A) blank incubation buffer (red) and buffer spiked

with DEAQ at LOQ (black) and (B) incubation sample: blank (red) and after 10 min

incubation of AQ at 0.5 gM (black) (m/z 328 -> 283, overlay offset = 0%).


05 10 15 20 25
Time (mm)









Linearity

Calibration curve demonstrated good linearity for the entire concentration range with a

mean correlation coefficient (R2) of 0.9969 + 0.0012.

Precision and accuracy

Precision was represented as the relative standard deviation (%RSD) whereas accuracy

was calculated as the percent deviation (% bias) from the respective nominal concentration. The

maximum acceptable limit for precision and accuracy was set at 15%. The intra-day and inter-

day precision and accuracy were within 7.9% and 4.3%, respectively, for all standards and QC

samples (Table 2-1). Thus, the present method was found to be highly reproducible and

demonstrated a high degree of accuracy.

Table 2-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for
analysis of DEAQ in 50 mM phosphate buffer, pH 7.4.



Concentration (nM)

Back calculated % RSD % Deviation
Nominal
(mean + SD)

Intraday

50.00 51.70 0.7 1.4 3.4

500.0 492.8 9.1 1.8 -1.4

1200 1181 +28 2.4 -1.5

Interday

50.00 51.20 3.6 7.0 2.5

500.0 487.9 + 13 2.6 -2.4

1200 1209 +83 6.9 0.8









Autosampler stability

As the autosampler was maintained at 10C during the run, stability of analytes at 10C

was determined by reanalyzing the same standards and QC samples after 36 hours. Both DEAQ

and DEAQ-d3 were found to be stable at 10C for at least 36 hours.

Metabolism of AQ in Pooled Human Liver Microsomes

AQ was incubated with pHLM at nine different concentrations (0.5 -80 [tM). The linearity

of AQ metabolism with respect to the microsomal protein content was studied at five protein

concentrations (0.01-0.2 mg/ml). In order to avoid non-specific protein binding, the lowest

protein concentration that produced quantifiable metabolite (0.1 mg/ml) was selected. Formation

of DEAQ was linear up to 20 minutes. Considering the photosensitivity of AQ, samples were

protected from light. Following the incubation of AQ in pHLM, DEAQ was detected by using

the validated method. The rate of formation of DEAQ was measured as the index of CYP2C8

enzyme activity (Li et al., 2002; Walsky and Obach, 2004; Turpeinen et al., 2005; Walsky et al.,

2005; Dixit et al., 2007; O'Donnell et al., 2007). When modeled by using GraphPad Prism

(version 4), the formation of DEAQ exhibited typical Michaelis-Menten kinetics (Figure 2-2)

with the maximal rate of formation of DEAQ (Vmax) of 2060 94 pmol/min/mg protein and the

concentration of AQ at half maximal velocity (Ki) was 5.78 0.94. In a recent study, Li et al.,

characterized the disappearance of AQ as well as the formation of DEAQ in various expressed

CYP enzymes and pHLM. They reported AQ to be a high affinity and turnover probe substrate

of CYP2C8. Our results are in agreement with the findings of the study conducted by Li et al

recommending the use of DEAQ formation as a CYP2C8 specific probe reaction (Table 2-2) (Li

et al., 2002; Li et al., 2003; Walsky and Obach, 2004). Thus, the present method was

successfully applied to in vitro drug metabolism studies.














4-



cc t


2500-


2000-


1500-


1000.


500-


0 25 50 75
Amodiaquine (pM)


Figure 2-2.Plot of initial velocity versus amodiaquine concentration for the formation of
desethylamodiaquine in pHLM (n=2).


Table 2-2.Enzyme kinetic parameters of AQ in pHLM.


In house

Walsky and Obach, 2004

Li et al., 2003

Li et al., 2002


Km + SE

(FM)

5.8 + 0.94

1.9 + 0.06

3.4

2.4


Vmax
(pmol/min/mg protein)

2060 + 94

1480 + 20

1696

1462


Conclusion

Application of HILIC technique to separate DEAQ resulted in a simple and robust

LC/MS/MS based method. The method was validated with respect to selectivity, carry over,

matrix effects, linearity, precision, accuracy and autosampler stability. Enzyme kinetic









parameters obtained by incubating AQ with pHLM in presence of NADPH regenerating system

were in accordance with the available literature. Therefore, the present method could be applied

for future CYP2C8 drug metabolism studies.









CHAPTER 3
DETERMINATION OF HYDROXYPIOGLITAZONE (M-IV) BY LC/MS/MS:
APPLICATION TO IN VITRO DRUG METABOLISM STUDIES

Introduction

Cytochrome P450 2C8 plays an important role in the metabolism of various drugs

including paclitaxel (Dai et al., 2001; Bahadur et al., 2002), amodiaquine (Li et al., 2002),

troglitazone (Yamazaki et al., 1999), rosiglitazone (Baldwin et al., 1999), pioglitazone (Deng et

al., 2005), repaglinide (Bidstrup et al., 2003; Kajosaari et al., 2005), cerivastatin (Backman et al.,

2002), amiodarone (Soyama et al., 2002), verapamil (Busse et al., 1995), and endogenous

substances like all-trans retinoic acid (McSorley and Daly, 2000) and arachidonic acid (Dai et

al., 2001). Therefore, it is important to know whether a new drug candidate is a substrate or an

inhibitor of CYP2C8. Considering the contribution of CYP2C8 in the metabolism of various

drugs and the impact of inhibition of CYP2C8 on the pharmacokinetics of its substrates, it has

been added to the panel of CYP enzymes for reaction phenotyping studies. Until recently, there

was a lack of information about CYP2C8 specific reactions that could be used to quantify the

activity of this enzyme.

In vivo pharmacokinetic studies as well as in vitro experiments with expressed CYP2C8

and pHLM have shown that AQ is almost exclusively metabolized to DEAQ by CYP2C8.

Therefore, amodiaquine desethylation is used as a CYP2C8 specific marker reaction to quantify

CYP2C8 activity in vitro (Li et al., 2002). However, AQ cannot be used as in in vivo probe of

CYP2C8 as it was withdrawn from the US market due to its intrinsic toxicity. Thus, there is a

need to identify a CYP2C8 specific probe substrate that could be used for in vitro as well as in

vivo reaction phenotyping studies.

Pioglitazone, a thiazolidinedione antidiabetic agent, is widely used in the treatment of non-

insulin dependent diabetes mellitus either as monotherapy or in combination with other









hypoglycemic agents (e.g., metformin, sulfonylurea or insulin). Like other Peroxisome

Proliferator Activated Receptor y (PPARy) agonists, pioglitazone mediates its antidiabetic effects

by increasing insulin stimulated glucose uptake in peripheral tissues as well as the ability of

insulin to suppress endogenous glucose production in the liver (Yki-Jarvinen, 2004). In vitro

studies in human liver microsomes have shown that PIO (Figure 3-1) undergoes extensive

metabolism in the liver primarily by CYP2C8 and CYP3A4 to five primary metabolites (M-I, M-

II, M-IV, M-VI and M-VII). Further, M-IV (Figure 3-1) is oxidized to a ketone to form M-III,

whereas oxidation of M-V leads to formation of M-VI (Shen et al., 2003; Baughman et al., 2005;

Jaakkola et al., 2006). In humans, M-III and M-IV are found to be the major metabolites with

about 40-60% hypoglycemic potency thus, contributing significantly to the pharmacological

activity of PIO.




S S
oN N oN0
H H
CH3 CH3
HO


Figure 3-1.Chemical structures of (A) pioglitazone and (B) hydroxypioglitazone (M-IV)

Studies with various recombinant CYP isoforms as well as pHLM have shown that

montelukast, a potent and highly selective CYP2C8 inhibitor, significantly inhibited depletion of

PIO (IC50 = 0.51 pM) and more strongly inhibited formation of M-IV (IC50 = 0.18 pM)

indicating the major role of CYP2C8 in the formation of M-IV (Jaakkola et al., 2006). In human

pharmacokinetics study, gemfibrozil alone raised the mean area under the plasma concentration-

time curve (AUCo-o) of PIO 3.2-fold and prolonged its elimination half-life (ti/2) from 8.3 to 22.7









hours. It also decreased the AUCO-48 of M-III and M-IV by 42% and 45%, respectively. The

effect of gemfibrozil on the pharmacokinetics of PIO and its metabolites due to CYP2C8

inhibition could be attributed to the acylglucoronide of gemfibrozil. In the same study, a potent

CYP3A4 inhibitor itraconazole did not have any significant effect on the pharmacokinetics of

PIO or either of its metabolites indicating a minor role of CYP3A4 in the metabolism of PIO and

formation of M-IV (Jaakkola et al., 2005). In a recent study, trimethoprim, a known inhibitor of

CYP2C8, was found to increase AUCo-,, of PIO by 42% and reduced the apparent formation rate

of M-IV by 27% validating the major role of CYP2C8 in the formation of M-IV (Tornio et al.,

2007). From above mentioned in vitro and in vivo drug interaction studies it is evident that

CYP2C8 plays a major role in the formation of M-IV. Therefore, formation of M-IV could be

employed as a marker reaction for quantification of CYP2C8 activity in reaction phenotyping

studies.

Several analytical methods are available for the quantification of M-IV in various

biological fluids (e.g., plasma and urine) (Kiyota et al., 1997; Lin et al., 2003; Deng et al., 2005;

Jaakkola et al., 2005; Tornio et al., 2007) and subcellular fractions (e.g., recombinant CYP

enzymes and liver microsomes) (Shen et al., 2003; Baughman et al., 2005; Jaakkola et al., 2006;

Tomio et al., 2007). Most of the LC/MS/MS based methods were developed with the purpose of

identification and characterization of various metabolites of PIO and therefore, involve slow

mobile phase gradients with long run times. Many of these methods assigned arbitrary units to

M-IV due to the lack of metabolite standards at the time of development. More recent

LC/MS/MS based methods used in drug interaction studies in humans involve sample processing

by solid phase extraction and suffer from the disadvantage of very long run times. Analytical

methods developed by Lin et al and Deng et al separate PIO and its metabolites by isocratic









elution and quantify M-IV based on metabolite standards (Lin et al., 2003; Deng et al., 2005).

Although both of these methods could be successfully employed for quantification of M-IV

(LOQ of M-IV 0.5 and 1.1 ng/ml, respectively) in pharmacokinetic studies in humans, they are

not sensitive enough to be used for in vitro enzyme assays and inhibition studies. Thus, the

purpose of this study was to develop a rapid, sensitive and robust method for determination of

M-IV that could be easily applied to in vitro drug metabolism studies.

Experimental

Chemicals and Reagents

P-Nicotinamide adenine dinucleotide phosphate (3-NADP), glucose-6-phosphate, glucose-

6-phosphate dehydrogenase, magnesium chloride and ammonium acetate were procured from

Sigma (St. Louis, MO, USA). Pioglitazone hydrochloride, M-IV metabolite standard and the

stable labeled internal standard, M-IV-d4, were obtained from Torronto Research Chemicals Inc.

(North York, Ontario, Canada). Glucose 6-phosphate dehydrogenase solution was prepared by

dissolving lyophilized enzyme in 5 mM sodium citrate to produce 40 U/ml solution and was

stored at -20'C until use. Potassium phosphate was purchased from Fisher Scientific (Fair Lawn,

NJ, USA) and formic acid was obtained from Mallinckrodt baker Inc. (Phillipsburg, NJ, USA).

HPLC grade acetonitrile and methanol were purchased from EMD Chemicals (Gibbstown, NJ,

USA). All other chemicals used in the study were of analytical grade. Deionized water was

obtained from a Barnstead Nanopure Diamond UV Ultrapure Water System (Dubuque, IA,

USA). Pooled HLM were obtained from In Vitro technologies (Baltimore, MD, USA).

Preparation of M-IV Standards and Quality Control samples

Two sets of stock solutions were prepared in methanol at concentrations of 0.1 and 1.0

[tg/ml. One set of stock solutions was used to spike standards and the other set was used to spike

QC samples. Standards were prepared by spiking phosphate buffer (50 mM, pH 7.4) at seven









concentrations ranging from 0.1 to 20 ng/ml. For validation, QC samples were prepared by

spiking phosphate buffer (50 mM, pH 7.4) at three concentration levels (spiked at 0.5, 2.0 and 10

ng/ml). The standards and QC samples were stored at -20'C until analysis. The internal standard

solution was prepared by dissolving M-IV-d4 in methanol to produce a final concentration of 12

ng/ml and was stored at -20'C.

Sample Preparation

Acetonitrile (750 il) was added to 250 tl of standard solution, QC or incubation sample

followed by the addition of the internal standard solution in methanol (50 il; 12 ng/ml). Samples

were processed by liquid-liquid extraction using dichloromethane (2 ml) as the extraction

solvent. After shaking for 10 minutes on a horizontal shaker at low speed, samples were

centrifuged at 3200 g for 10 minutes. The top aqueous layer was aspirated and the organic layer

was transferred to glass tubes and dried under a stream of nitrogen at 50'C. The obtained residue

was redissolved in methanol: water (50:50, 150 tIl) and an aliquot was transferred to autosampler

vials.

LC/MS/MS Conditions

The LC system was comprised of a ThermoFinnigan Surveyor HPLC autosampler and

ThermoFinnigan Surveyor MS quaternary pump. Chromatographic separation was achieved on a

SYNERGI MAX-RP 80A (150 x 2.00 mm, 4ti, Phenomenex) analytical column. Gradient

elution was performed at a flow rate of 220 ptl/min using the following mobile phase system: A =

5 mM ammonium acetate and 0.1 % (v/v) formic acid and B = 5 mM ammonium acetate and 0.1

% (v/v) formic acid in methanol. The column was started at 40:60 of A:B and at 0.5 min, the

mobile phase composition was changed to 90% of B over 0.5 minutes and held for 1.2 min (2.2

min total) before returning to the starting conditions, which was held for 2.3 minutes (total run

time 5.0 minutes). The autosampler was maintained at 10C and 20 [tl of sample was injected on









the column. The mobile phase flow was diverted from the mass spectrometer to waste for the

first 1.0 min of run time to remove nonvolatile salts. After each injection, the needle was washed

and flushed with 500 .il of solution containing: acetonitrile:2-propanol:water (35:35:30 v/v) and

0.1% (v/v) formic acid.

The mass spectrometer was a TSQ Quantum Discovery triple quadrupole mass

spectrometer equipped with an ESI source. It was calibrated with a solution of polytyrosine-1, 3,

6 per manufacturer's instructions. The operating conditions were optimized by infusing M-IV in

the mobile phase in order to maximize the detector signal. ESI source was operated in the

positive mode and was set orthogonal to the ion transfer capillary tube.

For quantification, the TSQ quantum was operated in the MRM mode and the precursor-product

ion pair was 373 -* 150 m/z for M-IV and 377 -* 154 m/z for M-IV-d4. The acquisition

parameters were: spray voltage 4.0 kV, source CID -3 V, heated capillary temperature 325"C and

capillary offset 35 V. Nitrogen was used as a sheath and auxiliary gas set to 35 and 10 (arbitrary

units), respectively. The argon collision gas pressure was set to 1.5 mTorr. The collision energy

was 35 eV for the analyte as well as the internal standard. The peak FWHM was set at 0.2 Th

and 0.7 Th for Q1 and Q3, respectively. Scan width was fixed to 1.0 Th for both SRM channels

and scan time was set to 250 ms.

Chromatographic peaks were quantified using XcaliburTM software (version 1.4) and

calibration curves were constructed by linear regression of the peak area ratio of M-IV to that of

M-IV-d4 (Y-axis) and the nominal standard concentration (X-axis) with a weighting factor of

1/y2. The concentration in QCs and incubation samples was calculated by using the regression

equation of the calibration curve.









Validation

The newly developed analytical method was validated with respect to selectivity, carry

over, linearity, precision, accuracy and autosampler stability. For selectivity, samples of blank

incubation matrix were analyzed to check for lack of interference at the retention time of M-IV

and M-IV-d4. Carry-over was evaluated by placing vials of methanol at several locations in the

analysis set.

Calibration curves were constructed by plotting the ratio of the peak area of M-IV to that

of M-IV-d4 against the nominal M-IV concentration. Standards at all concentrations were

analyzed in duplicate except the LOQ, which was run in triplicate. To assess linearity, the

maximum allowable deviation of the back calculated concentration was set at 15% for all

standards and at 20% for LOQ.

The accuracy and precision of the assay was determined by the analysis of QC samples of

M-IV at concentrations of 0.5, 2.0 and 10 ng/ml. Twelve of each QC sample were analyzed on

the same day to determine intra-day precision and accuracy, and six of each QC sample on two

different occasions to assess inter-day precision and accuracy.

The stability of the samples in the autosampler was tested after the samples were left in the

autosampler for up to 48 hours by reanalyzing the standards and QC samples. Stability was

defined as less than 10% deviation in concentration from that on the day samples were

processed.

Incubation Conditions

Preliminary experiments were conducted to optimize the microsomal protein concentration

(0.05-0.4 mg/ml) and incubation time (5-20 minutes) in order to assure the linearity of M-IV

formation. Pioglitazone and pHLM (0.05 mg/ml) were mixed with phosphate buffer (50 mM, pH

7.4) and warmed at 37"C for five minutes. Incubations were commenced by addition of the









NADPH regenerating system, which consisted ofMgCl2 (assay concentration, 3.3 mM), NADP+

(1.25 mM), glucose 6-phosphate (3.3 mM) and glucose 6-phosphate dehydrogenase (0.32 U/ml)

in 5 mM sodium citrate solution. Final incubation volume was 250 il. After incubating for 10

minutes at 37"C, the reaction was terminated by addition of 750 [il of ice-cold acetonitrile

containing internal standard and samples were processed as described above.

Enzyme kinetic parameters were obtained by performing incubations at nine different

concentrations of PIO ranging from 0.25 iM to 25 riM. Pioglitazone was dissolved in

acetonitrile and methanol (90:10 v/v, final organic solvent concentration of 0.8% v/v

corresponding to final methanol content 0.08%). Microsomes were stored at -80'C and thawed

immediately before use. Polypropylene microcentrifuge tubes were used to store PIO stocks and

microsomal incubations were conducted in polypropylene tubes (4 ml) to minimize any non-

specific binding. All incubations were performed in duplicate.

Results and Discussion

Method Development

Pioglitazone is primarily metabolized by CYP2C8 and CYP3A4 to four hydroxylated (M-

II, M-IV, M-VII and M-VIII) and various other metabolites. Baughman et al have identified M-

II, M-IV, M-VII and M-VIII in freshly isolated human hepatocytes and in pHLM (Baughman et

al., 2005). Shen et al. (Shen et al., 2003) also reported formation of M-VII along with M-IV in

various preclinical species and pHLM. In the LC/MS/MS based method described by Shen et al.,

M-VII elutes very close (retention time 38.3 min) to M-IV (40.2 min) in a total of 90 min run

time (Shen et al., 2003). Although these metabolites differ in the position of hydroxylation they

have similar fragmentation pattern and therefore, possess the same transition pair of Q1 and Q3

(373 -* 150 m/z). Therefore, it was important to separate M-IV from M-VII on the analytical

column. With the purpose of shortening the run time while achieving good baseline resolution









between M-IV and M-VII, we selected SYNERGI MAX-RP 80A (150 x 2.00 mm, 4 i,

Phenomenex) column. We were able to achieve baseline resolution keeping the run time

relatively short (5 minutes). M-IV eluted at 2.7 minutes on the column.

A mobile phase consisting of ammonium acetate buffer was used to aid ionization of M-

IV. Addition of 0.1% (v/v) formic acid in the mobile phase also enhanced ionization and

improved the peak shape of M-IV. A full scan mass spectrum of M-IV was obtained in the

positive and negative mode. The most abundant parent ion of M-IV (373 m/z) was obtained in

the positive mode and the specific ion pair (373 -* 150 m/z) was selected based on the intensities

of three most abundant product ions. Thus, for quantification purpose, the TSQ quantum was

operated in MRM mode and the precursor-product ion pair of 373 -* 150 m/z and 377 -* 154

m/z was followed for M-IV and M-IV-d4, respectively. In order to improve sensitivity of the

method, M-IV was extracted by a liquid-liquid extraction method. Various solvents including

acetonitrile, methyl tert butyl ether, dichloromethane, ethyl acetate and their combinations were

used as extraction solvents. The highest and most consistent recovery was with dichloromethane

(lower limit of quantification, LOQ 0.1 ng/ml), which was selected as the extraction solvent for

future experiments. Considering the high sensitivity, simplicity of sample processing and short

run time, the present method could potentially be applied to drug metabolism assays in future.

Method Validation

Validation of the assay method was conducted according to the FDA guidelines with

respect to selectivity, carry over, linearity, precision, accuracy and autosampler stability

(http://www.fda.gov/cder/guidance).

Selectivity, carry over and matrix effect

To determine the selectivity of the method, blank microsomal incubation samples were

used to investigate the potential interference due to the endogenous compounds in the matrix.












The present method demonstrated high degree of selectivity by means of MRM mode. A clear


baseline was observed without any significant interference at the retention times of M-IV and M-


IV-d4. The ratio of signal to noise obtained from an extracted standard at LOQ (0.1 ng/ml) was


at least 50 for M-IV. Representative chromatograms of (A) blank buffer and buffer spiked with


M-IV at the low QC (0.5 ng/ml) and (B) blank incubation sample and a sample after 10 minute


incubation of PIO at 10 [tM are depicted in Figure 3-2. No carry over was observed in any of the


blank samples.

A


7 576

70-
65
$ 60-

50-




2 0


5 <2 031 054 08 9 1 /2 1/94 10


























Figure 3-2.Representative chromatograms of (A) blank buffer (red) and buffer spiked with OH-
PI at low QC (black) and (B) incubation sample: blank (red) and after 10 min
incubation ofPI at 10 (black) (mz 373 150, overlay offset ).
85
8 2 73



605


455

35{






00 05 1 0 1 5 20 25 30 35


Figure 3-2. Representative chromatograms of (A) blank buffer (red) and buffer spiked with OH-
PIO at low QC (black) and (B) incubation sample: blank (red) and after 10 min
incubation of PIO at 10lM (black) (m/z 373 -> 150, overlay offset = 0%).









Linearity

Calibration curves were linear in the concentration range of 0.1 20 ng/ml using a linear

regression equation with a weighting factor of 1/y2. The mean correlation coefficient (R2) of the

calibration curves was 0.9967.

Precision and accuracy

Precision was represented as the relative standard deviation (%RSD) whereas accuracy

was calculated as the percent deviation (% bias) from the respective nominal concentration. The

maximum acceptable limit for precision and accuracy was set at 15%. The intra-day and inter-

day precision and accuracy were within 4.0% and 10%, respectively, for all standards and QC

samples (Table 3-1). Thus, the present method was found to be highly reproducible and

demonstrated a high degree of accuracy.

Table 3-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for
analysis of M-IV in 50 mM phosphate buffer, pH 7.4.


Concentration (ng/ml)
Back calculated
Nominal Bak cala% RSD % Deviation
(mean SD)
Intraday
0.50 0.48 + 0.02 1.4 3.4
2.00 2.10 + 0.05 1.8 -1.4
10 9.0 + 0.4 2.4 -1.5
Interday
0.50 0.48 + 0.02 3.6 -4.5
2.00 2.07+ 0.06 3.0 3.7
10.0 9.2 + 0.4 3.9 -7.7


Autosampler stability

The processed stability of analytes was determined by reanalyzing the same standards and

QC samples after 48 hours in the autosampler maintained at 100C. Both M-IV and M-IV-d4 were

found to be stable at 10C for at least 48 hours.









Formation of M-IV in Pooled Human Liver Microsomes

PIO was incubated with pHLM at nine different concentrations (0.25-25 pM). The

linearity of M-IV formation with respect to the microsomal protein concentration was studied at

five protein concentrations (0.05-0.4 mg/ml). In order to avoid non-specific protein binding, the

lowest protein concentration that produced quantifiable metabolite (0.05 mg/ml) was selected.

Formation of M-IV was linear up to 20 minutes and was dependent on the presence of NADPH

regenerating system. Following the incubation of PIO in pHLM, the concentration of M-IV was

determined by using the newly validated method. The rate of formation of M-IV was measured

as an index of CYP2C8 enzyme activity and data were analyzed by nonlinear regression using

GraphPad Prism (version 4). The formation of M-IV exhibited typical Michaelis-Menten kinetics

(Figure 3-3) with the maximal rate of formation of M-IV (Vmax) of 150.3 13.9 pmol/min/mg

protein and the concentration of PIO at half maximal velocity (Km) was 8.71 + 1.9 riM, which

are in agreement with the present literature (Table 3-2) (Tomio et al., 2007). Thus, the present

method was successfully applied to in vitro drug metabolism studies.


125-

100-

I 75-
Oa 50-





0 10 20 30
Pioglitazone (VM)


Figure 3-3.Plot of initial velocity versus pioglitazone concentration for the formation of
hydroxypioglitazone (M-IV) in pHLM (n=2).










Table 3-2.Enzyme kinetic parameters of M-IV formation in pHLM.

Km SE Vmax
(lM) (pmol.min-.mg protein1)
In house 8.71 + 1.90 150.3 13.9
Tornio et al., 2007 9.8 640


Conclusion

A LC/MS/MS based method for determination of M-IV, the CYP2C8 specific metabolite

of PIO was developed and validated. The method is sensitive (LOQ 0.1 ng/ml) and robust with a

very short run time (5 minutes). It was validated with respect to selectivity, carry over, matrix

effects, linearity, precision, accuracy and autosampler stability. Enzyme kinetic parameters

obtained by incubating PIO with pHLM in presence of NADPH regenerating system were in

accordance with the available literature. Therefore, the present method could be applied for

future in vitro reaction phenotyping studies of CYP2C8.









CHAPTER 4
DETERMINATION OF IC5o IN POOLED HUMAN LIVER MICROSOMES USING M-IV
FORMATION AS A CYP2C8 SPECIFIC REACTION

Introduction

It has become increasingly clear that the inhibition of CYP enzymes is often the key

mechanism underlying drug-drug interactions leading to an ADR. In many cases, the drug

interaction occurs via alterations in the activity of CYP enzymes. In other words, one drug (the

perpetrator) alters the activity of an enzyme that is responsible for the metabolism of another

drug (the victim or object), thus affecting its metabolic clearance. If the second drug has a wide

therapeutic window, then the interaction might be clinically insignificant. However, if the victim

drug has a narrow margin of safety, then the inhibition of its metabolism might result in drug

related toxicity. On the other hand, induction of metabolism of the victim drug by the perpetrator

might cause subtherapeutic effects leading to failure of drug therapy.

CYP2C8 plays an important role in the metabolism of various drugs including paclitaxel

(Dai et al., 2001; Bahadur et al., 2002), amodiaquine (Li et al., 2002), troglitazone (Yamazaki et

al., 1999), rosiglitazone (Baldwin et al., 1999), pioglitazone (Deng et al., 2005), repaglinide

(Bidstrup et al., 2003; Niemi et al., 2003b), cerivastatin (Backman et al., 2002), amiodarone

(Soyama et al., 2002) and verapamil (Busse et al., 1995). Reports of CYP2C8 inhibition by

gemfibrozil have shown an increase in rosiglitazone AUCo-0, from 1.8 to 2.8-fold (Niemi et al.,

2003a), repaglinide AUCo-0, from 5.5-15-fold (Niemi et al., 2003b), and cerivastatin AUCo-0

from 1.3-20-fold (Backman et al., 2002). Cerivastatin was withdrawn from the market after

about 500 ADRs, half of which involved coadministration of the CYP2C8 inhibitor gemfibrozil.

In a recent study, Walsky et al. examined 209 frequently prescribed drugs and related

xenobiotics for CYP2C8 inhibition using AQ as a CYP2C8 specific probe. In order to evaluate

the potential of PIO as a CYP2C8 marker substrate, we studied the inhibition of CYP2C8 by five









drugs that were the part of the comprehensive study by Walsky et al. and other smaller studies

using different CYP2C8 substrates (Walsky et al., 2005).

Experimental

Chemicals and Reagents

Ketoconazole, terfenadine, P-estradiol, midazolam, 3-NADP, glucose-6-phosphate,

glucose-6-phosphate dehydrogenase, magnesium chloride and ammonium acetate were procured

from Sigma (St. Louis, MO, USA). Pioglitazone hydrochloride, M-IV metabolite standard and

the deuterated internal standard, M-IV-d4, were obtained from Toronto Research Chemicals Inc.

(North York, Ontario, Canada). Montelukast was purchased from LKT Labs (St. Paul, MN,

USA). Pooled HLM were obtained from In vitro Technologies (Baltimore, MD, USA). Glucose

6-phosphate dehydrogenase solution was prepared by dissolving lyophilized enzyme in 5 mM

sodium citrate to produce 40 U/ml solution and was stored at -20 C until use. Potassium

phosphate was purchased from Fisher Scientific (Fair Lawn, NJ, USA) and formic acid was

obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). HPLC grade acetonitrile and

methanol were purchased from EMD Chemicals (Gibbstown, NJ, USA). All other chemicals

used in the study were of analytical grade. Deionized water prepared by using a Barnstead

Nanopure Diamond UV Ultrapure Water System (Dubuque, IA, USA) was used throughout the

study.

Incubation Conditions

Preliminary experiments were conducted to optimize the microsomal protein concentration

and incubation time in order to assure the linearity of M-IV formation (Chapter 3). The mixture

containing pioglitazone (assay concentration, 7.5 pM) and pHLM (0.05 mg/ml) in phosphate

buffer (50 mM, pH 7.4) was mixed with the inhibitor solution (in methanol:acetonitrile, 10:90) at

various concentrations and warmed at 37 C for five minutes. The final incubation concentration









range that was studied for various inhibitors was as follows: ketoconazole (0.1-100 aM),

terfenadine (0.1-100 aM), 3-estradiol (0.1-600 aM), midazolam (0.1-600 gM), and montelukast

(0.1-1000 nM). Equivalent amount of solvent was added to the incubation containing no

inhibitor. Incubations were commenced by addition of the NADPH regenerating system, which

consisted ofMgCl2 (assay concentration, 3.3 mM), NADP+ (1.25 mM), glucose 6-phosphate

(3.3 mM) and glucose 6-phosphate dehydrogenase (0.32 U/ml) in 5 mM sodium citrate solution.

Final incubation volume was 250 pl and the content of total organic solvent was kept to the

minimum (1.2%). Control incubations were conducted without addition of NADPH regenerating

system to check any interference at the metabolite retention time. After incubating for 10

minutes at 37"C, the reaction was terminated by addition of 750 [il of ice-cold acetonitrile

followed by the addition of internal standard solution in methanol (50 il, 12 ng/ml). Samples

were processed as described in Chapter 3. Microsomes were stored at -80'C and thawed

immediately before use. All incubations were performed in duplicate.

Analysis of Hydroxypioglitazone (M-IV)

Concentrations of M-IV were determined as described in Chapter 3. Briefly, the

chromatographic separation was achieved on a SYNERGI MAX-RP 80A (150 x 2.00 mm, 4 i,

Phenomenex) analytical column by gradient elution. The mass spectrometer was a TSQ

Quantum Discovery triple quadrupole mass spectrometer equipped with ESI source. The ESI

source was operated in the positive mode and was set orthogonal to the ion transfer capillary

tube. For quantification, TSQ quantum was operated in MRM mode and the precursor-product

ion pair was 373 -* 150 m/z for M-IV and 377 -* 154 m/z for M-IV-d4. The acquisition

parameters were optimized to maximize the signal to noise ratio.










Data Analysis

Enzyme kinetic and inhibition data were obtained by nonlinear regression using GraphPad

Prism (San Diego, CA, USA). Data for IC50 determinations were typically fit to the following

equation:

I xl
% Control activity = 100 mx Equation 4-1
IC50 +I


in which I is the inhibitor concentration, Imax is the initial velocity in the presence of inhibitor,

IC5o is the inhibitor concentration that reduces initial velocity by 50%.

Results and Discussion

Five drugs namely, montelukast, ketoconazole, P-estradiol, midazolam and terfenadine

were examined for their potential to inhibit the formation of the CYP2C8 specific metabolite (M-

IV) in pHLM. Chemical structures of these inhibitors are shown in Figure 4-1.





0 OH

0v0"

N O SOH
NN H C0 N -


Ketoconazole Montelukast



N
OH N

N H Cl -N
OH F
OH H H
HO

Terfenadine Estradiol Midazolam


Figure 4-1. Structures of the compounds tested for inhibition of in vitro CYP2C8 activity.









The concentration of inhibitor that reduces the initial velocity by 50% (IC5o) was

determined by conducting the enzyme assay in the presence of seven different inhibitor

concentrations. The results of the present study demonstrate that all the test inhibitors inhibited

the activity of CYP2C8 in a concentration dependent manner when studied as a function of

formation of M-IV from PIO. IC50 plots of pioglitazone hydroxylase inhibition are shown in

Figure 4-2.


120-
S Ketoconazole
100- A Beta-estradiol

S80 Midazolam
+ Terfenadine
S 60 Montelukast



0-
201


0.001 0.01 0.1 1 10 100 1000
Inhibitor Cone. (jM)



Figure 4-2.IC50 plots of pioglitazone hydroxylase inhibition in pHLM by montelukast (circles),
ketoconazole (squares), terfenadine (diamonds), P-estradiol (triangles) and
midazolam (reverse triangles). Data points reflect the average for incubations run in
duplicate SE.


Montelukast was the most potent inhibitor of formation of M-IV with an IC50 of 57.16 +

12.12 nM (R2 = 0.8896) in pHLM. Walsky et al. examined the inhibition of CYP2C8 activity by

209 drugs and related compounds in expressed CYP2C8 as a function of AQ desethylation. They

also reported montelukast to be the most potent inhibitor of CYP2C8 activity with an IC50 of









19.6 nM in pHLM (Walsky et al., 2005). Our results are in accordance with another study by

Jaakkola et al. that examined the CYP2C8 inhibition potential of montelukast using pioglitazone

as a substrate (IC5o = 180 nM) (Jaakkola et al., 2006).

The antifungal agent ketoconazole was the next potent inhibitor of CYP2C8 activity with

an IC5o of 0.618 0.12 tM (R2 = 0.9039). In the present study, we found that terfenadine, a H1

receptor antagonist, inhibited the activity of CYP2C8 with an IC50 of 15.79 7.32 [M (R2 =

0.4247). Ketoconazole and terfenadine are known inhibitors of CYP3A4. Ketoconazole also

inhibited 6-a-hydroxylation of paclitaxel (Desai et al, 1998) and tolyl methylhydroxylation of

torsemide (Ong et al., 2000), both of which are CYP2C8 specific reactions, where as terfenadine

was shown to inhibit AQ desethylation (IC50 11.5 5.1 [M) (Walsky et al., 2005) as well as

tolyl methylhydroxylation of torsemide in expressed CYP2C8 (Ong et al., 2000). Inhibition of

CYP2C8 by ketoconazole and terfenadine could be explained by the fact that both enzymes,

CYP3A4 and CYP2C8, have a large active site and many CYP2C8 substrates are also

metabolized by CYP3A4. Walsky et al. reported a moderate inhibition of the metabolism of AQ

by P-estradiol in expressed CYP2C8 (21.5 5.8) (Walsky et al., 2005). In the current study, we

proved the moderate inhibitory potential of P-estradiol to inhibit CYP2C8 activity measured as

the formation rate of M-IV. Midazolam, a benzodiazepine drug, moderately inhibited CYP2C8

mediated hydroxylation of PIO (IC50 = 44.60 7.1). It also inhibited the formation of DEAQ

(Walsky et al., 2005) as well as formation of tolyl methylhydroxy torsemide in expressed

CYP2C8 (Ong et al., 2000). Midazolam is used as a maker substrate of CYP3A4, once again

indicating the overlap between the substrate and inhibitor profiles of CYP2C8 and 3A4.

Conclusion

We examined the potential of montekulast, ketoconazole, terfenadine, P-estradiol and

midazolam to inhibit the formation of M-IV which is CYP2C8 specific metabolite of PIO using









pHLM. All test inhibitors inhibited the M-IV formation in the concentration dependent manner

and the inhibitory potentials of each of them are comparable to that studied with other CYP2C8

substrates. Therefore, formation of M-IV could potentially be used as a CYP2C8 specific probe

reaction.









CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS

Drug-drug interactions due to induction or inhibition of DMEs significantly add to the

ADRs. In order to avoid any unforeseen interactions in the clinic, the FDA requires

pharmaceutical companies to identify the major drug metabolizing enzymes involved in the

elimination of a NCE as well as the effect of its coadministration on the pharmacokinetics of

marker substrates of DMEs that are responsible for its elimination. Common experimental

methods used to characterize the metabolism of a NCE and predict its drug-drug interactions

involve the use of enzyme specific chemical inhibitors, expressed enzymes, antibodies and

correlation analysis.

The polymorphic CYP2C8 is involved in the metabolism of paclitaxel, amodiaquine,

troglitazone, rosiglitazone, pioglitazone, repaglinide, cerivastatin, amiodarone and verapamil.

Conventionally, paclitaxel has been used as a CYP2C8 specific probe substrate for in vitro

reaction phenotying studies. Recently, AQ was found to be a high turnover and affinity substrate

of CYP2C8 and is used for in vitro drug metabolism studies however, it can not be administered

to humans due to its toxocity. Therefore, identification of a CYP2C8 specific substrate that can

be used for in vitro as we as in vivo drug interaction studies is needed. Hydroxylation of PIO to

form M-IV is primarily medicated by CYP2C8, therefore, formation of M-IV could be used as a

CYP2C8 specific probe reaction.

We developed a simple and robust LC/MS/MS based method using HILIC

chromatography to separate DEAQ. The method was validated with respect to selectivity, carry

over, matrix effects, linearity, precision, accuracy and autosampler stability. Enzyme kinetic

parameters obtained by incubating AQ with pooled HLM in presence of NADPH regenerating









system were in accordance with the available literature. Therefore, the present method could be

applied for future CYP2C8 drug metabolism studies.

A LC/MS/MS based method for determination of M-IV, the CYP2C8 specific metabolite

of PIO was developed and validated. The method is sensitive (LOQ 0.1 ng/ml) and robust with a

very short run time (5 minutes). It was validated with respect to selectivity, carry over, matrix

effects, linearity, precision, accuracy and autosampler stability. Enzyme kinetic parameters

obtained by incubating PIO with pooled HLM in presence of NADPH regenerating system were

in accordance with the available literature. Therefore, the present method could be applied for

future in vitro reaction phenotyping studies of CYP2C8.

We examined the potential of montekulast, ketoconazole, terfenadine, estradiol and

midazolam to inhibit the formation of M-IV which is CYP2C8 specific metabolite of PIO using

pooled human liver microsomes. All test inhibitors inhibited the M-IV formation in the

concentration dependent manner and the inhibitory potentials of each of them are comparable to

that studied with other CYP2C8 substrates. Therefore, formation of M-IV could potentially be

used as a CYP2C8 specific probe reaction.

Although, the presence of CYP2C8*3 allele reduced the intrinsic clearance of AQ in

human liver microsomes, its effect of AQ pharmacokinetics is unknown. In future, it will be

interesting to study the effect of CYP2C8*3/*3 genotype on the disposition of AQ. On the other

hand, CYP2C8*3 allele reduced the AUCo-, of PIO. However, its effect on PIO metabolism has

not been studied at enzymatic level. Further, we would like to study the effect of CYP2C8*3

allele on M-IV formation in genotyped human liver microsomes.









LIST OF REFERENCES


Bachmann KA, Ring BJ and Wrighton SA (2003) Drug-Drug Interactions and the Cytochrome
P450, in: Drug Metabolizing Enzymes: Cytochrome P450 and Other Enzymes in Drug
Discovery and Development (Lee JS, Obach RS and Fisher MB eds), FrontisMedia SA and
Marcel Dekker, New York.

Backman JT, Kyrklund C, Neuvonen M and Neuvonen PJ (2002) Gemfibrozil greatly increases
plasma concentrations of cerivastatin. Clin Pharmacol Ther 72:685-691.

Bahadur N, Leathart JB, Mutch E, Steimel-Crespi D, Dunn SA, Gilissen R, Houdt JV, Hendrickx
J, Mannens G, Bohets H, Williams FM, Armstrong M, Crespi CL and Daly AK (2002)
CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alpha-
hydroxylase activity in human liver microsomes. Biochem Pharmacol 64:1579-1589.

Basco LK, Ndounga M, Keundjian A and Ringwald P (2002) Molecular epidemiology of malaria
in cameroon. IX. Characteristics of recrudescent and persistent Plasmodium falciparum
infections after chloroquine or amodiaquine treatment in children. Am J Trop MedHyg
66:117-123.

Baldwin SJ, Clarke SE and Chenery RJ (1999) Characterization of the cytochrome P450
enzymes involved in the in vitro metabolism of rosiglitazone. Br J Clin Pharmacol 48:424-
432.

Baughman TM, Graham RA, Wells-Knecht K, Silver IS, Tyler LO, Wells-Knecht M and Zhao Z
(2005) Metabolic activation of pioglitazone identified from rat and human liver microsomes
and freshly isolated hepatocytes. DrugMetab Dispos 33:733-738.

Bell DJ, Nyirongo SK, Molyneux ME, Winstanley PA and Ward SA (2007) Practical HPLC
methods for the quantitative determination of common antimalarials in Africa. J Chromatogr
B Analyt Technol Biomed Life Sci 847:231-236.

Bidstrup TB, Bjomrsdottir I, Sidelmann UG, Thomsen MS and Hansen KT (2003) CYP2C8 and
CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the
insulin secretagogue repaglinide. Br J Clin Pharmacol 56:305-314.

Busse D, Cosme J, Beaune P, Kroemer HK and Eichelbaum M (1995) Cytochromes of the P450
2C subfamily are the major enzymes involved in the O-demethylation of verapamil in
humans. Naunyn Schmiedebergs Arch Pharmacol 353:116-121.

Cavaco I, Stromberg-Norklit J, Kaneko A, Msellem MI, Dahoma M, Ribeiro VL, Bjorkman A
and Gil JP (2005) CYP2C8 polymorphism frequencies among malaria patients in Zanzibar.
Eur J Clin Pharmacol 61:15-18.

Cresteil T, Monsarrat B, Alvinerie P, Treluyer JM, Vieira I and Wright M (1994) Taxol
metabolism by human liver microsomes: identification of cytochrome P450 isozymes
involved in its biotransformation. Cancer Res 54:386-392.









Dai D, Zeldin DC, Blaisdell JA, Chanas B, Coulter SJ, Ghanayem BI and Goldstein JA (2001)
Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and
arachidonic acid. Pharmacogenetics 11:597-607.

Deng LJ, Wang F and Li HD (2005) Effect of gemfibrozil on the pharmacokinetics of
pioglitazone. Eur J Clin Pharmacol 61:831-836.

Dixit V, Hariparsad N, Desai P and Unadkat JD (2007) In vitro LC-MS cocktail assays to
simultaneously determine human cytochrome P450 activities. Biopharm Drug Dispos 28:257-
262.

Dua VK, Gupta NC, Sharma VP and Subbarao SK (2004) Liquid chromatographic determination
of amodiaquine in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 803:371-
374.

Ferguson SS, Chen Y, LeCluyse EL, Negishi M and Goldstein JA (2005) Human CYP2C8 is
transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane
X receptor, glucocorticoid receptor, and hepatic nuclear factor 4alpha. Mol Pharmacol
68:747-757.

Food and Drug Administration. 1997. Guidance for Industry: Drug Metabolism/Drug Interaction
Studies in the Drug Development Process: Studies In Vitro.
http://www.fda. gov/cder/guidance/clin3.pdf

Food and Drug Administration. 1999. Guidance for Industry: In Vivo Drug Metabolism/Drug
Interaction Studies Study Design, Data Analysis, and Recommendations for Dosing and
Labeling. http: //www.fda.gov/cder/guidance/2635fnl.pdf

Food and Drug Administration. 2001 Guidance for Industry: Bioanalytical Method Validation.
http: www.fda. gov/cder/guidance/4252fnl.pdf

Frye RF (2004) Probing the world of cytochrome P450 enzymes. MolInterv 4:157-162.

Gitau EN, Muchohi SN, Ogutu BR, Githiga IM and Kokwaro GO (2004) Selective and sensitive
liquid chromatographic assay of amodiaquine and desethylamodiaquine in whole blood
spotted on filter paper. J Chromatogr B Analyt Technol Biomed Life Sci 799:173-177.

Hombhanje FW, Hwaihwanje I, Tsukahara T, Saruwatari J, Nakagawa M, Osawa H, Paniu MM,
Takahashi N, Lum JK, Aumora B, Masta A, Sapuri M, Kobayakawa T, Kaneko A and
Ishizaki T (2005) The disposition of oral amodiaquine in Papua New Guinean children with
falciparum malaria. Br J Clin Pharmacol 59:298-301.

Jaakkola T, Backman JT, Neuvonen M and Neuvonen PJ (2005) Effects of gemfibrozil,
itraconazole, and their combination on the pharmacokinetics of pioglitazone. Clin Pharmacol
Ther 77:404-414.









Jaakkola T, Laitila J, Neuvonen PJ and Backman JT (2006) Pioglitazone is metabolised by
CYP2C8 and CYP3A4 in vitro: potential for interactions with CYP2C8 inhibitors. Basic Clin
Pharmacol Toxicol 99:44-51.

Jewell H, Maggs JL, Harrison AC, O'Neill PM, Ruscoe JE and Park BK (1995) Role of hepatic
metabolism in the bioactivation and detoxication of amodiaquine. Xenobiotica 25:199-217.

Kajosaari LI, Laitila J, Neuvonen PJ and Backman JT (2005) Metabolism of repaglinide by
CYP2C8 and CYP3A4 in vitro: effect of fibrates and rifampicin. Basic Clin Pharmacol
Toxicol 97:249-256.

Kiyota Y, Kondo T, Maeshiba Y, Hashimoto A, Yamashita K, Yoshimura Y, Motohashi M and
Tanayama S (1997) Studies on the metabolism of the new antidiabetic agent pioglitazone.
Identification of metabolites in rats and dogs. Arzneimittelforschung 47:22-28.

Klose TS, Blaisdell JA and Goldstein JA (1999) Gene structure of CYP2C8 and extrahepatic
distribution of the human CYP2Cs. JBiochem Mol Toxicol 13:289-295.

Knupfer H, Schmidt R, Stanitz D, Brauckhoff M, Schonfelder M and Preiss R (2004) CYP2C
and IL-6 expression in breast cancer. Breast 13:28-34.

Laurent F, Saivin S, Chretien P, Magnaval JF, Peyron F, Sqalli A, Tufenkji AE, Coulais Y, Baba
H, Campistron G and et al. (1993) Pharmacokinetic and pharmacodynamic study of
amodiaquine and its two metabolites after a single oral dose in human volunteers.
Arzneimittelforschung 43:612-616.

Li XQ, Bjorkman A, Andersson TB, Ridderstrom M and Masimirembwa CM (2002)
Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by
CYP2C8: a new high affinity and turnover enzyme-specific probe substrate. JPharmacolExp
Ther 300:399-407.

Li XQ, Bjorkman A, Andersson TB, Gustafsson LL and Masimirembwa CM (2003)
Identification of human cytochrome P(450)s that metabolise anti-parasitic drugs and
predictions of in vivo drug hepatic clearance from in vitro data. Eur J Clin Pharmacol 59:429-
442.

Lin ZJ, Ji W, Desai-Krieger D and Shum L (2003) Simultaneous determination of pioglitazone
and its two active metabolites in human plasma by LC-MS/MS. JPharm BiomedAnal
33:101-108.

McSorley LC and Daly AK (2000) Identification of human cytochrome P450 isoforms that
contribute to all-trans-retinoic acid 4-hydroxylation. Biochem Pharmacol 60:517-526.

Minzi OM, Rais M, Svensson JO, Gustafsson LL and Ericsson O (2003) High-performance
liquid chromatographic method for determination of amodiaquine, chloroquine and their









monodesethyl metabolites in biological samples. J Chromatogr B Analyt Technol BiomedLife
Sci 783:473-480.

Mizushige K, Tsuji T and Noma T (2002) Pioglitazone: cardiovascular effects in prediabetic
patients. Cardiovasc Drug Rev 20:329-340.

Motten AG, Martinez LJ, Holt N, Sik RH, Reszka K, Chignell CF, Tonnesen HH and Roberts JE
(1999) Photophysical studies on antimalarial drugs. Photochem Photobiol 69:282-287.

Mount DL, Patchen LC, Nguyen-Dinh P, Barber AM, Schwartz IK and Churchill FC (1986)
Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid
chromatography with electrochemical detection. J Chromatogr 383:375-386.

Niemi M, Backman JT, Granfors M, Laitila J, Neuvonen M and Neuvonen PJ (2003a)
Gemfibrozil considerably increases the plasma concentrations of rosiglitazone. Diabetologia
46:1319-1323.

Niemi M, Backman JT, Neuvonen M and Neuvonen PJ (2003b) Effects of gemfibrozil,
itraconazole, and their combination on the pharmacokinetics and pharmacodynamics of
repaglinide: potentially hazardous interaction between gemfibrozil and repaglinide.
Diabetologia 46:347-351.

Nishimura M, Yaguti H, Yoshitsugu H, Naito S and Satoh T (2003) Tissue distribution of
mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time
reverse transcription PCR. Yakugaku Zasshi 123:369-375.

O'Donnell CJ, Grime K, Courtney P, Slee D and Riley RJ (2007) The development of a cocktail
CYP2B6, CYP2C8, and CYP3A5 inhibition assay and a preliminary assessment of utility in a
drug discovery setting. DrugMetab Dispos 35:381-385.

Ong CE, Coulter S, Birkett DJ, Bhasker CR and Miners JO (2000) The xenobiotic inhibitor
profile of cytochrome P4502C8. Br J Cin Pharmacol 50:573-580.

Parikh S, Ouedraogo JB, Goldstein JA, Rosenthal PJ and Kroetz DL (2007) Amodiaquine
metabolism is impaired by common polymorphisms in CYP2C8: implications for malaria
treatment in Africa. Clin Pharmacol Ther 82:197-203.

Pussard E, Verdier F, Blayo MC and Pocidalo JJ (1985a) [Biotransformation of amiodaquine and
prophylaxis of Plasmodium falciparum malaria]. C R Acad Sci III 301:383-385.

Pussard E, Verdier F, Faurisson F and Blayo MC (1985b) [Pharmacokinetics of amodiaquine and
prevention of Plasmodium falciparum malaria]. Bull Soc Pathol Exot Filiales 78:959-64.

Pussard E, Verdier F, Faurisson F, Scherrmann JM, Le Bras J and Blayo MC (1987) Disposition
of monodesethylamodiaquine after a single oral dose of amodiaquine and three regimens for
prophylaxis against Plasmodium falciparum malaria. Eur J Clin Pharmacol 33:409-414.









Rendic S and Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report summarizing
their reactions, substrates, inducers, and inhibitors. DrugMetab Rev 29:413-580.

Rower S, Bienzle U, Weise A, Lambertz U, Forst T, Otchwemah RN, Pfutzner A and
Mockenhaupt FP (2005) Short communication: high prevalence of the cytochrome P450
2C8*2 mutation in Northern Ghana. Trop MedInt Health 10:1271-1273.

Shen Z, Reed JR, Creighton M, Liu DQ, Tang YS, Hora DF, Feeney W, Szewczyk J, Bakhtiar R,
Franklin RB and Vincent SH (2003) Identification of novel metabolites of pioglitazone in rat
and dog. Xenobiotica 33:499-509.

Soyama A, Hanioka N, Saito Y, Murayama N, Ando M, Ozawa S and Sawada J (2002)
Amiodarone N-deethylation by CYP2C8 and its variants, CYP2C8*3 and CYP2C8 P404A.
Pharmacol Toxicol 91:174-178.

Taniguchi R, Kumai T, Matsumoto N, Watanabe M, Kamio K, Suzuki S and Kobayashi S (2005)
Utilization of human liver microsomes to explain individual differences in paclitaxel
metabolism by CYP2C8 and CYP3A4. JPharmacol Sci 97:83-90.

Thum T and Borlak J (2000) Gene expression in distinct regions of the heart. Lancet 355:979-
983.

Tomio A, Niemi M, Neuvonen PJ and Backman JT (2007) Trimethoprim and the CYP2C8*3
allele have opposite effects on the pharmacokinetics of pioglitazone. DrugMetab Dispos
36:(Fast forward Oct 3).

Trenholme GM, Williams RL, Patterson EC, Frischer H, Carson PE and Rieckmann KH (1974)
A method for the determination of amodiaquine. Bull World Health Organ 51:431-434.

Turpeinen M, Uusitalo J, Jalonen J and Pelkonen O (2005) Multiple P450 substrates in a single
run: rapid and comprehensive in vitro interaction assay. Eur JPharm Sci 24:123-132.

Venkatakrishnan K, von Moltke LL, Obach RS and Greenblatt DJ (2003) Drug metabolism and
drug interactions: application and clinical value of in vitro models. Curr DrugMetab 4:423-
459.

Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug
Metab Dispos 32:647-60.

Walsky RL, Gaman EA and Obach RS (2005) Examination of 209 drugs for inhibition of
cytochrome P450 2C8. J Clin Pharmacol 45:68-78.

Walle T, Walle UK, Kumar GN and Bhalla KN (1995) Taxol metabolism and disposition in
cancer patients. Drug Metab Dispos 23:506-512.









Winstanley P, Edwards G, Orme M and Breckenridge A (1987) The disposition of amodiaquine
in man after oral administration. Br J Clin Pharmacol 23:1-7.

Wienkers LC and Stevens JC (2003) Cytochrome P450 Reaction Phenotyping, in: Drug
Metabolizing Enzymes: Cytochrome P450 and Other Enzymes in Drug Discovery and
Development (Lee JS, Obach RS and Fisher MB eds), pp 255-310, FrontisMedia SA and
Marcel Dekker Inc, New York.

Yamazaki H, Shibata A, Suzuki M, Nakajima M, Shimada N, Guengerich FP and Yokoi T
(1999) Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450
2C8 and P-450 3A4 in human liver microsomes. DrugMetab Dispos 27:1260-1266.

Yki-Jarvinen H (2004) Thiazolidinediones. NEngl JMed 351:1106-1118.









BIOGRAPHICAL SKETCH

Prajakta Dravid was born in Maharashtra, India. In 2000, she received her B.S. in Pharmacy

from University of Pune, India. During her bachelors, she developed an interest in research and

development and followed her interest by getting M.S. degree in Pharmaceutics from the

National Institute of Pharmaceutical Education and Research (NIPER), India. Chasing her

interest in the area of pharmacokinetics and drug metabolism, Prajakta joined the Bioanalysis,

Drug Metabolism and Pharmacokinetics Department at Dr. Reddy's Laboratories in Hyderabad,

India. At Dr. Reddy's she gained extensive experience in the field of bioanalysis and preclinical

pharmacokinetics. After two years of industrial experience, she joined the PhD program at the

Department of Pharmaceutics, University of Florida in the August of 2004. Under the

supervision of Dr. Reginald Frye, she looked at the reaction phenotyping aspects of Cytochrome

P450 2C8 with a focus on substrate selection and inhibition profile. She received M.S. in

Pharmacy from University of Florida in December 2007 and pursuing her PhD in the same

program.





PAGE 1

1 CYTOCHROME P450 2C8 REACTION PHE NOTYPING: SUBSTRATE SELECTION AND INHIBITION PROFILE By PRAJAKTA V DRAVID A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Prajakta V Dravid

PAGE 3

3 To the sweet memories of my little heroes, Jayesh and Anay.

PAGE 4

4 ACKNOWLEDGMENTS Any project no matter how individual will almost certainly require input, assistance and encouragement from others, my project is no exception! I take this opportunity to thank my research supervisor, Dr. Reggie Frye, for his invaluable guidance and abundant help during my work on this project. This research project would not have been possible without his contribution and support. I would like to thank Dr. Gunther Hochhaus for being a member of my graduate committee. Members of Frye lab, Cheryl, Mohame d and Melonie, require a special mention. I would like to thank them for all the good times and laughter in the lab. I e xpress my gratitude to departments of Pharmacy Practice and Pharmace utics for providing me with financial support. Words fail to express my love and gratitude to my beloved parents and grandparents. I would like to thank them for their unconditi onal support and encouragement. Needless to mention, I would not have seen this day wit hout the emotional support provided by my husband, Anand. He patiently embraced the lonely time an d encouraged me to complete my education. I thank him for standing by me in every decision I took.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION................................................................................................................. .....13 Cytochrome P450 Reaction Phenotyping...............................................................................13 The Centrality of Cytochrome P450 Enzymes in Drug Metabolism......................................14 Cytochrome P450 2C8............................................................................................................ 15 Paclitaxel: A Conventional Pr obe of CYP2C8 Activity.........................................................16 Amodiaquine: A High Affinity and Tu rnover Probe Substrate of CYP2C8..........................17 Pioglitazone as Probe Substrate of CYP2C8..........................................................................19 The Scope of Present Work....................................................................................................22 2 DETERMINATION OF N-DESETHYLAMODIAQUINE BY LC/MS/MS: APPLICATION TO IN VITRO DRUG METABOLISM STUDIES....................................23 Introduction................................................................................................................... ..........23 Experimental................................................................................................................... ........25 Chemicals and Reagents..................................................................................................25 Preparation of DEAQ Standards and Quality Control Samples......................................25 Sample Preparation..........................................................................................................26 LC/MS/MS Conditions....................................................................................................26 Validation..................................................................................................................... ...27 Incubation Conditions.....................................................................................................28 Data Analysis.................................................................................................................. .29 Results and Discussion......................................................................................................... ..29 Method Development......................................................................................................29 Method Validation...........................................................................................................30 Selectivity, carry over and matrix effect..................................................................30 Linearity...................................................................................................................32 Precision and accuracy.............................................................................................32 Autosampler stability...............................................................................................33 Metabolism of AQ in Pooled Human Liver Microsomes...............................................33 Conclusion..................................................................................................................... .........34 3 DETERMINATION OF HYDROXYPIOGL ITAZONE (M-IV) BY LC/MS/MS: APPLICATION TO IN VITRO DRUG METABOLISM STUDIES....................................36

PAGE 6

6 Introduction................................................................................................................... ..........36 Experimental................................................................................................................... ........39 Chemicals and Reagents..................................................................................................39 Preparation of M-IV Standards and Quality Control samples.........................................39 Sample Preparation..........................................................................................................40 LC/MS/MS Conditions....................................................................................................40 Validation..................................................................................................................... ...42 Incubation Conditions.....................................................................................................42 Results and Discussion......................................................................................................... ..43 Method Development......................................................................................................43 Method Validation...........................................................................................................44 Selectivity, carry over and matrix effect..................................................................44 Linearity...................................................................................................................46 Precision and accuracy.............................................................................................46 Autosampler stability...............................................................................................46 Formation of M-IV in Pooled Human Liver Microsomes...............................................47 Conclusion..................................................................................................................... .........48 4 DETERMINATION OF IC50 IN POOLED HUMAN LIVER MICROSOMES USING MIV FORMATION AS A CYP2 C8 SPECIFIC REACTION...................................................49 Introduction................................................................................................................... ..........49 Experimental................................................................................................................... ........50 Chemicals and Reagents..................................................................................................50 Incubation Conditions.....................................................................................................50 Analysis of Hydroxypioglitazone (M-IV).......................................................................51 Data Analysis.................................................................................................................. .52 Results and Discussion......................................................................................................... ..52 Conclusion..................................................................................................................... .........54 5 CONCLUSIONS AND FUTURE DIRECTIONS......................................................................56 LIST OF REFERENCES............................................................................................................. ..58 BIOGRAPHICAL SKETCH.........................................................................................................64

PAGE 7

7 LIST OF TABLES Table page 1-1.Substrates and inhibitors of CY P2C8 for in vitro experiments...............................................16 2-1.Intraday (n=6) and Interday (n=18) prec ision (%RSD) and accuracy (% deviation) for analysis of DEAQ in 50 mM phosphate buffer, pH 7.4.....................................................32 2-2.Enzyme kinetic parameters of AQ in pHLM...........................................................................34 3-1.Intraday (n=6) and Interday (n=18) prec ision (%RSD) and accuracy (% deviation) for analysis of M-IV in 50 mM phosphate buffer, pH 7.4.......................................................46 3-2.Enzyme kinetic parameters of M-IV formation in pHLM......................................................48

PAGE 8

8 LIST OF FIGURES Figure page 1-1.Proposed structures of pioglitazone metabolites in pHLM.....................................................21 2-1.Representative chromatogram s of (A) blank incubation bu ffer (red) and buffer spiked with DEAQ at LOQ (black) and (B) incuba tion sample: blank (red) and after 10 min incubation of AQ at 0.5 M (black) (m/z 328 283, overlay offset = 0%)....................31 2-2.Plot of initial velocity versus amodia quine concentration for the formation of desethylamodiaquine in pHLM (n=2)................................................................................34 3-1.Chemical structures of (A) pioglit azone and (B) hydroxypioglitazone (M-IV)......................37 3-2.Representative chromatogram s of (A) blank buffer (red) and buffer spiked with OH-PIO at low QC (black) and (B) incubation sample: blank (red) and after 10 min incubation of PIO at 10M (black) ( m/z 373 150, overlay offset = 0%)......................45 3-3.Plot of initial velocity versus pioglit azone concentration for the formation of hydroxypioglitazone (M-IV) in pHLM (n=2)....................................................................47 4-1.Structures of the compounds tested for inhibition of in vitro CYP2C8 activity.....................52 4-2.IC50 plots of pioglitazone hydroxylase inhib ition in pHLM by montelukast (circles), ketoconazole (squares), terfenadine (diamonds), -estradiol (triangles) and midazolam (reverse triangles). Data points reflect the average for incubations run in duplicate SE................................................................................................................. ...53

PAGE 9

9 LIST OF ABBREVIATIONS -NADP: -nicotinamide adenine dinucleotide phosphate ADR: Adverse drug reaction AQ: Amodiaquine AUC0: Area under the plasma concentration time curve CYP: Cytochrome P450 DEAQ: N -desethylamodiaquine DME: Drug metabolizing enzyme DMSO: Dimethyl sulfoxide ESI: Electrospray ionization FDA: Food and Drug Administration FWHM: Full width at half maximum HILIC: Hydrophilic interaction chromatography HPLC: High pressure li quid chromatography IC50: Concentration of the inhibitor that reduces the maximum initial velocity to 50% LC/MS/MS: Liquid chromatography tandem mass spectrometry LOQ: Limit of quantification M-IV Hydroxypioglitazone MRM: Multiple reaction monitoring NCE: New chemical entity NIDDM: Non insulin dependent diabetes mellitus PHLM: Pooled human liver microsomes PIO: Pioglitazone PPAR: Peroxisome prolifer ator activated receptor

PAGE 10

10 QC: Quality control RSD: Relative standard deviation SRM: Single reaction monitoring T1/2 Terminal elimination half-life UV: Ultraviolet WHO: World Health Organization

PAGE 11

11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science in Pharmacy CYTOCHROME P450 2C8 REACTION PHE NOTYPING: SUBSTRATE SELECTION AND INHIBITION PROFILE By Prajakta V Dravid December 2007 Chair: Reginald Frye Major: Pharmaceutical Sciences About 20% of the adverse drug reactions are related to drug-drug interactions caused by inhibition or induction of a drug metabolizing en zyme determining the elimination of a drug by another concomitantly administ ered drug. These drug-drug intera ctions could be predicted by identifying the major drug metabolizing enzyme s responsible for the metabolism of a new chemical entity. The reaction phenotyping of Cytochrome P450 2C8 (CYP2C8) did not gain enough attention due to the lack of an enzyme specific pr obe substrate and inhibitor. Paclitaxel is used as a conventional probe substrate of CYP2C8 in vitro Recently, amodiaquine (AQ) is reported to be a high affinity and turnover pr obe substrate of CYP2C8. However, it is not ideal for clinical drug metabolism studies due to its toxicity and very long elimination half-life of its CYP2C8 specific metabolite, N-desethyl amodiaquine (DEAQ). Pioglitazone (PIO) hydroxylation to form M-IV is also a CYP2 C8 specific reaction. We recognized a need for development of a LC/MS/MS based analytical method for determination of DEAQ and M-IV. The present work involves the development and validation of analytical methods for quantification of these two CYP2C8 specific metabolites. Further, we

PAGE 12

12 successfully applied these methods to in vitro drug metabolism studies using pooled human liver microsomes. The potential of M-IV formati on as a CYP2C8 specific probe reaction was evaluated by studying the inhibition of m ontelukast, ketoconazole, terfenadine, -estradiol and midazolam and comparing their IC50 values to those obtained by using other CYP2C8 specific probe reactions.

PAGE 13

13 CHAPTER 1 INTRODUCTION Cytochrome P450 Reaction Phenotyping Drug-drug interactions pose a si gnificant health concern ca using approximately 20% of adverse drug reactions (ADRs) (Backmann et al., 2003). Clea rly, the probability of ADRs associated with drug-drug interactions increase s as the number of concomitantly administered drugs increase (Bachmann et al., 2003). Inhibiti on of one or more drug metabolizing enzymes (DMEs) could result in an increased exposure of the parent drug and therefore, an increased chance of observing drug related toxicity. On th e other hand, induction of a DME might result in subtherapeutic response leading to failure of drug therapy (Venkatakris hnan et al., 2003; Frye, 2004). In order to avoid any unforeseen intera ctions in the clinic, the Food and Drug Administration (FDA) requires pharmaceutical companies to identify drug metabolizing enzymes involved in the elimination of a new chem ical entity (NCE) as well as the effect of its coadministration on the pharmacokinetics of marker substrates of DMEs that are responsible for its elimination (http://www.fda.gov/cder/guidan ce). Common experimental methods to identify the drug metabolizing enzymes responsible for th e metabolism of the NCE are as follows: (1) Chemical inhibitors: In order to determin e the contribution of a particular DME, the metabolism of the NCE is studied in presence of an enzyme selective chemical inhibitor. In this case, selectivity could be defined by two factor s, the mechanism of inhibition and the relative affinity of both the inhibitor and the test substrate for the enzyme. The limiting factor in the use of chemical inhibitors for in vitro studies has historically been the lack of adequate selectivity of inhibition among cytochrome P450 (CYP) enzymes. (2) Expressed CYP enzymes: The ability of a pa nel of expressed CYP enzymes to metabolize a specific NCE reduces reaction ph enotyping to the simplest system of only one enzyme and a

PAGE 14

14 substrate. Although these studies give important qualitative information, the importance of each CYP enzyme in presence of other enzymatic pa thways is difficult to quantify thus providing incomplete information about overall metabolic fate of the NCE. (3) Antibodies: The use of anti-CY P antibodies as biological inhibito rs of enzyme activity allows the direct assessment of the role of specific CY P to the metabolism of the NCE in an enzyme mixture like pooled human liver microsomes (pHLM). A primary limitation of anti-CYP antibodies for reaction phenotyping is their crossreactivity with related CYPs. Additionally, the inhibition of reactions known to be highly specific to a particul ar CYP isoform is rarely 100% and a small fraction of the enzyme retains its function. (4) Correlation analysis: It involves the comparison of the inter-liver variability in the rate of formation of a specific drug metabolite with the measured activitie s towards CYP marker substrates. This approach warrants prior establ ishment of enzyme kinetic parameters, a thorough investigation of the correlation between the diffe rent CYP enzymes and the use of considerable number of liver samples with wide range of enzyme activities (Wienkers and Stevens, 2003). The potential of a NCE to inhibit the metabo lism of currently available drugs could be determined by studying the metabolism of an enzyme specific probe substrate in the presence of the NCE. The identification of substrat es that are selectively metabolized in vitro by specific CYPs allows for the investigation of the inhibition by the NCE. The results from in vitro inhibition studies, yielding IC50 or Ki values allows for the predic tion of the potential for the NCE to inhibit specific CYPs in vivo Information thus obtained is us ed by the clinician to design in vivo interaction studies that are releva nt to the NCE (Bachmann et al., 2003). The Centrality of Cytochrome P450 Enzymes in Drug Metabolism Cytochrome P450 constitutes a superfamily of drug metabolizing enzymes that contributes to the Phase I metabolism of appr oximately three-forths of all drugs (Wilkinson, 2001). From the

PAGE 15

15 drug metabolism point of view, the most impor tant CYPs in humans are CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4/5. The CYP2C subfamily consists of four members namely, CYP2C 8, CYP2C9, CYP2C18 and CYP2C19 and is the second most abundant CYP subfamily after CYP3A, representing about 20% of the total hepatic P450 (Montellano, Handbook of Drug Metabolism, 1999). Collectively, the CYP2C subfamily is responsible for the metabolism of about 20% of c linically prescribed drug s. Clinical importance of CYP2C9 and CYP2C19 is well known and ther efore, these two isoforms are extensively studied and always included in the battery of enzymes used in reac tion phenotyping studies during the development of a new chemical en tity. However, until recently, CYP2C8 was often neglected due to the limit ed knowledge of its importance as well as the lack of CYP2C8 specific probe substrate and inhibitor. Cytochrome P450 2C8 CYP2C8 is a major human hepatic P450, const ituting about 7% of total microsomal CYP content in the liver. It is res ponsible of at least 5% of dr ugs cleared by Phase I metabolism (Rendic and Di Carlo, 1997). Alt hough it is primarily expressed in the liver, CYP2C8 protein is also found in kidney, intestine, adrenal glands, ma mmary glands, ovary, heart, aorta as well as in breast cancer tumors (Klose et al., 1999; Thum and Borlak, 2000; Ni shimura et al., 2003; Knupfer et al., 2004). Ferguson et al. found that the pregnane X rece ptor constitutive androstane receptor and glucocorticoid rece ptor are involved in the regulation of CYP2C8 gene expression (Ferguson et al., 2005). In humans, the CYP2C8 gene is located on chromosome 10q24, spanning 31 kilobases and consisting of nine exons. Single nucleotide poly morphisms have been identified in exons 3, 5 and 8 (Klose et al., 1999). The most common varient alleles are CYP2C8*2 and CYP2C8*3 The protein product CYP2C8.2 contains Ile269Phe subs titution and is expressed in African American

PAGE 16

16 population with an allle frequency of 18%. CYP2C8*3 has two amino acid susbstitutions, Arg139Lys and Lys399Arg. It is most commonly f ound in Caucasians with an allele frequency of 13% and rarely expressed in African Amer icans (2%) (Bahadur et al., 2002). Interestingly, CYP2C8*3 is in incomplete genetic equilibrium with CYP2C9*2 CYP2C8 plays an important role in the meta bolism of various drugs including paclitaxel (Dai et al., 2001; Bahadur et al., 2002), amodiaquine (AQ) (Li et al., 2002), troglitazone (Yamazaki et al., 1999), rosiglitazo ne (Baldwin et al., 1999), pi oglitazone (PIO) (Deng et al., 2005), repaglinide (Deng et al., 2005), cerivastatin (Backman et al., 2002), amiodarone (Soyama et al., 2002) and verapamil (Busse et al., 1995). It also contributes to the metabolism of various endogenous substances like all-trans retinoic acid (McSorley a nd Daly, 2000) and arachidonic acid (Dai et al., 2001). Common substr ates and inhibitors used in in vitro drug metabolism experiments are listed in Table 1-1. Table 1-1.Substrates and inhibitors of CYP2C8 for in vitro experiments. Substrates Inhibitors Paclitaxel Gemfibrozil Torsemide Trimethoprim Cerivastatin Montelukast Repaglinide Quercetin Amodiaquine Thiazolidinediones Pioglitazone Paclitaxel: A Conventional Probe of CYP2C8 Activity Paclitaxel, an antineoplastic agent, was origin ally isolated from th e stem bark of the western yew, Taxus brevifolia It is a potent inhibitor of cell re plication, blocking the cells in the late G2 mitotic phase of the cell cycle, pres umably by stabilizing th e microtubule cytoskeleton. As a therapeutic antineoplastic agen t, paclitaxel is used in pati ents with breast, lung, esophageal, head and neck, and advanced platinum-refractor y ovarian carcinomas (Wa lle et al., 1995). In

PAGE 17

17 humans, paclitaxel is primarily metabolized by CYP2C8 to 6-hydroxypaclitaxel and to lesser extent by CYP3A4 to form 3-p-hydroxypaclitaxel. Further, both metabolites are hydroxylated to lead to dihydroxypaclitaxel. Paclitaxel and its meta bolites are secreted in the bile and feces. The ratio of 6-hydroxypaclitaxel to 3-p-hydroxypaclitaxel is 6:1 in bile (Cresteil et al., 1994). Similarly, in pHLM, the concentration of 6-hydroxypaclitaxel was about two-fold that of 3-phydroxypaclitaxel suggesting the major role of CYP2C8 in the elimination of paclitaxel (Taniguchi et al., 2005). Th erefore, formation of 6-hydroxypaclitaxel is widely used as a marker substrate of CYP2C8 activity in reac tion phenotyping studies. Dai et al. found that CYP2C8.2 exhibits two-fold higher Km for paclitaxel 6-hydroxylation (31 M) compared to that of CYP2C8.1 (15 M) whereas CYP2C8.3 had a turnover nu mber of 15% that of CYP2C8.1 for paclitaxel 6-hydroxylation (Dai et al., 2001). Other re search groups indicated a moderate decrease (31 to 84 %) in the en zyme activity by presence of CYP2C8*3 allele (Bahadur et al., 2002; Soyama et al., 2002). Amodiaquine: A High Affinity and Turnover Probe Substrate of CYP2C8 Amodiaquine a 4-aminoquinolone antimalarial dr ug, is clinically effective against certain chloroquine resistant strains of Plasmodium falciparum However, it is no longer recommended for prophylactic antimalarial therapy due to the hi gh risk of agranulocytosis and hepatitis caused by the reactive quinine-imine meta bolite. Although the global use of AQ has declined due to the intrinsic toxicity, it is still be ing used as a first-line drug in the treatment of uncomplicated falciparum malaria, especially in African c ountries. In 2005, World Health Organization (WHO) recommended the use of AQ in combination with artemisinin-based antima larial therapy due to higher clinical efficacy of the combination, revita lizing the interest in the use of AQ in treating malaria.

PAGE 18

18 In vivo pharmacokinetic studies have shown that the primary route of systemic elimination in humans is via the extensive first-pass metabolism to N -desethylamodiaquine (DEAQ). Li et al. characterized the metabolism of AQ in various expressed CYP enzymes and pHLM. They found that AQ is almost exclusively metabolized by CYP2C8 to produce DEAQ (Li et al., 2002). Therefore, AQ clearance and its metabolism to DEAQ could be used as a measure of CYP2C8 enzyme activity for in vitro reaction phenotyping st udies. Recently, Walsky et al. examined 209 frequently prescribed drugs and related xenobio tics for inhibition of CY P2C8 using amodiaquine N-desethylation as a CYP2C8 specific mark er reaction. Forty-eight compounds exhibiting greater than 50% inhibition were furt her evaluated for determination of IC50 using expressed CYP2C8. In pHLM, the leukotrien e receptor antagonist, montelukast was found to be the most potent inhibitor of CYP2C8 with an IC50 of 19 nM (Walsky et al., 2005). In a recent report, Parikh et al. found th at the presence of CYP2C8*2 allele increased the Km of AQ metabolism by three-fold and decreased intrinsic clearance by six-fold. They also reported a marked decrease in the AQ metabolism with the presence of CYP2C8*3 allele (Parikh et al., 2007). Various analytical methods are described fo r determination of DEAQ (Trenholme et al., 1974; Pussard et al., 1985; Mount et al., 1986; Pussard et al., 1987; Winstanley et al., 1987; Laurent et al., 1993; Li et al ., 2002; Minzi et al., 2003; Dua et al., 2004; Gitau et al., 2004; Walsky and Obach, 2004; Bell et al., 2007; Dixit et al., 2007; O'Donnell et al., 2007). Earlier methods have long run times with high mobile phas e flow rates and lack the sensitivity required for in vitro drug metabolism assays. Most of the r ecent methods use highly aqueous mobile phases in order to improve the retention of the polar DEAQ on the column, which is not ideal for detection by mass spectrometry.

PAGE 19

19 Pioglitazone as Probe Substrate of CYP2C8 Pioglitazone, a thiazolidinedione, is used in the treatment of non-insulin dependent diabetes mellitus (NIDDM) as monotherapy or in combination with other hypoglycemic agents (e.g., sulfonylureas, metformin and insulin). Like other thiazolidinediones (e.g., troglitazone and rosiglitazone), PIO mediates its hypoglycemic e ffects by activation of peroxisome proliferator activated receptor (PPAR) thereby enhancing the sensitivit y of insulin responsive tissues rather than increasing release of insulin from islet -cells. In clinical stud ies, PIO is shown to produce antihyperglycemic effects by increasing in sulin stimulated glucos e uptake in peripheral tissues as well as the ability of insulin to suppress endogenous glucose production in the liver. It was also shown to decrease plasma levels of insulin, thus reducing the risk of hypoglycemia. From the toxicity point of view, PIO has a much better safely profile as compared to troglitazone and rosiglitazone. Additionally, PIO is found to exert hypolipidemic effects by reducing the serum concentrations of free fatty acids (Mizushige et al., 2002). After oral administration (30 mg once daily), PIO undergoes minimal first pass metabolism in the gut and is almost completely absorbed wi th an absolute bioavaila bility of 83%. Although it is distributed to peripheral tissues, plasma c oncentrations are always higher than tissues concentrations indicating a small volume of di stribution due to high plasma protein binding (>97%). In humans, PIO undergoes extensive metabolism in the liver to form various hydroxylated and oxidized metabolites (M-I to M-VII). Oxidative cleavage of aliphatic C-O bond leads to formation of M-I. M-II and hydr oxypioglitazone (M-IV) are formed by the hydroxylation of aliphatic methylene group whereas terminal ethyl group is hydroxylated to give M-VII and is oxidized to form M-V. Oxidati on of the hydroxyl group in M-IV to a ketone generates M-III (Yki-Jarvinen, 2004).

PAGE 20

20 In vitro studies in pHLM have shown that PIO un dergoes extensive metabolism in the liver primarily by CYP2C8, with mi nor contribution from CYP3A4. Th e metabolites M-II, M-III and M-IV are pharmacologically active possessing about 40-60% anti-hyperglycemic potency as compared to PIO. In humans, M-III and M-IV are found to be the major metabolites with considerably longer te rminal half lives (t1/2, 26-28 hours) than that of PIO (mean t 5.8 hours), presumably contributing to the extended pha rmacological activity allowing once-daily administration of PIO. In another study, a poten t and highly selective CYP2C8 inhibitor, montelukast (1 M), significantl y inhibited depletion of PIO (IC50 = 0.51 M) and more strongly inhibited formation of M-IV (IC50 = 0.18 M), clearly indicating th e major role of CYP2C8 in the formation of M-IV (Jaakkola et al., 2006). In a clinical drug interaction study, gemfibrozil alone raised the mean area under the pl asma concentration-time curve (AUC0) of PIO 3.2-fold and prolonged its t1/2 from 8.3 to 22.7 hours. Additionally, it also decreased the AUC0-48 of M-III and M-IV by 42% and 45%, respectively. Howeve r, itraconazole, a potent CYP3A4 inhibitor, did not have any significant effect on the pharmac okinetics of PIO or either of its metabolites suggesting a minor role of CYP3A4 in the fo rmation of its major plasma metabolite M-IV (Jaakkola et al., 2005). In a recent report, To rnio et al., found that trimethoprim, a known inhibitor of CYP2C8 increased the AUC0of PIO by 42% and reduced the apparent formation rate of M-IV by 27%, validating the major contri bution of CYP2C8 in the formation of M-IV (Tornio et al., 2007). From the above mentioned in vitro and in vivo drug interaction studies it is evident that M-IV is almost exclusively form ed by CYP2C8. Therefor e, formation of M-IV could be employed as a marker reaction for the quantification of CYP2C8 activity for in vitro reaction phenotyping studies as well as clinical drug interaction st udies. The proposed structures of PIO metabolites in pHLM are depicted in Figure 1-1. Tornio et al. also investigated the effect

PAGE 21

21 of carrying the CYP2C8*3 allele on the pharmacokinetics of PIO. The weight-adjusted AUC0of PIO was 34% lower in the subjects with the CYP2C8*3/*3 genotype and 26% lower in case of subjects carrying CYP2C8*1/*3 genotype (Tornio et al., 2007). Figure 1-1.Proposed structures of pioglitazone metabolites in pHLM. Several analytical methods are available fo r the quantification of M-IV in various biological fluids (e.g., plasma and urine) (Kiyot a et al., 1997; Lin et al ., 2003; Deng et al., 2005; Jaakkola et al., 2005; Tornio et al., 2007) and subcellular fr actions (e.g., recombinant CYP enzymes and pHLM) (Shen et al., 2003; Baughman et al., 2005; Jaakkola et al., 2006; Tornio et al., 2007). Most of these methods involve slow mobile phase grad ients with long run times as they were developed for charac terization of all of the metabol ites. Many of the recent methods S N H O O O N CH3 OH S N H O O O N CH3 S N H O O OH S N H O O O N CH3 O S N H O O O N CH3 O H S N H O O O N C O OH S N H O O O N C O H O S N H O O O N CH2 OH Pioglitazone M-II M-I M-III M-IV M-V M-VI M-VII

PAGE 22

22 are either not sensitive enough for the detection of metabolites or assign arbitrary units to M-IV due to the lack of commercially available meta bolite standard at the time of development. The Scope of Present Work Considering the limitations of the current an alytical methods for determination of DEAQ and potential application of DEAQ formation as a CYP2C8 specific reaction for in vitro reaction phenotyping studies, there is a need for devel opment of a simple, sensitive and robust mass spectrometric method that could be easily appl ied to drug metabolism studies. Therefore, the current work involves development and validat ion of a liquid chroma tography tandem mass spectrometry (LC/MS/MS) based method for determ ination of DEAQ and its application to drug metabolism studies using pHLM. Despite the number of drug interaction studies involving PIO, until recently, very little was known about the affinity of PIO towards CYP2C8. Therefore, one of the aims of the present work is to characterize the formation of M-IV with respect to Michaelis-Menten kinetics in pHLM. In order to facilitate the detection of M-IV, we developed and validated a LC/MS/MS based method that can be applied to drug meta bolism studies. Additionally, the focus of the current work was to evaluate the potential of PI O as a CYP2C8 specific substrate by comparing the IC50 values of montel ukast, ketoconazole, -estradiol, midazolam and terfenadine with those obtained using AQ.

PAGE 23

23 CHAPTER 2 DETERMINATION OF N-DESETHYLAMODIAQUI NE BY LC/MS/MS: APPLICATION TO IN VITRO DRUG METABOLISM STUDIES Introduction Amodiaquine, a 4-aminoquinolone antimalarial drug, is clinically effec tive against certain chloroquine resistant strains of Plasmodium falciparum Although the global use of AQ has declined due to the the high risk of agranulocytosis and hepatiti s caused by the reactive quinineimine metabolite (Jewell et al., 1995), it is still bei ng used as a first-line drug in the treatment of uncomplicated falciparum malaria, especially in African countries (Basco et al., 2002; Cavaco et al., 2005; Hombhanje et al., 2005; Rower et al ., 2005). After oral administration, AQ undergoes rapid and extensive metabolism in the liver to form the pharmacologi cally active metabolite DEAQ, which is primarily responsib le for the antimalarial effect s (Winstanley et al., 1987). In humans, desethylation of AQ is the major pathwa y of elimination with other minor metabolites being 2-hydroxyl DEAQ and N-bi sdesethylAQ (Pussard et al ., 1985a; Pussard et al., 1987; Laurent et al., 1993; Jewell et al., 1995). Studies in pHLM and recombinant enzymes show that AQ desethylation is almost exclusively cataly zed by Cytochrome P450 2C8 (Li et al., 2002). Therefore, AQ is used as an enzyme-selectiv e probe substrate to quantify CYP2C8 enzyme activity in vitro (Li et al., 2002). Several analytical methods are available for quantification of DEAQ in various biological fluids (e.g., blood, plasma and urine) and subce llular fractions (e.g., pHLM) (Trenholme et al., 1974; Pussard et al., 1985b; Mount et al., 1986; Pu ssard et al., 1987; Wins tanley et al., 1987; Laurent et al., 1993; Li et al ., 2002; Minzi et al., 2003; Dua et al., 2004; Gitau et al., 2004; Walsky and Obach, 2004; Bell et al., 2007). Earl y reverse phase chromatographic methods suffered from poor retention of AQ and DEAQ, l ong run times and high mobile phase flow rates (Pussard et al., 1985b; Gitau et al., 2004). Anal ytical methods based on UV detection did not

PAGE 24

24 permit the accuracy and sensitivity required fo r the quantification of the analytes due to endogenous interferences from the biological matrices as a resu lt of poor baseline resolution between AQ and its metabolites. Additionally, al l of these methods involved tedious multiple extraction steps and large volumes of organic solvents (Pussard et al., 1987; Winstanley et al., 1987; Laurent et al., 1993; Minzi et al., 2003; Dua et al., 2004; G itau et al., 2004; Bell et al., 2007). Higher sensitivity was achieved by Trenhol me and coworkers through conversion of AQ to a fluorescent product by re fluxing it with borate buffe r. Although this normal phase chromatographic method improved sensitivity an d retention, it was found to be non-specific because the concentration of AQ was confounde d by its metabolites (Trenholme et al., 1974). Mount and coworkers developed the most sens itive method for assaying DEAQ in human blood and urine (LOQ 1 ng/ml) by employing electroc hemical detection (M ount et al., 1986). However, it involved lengthy extraction steps an d consumed high amounts of organic solvents making it unsuitable for analysis of large number of samples. Considering the prospects of the use of amodiaquine as a CYP2C8 probe subs trate in drug metabolism studies, high throughput LC/MS/MS based methods were developed for analysis of DEAQ (Li et al., 2002; Walsky and Obach, 2004; Turpeinen et al., 2005; Walsky et al., 2005; Dixit et al., 2007; O'Donnell et al., 2007). All of these methods use simple processing methods and are sensitive enough for determination of DE AQ concentration in in vitro assays as well as clin ical studies. However, DEAQ was separated on reverse pha se columns resulting in the use of highly aqueous mobile phase gradients to prolong re tention of DEAQ, which is not ideal for mass spectrometric detection. Additionally, they involved separation of DEAQ by gradient elution with long run times. Thus, a simple, sensitive and robust mass spectrometric method that could be easily applied to drug metabolism studies is needed.

PAGE 25

25 The purpose of the present work was to develop a LC/MS/MS method using hydrophilic interaction chromatography (HILIC) that i nvolved minimal sample preparation. HILIC chromatography yielded excellent separation of AQ from DEAQ by prolonging DEAQ retention time while using high proportions of organic solv ent in the mobile phase. The method was used to determine enzyme kinetic parameters for DEAQ formation in pHLM. Experimental Chemicals and Reagents Amodiaquine, -nicotinamide adenine dinucleotide phosphate ( NADP), glucose-6phosphate, glucose-6-phosphate dehydrogenase, magnesium chloride and ammonium acetate were purchased from Sigma (St. Louis, MO USA). The DEAQ meta bolite standard and deuterated internal standard, DEAQ-d3, were obtained from BD -Gentest (San Jose, CA, USA). Potassium phosphate, sodium citrate and dimet hyl sulfoxide (DMSO) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Glucose 6-phosphate dehydrogenase solution was prepared by dissolving lyophilized enzyme in 5 mM sodium citrate to produce 40 U/ml solution and was stored at -20C until use. All chemicals used in the study were of analytical grade. HPLC grade acetonitrile was obtained from EMD Chemicals (Gibbstown, NJ, USA). Deionized water was prepared by using a Barnstead Nanopure Diamond UV Ultrapure Water System (Dubuque, IA, USA). Pooled HLM were purchased from BD Biosciences (San Jose, CA, USA). Preparation of DEAQ Standards and Quality Control Samples Two sets of stock solutions were prepared in acetonitrile and water (50:50 v/v) at concentrations of 0.1 and 0.5 mM. On e set of stock solutions was us ed to spike standards and the other set was used to spike quali ty control (QC) samples. Sta ndards were prepared by spiking phosphate buffer (50 mM, pH 7.4) at seven concen trations ranging from 10 nM to 1500 nM. For validation, QC samples were prepared by spik ing phosphate buffer (50 mM, pH 7.4) at three

PAGE 26

26 concentration levels (50, 500 and 1200 nM). The st andards and QC samples were stored at -20C until analysis. The internal standard solu tion was prepared by dissolving DEAQ-d3 in acetonitrile to produce a final concentr ation of 200 nM and stored at 4 C. Sample Preparation The internal standard solution in aceton itrile (200 nM, 1400 l) was added to DEAQ standard or QC sample (250 l). After shaking for 2 minutes on a vortex shaker, samples were centrifuged at 20,817 g for 8 minutes. An aliquot of clear supernatant was tr ansferred to a 96 well plate and 10 l was injected on the column. All samples were protected from light exposure during processing in order to avoid ph otodecomposition (Motten et al., 1999). LC/MS/MS Conditions The LC system was comprised of a Thermo Finnigan Surveyor HPLC autosampler and ThermoFinnigan Surveyor MS quaternary pump. Chromatographic separation was achieved on a BETASIL Silica-100 (50 x 2.1 mm, 5, ThermoElectron Corporat ion) analytical column. Isocratic elution was performed at a flow rate of 220 l/min for 4.7 minutes using a mobile phase consisting of 5 mM ammonium acetate and 0.1 % (v/v) formic acid in water and 5 mM ammonium acetate and 0.1% (v/v) formic acid in acetonitrile (15:85 v/v). The autosampler was maintained at 10C and 10 l of sample was injected on the column. The mobile phase flow was diverted from the mass spectrometer to waste fo r the first 1.5 minutes of run time to remove nonvolatile salts. After each inj ection, the needle was washed and flushed with 1000 l of solution containing: acetonitrile:2-propanol:water (35:35:30 v/v) and 0.1% (v/v) formic acid. The mass spectrometer was a TSQ Quan tum Discovery triple quadrupole mass spectrometer equipped with electrospray ioniza tion (ESI) source. The mass spectrometer was calibrated with a solution of polytyrosine-1, 3, 6 per manufacturers instructions. The operating

PAGE 27

27 conditions were optimized by infusing DEAQ in the mobile phase in order to maximize the detector signal. ESI source was operated in po sitive mode and was set orthogonal to the ion transfer capillary tube. For quantification, the TSQ quantum was operate d in multiple reaction monitoring (MRM) mode and the precursor-produ ct ion pair was 328 283 m/z for DEAQ and 331 283 m/z for DEAQ-d3. The acquisition parameters were: spray voltage 4.0 kV, source CID -10 V, heated capillary temperature 325C and capillary offset 35V. Nitrogen was used as a sheath and auxiliary gas set to 35 and 10 (arbitrary units), respectively. The argon collision gas pressure was set to 1.5 mTorr. The collision energy was 24 eV for the analyte as well as the internal standard. The peak full width at half maximum (FWHM) was set at 0.2 Th and 0.7 Th for Q1 and Q3, respectively. Scan width was fixed to 0.1 Th fo r both SRM channels and scan time was set to 250 ms. Calibration curves were construc ted by linear regression of the p eak area ratio of analyte to that of the internal standard (Y-axis) and the nominal standard concentration (X-axis) with a weighting factor of 1/y2. Concentrations of QCs and incubati on samples were calculated by using the regression equation of the calibration curv e. Chromatographic peaks were quantified by using XcaliburTM software (version 1.4) and the peak area ratio of DEAQ to DEAQ-d3 was plotted against the nominal DEAQ concentrations. Validation The newly developed analytical method was va lidated with respect to selectivity, carry over, linearity, precision, accuracy and autosampler stability. For selectivity, samples of blank incubation ma trix were analyzed to check the lack of interference in the quantification of DEAQ. Ca rry-over was evaluated by placing vials of blank mobile phase at several locations in the analysis set.

PAGE 28

28 Standards at all concentrations were analyzed in duplicate except the LOQ, which was run in triplicate. To assess linearity, the maximum a llowable deviation of the back calculated concentration was set at 15% for all st andards and at 20% for the LOQ. The accuracy and precision of the assay was de termined by the analysis of QC samples of DEAQ at concentrations of 50.0, 500.0 and 1200.0 nM. Six of each QC sample were analyzed on the same day to determine intra-day precision an d accuracy, and on three di fferent occasions to assess inter-day precision and accuracy. Reanalysis of standards and QC samples tested the stability of the analyses. The stability of the samples in the autosampler was tested after the samples were left in the autosampler for up to 36 hours by reanalyzing the standards and QC sample s. Stability was defined as less than 10% deviation in concentration from that on the day samples were processed. Incubation Conditions Preliminary experiments were conducted to op timize the microsomal protein concentration (0.01-0.2 mg/ml) and incubation time (5-20 minutes) in order to assure the linearity of DEAQ formation Amodiaquine and pHLM (0.1 mg/ml) were mixed with phosphate buffer (50 mM, pH 7.4) and warmed at 37C for five minutes. Incubations were commenced by addition of the NADPH regenerating system, which consisted of MgCl2 (assay concentration, 3.3 mM), NADP+ (1.25 mM), glucose 6-phosphate (3.3 mM) and glucose 6-phosphate dehydrogenase (0.32 U/ml) in 5 mM sodium citrate solution. Final inc ubation volume was 250 l. After incubating for 10 minutes at 37C, the reaction was terminated by additi on of 1400 l of ice-cold acetonitrile containing DEAQ-d3 (0.28 nmol). Samples were processed as described above. Enzyme kinetic parameters were obtained by performing incubations at nine different concentrations of AQ ranging from 0.5 M to 80 M. AQ was dissolved in acetonitrile and water (50:50 v/v, final acetonitril e concentration of 0.4% v/v). Mi crosomes were stored at -80C

PAGE 29

29 and thawed immediately before us e. Polypropylene microcentrifuge tubes were used to store AQ stocks as well as to conduct the microsomal incubations. All incubations were performed in duplicate and were protected from light to av oid photodecomposition of AQ and the metabolite. Data Analysis Enzyme kinetic parameters were obtained by nonlinear regression using GraphPad Prism (San Diego, CA, USA). Data were typically f it to the following Michaelis-Menten equation: S K S V Vm max Equation 2-1 in which V is the initial velocity, Vmax is the maximal velocity, S is the substrate concentration and Km is the substrate concentration at half-maximal velocity. Results and Discussion Method Development In order to improve the retention of DEAQ a nd to avoid the use of highly aqueous mobile phases, we selected BETASIL Silica-100 (50 x 2.1 mm, 5 ) column, which separates analytes based on the principles of HILIC. It elutes analytes by passing a hydrophobic or mostly organic mobile phase across a neutral hydrop hilic stationary phase causing solutes to elute in order of increasing hydrophilicity resulti ng in better separation of highly polar compounds. Alternative to reverse phase chromatography, HILIC worked best for separation of DE AQ (retention time, 2.9 min) from AQ (retention time, 1.2 min) while stil l allowing use of 85% acetonitrile in the mobile phase. Ammonium acetate buffer was used to volatili ze the mobile phase and aid ionization of DEAQ. Addition of 0.1% (v/v) form ic acid in the mobile phase also enhanced ionization and improved the peak shape of DEAQ. ESI was chosen as the mode of ionization because it gave high signal intensity for DEAQ. A full scan mass spectrum of DEAQ was obtained in the

PAGE 30

30 positive and negative mode. The most abundant parent ion of DEAQ (328 m/z ) was obtained in the negative mode, which was selected for SRM s canning. Further, the frag mentation pattern of the precursor ion was obtained a nd a highly specific ion pair (328 283 m/z ) was selected based on the intensities of three most abundant product ions. Thus, for quantification purpose, TSQ quantum was operated in MRM mode a nd the precursor-product ion pair of 328 283 m/z and 331 283 m/z was followed for DEAQ and DEAQ-d3, resp ectively. In order to minimize the sample preparation time, a one step protein precipitation method was u tilized by addition of a solution of internal standard in acetonitrile followed by a short mixing and centrifugation step. Considering the simplicity of sample processing, the present method could potentially be applied to a high throughput drug metabolism assay. Method Validation Validation of the assay method was conducte d according to the FDA guidelines with respect to selectivity, carry over, linearity, precision, accuracy and autosampler stability (http://www.fda.gov/cder/guidance) For validation purposes, QC samples at low, medium and high concentrations were prepared independently and six of each QC sample was analyzed on three occasions. Selectivity, carry over and matrix effect To determine the selectivity of the method, blank microsomal incubation samples were used to investigate the potentia l interferences due to the endo genous compounds in the matrix. A clear baseline was observed without any significant interference at the retention times of DEAQ and DEAQ-d3. Representative chromatograms of (A ) blank buffer and buffer spiked with DEAQ at LOQ (10 nM) and (B) blank incubation sample and a sample after 10 minute incubation of AQ at 0.5 M are depicted in Figure 2-1. No carry over was observed in any of the blank samples. The potential of ion suppression or enhancement due to the matrix components was evaluated for

PAGE 31

31 DEAQ and DEAQ-d3. No significant matrix effect was observed in microsomal incubations, as the use of a deuterated internal standard co mpensated for any variation in matrix effect. Figure 2-1.Representative chromatograms of (A) blank incubation buffer (red) and buffer spiked with DEAQ at LOQ (black) and (B) incuba tion sample: blank (red) and after 10 min incubation of AQ at 0.5 M (black) (m/z 328 283, overlay offset = 0%). A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 2.86 1.35 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 3.02 B

PAGE 32

32 Linearity Calibration curve demonstrated good linearity for the entire concentration range with a mean correlation coefficient (R2) of 0.9969 0.0012. Precision and accuracy Precision was represented as the relative sta ndard deviation (%RSD) whereas accuracy was calculated as the percent deviation (% bias) from the respective nominal concentration. The maximum acceptable limit for precision and accuracy was set at 15%. The intra-day and interday precision and accuracy were within 7.9% an d 4.3%, respectively, for all standards and QC samples (Table 2-1). Thus, the present me thod was found to be hi ghly reproducible and demonstrated a high degree of accuracy. Table 2-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for analysis of DEAQ in 50 mM phosphate buffer, pH 7.4. Concentration (nM) Nominal Back calculated (mean SD) % RSD % Deviation Intraday 50.00 51.70 0.7 1.4 3.4 500.0 492.8 9.1 1.8 -1.4 1200 1181 28 2.4 -1.5 Interday 50.00 51.20 3.6 7.0 2.5 500.0 487.9 13 2.6 -2.4 1200 1209 83 6.9 0.8

PAGE 33

33 Autosampler stability As the autosampler was maintained at 10oC during the run, stability of analytes at 10oC was determined by reanalyzing the same standa rds and QC samples after 36 hours. Both DEAQ and DEAQ-d3 were found to be stable at 10oC for at least 36 hours. Metabolism of AQ in Pooled Human Liver Microsomes AQ was incubated with pHLM at nine different concentrations (0.5 -80 M). The linearity of AQ metabolism with respect to the microsomal protein content was st udied at five protein concentrations (0.01-0.2 mg/ml). In order to avoid non-specific protei n binding, the lowest protein concentration that produced quantifiable metabolite (0.1 mg/ml) was selected. Formation of DEAQ was linear up to 20 minutes. Consideri ng the photosensitivity of AQ, samples were protected from light. Following the incubation of AQ in pHLM, DEAQ was detected by using the validated method. The rate of formation of DEAQ was measured as the index of CYP2C8 enzyme activity (Li et al., 2002; Walsky and Ob ach, 2004; Turpeinen et al., 2005; Walsky et al., 2005; Dixit et al., 2007; O'Donnell et al., 2007) When modeled by using GraphPad Prism (version 4), the formation of DEAQ exhibited ty pical Michaelis-Menten ki netics (Figure 2-2) with the maximal rate of formation of DEAQ (Vmax) of 2060 94 pmol/min/mg protein and the concentration of AQ at ha lf maximal velocity (Km) was 5.78 0.94. In a recent study, Li et al., characterized the disappearance of AQ as well as the formation of DEAQ in various expressed CYP enzymes and pHLM. They reported AQ to be a high affinity and tu rnover probe substrate of CYP2C8. Our results are in agreement with the findings of the study conducted by Li et al recommending the use of DEAQ formation as a CY P2C8 specific probe reac tion (Table 2-2) (Li et al., 2002; Li et al., 2003; Walsky and Obach, 2004). Thus, the present method was successfully applied to in vitro drug metabolism studies.

PAGE 34

34 Figure 2-2. Plot of initial ve locity versus amodiaquine conc entration for the formation of desethylamodiaquine in pHLM (n=2). Table 2-2.Enzyme kinetic parameters of AQ in pHLM. Km SE (M) Vmax (pmol/min/mg protein) In house 5.8 0.94 2060 94 Walsky and Obach, 2004 1.9 0.06 1480 20 Li et al., 2003 3.4 1696 Li et al., 2002 2.4 1462 Conclusion Application of HILIC techni que to separate DEAQ resulted in a simple and robust LC/MS/MS based method. The method was validated with respect to selectivity, carry over, matrix effects, linearity, precision, accuracy and autosampler stability. Enzyme kinetic 0 25 50 75 100 0 500 1000 1500 2000 2500Amodiaquine (M)Initial Velocity (pmol/min/mg protein)

PAGE 35

35 parameters obtained by incubati ng AQ with pHLM in presence of NADPH regenerating system were in accordance with the ava ilable literature. Therefore, the present method could be applied for future CYP2C8 drug metabolism studies.

PAGE 36

36 CHAPTER 3 DETERMINATION OF HYDROXYPIOGLIT AZONE (M-IV) BY LC/MS/MS: APPLICATION TO IN VITRO DRUG METABOLISM STUDIES Introduction Cytochrome P450 2C8 plays an important ro le in the metabolism of various drugs including paclitaxel (Dai et al., 2001; Bahadur et al., 2002), amodiaquine (Li et al., 2002), troglitazone (Yamazaki et al., 1999), rosiglitazone (Baldwin et al ., 1999), pioglitazone (Deng et al., 2005), repaglinide (Bidstrup et al., 2003; Kajo saari et al., 2005), ceriv astatin (Backman et al., 2002), amiodarone (Soyama et al., 2002), verapa mil (Busse et al., 1995), and endogenous substances like all-trans retinoic acid (McSorley and Dal y, 2000) and arachidonic acid (Dai et al., 2001). Therefore, it is important to know whet her a new drug candidate is a substrate or an inhibitor of CYP2C8. Consideri ng the contribution of CYP2C8 in the metabolism of various drugs and the impact of inhibition of CYP2C8 on the pharmacokinetics of its substrates, it has been added to the panel of CYP enzymes for re action phenotyping studies. Until recently, there was a lack of information about CYP2C8 specific reactions that could be used to quantify the activity of this enzyme. In vivo pharmacokinetic studies as well as in vitro experiments with expressed CYP2C8 and pHLM have shown that AQ is almost exclusively metabolized to DEAQ by CYP2C8. Therefore, amodiaquine desethylation is used as a CYP2C8 specific marker reaction to quantify CYP2C8 activity in vitro (Li et al., 2002). However, AQ cannot be used as in in vivo probe of CYP2C8 as it was withdrawn from the US market due to its intrin sic toxicity. Thus, there is a need to identify a CYP2C8 specific probe substrate that could be used for in vitro as well as in vivo reaction phenotyping studies. Pioglitazone, a thiazolidinedione antidiabetic agent, is widely used in the treatment of noninsulin dependent diabetes mellitus either as monotherapy or in combination with other

PAGE 37

37 hypoglycemic agents (e.g., metf ormin, sulfonylurea or insulin). Like other Peroxisome Proliferator Activated Receptor (PPAR ) agonists, pioglitazone mediat es its antidiabetic effects by increasing insulin stimulated glucose uptake in peripheral tissues as well as the ability of insulin to suppress endogenous glucose pr oduction in the liver (Yki-Jarvinen, 2004). In vitro studies in human liver microsomes have show n that PIO (Figure 3-1) undergoes extensive metabolism in the liver primarily by CYP2C8 and CYP3A4 to five primary metabolites (M-I, MII, M-IV, M-VI and M-VII). Further, M-IV (Figure 3-1) is oxidized to a ketone to form M-III, whereas oxidation of M-V leads to formation of M-VI (Shen et al., 2003 ; Baughman et al., 2005; Jaakkola et al., 2006). In humans, M-III and M-IV are found to be the major metabolites with about 40-60% hypoglycemic potency thus, contri buting significantly to the pharmacological activity of PIO. Figure 3-1.Chemical structures of (A) piogl itazone and (B) hydroxypioglitazone (M-IV) Studies with various recombinant CYP isof orms as well as pHLM have shown that montelukast, a potent and highly se lective CYP2C8 inhibitor, signi ficantly inhibited depletion of PIO (IC50 = 0.51 M) and more strongly i nhibited formation of M-IV (IC50 = 0.18 M) indicating the major role of CYP2 C8 in the formation of M-IV (Jaakkola et al., 2006). In human pharmacokinetics study, gemfibrozil alone raised the mean area under the plasma concentrationtime curve (AUC0) of PIO 3.2-fold and prolonged its elimination half-life (t1/2) from 8.3 to 22.7 S N H O O O N CH3 S N H O O O N CH3 O H

PAGE 38

38 hours. It also decreased the AUC0-48 of M-III and M-IV by 42% and 45%, respectively. The effect of gemfibrozil on the pharmacokinetics of PIO and its metabolites due to CYP2C8 inhibition could be attributed to the acylglucoronide of gemfibro zil. In the same study, a potent CYP3A4 inhibitor itraconazole did not have any significant effect on the pharmacokinetics of PIO or either of its metabolites indicating a minor role of CYP3A4 in the metabolism of PIO and formation of M-IV (Jaakkola et al., 2005). In a recent study, trimethoprim, a known inhibitor of CYP2C8, was found to increase AUC0of PIO by 42% and reduced th e apparent formation rate of M-IV by 27% validating the major role of CYP2 C8 in the formation of M-IV (Tornio et al., 2007). From above mentioned in vitro and in vivo drug interaction studies it is evident that CYP2C8 plays a major role in the formation of M-IV. Therefore, formation of M-IV could be employed as a marker reaction for quantificati on of CYP2C8 activity in reaction phenotyping studies. Several analytical methods are available fo r the quantification of M-IV in various biological fluids (e.g., plasma and urine) (Kiyot a et al., 1997; Lin et al ., 2003; Deng et al., 2005; Jaakkola et al., 2005; Tornio et al., 2007) and subcellular fr actions (e.g., recombinant CYP enzymes and liver microsomes) (Shen et al., 2003; Baughman et al., 2005; Jaakkola et al., 2006; Tornio et al., 2007). Most of the LC/MS/MS ba sed methods were developed with the purpose of identification and characterization of various me tabolites of PIO and therefore, involve slow mobile phase gradients with long run times. Many of these methods assigned arbitrary units to M-IV due to the lack of metabolite standard s at the time of development. More recent LC/MS/MS based methods used in drug interactio n studies in humans i nvolve sample processing by solid phase extraction and suffer from the disa dvantage of very long run times. Analytical methods developed by Lin et al and Deng et al separate PIO and its metabolites by isocratic

PAGE 39

39 elution and quantify M-IV base d on metabolite standards (Lin et al., 2003; Deng et al., 2005). Although both of these methods could be succes sfully employed for quantification of M-IV (LOQ of M-IV 0.5 and 1.1 ng/ml, re spectively) in pharmacokinetic studies in humans, they are not sensitive enough to be used for in vitro enzyme assays and inhi bition studies. Thus, the purpose of this study was to develop a rapid, se nsitive and robust method for determination of M-IV that could be easily applied to in vitro drug metabolism studies. Experimental Chemicals and Reagents -Nicotinamide adenine dinucleotide phosphate ( NADP), glucose-6-phosphate, glucose6-phosphate dehydrogenase, magnesium chloride and ammonium acetate were procured from Sigma (St. Louis, MO, USA). Pioglitazone hydroc hloride, M-IV metabol ite standard and the stable labeled internal standard, M-IV-d4, were obtained from Torronto Re search Chemicals Inc. (North York, Ontario, Canada). Glucose 6-phos phate dehydrogenase solution was prepared by dissolving lyophilized enzyme in 5 mM sodium citrate to produce 40 U/ml solution and was stored at -20C until use. Potassium phosphate was purchased from Fisher Scientific (Fair Lawn, NJ, USA) and formic acid was obtained from Mall inckrodt baker Inc. (Phillipsburg, NJ, USA). HPLC grade acetonitrile and methanol were pur chased from EMD Chemicals (Gibbstown, NJ, USA). All other chemicals used in the study were of analytical grade. Deionized water was obtained from a Barnstead Nanopure Diamond UV Ultrapure Water System (Dubuque, IA, USA). Pooled HLM were obtained from In Vitro technologies (Baltimore, MD, USA). Preparation of M-IV Standard s and Quality Control samples Two sets of stock solutions we re prepared in methanol at concentrations of 0.1 and 1.0 g/ml. One set of stock solutions was used to sp ike standards and the other set was used to spike QC samples. Standards were prepared by spik ing phosphate buffer (50 mM, pH 7.4) at seven

PAGE 40

40 concentrations ranging from 0.1 to 20 ng/ml. Fo r validation, QC samples were prepared by spiking phosphate buffer (50 mM, pH 7.4) at thre e concentration levels (spiked at 0.5, 2.0 and 10 ng/ml). The standards and QC samples were stored at -20C until analysis. The internal standard solution was prepared by dissolving M-IV-d4 in methanol to produce a final concentration of 12 ng/ml and was stored at -20C. Sample Preparation Acetonitrile (750 l) was added to 250 l of standard soluti on, QC or incubation sample followed by the addition of the inte rnal standard solution in meth anol (50 l; 12 ng/ml). Samples were processed by liquid-liquid extraction usi ng dichloromethane (2 ml) as the extraction solvent. After shaking for 10 minutes on a hor izontal shaker at low speed, samples were centrifuged at 3200 g for 10 minutes. The top aqueous layer was aspirated and the organic layer was transferred to glass tubes and dr ied under a stream of nitrogen at 50C. The obtained residue was redissolved in methanol: water (50:50, 150 l) and an aliquot was transferred to autosampler vials. LC/MS/MS Conditions The LC system was comprised of a Thermo Finnigan Surveyor HPLC autosampler and ThermoFinnigan Surveyor MS quaternary pump. Chromatographic separation was achieved on a SYNERGI MAX-RP 80A (150 x 2.00 mm, 4, Phen omenex) analytical column. Gradient elution was performed at a flow rate of 220 l/min using the following mobile phase system: A = 5 mM ammonium acetate and 0.1 % (v/v) formic acid and B = 5 mM ammonium acetate and 0.1 % (v/v) formic acid in methanol. The column wa s started at 40:60 of A: B and at 0.5 min, the mobile phase composition was changed to 90% of B over 0.5 minutes and held for 1.2 min (2.2 min total) before returning to the starting condi tions, which was held for 2.3 minutes (total run time 5.0 minutes). The autosampler was maintained at 10C and 20 l of sample was injected on

PAGE 41

41 the column. The mobile phase flow was diverted from the mass spectrometer to waste for the first 1.0 min of run time to remove nonvolatile salts. After each injection, the needle was washed and flushed with 500 l of solu tion containing: acetoni trile:2-propanol:water (35:35:30 v/v) and 0.1% (v/v) formic acid. The mass spectrometer was a TSQ Quan tum Discovery triple quadrupole mass spectrometer equipped with an ESI source. It was calibrated with a soluti on of polytyrosine-1, 3, 6 per manufacturers instructions. The operating conditions were optimized by infusing M-IV in the mobile phase in order to maximize the de tector signal. ESI source was operated in the positive mode and was set orthogonal to the ion transfer capillary tube. For quantification, the TSQ quantum was operate d in the MRM mode and the precursor-product ion pair was 373 150 m/z for M-IV and 377 154 m/z for M-IV-d4. The acquisition parameters were: spray voltage 4.0 kV, source CID -3 V, heated capillary temperature 325C and capillary offset 35 V. Nitrogen was used as a shea th and auxiliary gas set to 35 and 10 (arbitrary units), respectively. The argon collision gas pressu re was set to 1.5 mTorr. The collision energy was 35 eV for the analyte as we ll as the internal standard. The peak FWHM was set at 0.2 Th and 0.7 Th for Q1 and Q3, respectively. Scan width was fixed to 1.0 Th for both SRM channels and scan time was set to 250 ms. Chromatographic peaks were quantified using XcaliburTM software (version 1.4) and calibration curves were constructed by linear regression of the peak ar ea ratio of M-IV to that of M-IV-d4 (Y-axis) and the nominal standard concen tration (X-axis) with a weighting factor of 1/y2. The concentration in QCs and incubation sa mples was calculated by using the regression equation of the calibration curve.

PAGE 42

42 Validation The newly developed analytical method was va lidated with respect to selectivity, carry over, linearity, precision, accuracy and autosample r stability. For selectivity, samples of blank incubation matrix were analyzed to check for lack of interference at the retention time of M-IV and M-IV-d4. Carry-over was evaluated by placing vi als of methanol at several locations in the analysis set. Calibration curves were construc ted by plotting the ratio of the peak area of M-IV to that of M-IV-d4 against the nominal M-IV concentr ation. Standards at all concentrations were analyzed in duplicate except the LOQ, which wa s run in triplicate. To assess linearity, the maximum allowable deviation of the back calcu lated concentration was set at 15% for all standards and at 20% for LOQ. The accuracy and precision of the assay was de termined by the analysis of QC samples of M-IV at concentrations of 0.5, 2.0 and 10 ng/ml. Twelve of each QC sample were analyzed on the same day to determine intra-day precision an d accuracy, and six of ea ch QC sample on two different occasions to assess inter-day precision and accuracy. The stability of the samples in the autosampler was tested after the samples were left in the autosampler for up to 48 hours by reanalyzing th e standards and QC sa mples. Stability was defined as less than 10% deviation in concen tration from that on the day samples were processed. Incubation Conditions Preliminary experiments were conducted to op timize the microsomal protein concentration (0.05-0.4 mg/ml) and incubation time (5-20 minutes) in order to assure th e linearity of M-IV formation. Pioglitazone and pHLM (0.05 mg/ml) were mixed with phosphate buffer (50 mM, pH 7.4) and warmed at 37C for five minutes. Incubations we re commenced by addition of the

PAGE 43

43 NADPH regenerating system, which consisted of MgCl2 (assay concentration, 3.3 mM), NADP+ (1.25 mM), glucose 6-phosphate (3.3 mM) and glucose 6-phosphate dehydrogenase (0.32 U/ml) in 5 mM sodium citrate solution. Final inc ubation volume was 250 l. After incubating for 10 minutes at 37C, the reaction was terminated by additi on of 750 l of ice-cold acetonitrile containing internal standard and sample s were processed as described above. Enzyme kinetic parameters were obtained by performing incubations at nine different concentrations of PIO ranging from 0.25 M to 25 M. Pioglitazone was dissolved in acetonitrile and methanol (90:10 v/v, final or ganic solvent concentration of 0.8% v/v corresponding to final methanol content 0.08%). Microsomes were stored at -80C and thawed immediately before use. Polypropylene microcentrif uge tubes were used to store PIO stocks and microsomal incubations were conducted in po lypropylene tubes (4 ml) to minimize any nonspecific binding. All incubations were performed in duplicate. Results and Discussion Method Development Pioglitazone is primarily metabolized by CY P2C8 and CYP3A4 to four hydroxylated (MII, M-IV, M-VII and M-VIII) and various other me tabolites. Baughman et al have identified MII, M-IV, M-VII and M-VIII in freshly isolated human hepatocytes and in pHLM (Baughman et al., 2005). Shen et al. (Shen et al., 2003) also reported formation of MVII along with M-IV in various preclinical species and pHLM. In the LC/M S/MS based method described by Shen et al., M-VII elutes very close (retention time 38.3 min) to M-IV (40.2 min) in a total of 90 min run time (Shen et al., 2003). Although these metabolites differ in the position of hydroxylation they have similar fragmentation pattern and therefore, possess the same transition pair of Q1 and Q3 (373 150 m/z ). Therefore, it was important to sepa rate M-IV from M-VII on the analytical column. With the purpose of shortening the run time while achieving goo d baseline resolution

PAGE 44

44 between M-IV and M-VII, we selected SYNERGI MAX-RP 80A (150 x 2.00 mm, 4 Phenomenex) column. We were able to achieve baseline resolution keeping the run time relatively short (5 minutes). M-IV el uted at 2.7 minutes on the column. A mobile phase consisting of ammonium acet ate buffer was used to aid ionization of MIV. Addition of 0.1% (v/v) formic acid in the mobile phase also enhanced ionization and improved the peak shape of M-IV. A full scan mass spectrum of M-IV was obtained in the positive and negative mode. The most abundant parent ion of M-IV (373 m/z ) was obtained in the positive mode and th e specific ion pair (373 150 m/z ) was selected based on the intensities of three most abundant product ions. Thus, fo r quantification purpose, the TSQ quantum was operated in MRM mode and the pr ecursor-product ion pair of 373 150 m/z and 377 154 m/z was followed for M-IV and M-IV-d4, respectivel y. In order to improv e sensitivity of the method, M-IV was extracted by a liquid-liquid extraction method. Various solvents including acetonitrile, methyl tert butyl ether, dichlorometh ane, ethyl acetate and their combinations were used as extraction solvents. The highest and most consistent recovery was with dichloromethane (lower limit of quantification, LOQ 0.1 ng/ml), wh ich was selected as the extraction solvent for future experiments. Considering the high sensi tivity, simplicity of sample processing and short run time, the present method could potentially be applied to drug metabolism assays in future. Method Validation Validation of the assay method was conducte d according to the FDA guidelines with respect to selectivity, carry over, linearity, precision, accuracy and autosampler stability (http://www.fda.gov/cder/guidance). Selectivity, carry over and matrix effect To determine the selectivity of the method, blank microsomal incubation samples were used to investigate the potent ial interferences due to the e ndogenous compounds in the matrix.

PAGE 45

45 The present method demonstrated high degree of selectivity by means of MRM mode. A clear baseline was observed without a ny significant interference at the retention times of M-IV and MIV-d4. The ratio of signal to noise obtained from an extracted standard at LOQ (0.1 ng/ml) was at least 50 for M-IV. Representative chromatogr ams of (A) blank buffer and buffer spiked with M-IV at the low QC (0.5 ng/ml) and (B) blank incubation sample and a sample after 10 minute incubation of PIO at 10 M are depicted in Figure 3-2. No carry over was observed in any of the blank samples. Figure 3-2. Representative chromatograms of (A ) blank buffer (red) and buffer spiked with OHPIO at low QC (black) and (B) incubati on sample: blank (red) and after 10 min incubation of PIO at 10M (black) ( m/z 373 150, overlay offset = 0%). A B 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 2.76 1.14 2.46 3.65 1.94 3.85 2.10 1.82 0.31 0.89 0.54 2.73 3.24 3.14 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance 2.73 2.19 3.69 3.49 1.141.32 1.71 0.13 0.75 0.59 2.74 3.69 3.06 2.17 1.96

PAGE 46

46 Linearity Calibration curves were linear in the concentr ation range of 0.1 20 ng/ml using a linear regression equation with a weighting factor of 1/y2. The mean correlation coefficient (R2) of the calibration curves was 0.9967. Precision and accuracy Precision was represented as the relative sta ndard deviation (%RSD) whereas accuracy was calculated as the percent deviation (% bias) from the respective nominal concentration. The maximum acceptable limit for precision and accuracy was set at 15%. The intra-day and interday precision and accuracy were within 4.0% an d 10%, respectively, for all standards and QC samples (Table 3-1). Thus, the present me thod was found to be hi ghly reproducible and demonstrated a high degree of accuracy. Table 3-1.Intraday (n=6) and Interday (n=18) precision (%RSD) and accuracy (% deviation) for analysis of M-IV in 50 mM phosphate buffer, pH 7.4. Concentration (ng/ml) Nominal Back calculated (mean SD) % RSD % Deviation Intraday 0.50 0.48 0.02 1.4 3.4 2.00 2.10 0.05 1.8 -1.4 10 9.0 0.4 2.4 -1.5 Interday 0.50 0.48 0.02 3.6 -4.5 2.00 2.07 0.06 3.0 3.7 10.0 9.2 0.4 3.9 -7.7 Autosampler stability The processed stability of analytes was dete rmined by reanalyzing the same standards and QC samples after 48 hours in the autosampler maintained at 10oC. Both M-IV and M-IV-d4 were found to be stable at 10oC for at least 48 hours.

PAGE 47

47 Formation of M-IV in Pool ed Human Liver Microsomes PIO was incubated with pHLM at ni ne different concentrations (0.25-25 M). The linearity of M-IV formation with respect to the microsomal prot ein concentration was studied at five protein concentrations ( 0.05-0.4 mg/ml). In order to avoid non-specific protein binding, the lowest protein concentration that produced qua ntifiable metabolite (0.05 mg/ml) was selected. Formation of M-IV was linear up to 20 minutes and was dependent on the presence of NADPH regenerating system. Following the incubation of PIO in pHLM, the concentration of M-IV was determined by using the newly validated method. The rate of formation of M-IV was measured as an index of CYP2C8 enzyme activity and da ta were analyzed by nonlinear regression using GraphPad Prism (version 4). The formation of M-IV exhibited typical Michaelis-Menten kinetics (Figure 3-3) with the maximal rate of formation of M-IV (Vmax) of 150.3 13.9 pmol/min/mg protein and the concentration of PIO at half maximal velocity (Km) was 8.71 1.9 M, which are in agreement with the presen t literature (Table 3-2) (Torni o et al., 2007). Thus, the present method was successfully applied to in vitro drug metabolism studies. Figure 3-3. Plot of initial velo city versus pioglitazone con centration for th e formation of hydroxypioglitazone (M-IV) in pHLM (n=2). 0 10 20 30 0 25 50 75 100 125Pioglitazone (M)Initial Velocity (pmol.min-1.mg protein-1)

PAGE 48

48 Table 3-2.Enzyme kinetic parameters of M-IV formation in pHLM. Km SE (M) Vmax (pmol.min-1.mg protein-1) In house 8.71 1.90 150.3 13.9 Tornio et al., 2007 9.8 640 Conclusion A LC/MS/MS based method for determination of M-IV, the CYP2C8 specific metabolite of PIO was developed and validated. The method is sensitive (LOQ 0.1 ng/ml) and robust with a very short run time (5 minutes). It was validated with respect to selectivity, carry over, matrix effects, linearity, precision, accu racy and autosampler stability. Enzyme kinetic parameters obtained by incubating PIO with pHLM in pres ence of NADPH regenerating system were in accordance with the available lite rature. Therefore, the present method could be applied for future in vitro reaction phenotyping studies of CYP2C8.

PAGE 49

49 CHAPTER 4 DETERMINATION OF IC50 IN POOLED HUMAN LIVER MICROSOMES USING M-IV FORMATION AS A CYP2C8 SPECIFIC REACTION Introduction It has become increasingly clear that the inhibition of CYP enzymes is often the key mechanism underlying drug-drug interactions lead ing to an ADR. In many cases, the drug interaction occurs via alterations in the activity of CYP enzymes. In other words, one drug (the perpetrator) alters the activity of an enzyme that is re sponsible for the metabolism of another drug (the victim or object), thus affecting its me tabolic clearance. If the second drug has a wide therapeutic window, then th e interaction might be c linically insignificant. Ho wever, if the victim drug has a narrow margin of safety, then the in hibition of its metabolism might result in drug related toxicity. On the other hand, induction of metabolism of th e victim drug by the perpetrator might cause subtherapeutic effects l eading to failure of drug therapy. CYP2C8 plays an important role in the meta bolism of various drugs including paclitaxel (Dai et al., 2001; Bahadur et al ., 2002), amodiaquine (Li et al., 2002), troglitazone (Yamazaki et al., 1999), rosiglitazone (Baldwin et al., 1999), pioglitazone (D eng et al., 2005), repaglinide (Bidstrup et al., 2003; Niemi et al., 2003b), cerivastatin (Backm an et al., 2002), amiodarone (Soyama et al., 2002) and verapamil (Busse et al., 1995). Reports of CYP2C8 inhibition by gemfibrozil have shown an increase in rosiglitazone AUC0from 1.8 to 2.8-fold (Niemi et al., 2003a), repaglinide AUC0from 5.5-15-fold (Niemi et al., 2003b), and cerivastatin AUC0from 1.3-20-fold (Backman et al., 2002). Cerivast atin was withdrawn from the market after about 500 ADRs, half of which involved coadminist ration of the CYP2C8 inhibitor gemfibrozil. In a recent study, Walsky et al. examined 209 frequently prescribed drugs and related xenobiotics for CYP2C8 i nhibition using AQ as a CYP2C8 specific probe. In order to evaluate the potential of PIO as a CYP2C8 marker substrate, we studied th e inhibition of CYP2C8 by five

PAGE 50

50 drugs that were the part of the comprehensive study by Walsky et al. a nd other smaller studies using different CYP2C8 substr ates (Walsky et al., 2005). Experimental Chemicals and Reagents Ketoconazole, terfenadine, -estradiol, midazolam, NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, magnesium ch loride and ammonium acetate were procured from Sigma (St. Louis, MO, US A). Pioglitazone hydrochloride, M-IV metabolite standard and the deuterated internal standar d, M-IV-d4, were obtained from To ronto Research Chemicals Inc. (North York, Ontario, Canada). Montelukast wa s purchased from LKT Labs (St. Paul, MN, USA). Pooled HLM were obtained from In vitro Technologies (Baltimo re, MD, USA). Glucose 6-phosphate dehydrogenase soluti on was prepared by dissolving lyophilized enzyme in 5 mM sodium citrate to produce 40 U/ml solution and was stored at -20C until use. Potassium phosphate was purchased from Fisher Scientific (Fair Lawn, NJ, USA) and formic acid was obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). HPLC grade acetonitrile and methanol were purchased from EMD Chemicals (Gibbstown, NJ, USA). All other chemicals used in the study were of analytical grade. Deionized water prepared by using a Barnstead Nanopure Diamond UV Ultrapure Water System (Dubuque, IA, USA) was used throughout the study. Incubation Conditions Preliminary experiments were conducted to op timize the microsomal protein concentration and incubation time in order to assure the linear ity of M-IV formation (Chapter 3). The mixture containing pioglitazone (assay concentration, 7.5 M) and pHLM (0.05 mg/ml) in phosphate buffer (50 mM, pH 7.4) was mixed with the inhibito r solution (in methanol:acetonitrile, 10:90) at various concentrations and warmed at 37C for five minutes. The fina l incubation concentration

PAGE 51

51 range that was studied for various inhib itors was as follows: ketoconazole (0.1-100 M), terfenadine (0.1-100 M), -estradiol (0.1-600 M), midazolam (0.1-600 M), and montelukast (0.1-1000 nM). Equivalent amount of solvent was added to the incubation containing no inhibitor. Incubations were commenced by a ddition of the NADPH regenerating system, which consisted of MgCl2 (assay concentration, 3.3 mM), NADP + (1.25 mM), glucose 6-phosphate (3.3 mM) and glucose 6-phosphate dehydrogenase ( 0.32 U/ml) in 5 mM sodium citrate solution. Final incubation volume was 250 l and the conten t of total organic solv ent was kept to the minimum (1.2%). Control incubations were con ducted without addition of NADPH regenerating system to check any interference at the meta bolite retention time. After incubating for 10 minutes at 37C, the reaction was terminated by additi on of 750 l of ice-cold acetonitrile followed by the addition of internal standard so lution in methanol (50 l, 12 ng/ml). Samples were processed as described in Chapter 3. Microsomes were stored at -80C and thawed immediately before use. All incubations were performed in duplicate. Analysis of Hydroxypioglitazone (M-IV) Concentrations of M-IV were determined as described in Chapter 3. Briefly, the chromatographic separation was achieved on a SYNERGI MAX-RP 80A (150 x 2.00 mm, 4 Phenomenex) analytical column by gradient elution. The mass spectrometer was a TSQ Quantum Discovery triple quadrupole mass spect rometer equipped with ESI source. The ESI source was operated in the positive mode and was set orthogonal to the i on transfer capillary tube. For quantification, TSQ qua ntum was operated in MRM mode and the precursor-product ion pair was 373 150 m/z for M-IV and 377 154 m/z for M-IV-d4. The acquisition parameters were optimized to maximize the signal to noise ratio.

PAGE 52

52 Data Analysis Enzyme kinetic and inhibition data were obt ained by nonlinear regression using GraphPad Prism (San Diego, CA, USA). Data for IC50 determinations were typically fit to the following equation: I IC I I 100 activity Control %50 max Equation 4-1 in which I is the inhibito r concentration, Imax is the initial velo city in the presence of inhibitor, IC50 is the inhibitor concentration th at reduces initia l velocity by 50%. Results and Discussion Five drugs namely, montelukast, ketoconazole, -estradiol, midazolam and terfenadine were examined for their potential to inhibit th e formation of the CYP2C8 specific metabolite (MIV) in pHLM. Chemical structures of th ese inhibitors are shown in Figure 4-1. Figure 4-1. Structures of the comp ounds tested for inhibition of in vitro CYP2C8 activity. N N N Cl F MidazolamCl Cl O O N N H O N N O KetoconazoleOH O H H H H EstradiolOH N OH TerfenadineS O OH OH N Cl Montelukast

PAGE 53

53 The concentration of inhibitor that reduces the initial velocity by 50% (IC50) was determined by conducting the enzyme assay in the presence of seven different inhibitor concentrations. The results of the present study dem onstrate that all the test inhibitors inhibited the activity of CYP2C8 in a c oncentration dependent manner when studied as a function of formation of M-IV from PIO. IC50 plots of pioglitazone hydroxyl ase inhibition are shown in Figure 4-2. Figure 4-2. IC50 plots of pioglitazone hydroxyl ase inhibition in pHLM by montelukast (circles), ketoconazole (squares), terfenadine (diamonds), -estradiol (triangles) and midazolam (reverse triangles). Data points reflect the average for incubations run in duplicate SE. Montelukast was the most potent inhibito r of formation of M-IV with an IC50 of 57.16 12.12 nM (R2 = 0.8896) in pHLM. Walsky et al. examined the inhibition of CYP2C8 activity by 209 drugs and related compounds in expressed CYP2 C8 as a function of AQ desethylation. They also reported montelukast to be the most potent inhibitor of CYP2C8 activity with an IC50 of 0.001 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 120 Ketoconazole Beta-estradiol Midazolam Terfenadine Montelukast Inhibitor Conc. ( M)% Control activity

PAGE 54

54 19.6 nM in pHLM (Walsky et al., 2005). Our resu lts are in accordance with another study by Jaakkola et al. that examined the CYP2C8 inhib ition potential of montelukast using pioglitazone as a substrate (IC50 = 180 nM) (Jaak kola et al., 2006). The antifungal agent ketoconazole was the next potent inhibitor of CYP2C8 activity with an IC50 of 0.618 0.12 M (R2 = 0.9039). In the present study, we found that terfenadine, a H1 receptor antagonist, inhibited the ac tivity of CYP2C8 with an IC50 of 15.79 7.32 M (R2 = 0.4247). Ketoconazole and terfenadine are known i nhibitors of CYP3A4. Ketoconazole also inhibited 6-hydroxylation of paclitaxel (Desai et al, 1998) and tolyl methylhydroxylation of torsemide (Ong et al., 2000), both of which are CY P2C8 specific reactions, where as terfenadine was shown to inhibit AQ desethylation (IC50 11.5 5.1 M) (Walsky et al., 2005) as well as tolyl methylhydroxylation of torsemide in expres sed CYP2C8 (Ong et al., 2000). Inhibition of CYP2C8 by ketoconazole and terfenadine could be explained by the fact that both enzymes, CYP3A4 and CYP2C8, have a large active site and many CY P2C8 substrates are also metabolized by CYP3A4. Walsky et al. reported a moderate inhibi tion of the metabolism of AQ by -estradiol in expressed CYP2C8 (21.5 5.8) (Walsky et al., 2005). In the current study, we proved the moderate in hibitory potential of -estradiol to inhibit CYP2 C8 activity measured as the formation rate of M-IV. Midazolam, a be nzodiazepine drug, moderately inhibited CYP2C8 mediated hydroxylation of PIO (IC50 = 44.60 7.1). It also inhibited the formation of DEAQ (Walsky et al., 2005) as well as formation of tolyl methylhydroxy torsemide in expressed CYP2C8 (Ong et al., 2000). Midazolam is used as a maker substrate of CYP3A4, once again indicating the overlap between the substrate and inhibitor profiles of CYP2C8 and 3A4. Conclusion We examined the potential of montek ulast, ketoconazole, terfenadine, -estradiol and midazolam to inhibit the formation of M-IV wh ich is CYP2C8 specific metabolite of PIO using

PAGE 55

55 pHLM. All test inhibitors inhibited the M-IV formation in the concentration dependent manner and the inhibitory potentials of each of them are comparable to that studied with other CYP2C8 substrates. Therefore, formation of M-IV could potentially be used as a CYP2C8 specific probe reaction.

PAGE 56

56 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Drug-drug interactions due to induction or i nhibition of DMEs significantly add to the ADRs. In order to avoid any unforeseen inte ractions in the clinic, the FDA requires pharmaceutical companies to identify the major drug metabolizing enzymes involved in the elimination of a NCE as well as the effect of its coadminist ration on the pharmacokinetics of marker substrates of DMEs that are respons ible for its elimination. Common experimental methods used to characterize the metabolism of a NCE and predict its drug-drug interactions involve the use of enzyme specific chemical inhibitors, expressed en zymes, antibodies and correlation analysis. The polymorphic CYP2C8 is involved in th e metabolism of paclitaxel, amodiaquine, troglitazone, rosiglitazone, pioglit azone, repaglinide, cerivastati n, amiodarone and verapamil. Conventionally, paclitaxel has been used as a CYP2C8 specific probe substrate for in vitro reaction phenotying studies. Recently, AQ was found to be a high turnover and affinity substrate of CYP2C8 and is used for in vitro drug metabolism studies however it can not be administered to humans due to its toxocity. Therefore, identi fication of a CYP2C8 specific substrate that can be used for in vitro as we as in vivo drug interaction studies is n eeded. Hydroxylation of PIO to form M-IV is primarily medicated by CYP2C8, ther efore, formation of M-IV could be used as a CYP2C8 specific probe reaction. We developed a simple and robust LC/MS/MS based method using HILIC chromatography to separate DEAQ. The method was validated with respect to selectivity, carry over, matrix effects, linearity, precision, accura cy and autosampler stability. Enzyme kinetic parameters obtained by incubating AQ with pooled HLM in presence of NADPH regenerating

PAGE 57

57 system were in accordance with the available literature. Therefore, the present method could be applied for future CYP2C8 drug metabolism studies. A LC/MS/MS based method for determination of M-IV, the CYP2C8 specific metabolite of PIO was developed and validated. The method is sensitive (LOQ 0.1 ng/ml) and robust with a very short run time (5 minutes). It was validated with respect to selectivity, carry over, matrix effects, linearity, precision, accu racy and autosampler stability. Enzyme kinetic parameters obtained by incubating PIO with pooled HLM in pr esence of NADPH regenerating system were in accordance with the available literature. Ther efore, the present method could be applied for future in vitro reaction phenot yping studies of CYP2C8. We examined the potential of montekulast, ketoconazole, terfenadine, estradiol and midazolam to inhibit the formation of M-IV wh ich is CYP2C8 specific metabolite of PIO using pooled human liver microsomes. All test inhib itors inhibited the M-IV formation in the concentration dependent manner and the inhibitory potentials of each of them are comparable to that studied with other CYP2C8 substrates. Ther efore, formation of M-IV could potentially be used as a CYP2C8 specific probe reaction. Although, the presence of CYP2C8*3 allele reduced the intrinsic clearance of AQ in human liver microsomes, its effect of AQ pha rmacokinetics is unknown. In future, it will be interesting to st udy the effect of CYP2C8*3/*3 genotype on the disposition of AQ. On the other hand, CYP2C8*3 allele reduced the AUC0of PIO. However, its effect on PIO metabolism has not been studied at enzymatic level. Furt her, we would like to study the effect of CYP2C8*3 allele on M-IV formation in genotyped human liver microsomes.

PAGE 58

58 LIST OF REFERENCES Bachmann KA, Ring BJ and Wrighton SA (2003) Dr ug-Drug Interactions and the Cytochrome P450, in: Drug Metabolizing Enzymes: Cytochrome P450 and Other Enzymes in Drug Discovery and Development (Lee JS, Obach RS and Fisher MB eds), FrontisMedia SA and Marcel Dekker, New York. Backman JT, Kyrklund C, Neuvonen M and Neuvonen PJ (2002) Gemfibrozil greatly increases plasma concentrations of cerivastatin. Clin Pharmacol Ther 72: 685-691. Bahadur N, Leathart JB, Mutch E, Steimel-Cres pi D, Dunn SA, Gilissen R, Houdt JV, Hendrickx J, Mannens G, Bohets H, Williams FM, Ar mstrong M, Crespi CL and Daly AK (2002) CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alphahydroxylase activity in human liver microsomes. Biochem Pharmacol 64: 1579-1589. Basco LK, Ndounga M, Keundjian A and Ringwald P (2002) Molecular epid emiology of malaria in cameroon. IX. Characteristics of recrudescent and persiste nt Plasmodium falciparum infections after chloroquine or am odiaquine treatment in children. Am J Trop Med Hyg 66: 117-123. Baldwin SJ, Clarke SE and Chenery RJ (1999) Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone. Br J Clin Pharmacol 48: 424432. Baughman TM, Graham RA, Wells-Knecht K, Silv er IS, Tyler LO, Wells -Knecht M and Zhao Z (2005) Metabolic activation of pi oglitazone identified from ra t and human liver microsomes and freshly isolated hepatocytes. Drug Metab Dispos 33: 733-738. Bell DJ, Nyirongo SK, Molyneux ME, Winstanley PA and Ward SA (2007) Practical HPLC methods for the quantitative determinati on of common antimalarials in Africa. J Chromatogr B Analyt Technol Biomed Life Sci 847: 231-236. Bidstrup TB, Bjornsdottir I, Sidelmann UG, Th omsen MS and Hansen KT (2003) CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide. Br J Clin Pharmacol 56: 305-314. Busse D, Cosme J, Beaune P, Kroemer HK and Eichelbaum M (1995) Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans. Naunyn Schmiedebergs Arch Pharmacol 353: 116-121. Cavaco I, Stromberg-Norklit J, Kaneko A, Mse llem MI, Dahoma M, Ribeiro VL, Bjorkman A and Gil JP (2005) CYP2C8 polymorphism freque ncies among malaria patients in Zanzibar. Eur J Clin Pharmacol 61: 15-18. Cresteil T, Monsarrat B, Alvinerie P, Trel uyer JM, Vieira I and Wright M (1994) Taxol metabolism by human liver microsomes: iden tification of cytochrome P450 isozymes involved in its biotransformation. Cancer Res 54: 386-392.

PAGE 59

59 Dai D, Zeldin DC, Blaisdell JA, Chanas B, C oulter SJ, Ghanayem BI and Goldstein JA (2001) Polymorphisms in human CYP2C8 decrease me tabolism of the anticancer drug paclitaxel and arachidonic acid. Pharmacogenetics 11: 597-607. Deng LJ, Wang F and Li HD (2005) Effect of gemfibrozil on the pharmacokinetics of pioglitazone. Eur J Clin Pharmacol 61: 831-836. Dixit V, Hariparsad N, Desai P and Unadkat JD (2007) In vitro LC-MS cocktail assays to simultaneously determine human cytochrome P450 activities. Biopharm Drug Dispos 28: 257262. Dua VK, Gupta NC, Sharma VP and Subbarao SK (2004) Liquid chromatographic determination of amodiaquine in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 803: 371374. Ferguson SS, Chen Y, LeCluyse EL, Negishi M and Goldstein JA (2005) Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4alpha. Mol Pharmacol 68: 747-757. Food and Drug Administration. 1997. Guidance for Industry: Drug Metabolism/Drug Interaction Studies in the Drug Devel opment Process: Studies In Vitro http://www.fda.gov/cder/guidance/clin3.pdf Food and Drug Administration. 1999. Guidance for Industry: In Vivo Drug Metabolism/Drug Interaction Studies Study Design, Data An alysis, and Recommendations for Dosing and Labeling. http://www.fda.gov/cder/guidance/2635fnl.pdf Food and Drug Administrati on. 2001 Guidance for Industry: Bioanalytical Method Validation http://www.fda.gov/cder/guidance/4252fnl.pdf Frye RF (2004) Probing the world of cytochrome P450 enzymes. Mol Interv 4: 157-162. Gitau EN, Muchohi SN, Ogutu BR, Githiga IM and Kokwaro GO (2004) Selective and sensitive liquid chromatographic assay of amodiaquine and desethylamodiaqui ne in whole blood spotted on filter paper. J Chromatogr B Analyt Technol Biomed Life Sci 799: 173-177. Hombhanje FW, Hwaihwanje I, Tsukahara T, Saru watari J, Nakagawa M, Osawa H, Paniu MM, Takahashi N, Lum JK, Aumora B, Masta A, Sapuri M, Kobayakawa T, Kaneko A and Ishizaki T (2005) The disposition of oral amodiaquine in Papua New Guinean children with falciparum malaria. Br J Clin Pharmacol 59: 298-301. Jaakkola T, Backman JT, Neuvonen M and Neuvone n PJ (2005) Effects of gemfibrozil, itraconazole, and their co mbination on the pharmacokinetics of pioglitazone. Clin Pharmacol Ther 77: 404-414.

PAGE 60

60 Jaakkola T, Laitila J, Neuvonen PJ and Backma n JT (2006) Pioglitazone is metabolised by CYP2C8 and CYP3A4 in vitro : potential for interactions with CYP2C8 inhibitors. Basic Clin Pharmacol Toxicol 99: 44-51. Jewell H, Maggs JL, Harrison AC, O'Neill PM, Rusc oe JE and Park BK (1995) Role of hepatic metabolism in the bioactivation and detoxication of amodiaquine. Xenobiotica 25: 199-217. Kajosaari LI, Laitila J, Neuvonen PJ and Back man JT (2005) Metabolism of repaglinide by CYP2C8 and CYP3A4 in vitro : effect of fibrates and rifampicin. Basic Clin Pharmacol Toxicol 97: 249-256. Kiyota Y, Kondo T, Maeshiba Y, Hashimoto A, Yamashita K, Yoshimura Y, Motohashi M and Tanayama S (1997) Studies on the metabolism of the new antidiabetic agent pioglitazone. Identification of metabo lites in rats and dogs. Arzneimittelforschung 47: 22-28. Klose TS, Blaisdell JA and Goldstein JA (1999) Gene structure of CYP2C8 and extrahepatic distribution of the human CYP2Cs. J Biochem Mol Toxicol 13: 289-295. Knupfer H, Schmidt R, Stanitz D, Brauckho ff M, Schonfelder M and Preiss R (2004) CYP2C and IL-6 expression in breast cancer. Breast 13: 28-34. Laurent F, Saivin S, Chretien P, Magnaval JF, Peyron F, Sqalli A, Tufenkji AE, Coulais Y, Baba H, Campistron G and et al. (1993) Phar macokinetic and pharmacodynamic study of amodiaquine and its two metabolites afte r a single oral dose in human volunteers. Arzneimittelforschung 43: 612-616. Li XQ, Bjorkman A, Andersson TB, Ridde rstrom M and Masimirembwa CM (2002) Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnove r enzyme-specific probe substrate. J Pharmacol Exp Ther 300: 399-407. Li XQ, Bjorkman A, Andersson TB, Gu stafsson LL and Masimirembwa CM (2003) Identification of human cytochrome P(450)s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data. Eur J Clin Pharmacol 59: 429442. Lin ZJ, Ji W, Desai-Krieger D and Shum L ( 2003) Simultaneous determination of pioglitazone and its two active metabolites in human plasma by LC-MS/MS. J Pharm Biomed Anal 33: 101-108. McSorley LC and Daly AK (2000) Identification of human cytochrome P450 isoforms that contribute to all-trans-re tinoic acid 4-hydroxylation. Biochem Pharmacol 60: 517-526. Minzi OM, Rais M, Svensson JO, Gustafss on LL and Ericsson O (2003) High-performance liquid chromatographic method for determina tion of amodiaquine, chloroquine and their

PAGE 61

61 monodesethyl metabolites in biological samples. J Chromatogr B Analyt Technol Biomed Life Sci 783: 473-480. Mizushige K, Tsuji T and Noma T (2002) Pioglit azone: cardiovascular effects in prediabetic patients. Cardiovasc Drug Rev 20: 329-340. Motten AG, Martinez LJ, Holt N, Sik RH, Reszka K, Chignell CF, Tonnesen HH and Roberts JE (1999) Photophysical studies on antimalarial drugs. Photochem Photobiol 69: 282-287. Mount DL, Patchen LC, Nguyen-Dinh P, Barber AM, Schwartz IK and Churchill FC (1986) Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection. J Chromatogr 383: 375-386. Niemi M, Backman JT, Granfors M, Laiti la J, Neuvonen M and Neuvonen PJ (2003a) Gemfibrozil considerably increases the pl asma concentrations of rosiglitazone. Diabetologia 46: 1319-1323. Niemi M, Backman JT, Neuvonen M and Neuvonen PJ (2003b) Effects of gemfibrozil, itraconazole, and their comb ination on the pharmacokinetics and pharmacodynamics of repaglinide: potentially hazardous interac tion between gemfibrozil and repaglinide. Diabetologia 46: 347-351. Nishimura M, Yaguti H, Yoshitsugu H, Naito S and Satoh T (2003) Ti ssue distribution of mRNA expression of human cytochrome P450 is oforms assessed by high -sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 123: 369-375. O'Donnell CJ, Grime K, Courtney P, Slee D and R iley RJ (2007) The development of a cocktail CYP2B6, CYP2C8, and CYP3A5 inhibition assay a nd a preliminary assessment of utility in a drug discovery setting. Drug Metab Dispos 35: 381-385. Ong CE, Coulter S, Birkett DJ, Bhasker CR a nd Miners JO (2000) Th e xenobiotic inhibitor profile of cytochrome P4502C8. Br J Clin Pharmacol 50: 573-580. Parikh S, Ouedraogo JB, Goldstein JA, Rosentha l PJ and Kroetz DL (2007) Amodiaquine metabolism is impaired by common polymorphism s in CYP2C8: implications for malaria treatment in Africa. Clin Pharmacol Ther 82: 197-203. Pussard E, Verdier F, Blayo MC and Pocidalo JJ (1985a) [Biotransformation of amiodaquine and prophylaxis of Plasmodium falciparum malaria]. C R Acad Sci III 301: 383-385. Pussard E, Verdier F, Faurisson F and Blayo MC (1985b) [Pharmacokinetic s of amodiaquine and prevention of Plasmodium falciparum malaria]. Bull Soc Pathol Exot Filiales 78:959-64. Pussard E, Verdier F, Faurisson F, Scherrmann JM, Le Bras J and Blay o MC (1987) Disposition of monodesethylamodiaquine afte r a single oral dose of amodia quine and three regimens for prophylaxis against Plasmodi um falciparum malaria. Eur J Clin Pharmacol 33: 409-414.

PAGE 62

62 Rendic S and Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29: 413-580. Rower S, Bienzle U, Weise A, Lambertz U, Forst T, Otchwemah RN, Pfutzner A and Mockenhaupt FP (2005) Short communication: high prevalence of the cytochrome P450 2C8*2 mutation in Northern Ghana. Trop Med Int Health 10: 1271-1273. Shen Z, Reed JR, Creighton M, Liu DQ, Tang YS, Hora DF, Feeney W, Szewczyk J, Bakhtiar R, Franklin RB and Vincent SH (2003) Identifica tion of novel metabolites of pioglitazone in rat and dog. Xenobiotica 33: 499-509. Soyama A, Hanioka N, Saito Y, Murayama N, Ando M, Ozawa S and Sawada J (2002) Amiodarone N-deethylation by CYP2C8 and its variants, CYP2C8*3 and CYP2C8 P404A. Pharmacol Toxicol 91: 174-178. Taniguchi R, Kumai T, Matsumoto N, Watanabe M, Kamio K, Suzuki S and Kobayashi S (2005) Utilization of human liver microsomes to expl ain individual differences in paclitaxel metabolism by CYP2C8 and CYP3A4. J Pharmacol Sci 97: 83-90. Thum T and Borlak J (2000) Gene expressi on in distinct regions of the heart. Lancet 355: 979983. Tornio A, Niemi M, Neuvonen PJ and Backma n JT (2007) Trimethoprim and the CYP2C8*3 allele have opposite e ffects on the pharmacokine tics of pioglitazone. Drug Metab Dispos 36: (Fast forward Oct 3). Trenholme GM, Williams RL, Patterson EC, Fris cher H, Carson PE and Rieckmann KH (1974) A method for the determination of amodiaquine. Bull World Health Organ 51: 431-434. Turpeinen M, Uusitalo J, Jalonen J and Pelkone n O (2005) Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay. Eur J Pharm Sci 24: 123-132. Venkatakrishnan K, von Moltke LL, Obach RS a nd Greenblatt DJ (2003) Drug metabolism and drug interactions: applicati on and clinical value of in vitro models. Curr Drug Metab 4: 423459. Walsky RL and Obach RS (2004) Validated as says for human cytochrome P450 activities. Drug Metab Dispos 32: 647-60. Walsky RL, Gaman EA and Obach RS (2005) Ex amination of 209 drugs for inhibition of cytochrome P450 2C8. J Clin Pharmacol 45: 68-78. Walle T, Walle UK, Kumar GN and Bhalla KN (1995) Taxol metabolism and disposition in cancer patients. Drug Metab Dispos 23: 506-512.

PAGE 63

63 Winstanley P, Edwards G, Orme M and Brecken ridge A (1987) The disposition of amodiaquine in man after oral administration. Br J Clin Pharmacol 23: 1-7. Wienkers LC and Stevens JC (2003) Cytochrome P450 Reaction Phenotyping, in: Drug Metabolizing Enzymes: Cytochrome P450 and Other Enzymes in Drug Discovery and Development (Lee JS, Obach RS and Fisher MB eds), pp 255-310, FrontisMedia SA and Marcel Dekker Inc, New York. Yamazaki H, Shibata A, Suzuki M, Nakajima M, Shimada N, Guengerich FP and Yokoi T (1999) Oxidation of troglitazone to a quinone-t ype metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes. Drug Metab Dispos 27: 1260-1266. Yki-Jarvinen H (2004) Thiazolidinediones. N Engl J Med 351: 1106-1118.

PAGE 64

64 BIOGRAPHICAL SKETCH Prajakta Dravid was born in Maharashtra, Indi a. In 2000, she received her B.S. in Pharmacy from University of Pune, India. During her bachel ors, she developed an in terest in research and development and followed her interest by get ting M.S. degree in Pharmaceutics from the National Institute of Pharmaceutical Education and Research (NIPER), India. Chasing her interest in the area of pharmac okinetics and drug metabolism, Pr ajakta joined the Bioanalysis, Drug Metabolism and Pharmacokinetics Department at Dr. Reddys Laborat ories in Hyderabad, India. At Dr. Reddys she gained extensive experi ence in the field of bioa nalysis and preclinical pharmacokinetics. After two years of industrial e xperience, she joined the PhD program at the Department of Pharmaceutics, University of Florida in the August of 2004. Under the supervision of Dr. Reginald Fr ye, she looked at the reaction phe notyping aspects of Cytochrome P450 2C8 with a focus on substrate selection and inhibition profile. She received M.S. in Pharmacy from University of Florida in December 2007 and pursuing her PhD in the same program.


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101203_AAAAFC INGEST_TIME 2010-12-04T02:58:00Z PACKAGE UFE0021832_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2390 DFID F20101203_AACEBP ORIGIN DEPOSITOR PATH dravid_p_Page_12thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
e181e72ad021994a52f9fa870a0c9fd2
SHA-1
c2fb4608492070ea0c7e7ab0bf56a21ef3a580b1
1053954 F20101203_AACDUS dravid_p_Page_22.tif
b7821dde465c03e6da085813cc72adb4
f3f851e43c2791f5e3b173b4c413258fee205b80
76243 F20101203_AACDPV dravid_p_Page_17.jpg
1aa9f6548547c8a1a6738eb712a30830
6b0a82fd1e3f697542696c153f6aa2bc550fa06b
2029 F20101203_AACDZP dravid_p_Page_33.txt
485f854a786c98e9977a3370662042c6
aa70a90db2ce34ed02a79483e0db5fcf0ee7d7df
14801 F20101203_AACEBQ dravid_p_Page_09.QC.jpg
ce6f926c42bbbd8e3df17eed33eef33f
19c25cd26e7a478d676833bd22e18ae5b6603c7e
F20101203_AACDUT dravid_p_Page_23.tif
ed30ec7e12ea5208cca697a3eb11e4c3
601811632ad81da10507acfa636d819a87a2daf4
950 F20101203_AACDZQ dravid_p_Page_34.txt
97c9996cc582f38ae21b888855486e25
a3b2d0c16dc2ce58f4cfc0083034b299628d98d9
24986 F20101203_AACEBR dravid_p_Page_33.QC.jpg
7d4ddfc15c9b751ba0ebb3ad6c6d7c32
36af5f3a215fc7352accc3449049b4076c99ae19
F20101203_AACDUU dravid_p_Page_24.tif
a1f810c09d9421d56db5802bcd5b4fb4
efffa5e86eadb3055d7c6976603ab8a84c85b2bd
76178 F20101203_AACDPW dravid_p_Page_18.jpg
56b217e4e1230c37cda784afbbd890f0
4f9297a617966279c3e1ab7b8e0e18c4e6a91be9
250 F20101203_AACDZR dravid_p_Page_35.txt
e10f61655185bae53adabed07554aecd
33b620371080da4a11c4fffea99e754685c25317
5048 F20101203_AACEBS dravid_p_Page_52thm.jpg
7297d059f306d9d8fbaf39a02c1fc1ec
c42f1dde308cc9f12bee30268fefd5a5411688b7
25271604 F20101203_AACDUV dravid_p_Page_25.tif
f491d55e6aa6d16eedfb3f739a410740
7e12fb14c9613061ab19492a3b8a698e6d664eee
76695 F20101203_AACDPX dravid_p_Page_19.jpg
e4b9c5938ff57424a2f98ff4a15f938e
6ba42534af30c98a91c9801b21dbca96df293773
2134 F20101203_AACDZS dravid_p_Page_36.txt
178ca98337be207148f0aba77f0e4cdf
48ce674bf7c92aa503511e4aab12b2fe6eaff12c
6827 F20101203_AACEBT dravid_p_Page_18thm.jpg
2cac08e902650b600515ef78601b87c7
955fac7a17653e110591ccdc30e9e440e6871be8
F20101203_AACDUW dravid_p_Page_26.tif
6457a1ea2ffb25e230fd25abf99eaf4a
3f70296de1071ffddcd05d49cb6d1f915da23f9d
81725 F20101203_AACDPY dravid_p_Page_20.jpg
ad24d30b9131f0b35694197a1a592ddf
c4fd906986cd22eeb3281a3f25647d3a7d2cfedc
1971 F20101203_AACDZT dravid_p_Page_37.txt
620b17dd65a4cc4fe709906e33ab3e80
b75adf5df44f32bbc833b73b5417d967aa5fd3be
F20101203_AACDUX dravid_p_Page_27.tif
34c8e752c3be35458f19f842225f7873
8102fdb2abecfaef498a5d57754294a8f9ad43d3
59554 F20101203_AACDPZ dravid_p_Page_22.jpg
e04c6abc19e6c03ecfd3653c23b73111
7062f08e4e25ec372c0cf3c184ab5a92911b88e3
2132 F20101203_AACDZU dravid_p_Page_38.txt
4e1804102aa6d84da2c40608b0a052b6
fb53a8534015e53dd471ca022f694e29f39628b5
4875 F20101203_AACEBU dravid_p_Page_10.QC.jpg
7b816d66e76338dd9e1b9399ad6d29cd
f21a61afa11d6d4c7c0bde1d17e23a819ca4084e
F20101203_AACDUY dravid_p_Page_28.tif
5fc0818ae32724e40898c0dd5ac050c4
52695b6c6d99f87bea72147bb19bdcb88c6495f5
2102 F20101203_AACDZV dravid_p_Page_39.txt
fb73f4dd621f649aac8208e94d275886
b193e8af4957d5a15adc5baed0c68b61d52393cc
7207 F20101203_AACEBV dravid_p_Page_25thm.jpg
18680c77800337cc1333a4f6edbc9bab
5b22477893c66f60c08018c29682a1e95227e459
F20101203_AACDUZ dravid_p_Page_29.tif
4887eebbb9e4c20650780e6455725c99
1e4d472b408e24b24226ec00b4c5174f16f50021
2025 F20101203_AACDZW dravid_p_Page_40.txt
8074dc8ebf32e02eec794a36b5161a38
ff26f59de05a099cfeedd2ff3a8269d6e4cc7010
3385 F20101203_AACEBW dravid_p_Page_03.QC.jpg
017380508c400618c482f0d1240f8f03
9177e713a6162ff881c1aea707e972f155df5c7c
113740 F20101203_AACDSA dravid_p_Page_13.jp2
d206d0ac4ba4c8b413bcde48f69c382d
df6273ee454335349668cc3f33ea56abfd314cbb
1919 F20101203_AACDZX dravid_p_Page_41.txt
1d5c86810adea3494d1a0ef62f09b853
e4e6243a6e745519ccac749a6b4f0883ca88059e
7185 F20101203_AACEBX dravid_p_Page_62thm.jpg
231bb9ed424d174f773e84cd9ce4db23
3fd8936a334d29e122a28e875ed0f171de2cb875
121062 F20101203_AACDSB dravid_p_Page_14.jp2
95fdcd7e79ef1ec5a142eeda22ffc9b5
96a53bbfeb1f8245bdbf1480eea636c45d8f33da
1903 F20101203_AACDZY dravid_p_Page_42.txt
b00e8b81ee147ea58b1492684d35b95c
0cb5a85ba6e33e9ee7b48459f6b29a87b5542a15
16319 F20101203_AACEBY dravid_p_Page_52.QC.jpg
e0dd232be6951d1013b9b6c4a608b066
c3438e9c89e41eba33e5c524e525ca6621f95ef1
118199 F20101203_AACDSC dravid_p_Page_15.jp2
9449bf44819d0f2c1b1900e6209e3c99
c30164784999aefe1068e1d1150a6e5a6ea7f92a
54711 F20101203_AACDXA dravid_p_Page_24.pro
564f467a81a3b11b057b4dc2f441256e
c7ac8c281b8fe1519edaf83b012b86f213352a3a
F20101203_AACDZZ dravid_p_Page_43.txt
5d78e3fe37143edf5d1021d21c86cb02
c2012d25b66ae453f18c8bdebe16187034095946
7046 F20101203_AACEBZ dravid_p_Page_20thm.jpg
7c5824fd89c5a77254c95eed4839223b
f8026d91812a6df7954d56b34c080f1a4b4b557e
105184 F20101203_AACDSD dravid_p_Page_16.jp2
737949d0bd83276baca6ab96efac15b6
f44f28107ac5429b5b343c7bf1a65e97f38b8a33
51328 F20101203_AACDXB dravid_p_Page_25.pro
51d0429c21c35b618be4b23b94debd37
d21c9a3140c01d6007478850066d36b1c1793df1
113856 F20101203_AACDSE dravid_p_Page_18.jp2
71ea7c550ea5b8507e4a013ab9c22830
b71717d92a60d762aba8f822052b6034fc2ef834
7314 F20101203_AACEEA dravid_p_Page_14thm.jpg
f643b9a7f0314d7f47af1d0ec9c402d8
ca4112d41b2766778f236dda207414d24a77e8b1
50572 F20101203_AACDXC dravid_p_Page_26.pro
1fce2470c3848b35b515f4b703f8fa69
7954d76b45e841943639377a24be6d76801abc3f
115370 F20101203_AACDSF dravid_p_Page_19.jp2
fb3a89e0a84aff438271bc4dca93658b
c99470b4f28e0ca4d8df8441f58fd1e5cb169462
7001 F20101203_AACEEB dravid_p_Page_15thm.jpg
b97a309916249d5cd5a1394479ca02fb
acdb983d1a2b6929b81b87aba7dc10f8c1b385bc
49622 F20101203_AACDXD dravid_p_Page_27.pro
b907a0b33355cfaa97e27b44bed500d0
68271a0404474455ac8fbc17c3efb66acc8a938b
120750 F20101203_AACDSG dravid_p_Page_20.jp2
fc9e37f3f434645cc3a9174dbc366b16
c4f25a43e10dde86ad52ab7c1ba9cff8f508c62d
6851 F20101203_AACEEC dravid_p_Page_17thm.jpg
4d7027b390f223270f835557599b3546
a7edd4da70cb63d934d4e4eecc15f07bcd5b438e
53798 F20101203_AACDXE dravid_p_Page_28.pro
550a84115b3be21dbd9a5c4a43d9e734
e877524f5b16e5124aa29fcf81cbeba0ad030211
76212 F20101203_AACDSH dravid_p_Page_21.jp2
5e95f1b6c74c7b05983cbe1acfdda385
0f30ace2a61d328b109392729db5ed8f94b544e6
25115 F20101203_AACEED dravid_p_Page_18.QC.jpg
e16fbf9f076563f25a3a107aa8d38661
5a9cbdaf3ec15e5d33d2c9c310a70ed941b98769
46831 F20101203_AACDXF dravid_p_Page_29.pro
80568ec632fcf8b87ce7e8dfdfb7d1a6
7c5427b1b9b7554f5be9588e083cf761d8a5faaa
86864 F20101203_AACDSI dravid_p_Page_22.jp2
b0ea5669399e5156d3e61602f491fc01
1979f08ce80f1df4a904712d8ef7a7059c7537ac
25086 F20101203_AACEEE dravid_p_Page_19.QC.jpg
e1a0e69abd68c2e23a00d62647d97353
aea817b76a26e61f0a6b8b9cc0f215f75b14cb7a
52667 F20101203_AACDXG dravid_p_Page_30.pro
448655616d279366c8f674ed46e63fcc
7c339a2a56de25e11987e9ed549fc46c5e717839
118207 F20101203_AACDSJ dravid_p_Page_23.jp2
5c530260c31057c97f4ed36b62ea3184
0e4923f1b0e84f472736246bd74b0b281a59bf12
26959 F20101203_AACEEF dravid_p_Page_20.QC.jpg
35436b84b79dba30c3d17dee0fceb775
98fef24e1c11b027e670e48dae7ef0e492f45b09
31034 F20101203_AACDXH dravid_p_Page_32.pro
279794fc57bc7adbd9313fe077bd4b65
80593f23e38395b2c04bcee27490bfa9d1190f20
119197 F20101203_AACDSK dravid_p_Page_24.jp2
35dc0d860bfb12b8de3e25832fc37262
3693c341665a4efa825eed21f72afdb2e8221a7b
17484 F20101203_AACEEG dravid_p_Page_21.QC.jpg
eab4f84197ecd34a68b920f33f8d15a8
90fc43dc2d871dcacf47a0f7a1f59d5ccdec7f2d
6217 F20101203_AACDXI dravid_p_Page_35.pro
f5408c2bbbc63cd58f10d3777e52ff91
b27bdbb176f4d33dd8d58bdbc88308e07f0d7052
1051977 F20101203_AACDSL dravid_p_Page_25.jp2
a5b14de75c19e679523355d20e9e6078
55f18f2daffd437be0083d7225d89d0da199eef4
5274 F20101203_AACEEH dravid_p_Page_21thm.jpg
588f524d392bf9c93c42003fe70a7da9
92d1c9e138930d7b7a0e282b2c8c7ff817f6f6a6
51812 F20101203_AACDXJ dravid_p_Page_36.pro
5c081806a4b1d366fa107f4422b607ad
8f9cbf9ff808ca2437ba1dcc26b9926c4347359a
111560 F20101203_AACDSM dravid_p_Page_26.jp2
ed37379e423e1a6153c79e0e67b17b44
227d62d740faa11e62fbb4a3598c2ceea4eef268
7149 F20101203_AACEEI dravid_p_Page_23thm.jpg
4e9f442a10332d8743defa3ef1d7165e
fc213608e47f01a094dfb5e413d2565b0a313743
45264 F20101203_AACDXK dravid_p_Page_37.pro
c41bc99df4ee0401d031d89f414764c7
0847ee7a7f8310cdf07fb0b1162903de9a1f26f4
1051944 F20101203_AACDSN dravid_p_Page_27.jp2
0fa7a3eb04b08494df2a6105f4d5e4b0
39a63d79a30f71fcb5ce42929b7f3cd30c9ff979
25836 F20101203_AACEEJ dravid_p_Page_24.QC.jpg
d07f10908df5374a86dc6e3e4679dbed
279711515e120d79293e9172d9d5b1908115d536
54438 F20101203_AACDXL dravid_p_Page_38.pro
01e0a9061475d43c8b891f2bb5aa7687
80d2b78dae12384be2359df78f6a369e3e926b3e
118709 F20101203_AACDSO dravid_p_Page_28.jp2
04d375256d729f55ce32e22f9e19544e
6fbd96e42134c4eced27b06aa97ea008e8baaad2
52563 F20101203_AACDXM dravid_p_Page_39.pro
3eabcc96090306f6ff68ecc7bdd2f2e3
f87a25dda683bf1794b8f74056ffd9d601d7af14
101889 F20101203_AACDSP dravid_p_Page_29.jp2
5beeaf42c54c9ac25c68c5eab6de3a4e
a2d54348d3b32c2a44b620fd57f279bf57566bc8
6864 F20101203_AACEEK dravid_p_Page_24thm.jpg
92b62c72b704dad6a24f5233fb263168
391f003ef401119817d1987750c1de0e13c3cf9f
51432 F20101203_AACDXN dravid_p_Page_40.pro
445f83fd8455acf777dd654e33404017
51aac7a9669ec2aa6d4356c266173699eac61245
115626 F20101203_AACDSQ dravid_p_Page_30.jp2
1327c6cfcb5a57f56d2b1be71f60565e
7c6d82740981eaaa4237cb433530797c8d140d47
25978 F20101203_AACEEL dravid_p_Page_28.QC.jpg
2cd6e90cf49fa37ee7d630193cd4400c
1c1d753bd2f994c05a91ccd1b686141b9db7b51d
48506 F20101203_AACDXO dravid_p_Page_41.pro
bcb0f3ada708db097e1878d8855659c3
441d81eb6d67e9fd53da9bb4ab41554d55a60f03
450627 F20101203_AACDSR dravid_p_Page_31.jp2
7c066edc891eae9a3e8c8db0495553aa
d4b0cff39315bbb60c9e92e183d586345657367d
6895 F20101203_AACEEM dravid_p_Page_28thm.jpg
2178d8853fbd4a790251ecc1a5904372
0c623e6cf3efabb74656eb67e0908a0fa345c1be
47847 F20101203_AACDXP dravid_p_Page_42.pro
e0a951af3ac753cad639509a2279f5a5
576d4d03afb5c40cee412fad87def8d72190cd60
68347 F20101203_AACDSS dravid_p_Page_32.jp2
9b192bbf09fee9866e788ad7b8478002
fcc5abf8f487dce7a8bde74952df548c7ddceb23
6548 F20101203_AACEEN dravid_p_Page_29thm.jpg
3a8fdc11018abb436b91aac837b3254b
e5a87e43324fdfaf9e26137362d7b4cf2a782276
53513 F20101203_AACDXQ dravid_p_Page_43.pro
96a7d0a785d99079ddef885de89186e2
0a592fa33c0d6348f1afaf538ac430bb93a3a967
113766 F20101203_AACDST dravid_p_Page_33.jp2
4e8fea8f57523dcc32709319f2afda28
93574705bab8756d77555a5db85c4397fa5a518f
7038 F20101203_AACEEO dravid_p_Page_30thm.jpg
2074d07e8e147b2aa55f3d5b2c286ef4
ac446ca288de54133f536eedea3b60229cc4384b
52732 F20101203_AACDXR dravid_p_Page_44.pro
39d075baf0fdc4653e3196ec543b20b9
d1edbd27ec9b39306599023daaa01c7800954313
51926 F20101203_AACDSU dravid_p_Page_34.jp2
a39d756bb0547a6c52a3fb963a7fa3f4
97a71c68c1fde00f92fb9068320492054f3267bd
6776 F20101203_AACEEP dravid_p_Page_33thm.jpg
aabfc4e4e5b96e990ba42f9ae809362b
4b366c3ad13116491c356d7af9e2b3c9e4644d63
32094 F20101203_AACDXS dravid_p_Page_45.pro
a3ebad69f0666c4cef44976029953686
a76682861d5d36e5ef7492f736c6a0adf8a73d52
17616 F20101203_AACDSV dravid_p_Page_35.jp2
01cd3a923daa04fb543bbb63898c179e
c9e2ab13f785a446689ff2ca43e62e1fd9b6dc9d
4555 F20101203_AACEEQ dravid_p_Page_34thm.jpg
f2f8cc2a2133f530e3e31e03e790e023
64b383dd4b5c1172d1bff9b3e59d1080c45404c1
40263 F20101203_AACDXT dravid_p_Page_46.pro
7598d375ea36b917dee5cc221e69be44
818258fbf4ca7f5f7ddddb7454019d5e67ea1a7d
114512 F20101203_AACDSW dravid_p_Page_36.jp2
6870edbd181cde5c178719ab363b5313
be2147c3b93367851cfde2fbcebab40fec77a54d
5797 F20101203_AACEER dravid_p_Page_35.QC.jpg
02eb9511e8b6f096ca186c684097cfdc
9ece2eaa425877aee468a24063fe5b2071958d49
38206 F20101203_AACDXU dravid_p_Page_47.pro
37de450c95f87791583f3543f77c5f8f
68e243739c013e0aa343a91426fd82cd91eeb503
99662 F20101203_AACDSX dravid_p_Page_37.jp2
2f79b59654855df13dd6a5e5dc212c6e
16c1e36be579ff16c6b8e685f7f955e2e03c1896
25378 F20101203_AACEES dravid_p_Page_36.QC.jpg
417631799d6ee68adf4cc14c26593ecf
38747d5a53d25d5f3e76997a63d5738b94274616
21494 F20101203_AACDXV dravid_p_Page_48.pro
8a6e92c780ac84d2a87cdaddba05438f
94b7536bf6114d41a5863a9ac8d6abf1b910a5d4
117368 F20101203_AACDSY dravid_p_Page_38.jp2
10f08b8261a7fee1b432e57778398ceb
cc10665669a05c1da4dc04b954d194a8038ed05b
7054 F20101203_AACEET dravid_p_Page_36thm.jpg
f7d1701b8d0e604a1dd44a4ab372cd64
7003be0801e032e15ef1552bdec7deba26764c3b
55194 F20101203_AACDXW dravid_p_Page_49.pro
aa8a98f95d105465fdbf6ef56c9509b0
01086ac9e74d6f37a6d0ccbbf09dc9e88c091357
80915 F20101203_AACDQA dravid_p_Page_23.jpg
2df5fa89412888e85c78b47aef2d7d3d
0004dd9fe2e8a60406e154c811e901c3c8e88c4d
22587 F20101203_AACEEU dravid_p_Page_37.QC.jpg
c6db9b8169cbaf4ab3f971b0d19b5ec7
897f0be126bdee8a7f912bc92192d7b0f77ff0dc
48953 F20101203_AACDXX dravid_p_Page_50.pro
2b7edd90680c1924774f8da6ea2baf90
0493679556b3a817d98b2f432494f96f302288fc
79350 F20101203_AACDQB dravid_p_Page_24.jpg
78fa8f5fb67870acbca5bfc04af0ebf5
605f8bd8d2b9348d9ff11e62626ea41be2688fee
116565 F20101203_AACDSZ dravid_p_Page_39.jp2
600c9960f8f041d07895f0a662a8d334
a667ab2aed68ef0ef6932c58a0e98b2eb34aa6dc
26345 F20101203_AACEEV dravid_p_Page_39.QC.jpg
566b15339f1541a72c8224b6fc8a053a
6c3dbc51f5a4475d30c70c220b22b7785a052e63
49302 F20101203_AACDXY dravid_p_Page_51.pro
f14528e4c57715d23ffdb9ef416b0734
34a3ab49f4179ab4ec5193933aa535cc3ef7894c
87213 F20101203_AACDQC dravid_p_Page_25.jpg
4878f83927d08b34488049380a7a8cc9
7ea57fe1ecaf178686a70df88282df9dff086624
22753 F20101203_AACEEW dravid_p_Page_42.QC.jpg
92e589bd52d3aedd559a4a85523149f3
40e9d8efca17fede1b1dc4ac28c13ec6caaa7041
30185 F20101203_AACDXZ dravid_p_Page_52.pro
c21ed96725a24ec95f34f601413a0f2e
2b8ba0f8324cf5588a95774c965b4bc417acc6cc
73490 F20101203_AACDQD dravid_p_Page_26.jpg
184f250d4f1ddfe5eaedd59e8a7e9010
c3b2c97c0cbb2fb15401f1e405ea0bbcf88c8c77
F20101203_AACDVA dravid_p_Page_31.tif
b06b07a367c2e1e37cc18a8870c60fbc
3c19376242b659926f594015a049e18f313e4944
6216 F20101203_AACEEX dravid_p_Page_42thm.jpg
2b075481f03b01f31e725af83f0c3735
d2f1ee9438d4630960b301b80240ff0a3745f66d
80026 F20101203_AACDQE dravid_p_Page_27.jpg
07dec24c6b61e2447e64b76558ea0a86
3358b140c2361623718a5dad57494ec74a18acf6
F20101203_AACDVB dravid_p_Page_32.tif
3f59a4bbe69e6159470e67577b6787e4
83f4d10ee092665106d49c7df6cfede519624736
14733 F20101203_AACEEY dravid_p_Page_45.QC.jpg
6a7faed4e34b8e8fb1142b20d6c1a718
334285bee97d9ba8a9d25ac83e87268eb68fb418
78729 F20101203_AACDQF dravid_p_Page_28.jpg
4e3a999f471072093f230c7b14ff46ea
ae0368333718d76c486888baf7a7245c9322b6ea
7174 F20101203_AACECA dravid_p_Page_60thm.jpg
4486dbf13f45e274c617c26dce68a92a
ac24e307ce4b34e502166e10730d67bba6314803
F20101203_AACDVC dravid_p_Page_33.tif
22b66dd53e82e0f6242cb8ca934a1c8d
1605520ad2e59207ffed0ece1223dad896719814
18164 F20101203_AACEEZ dravid_p_Page_46.QC.jpg
fa887891ed67a83087faed9928128de5
4cda8d872bb82f2344421fc6ed4b88bc6e171f04
67964 F20101203_AACDQG dravid_p_Page_29.jpg
707695d6826b71dcea3ad71d1cc01d02
c675b8a72eaae6cdf252779242dbb68b2b846cbc
6761 F20101203_AACECB dravid_p_Page_50thm.jpg
f635ab6248440c77abdce6f793b67851
edbba4cd100712ff41fdb8b0b29d1903e7274b8f
F20101203_AACDVD dravid_p_Page_34.tif
d78dd5f6277e4152f6ad025f31a41f7f
2b65a9257336845ef16d97e8eac6a983a9ff4e01
78324 F20101203_AACDQH dravid_p_Page_30.jpg
2d45f36c78a86c2ed0412f96fb5b3ab5
3c3987e582e3222c59865ae7d0a4bd9f89e532f9
22627 F20101203_AACECC dravid_p_Page_57.QC.jpg
d35bf4a19f45ad60ebb480be73d37191
a762d606170a8f5832576661f47567d3f410fd16
F20101203_AACDVE dravid_p_Page_35.tif
d4519c4de66a5985029704043d6d6367
e57e42b61bb72cdba79f69ea12b8a796fef6067a
36152 F20101203_AACDQI dravid_p_Page_31.jpg
b316a70db53aa721b32e41732efb1230
4f68a2e78804aa98c1bb38cf19e5bf0fb350977b
5471 F20101203_AACECD dravid_p_Page_46thm.jpg
c998d9fc5332b3ef477f2b48ed2e91fb
dad84a41bb3f845f870d6f658d2fb1c74b2f030a
F20101203_AACDVF dravid_p_Page_36.tif
b00606b2276dab4c5f270a2ddcacf353
8430883d7ed5b8b1d5ec84f4c855a8aa50d440dd
48525 F20101203_AACDQJ dravid_p_Page_32.jpg
997859ed51cbc33f37a9f06c3830fea5
846804ad2121d5f91ae61f0b560b3f1e0cba284b
24677 F20101203_AACECE dravid_p_Page_50.QC.jpg
f210c48dfb63cf3c21ab1ff6f845b2d8
45cefd9f8e36422ef8ce902be621e2e2af27ff36
F20101203_AACDVG dravid_p_Page_37.tif
46e1ed96141068beee5c63f0b8344772
445bd7f0f6d80fed0d874c2e52f51a4929ce73ae
76021 F20101203_AACDQK dravid_p_Page_33.jpg
9bc4c46ebf2f1ced35c94f4569b2f531
94362e78f449ad38138ae6057a6a4c4be7dcb256
6682 F20101203_AACECF dravid_p_Page_41thm.jpg
54d5d650f38283c84ffb5aa4d36cbce9
d3c61be3e8e562a9176557677bd8c354b5920f0c
F20101203_AACDVH dravid_p_Page_38.tif
156e5a95c364e8217da26831c63e356c
0cbed022d96406d546bc635a4ae1c353ca091ee0
40410 F20101203_AACDQL dravid_p_Page_34.jpg
3a5be1f8ba2d7e78f3acd0c89afd73d5
467ece12895a563c3309b6bfe75b1255fc3b2403
4364 F20101203_AACECG dravid_p_Page_09thm.jpg
5aafac4581c545a68e14c209923c32ee
e3f3592a067791704bd1825ef451c33ed83c18d4
F20101203_AACDVI dravid_p_Page_39.tif
abcd06b684ab4a742ad7abbae6d1d49b
04de2d05c842102cef3fe41d0e580e58d87cd410
16853 F20101203_AACDQM dravid_p_Page_35.jpg
fe440b3adf6172a2f22b63ac5eaec162
44274517ca1e688fae1da490765b100f437beef3
25279 F20101203_AACECH dravid_p_Page_30.QC.jpg
725d69fd112c5eff81d230816db2f919
00b5afe6205a2e7a41bf6d76dce6d4be511a11d0
F20101203_AACDVJ dravid_p_Page_40.tif
1f4cecc50b05d086415a57d4af8877e6
0cf1b7442856979ab1ba66e587f2419f12a0ff32
77162 F20101203_AACDQN dravid_p_Page_36.jpg
88efd0619f1b7b5d615891aa2b351c70
9b2394b6b4a0f90c74d012763faee86dc9aae7c5
F20101203_AACDVK dravid_p_Page_41.tif
9fc5b6b7095fde76302bdb3371fccaba
f228370b730d1db4e0a789d11a607899036d7cee
67023 F20101203_AACDQO dravid_p_Page_37.jpg
816761889fd59ee43f9f65414d652595
9a275dd47de4d55998aa38f5f060d33d8d4c48f0
22048 F20101203_AACECI dravid_p_Page_53.QC.jpg
2e735c4edfb72a26a817586f22849f50
ac1f0f6ddfe6d9996f4ec1e6bab57802930d8482
F20101203_AACDVL dravid_p_Page_42.tif
f384da09821179c69b299ec11ee426b8
66f642213dbed5be4595fbd474d64b2ebcb6b012
78349 F20101203_AACDQP dravid_p_Page_39.jpg
610037e87bb8a5e372741082db69ed44
286ee147771b9eb3f20dfa4b83a59832759f9b8b
9335 F20101203_AACECJ dravid_p_Page_07.QC.jpg
52d3b24c6e9bcfe3aacb6908907a8ea5
e414cff72ea7fd4bbd65126c07d76c59050ed826
F20101203_AACDVM dravid_p_Page_43.tif
44ba6457f547e373caaad8be3c5bb595
5e49b29aa9f563d5a6518f535801799f20860aef
74470 F20101203_AACDQQ dravid_p_Page_40.jpg
e24d56d372a444cc99077400714484b8
e72f67ccf994b41d5481cae108ae968e1ad1680b
25213 F20101203_AACECK dravid_p_Page_17.QC.jpg
c689c2313efbafeff49869bb7d1c9274
a376e3a31b02a7a63d80bbd5d704dcab1ea2a263
F20101203_AACDVN dravid_p_Page_44.tif
2b9743dd72a4c68f9438e6ec389895e0
4d44f3989da215a05dafc4323e656ad0266dd62b
70129 F20101203_AACDQR dravid_p_Page_41.jpg
892dbf227e673699a69004df73d5ea5b
91d2817dcb129a472a920e5750d0d240d60611bc
23178 F20101203_AACECL dravid_p_Page_16.QC.jpg
145759029caaa05d585174c06d2d8134
48d8b2fe567f25a9189269d7cba7e5668ebb23ce
F20101203_AACDVO dravid_p_Page_45.tif
398ddd76fc8daf4f1bdaa3599a381fba
89272ed7deb375b610fe0049a3b84b36c81c966b
69844 F20101203_AACDQS dravid_p_Page_42.jpg
e75ab837c17ba4ecf8bdc312b130d353
64eb0ffd4bd70d629b4614e39ec4b68236313cf5
6969 F20101203_AACECM dravid_p_Page_39thm.jpg
bba4a5c88af413e0608934c89560d529
be78996502fe68a678bec98dc25e87e04d4e3aef
F20101203_AACDVP dravid_p_Page_46.tif
70efa2063974b6c2b25d0a69e1e86d34
ee81e9a51e51bc40517a70ab368243ce9bc430c2
77311 F20101203_AACDQT dravid_p_Page_43.jpg
78e1f677c88b5ea3f36c469c5ea72cbf
f68190ffa089c03dd8ec5ab5892e70c10dd2c20f
10547 F20101203_AACECN dravid_p_Page_63.QC.jpg
3c04d5ea37007622f8f179e2be066807
d578b5a010a9bfa5e275cf464f0a206fc4c5bec9
F20101203_AACDVQ dravid_p_Page_47.tif
d9f159d73ecaba94fa226ca88e10c527
ddd40957dfffdc48039654263c7945a7293cd43f
75882 F20101203_AACDQU dravid_p_Page_44.jpg
2b9d6a6ab91d32e0ad48e34e73352745
f9f7120b8a7d6a82b5437ea57fbeefaa819e9335
3469 F20101203_AACECO dravid_p_Page_31thm.jpg
6d836325a48c9d39c9306716083e0119
01aad53fe43544caaf6f54655ffb20244a473a1a
F20101203_AACDVR dravid_p_Page_48.tif
c7d8eae401c91a98a2c56095b7fd1e7a
79429cad28f4283f4ba64aea797d86e6d3d3391e
46732 F20101203_AACDQV dravid_p_Page_45.jpg
2cfc0afc7b88bf8c4ba83ef3ee9bc004
04c4b5f89c06b6189b246bc8b2a820df32482adb
28807 F20101203_AACECP dravid_p_Page_61.QC.jpg
0a3adbb58fbc128fd7efdf8a293e4b43
057db2c1a5cb7e4b8787fac522d64927ef9e0925
F20101203_AACDVS dravid_p_Page_50.tif
deebde29e4a5c07d9fb1f6df2f3f8768
25e5f16e3341b976ac22d37f3b16ce49ceb64d3b
58761 F20101203_AACDQW dravid_p_Page_46.jpg
575aa09df9b691762eeb9a41ef04b34e
0c8824e6404b552809a22dd6b9a7611bae658056
5916 F20101203_AACECQ dravid_p_Page_11thm.jpg
3a8bf37f9d8cdf298961f3b8f7a20d58
9544295dec8d1123b33bb160194b4a141fac545d
F20101203_AACDVT dravid_p_Page_52.tif
c0a69e4fbbfa96ff7643587709085cc7
f6c659e51effa97f392b2571497e83e5280d6050
26106 F20101203_AACECR dravid_p_Page_15.QC.jpg
a09824c65c24e5b169daff4487a6faee
86498c0827bfd14c79904b93254fcc4ccbf8f6ac
F20101203_AACDVU dravid_p_Page_53.tif
98edc35054240775d79680bc43f8ecf5
a92e987b0d500aa95b7e1306a134fb2102a524d9
60033 F20101203_AACDQX dravid_p_Page_47.jpg
f458da94b9b44ee87f679d8c4178a4e0
24e5a7b044718cebb26c9c473e5d7d1abf2392a5
6819 F20101203_AACECS dravid_p_Page_38thm.jpg
033795a759488dedb3a0088b28f973c4
0ab7d1fe944329da35eaa0b39ea7596512d07c03
F20101203_AACDVV dravid_p_Page_54.tif
daf99c9ac5bb1897698d6c03130b6272
06f42bac819f47d3deac4181f783abfe9e3823a3
6352 F20101203_AACECT dravid_p_Page_57thm.jpg
254d967bbc518c596717703e030be03c
847c4f5b701fe185c99d9fdcfbdad06afb4b4c5a
F20101203_AACDVW dravid_p_Page_56.tif
d42c7e4171d8981466cc5f02c5d311bc
01b3e50e79f92767d98b09cad1d851e5c46399fb
36469 F20101203_AACDQY dravid_p_Page_48.jpg
5adfd0dfcb16d2e4b9b08230fab14c04
ec1ffc2d0b90844ebafef30e67b0eb15ded6693f
6923 F20101203_AACECU dravid_p_Page_40thm.jpg
1f562083ea89edd6fa341f43b247b377
07032556b75cfd88dfc24d6a26528f939c3af427
F20101203_AACDVX dravid_p_Page_57.tif
8055059321217b31b87e9d30e474acb4
ad7ea004c966ea34b12c516a93c65ee29812fbc5
5146 F20101203_AACDOB dravid_p_Page_32thm.jpg
f1d025d42379e82873b4ff2433061571
f96d91864b3ec06f275d85e996fea85fc53a2ad3
80593 F20101203_AACDQZ dravid_p_Page_49.jpg
e47341d485409c4184edb73e2ac4d151
b7fd6da63d50d2ebb35033e3b93a9d253ee79ddb
27361 F20101203_AACECV dravid_p_Page_25.QC.jpg
71a7a46c10dc1d109136116e49319400
9769a8357f03016e17397a8a820488b80a85b07c
F20101203_AACDVY dravid_p_Page_58.tif
112bdb97c9b2339f62f2331815ef28fb
1c2c9c9096a75f6e232e2c1eb79f0e02a73b0130
F20101203_AACDOC dravid_p_Page_04.tif
c703af3c46bf113592e8003d860b7534
d50395063d56b08e6a8668b155f088096c1f6850
24203 F20101203_AACECW dravid_p_Page_40.QC.jpg
43cb79562b86e1d9ccd722750d026bc0
27aff576b5428ac375f9888baeeacbe6f09f87b6
112851 F20101203_AACDTA dravid_p_Page_40.jp2
515edbda36b6174c20a4fa758eaa1846
5dfbd3213af264501e3ba35235b244c30c472423
F20101203_AACDVZ dravid_p_Page_59.tif
86d64160e43ad29e2fbcffc709bf3a55
63720c55bfb65e56522a20f22b50fdac321d7a8d
F20101203_AACDOD dravid_p_Page_51.tif
9f345b8b09b21f5fb018f550205f9a52
3c746a16ddd093c3b6e6ad411e8cb99a6804fc38
6926 F20101203_AACECX dravid_p_Page_27thm.jpg
633ca64a7f3eb42d39f0a7c8f520efe6
41379295d9f4143a023d51f80418421bd6357a61
105427 F20101203_AACDTB dravid_p_Page_41.jp2
00c37f58b3a506266eda5de89cd77798
68c198780dae396392dacbf6e977543929ccf9af
22540 F20101203_AACDOE dravid_p_Page_29.QC.jpg
e644d94cece120dc5686743160ac127a
ac8736c37c54968b7a1b214dae561dcc08b4a621
22900 F20101203_AACECY dravid_p_Page_56.QC.jpg
0c493fd19fabe0e8370a18cc880e3d94
f3bb3b232abadffbd7e4bb487317d3fe0ff63a6d
104827 F20101203_AACDTC dravid_p_Page_42.jp2
28b3b3db42998bc8028eba50a72ba729
d6712765c0bbbd82af967316cded159e26a4faaa
51313 F20101203_AACDOF dravid_p_Page_33.pro
c4b9e809043d47bde71c3aeb4637ddb4
fc94d78df60fbc94286be05965a48c4821c85152
2082 F20101203_AACEAA dravid_p_Page_44.txt
770decb29a22c7b961c9ab4ebeca5108
a5dcdefb5bce5cf49a1ff2b3764c505193487be5
26189 F20101203_AACECZ dravid_p_Page_38.QC.jpg
882d6de1d9bba7a2d85464538c5dc0fa
0eb7d3d90ee0217c5f796b63f6502cd3f3b65904
116225 F20101203_AACDTD dravid_p_Page_43.jp2
4a7bdce058afb006e35a7e73099e8297
675913926a6c64903ff65c5788dcda3c26d3a0ee
13573 F20101203_AACDOG dravid_p_Page_34.QC.jpg
d84d398de13221418b4efe7499aca4b2
46e3a32fd63b2b0938a05fcdb8374292f74ba0f6
36404 F20101203_AACDYA dravid_p_Page_53.pro
d13ed92f6baaaaaa859a98516d3d7f47
8e6592dc133d42543fb219ec23e67fa123919540
1703 F20101203_AACEAB dravid_p_Page_45.txt
35e5b490cc27ee22773f9e1b6d752bd8
8f9240857783679ddf8ba86445f08d55b5b5f913
113817 F20101203_AACDTE dravid_p_Page_44.jp2
37c44c2b80adf3221b5ba10847caed3f
4bd6c6afcab42e959238fb1585f497e178c98fbd
57038 F20101203_AACDOH dravid_p_Page_14.pro
742251a7321c27eab0bf4bf840cb23ad
13d84d026bc8a326e1c90913618c11b2219f7be7
55103 F20101203_AACDYB dravid_p_Page_54.pro
98893230ef84234abc08fd36724b0c76
401f860ffe3c22b74da3cb22ff5133acbce47358
1885 F20101203_AACEAC dravid_p_Page_46.txt
aac14cfab150ebd847cf365cdfaa6830
9ea5e109232005a176fe7775c01e5cbd43306a19
20308 F20101203_AACEFA dravid_p_Page_47.QC.jpg
db6d74058a5b7e5eee42a51744b3886b
c3899a24a4f979656d39dc4a457cea2804a10da3
650966 F20101203_AACDTF dravid_p_Page_45.jp2
43eb377bbc8c4758d74ce4956512e9b3
3ccc5f628e13821f9b7d6bd8f71e7e77052a789e
26436 F20101203_AACDOI dravid_p_Page_59.QC.jpg
66d6f219d267b0ae36ee0d68e482b9ed
f71e0f0cf35dfbea09c71e09858d77fee10b3fae
7896 F20101203_AACDYC dravid_p_Page_55.pro
86b9a1cbba0f14411304b3251c60e239
2901e3b2d04e934b9bb41663208bd426921a2ebc
1695 F20101203_AACEAD dravid_p_Page_47.txt
b8e3133b4faa407859e923e8b660a0bb
53f55e9c0b8214bf5e791d937e8feb7e06d35cb9
23891 F20101203_AACEFB dravid_p_Page_51.QC.jpg
0ff934d2fdd5e965f2d2af36d15345e8
a27d085106818933beab60f6d9804520937a723d
84122 F20101203_AACDTG dravid_p_Page_46.jp2
940c80edc341bf96031ba4130fa3514b
57980f58d452f68048c0c71c81763528ed33a99f
F20101203_AACDOJ dravid_p_Page_49.tif
ec43aeac76d28b8f86c4867cd9c2135f
50a5a0de47c58491d74bca0a5d93599c156e53e4
48227 F20101203_AACDYD dravid_p_Page_56.pro
1a51a2fe09dda3542983324b613d2fc8
60323619e37e9c50232a93570f31662ed1fa2abe
1959 F20101203_AACEAE dravid_p_Page_50.txt
89452b0d53c105dcfbbed3c0a2c61efd
60b4ddfbe0e156f39788bbc2c59c9057d88cd842
6603 F20101203_AACEFC dravid_p_Page_51thm.jpg
4ab5a5408ada591994fa130d94460300
3adde49fb51023211ef3efa49d543c7a14135087
87333 F20101203_AACDTH dravid_p_Page_47.jp2
a9d94e89c57b20ae08f2eca2b3b0ed70
059b4a87b14c2241a524a09a9fd9dd2e3d255475
412 F20101203_AACDOK dravid_p_Page_12.txt
5ce7657885df0a825990035db2501eea
7d1e82fffcdb9a95375ea0e8b1aad53e18571f91
46651 F20101203_AACDYE dravid_p_Page_57.pro
43e8efbefac6e4f380665526fcd46677
5f6e6f15be9e4c85e0e337d71148ab14bef3acd1
1944 F20101203_AACEAF dravid_p_Page_51.txt
734071e9b3c6eb8deebee7dde1592117
76d544defcb44bbf6a66ea97816b5a58283a6c78
6260 F20101203_AACEFD dravid_p_Page_53thm.jpg
7b25bb3be36ffb187a1f3974ae303e9b
1b1eaeafd4bb8c3aa98111d2eb0ff37b7c616d7b
49430 F20101203_AACDTI dravid_p_Page_48.jp2
ca0648cf83dc8331c9e136bd9dbfda38
f6aa8446f6d184d5f215fbe0c5491aefca19cc73
1753 F20101203_AACDOL dravid_p_Page_53.txt
2af2a739f189c0dfd44f0742007e6393
41a02fe2f1d7e8e659ff18dbae1f379cd6657e81
69180 F20101203_AACDYF dravid_p_Page_58.pro
8788ea2a57c229014b669a907dc79e40
2d89d5f1aec62ffde518edd3907afec0732ea823
26880 F20101203_AACEFE dravid_p_Page_54.QC.jpg
f79051331f6e22ca377f12c35acbe3ff
4a6d8cdea033061094a6ebfee7c8544293d26488
119644 F20101203_AACDTJ dravid_p_Page_49.jp2
5d0653bc5713f5383cef21d1671173b1
ac33ef8dc826b8292bf8d3ff77e8e22c85134e4a
F20101203_AACDOM dravid_p_Page_30.tif
8b3f0b346508a10cc69da3061b3d8730
fff8480cf0115e79e722e37a50d2a9f02f7ffdf5
63583 F20101203_AACDYG dravid_p_Page_59.pro
4fd2913183659122462a48fb17a5dd63
c7b8872ca6e1b90851f8e0d8c49081c68d389e2a
1789 F20101203_AACEAG dravid_p_Page_52.txt
dd92c8ba58b17acc81482f289c957cfe
d11ff59d53e33fde9e29eb8814e8a3592d006581
7272 F20101203_AACEFF dravid_p_Page_54thm.jpg
ac26febac489447627cbc647f4e11779
dbdb0b1c058d43026f81cf5742d5e5470569990c
109923 F20101203_AACDTK dravid_p_Page_50.jp2
383adf1984d62134fc10aa6f71fcbc6e
5fef0ba23980e97aa8c3908f84c813967342bd0e
115354 F20101203_AACDON dravid_p_Page_17.jp2
df0f101fa73929d6206c29a90c0015e3
5e35a11ca6acc2d3cefc3e577383d99120d10167
65386 F20101203_AACDYH dravid_p_Page_60.pro
cabc8df95cf1d433aa14d9ebe207513d
4c1f857c1352bfa046f554009cdecf1cdc4425e4
2213 F20101203_AACEAH dravid_p_Page_54.txt
80033628de4312e4223ed2fdd490d7d8
e6098cbeb48289b9f5c9617809fb5fcc4cc4e8ad
6496 F20101203_AACEFG dravid_p_Page_56thm.jpg
61cb4105cf4bd9bc1685831638a2aa75
8f8a883916d236ba78d6633417b435fbaefac15a
109679 F20101203_AACDTL dravid_p_Page_51.jp2
1508d565b04efbc3508b4899d3a5e72a
2707d5d790664c07134121eb3e06dc9d5a3b8a77
19568 F20101203_AACDOO dravid_p_Page_34.pro
1f77f56b3894e41ff9c2a14e31ef830a
2f19f8ba27e6c477907bba5d5bba76bd0cfe9f36
68636 F20101203_AACDYI dravid_p_Page_61.pro
fa1ce269c39af8a55ca07b4cda36eb12
ac35fd7a759b5952a05d9c880bbbfd5d83d6d9eb
317 F20101203_AACEAI dravid_p_Page_55.txt
f2a5d640d0d06347fcf3eed2b65d2e05
2a4f5680dd06ffe9da84d2a3d7b5098628f36901
7508 F20101203_AACEFH dravid_p_Page_58thm.jpg
163c1612e2869385512aeb07d926a765
fa1ff76ba3cf845b9d5491472d405b2ef7fc39b1
68623 F20101203_AACDTM dravid_p_Page_52.jp2
fa5cb2d0e4534266e9f90bb918eae277
4ae3af5dd32bc5a0ecc2a198948fb147eeefae3a
1283 F20101203_AACDOP dravid_p_Page_04.txt
9938dc547ab02cec438d2243781a60df
ec54dab35711b975cf07f9c6f09bc690d8d22a6f
59466 F20101203_AACDYJ dravid_p_Page_62.pro
2edffbf84c3e99797250a36c9f6835bc
d2c74c2c1d354ec56d8f280f35208e9ac4026b1e
1967 F20101203_AACEAJ dravid_p_Page_56.txt
0f78009c676f4874a100093ccb0361a9
d1b177af1ea8a8c91e9884051675c3e51709ee80
27708 F20101203_AACEFI dravid_p_Page_60.QC.jpg
fbeb99ad62eced3bd6abc580da542276
1c6fffd3b4270d2f5b6eb06476f57580a6a2f330
1017826 F20101203_AACDTN dravid_p_Page_53.jp2
ce3ed21069bedc6c784dbe4cb9b57dcf
0f83f4f9ea7b6fba8aa42efea0ab48667c741d1a
1037 F20101203_AACDOQ dravid_p_Page_48.txt
4de314570eadb7e9734372531c48ba52
ebca28490066c6eeaee7f9da9b640dbd7fb59bc8
19609 F20101203_AACDYK dravid_p_Page_63.pro
5b707d8ef04aaa4c1360daed36876149
013c94397f274ff47f320c7bec8086ec9c8f463e
1856 F20101203_AACEAK dravid_p_Page_57.txt
1ea13e8a3a8c780f34461b7436902d5f
921c935b83ac6fa9b3ee198d840a093ed5d13438
7719 F20101203_AACEFJ dravid_p_Page_61thm.jpg
b024cf695573132b070c08a68e730e78
35acb513797a2fdffc80067e896835bc4c955e44
121074 F20101203_AACDTO dravid_p_Page_54.jp2
3b54304458af9f6b99b7d04848d24c90
6bd42d13a2a877ca72d04915af092e7e173da19c
F20101203_AACDOR dravid_p_Page_12.tif
96460ae67a98e7940200608dbe544cf2
c2339c2a222c255afe795d8206853aa1847b2e84
29653 F20101203_AACDYL dravid_p_Page_64.pro
95bb03e3e84c8d4acc73971d7531d1f7
f27edd2650906c6964d2ddc22bf3ab786e2c3094
2739 F20101203_AACEAL dravid_p_Page_58.txt
fdf639b4b149100ea6c1c4bd91bfa366
683d3f4130ef43b7257bdc9ca320b39b012b5373
3067 F20101203_AACEFK dravid_p_Page_63thm.jpg
7d234ecdd9cf834f394c4bd241c91cdd
72e1ae776ee0ba320f2fd7dcd7995642bc342446
20789 F20101203_AACDTP dravid_p_Page_55.jp2
bccf4d85f237ab9e4e883e2dac1b5c64
4cde2472815667337676ea023e720b662cadb3c1
10303 F20101203_AACDOS dravid_p_Page_12.pro
c268506bbc9814016b0e6599f5f09586
463ed3814f84cbceaa84c3bdbcdc5094cddf9f4b
506 F20101203_AACDYM dravid_p_Page_01.txt
b43db4b19d9c89300b7387e862cee878
af0faf84af54c5977e4df6d0e5e5e430851acbd9
2523 F20101203_AACEAM dravid_p_Page_59.txt
13dcd2161d927cc33dbc5cdf03bcc2cc
75ed1c553e820f7a383afebe4e7062366782b067
107551 F20101203_AACDTQ dravid_p_Page_56.jp2
9101759a88c33fb35826b0fe7344323b
e5d47c074d553122b7d370ebc168323973ed054c
17572 F20101203_AACDOT dravid_p_Page_31.pro
20bcacec5fbeb07db5a90800861074ee
865c0c417bff36545a045f44e2fda6a6e51edbb8
91 F20101203_AACDYN dravid_p_Page_02.txt
002716c964445f60ede1821da5582044
d09c941f86c5fe07e37049f76440371b1812d269
2596 F20101203_AACEAN dravid_p_Page_60.txt
2a7d4987facf41f8ee67c10018e38d75
802b8d5e416c203ca0e62a8d28c7058111a4e99b
4502 F20101203_AACEFL dravid_p_Page_64thm.jpg
9c93fdc6271aeaae07922b06a52487ab
f604455b9e84107d4377bc9abebb32911aee5682
103424 F20101203_AACDTR dravid_p_Page_57.jp2
74fbc332635f7ee2ca74fe4cab724109
0f4bea559b245cfa2332944dbdab499c36875792
F20101203_AACDOU dravid_p_Page_55.tif
c37d7b5ccd049ade97c26f602ddacdb4
dd4753b22647fb50ca0e6c7f0ed39d1298a955c7
124 F20101203_AACDYO dravid_p_Page_03.txt
6532d5d32f51ac1a03bc5f31e0d5aa41
03f5acf5e04ad8e76c5a4a5fc278fd9976267b32
2711 F20101203_AACEAO dravid_p_Page_61.txt
fe0d3a822447a3952ac10e5f2788899a
e729ecb9adb17ac81f0539d72ded588d03ac7ede
152588 F20101203_AACDTS dravid_p_Page_58.jp2
bf190a3b5419882e651cf033d455f74c
cd433d3a69837f509aa1a606da33b66e22855c87
3924 F20101203_AACDYP dravid_p_Page_05.txt
e1b6b93bf35e2f2f386a0481e8034f01
22f5954981a5a7c94564e77fa6af46a71c312cee
2366 F20101203_AACEAP dravid_p_Page_62.txt
9a70fc8ce31835545cd68cb09edacaff
5ebfb2f0cb1042abd3751b431ca23be3b2f6fdb5
141990 F20101203_AACDTT dravid_p_Page_59.jp2
74a43e787b6f5c1f22590bcda3229685
10433fec684fde42289ad5dbb7180fce5e904e79
23441 F20101203_AACDOV dravid_p_Page_41.QC.jpg
fdf511ec8f15fe3115e4b703ebd01aaa
c8fc9b78bdb6cc118ec6b80ee4f641c90f2b063e
3574 F20101203_AACDYQ dravid_p_Page_06.txt
eaf2a569db9e583ab5d0b03193648757
14027dc8d1fb098f9de56a576b8bb86036e22a73
785 F20101203_AACEAQ dravid_p_Page_63.txt
b650bac1aeb3f8c99d24276ce85f2bb0
e40cb1fa753e57d37376bf8ec9598709b5d71ebe
144061 F20101203_AACDTU dravid_p_Page_60.jp2
8dd84dab93ef0fc9f31439462e0f588e
bcb2d485b02eb96d8f545361d928c6a2c5777f68
2053 F20101203_AACDOW dravid_p_Page_35thm.jpg
cca744b773a0b9d25f3fbb4f76c5b5e5
559a489b36d9cc464eea0c49861bc72736e32b09
809 F20101203_AACDYR dravid_p_Page_07.txt
bd760ccfdd13fb4dd9926f671007e923
9ac987827a11c67c6026549313640cea767605a9
1209 F20101203_AACEAR dravid_p_Page_64.txt
ac896011b9c6726da5ff6a5c86a96d37
61791656655f87ce192e017e19d6f86dc6ed775e
2225 F20101203_AACDOX dravid_p_Page_23.txt
bbfd34f2a7691f293db093e2388daeec
8ffce216ba7a50c3e36e8a064979a390bf5d0f21
1798 F20101203_AACDYS dravid_p_Page_08.txt
6246a7f1d2571304a8549158f42ed417
c276b6b8897c906845f567af3a170ed037ff17cf
523721 F20101203_AACEAS dravid_p.pdf
458f908d93e2261b447b81b72563a827
259e57786bb87f601bb6b04a4481e4a483cd8296
149556 F20101203_AACDTV dravid_p_Page_61.jp2
1edf9618403f7662b0ce2b932cccf26d
7423e4dbea0ffbcedc8e55a1e3112d08ce7dc69d
25016 F20101203_AACDOY dravid_p_Page_01.jp2
ac894058224e7297a66d0789e592a837
cce2a5d51200f63756c7f485c0fc034bd749d3c5
1044 F20101203_AACDYT dravid_p_Page_09.txt
6674efd86533500bcbf108142b108e58
1fcf515888a63dbbc061a8ba1a26f9be32b419c5
5818 F20101203_AACEAT dravid_p_Page_47thm.jpg
0bf80e13a5c8c3f5c670db6e7a21b378
ecd4a02d5e4f11028a4f32ad78204216c5cdc8b0
133632 F20101203_AACDTW dravid_p_Page_62.jp2
2a553d28e94622ea599f7088c94176a2
d635f81c124df6a5904c74c50a08794b00d67c13
52330 F20101203_AACDOZ dravid_p_Page_21.jpg
4b2d854f72c9bd02424f45397d2ad5a8
1153b819e45d55890c11cd7c912b94dd4106eec3
294 F20101203_AACDYU dravid_p_Page_10.txt
24b8240dd72c8ecfd334989e0332a40a
b15fc499a3aa56f1a5993e7fd1698fea783f8e32
6894 F20101203_AACEAU dravid_p_Page_44thm.jpg
b4966191de014c44bab5844b9112d69f
2b71c11ea04e255bcb6c02bae4bca69f77e4b8b3
48109 F20101203_AACDTX dravid_p_Page_63.jp2
63c3e53a1232907bb5f7062aaf58cad2
ff88e948ed2c545be74ccf8fa9d83f3c48ff652b
1874 F20101203_AACDYV dravid_p_Page_11.txt
c1d6d56ee577f8d7ad61278657db608e
4119f67acbd90a78da322788e9944a4030d1cdaa
23442 F20101203_AACEAV dravid_p_Page_05.QC.jpg
f2851697b7959a98ad5f85edabf95b52
1c548ba187c7e080a948f1310f37273f473d74b4
68078 F20101203_AACDTY dravid_p_Page_64.jp2
2e0cb65e8a65dddffb1db8f3e07dd4ae
023dec329fd364dd6307dfc678c175f141393e4e
2150 F20101203_AACDYW dravid_p_Page_13.txt
962b831f95af035478ae496f1f11416e
294da7acd67390206f76b78a056c098391d8ad8a
6711 F20101203_AACEAW dravid_p_Page_19thm.jpg
3ca0395b26dcff2277b5a4ebd3f6fc32
2fbfa7b96b6ad28d476cd000f2acd0cbe1a3e668
73965 F20101203_AACDRA dravid_p_Page_50.jpg
d4cb8a55f0bf00ae4f7cba88cfb6d7cc
8e9c4cd8a40c4048999fbf46edb7af4332642b2a
F20101203_AACDTZ dravid_p_Page_01.tif
755429029949939e6629b0bb994976dd
41099dc25d16e22754694a48e814d2e99479a896
2243 F20101203_AACDYX dravid_p_Page_14.txt
748255cf5e9fb8894d8128a5b1a6e906
e9de4290464298a2f7e96ad8da74b8ba627d118c
5536 F20101203_AACEAX dravid_p_Page_22thm.jpg
52fc7d215c123d3f2c58d53996c1b12c
e2841fa6750d006b81721afdf899b3c0fa5f43cf
73139 F20101203_AACDRB dravid_p_Page_51.jpg
c5b0a28e2078a6586fa5c22262254aa4
d65c717e7b0eb2972ec9c547eb641272c8316ae6
2101 F20101203_AACDYY dravid_p_Page_15.txt
848ea3a06924162ee103e9f45b8ea851
97a9e0b4611564a2b152536db81497ed94f07c8a
15656 F20101203_AACEAY dravid_p_Page_64.QC.jpg
40ec71ddc8162e8b753a2273f15887d1
af520fac5d59092225ca439fcc980f68c5f36531
47233 F20101203_AACDRC dravid_p_Page_52.jpg
8a111e8adc98e163b337796fa40e7b27
08cc8e3ad906cc1c50f55e973fa4a4723f90d2de
F20101203_AACDWA dravid_p_Page_60.tif
ef0b548d51f05694e116a551a9ef7a7b
25882d23e6bdf2a77d11f72afb28816353c66b4e
2120 F20101203_AACDYZ dravid_p_Page_16.txt
fc1aad145be61ceeb9037a796636046e
37f462c117777b36386f82139052a1c1c1ad74d1
6778 F20101203_AACEAZ dravid_p_Page_13thm.jpg
2f2d3cc50c47c7171fa735598a6cd896
c080abd5be1214a963cbf0a245241ae5fe247dd9
69184 F20101203_AACDRD dravid_p_Page_53.jpg
31f1832c486bd6603ba158753000d96f
7c8c9838eeb63d52d745ee91a6ef85eca51509c6
F20101203_AACDWB dravid_p_Page_61.tif
157fe7c21f8e49d10a886dceaf8849e1
d30c8c1922a899a63d1901dea9abb58942d32870
81184 F20101203_AACDRE dravid_p_Page_54.jpg
f8e7749c8e70ff3733eef0ff7ce636f8
7211898fc251793598d2c32b6c71c5814854841c
F20101203_AACDWC dravid_p_Page_62.tif
83b2d0e379bc35210c58c96e27a07a6e
fb3104f6df72a3605db59feed3d86eda87c63cfe
18623 F20101203_AACDRF dravid_p_Page_55.jpg
482b92dd885b304978386d0c57ccb6e1
d213745743b36c26b2bc9944256ce9c5cef595d0
27977 F20101203_AACEDA dravid_p_Page_58.QC.jpg
f6b046ea0eddcd22734826d21809ede5
9cd7af6fda02653b61aaabfb46b9932e7e17ed8f
F20101203_AACDWD dravid_p_Page_63.tif
7f8f341b13c0a267f773fe836d60d161
2323845cf5e3718452584090cbb2b789c69eecb6
71423 F20101203_AACDRG dravid_p_Page_56.jpg
1cb1017028729084a859db710b79dc6b
addb0874e925b97ba1c92fcdf30222dd3fbd98b9
3784 F20101203_AACEDB dravid_p_Page_48thm.jpg
682b52ed11c659335d2a41d0337eded8
9805d0a12283e20d09f78c24c710361673695c3b
F20101203_AACDWE dravid_p_Page_64.tif
e663c81b53f04ebf141926d15a877de3
e3f5f7fd31c964716e1c9a24e0631fa8d4406ed5
69258 F20101203_AACDRH dravid_p_Page_57.jpg
ac5604a7c49ed7e4b6717362bd895592
6b1ab4d11a86ae5a06a878f1535d51c55bd80b6e
26479 F20101203_AACEDC dravid_p_Page_23.QC.jpg
9c7d8f2990129509f5c8c8108562ab07
8e803ac9242c60afeac550ead6423fb1a903a086
8213 F20101203_AACDWF dravid_p_Page_01.pro
5dd1e51d361c42039b48270567e03f18
e02155eb2ba7b988bc8638e889627a11528d7ee8
96470 F20101203_AACDRI dravid_p_Page_58.jpg
6e63cc093bccdf05bc3e38ed666d3e41
52a72353e6046093ff1f7f462d218fb6034a8381
25491 F20101203_AACEDD dravid_p_Page_44.QC.jpg
fe73c982db7e8b2197c7b803531a941b
b05dc9e3a38ec1b1e2526f6f9949c8106d547385
911 F20101203_AACDWG dravid_p_Page_02.pro
0fb196af8f3b7d150b2e15ad8cb285d8
45bb82500c2d60f464f61cf989c5b4baa5cad327
90476 F20101203_AACDRJ dravid_p_Page_59.jpg
000c8437c7780a7267b7ca50a34175f9
1de6a84ad206adb175be66a5e06ff53f51bcef99
16076 F20101203_AACEDE dravid_p_Page_32.QC.jpg
c57c8d7ec5c62e4eb48a75313b37a980
c0ba881d87ef94d0ed79984e0c5acdcf6e7a5161
1819 F20101203_AACDWH dravid_p_Page_03.pro
b11dce76ec7afe33e786396fc26814b4
cdb45bb59c0cdd3cbe243c4b999c84c037120334
95969 F20101203_AACDRK dravid_p_Page_60.jpg
3a7e48f271b7638424c132fc28040199
8f6cf5b0805a221bc3580cf18b2f2e21bbac4e7f
18963 F20101203_AACEDF dravid_p_Page_06.QC.jpg
9cdb4c31017c30dc4e5fb4a112719ab2
a21a2c94b7fe3cabacb3e9b38d2a18d7acdaa74d
30968 F20101203_AACDWI dravid_p_Page_04.pro
f820add761af65dbc59c1c4daaca1de5
72c7a123c8dcd544ad91d2fad9bdea6d4e0f658f
100136 F20101203_AACDRL dravid_p_Page_61.jpg
2def1e021fe13b6b4f3e9be3b6c76a76
ee374288426d8be33ab41e4fdf7fd5de27ec20cb
6186 F20101203_AACEDG dravid_p_Page_16thm.jpg
f46c8e8431f3b9953320ccca2447785f
ffa48e8f10e57194940ad849fefafd41da84b3d3
95682 F20101203_AACDWJ dravid_p_Page_05.pro
920ab7f75cbe5264e4cb55be89ccb7ea
cb62a945370b9462e429c47087605234e8ad30ec
86655 F20101203_AACDRM dravid_p_Page_62.jpg
8d7f77130f17e621529f9b09633d2eaf
baf1686d9a96ada3c1de754459436effbc579e36
3081 F20101203_AACEDH dravid_p_Page_02.QC.jpg
7b92dad7945aa67822eccd03431958d0
abce6c199fba12e6a4ab75e81c518d506e5c1c95
85653 F20101203_AACDWK dravid_p_Page_06.pro
55c3b6eddcfc5a694f53016403216803
a45d775d98399bb80d7c2cf6249d451b02d16aab
34511 F20101203_AACDRN dravid_p_Page_63.jpg
00afe49466ce2db834f331eeaa51e973
47a79e507bd35055735a49dec8bd35e78fe47265
6372 F20101203_AACEDI dravid_p_Page_55.QC.jpg
d6447dd98f4ebda3556b08b16c4349f5
cefea5c48260b906de6d520d17d07e1dcd59bb1a
19226 F20101203_AACDWL dravid_p_Page_07.pro
609b8d1855890e8b53f277c4f007cc2c
a2ab82930f06ffb6569651cf3596a4f64199c22c
48502 F20101203_AACDRO dravid_p_Page_64.jpg
ef9f5640c45c3a4a8836ba7f2fdd0da9
7f970b7c994cd1a26e1902d6d25d5b492c115007
43383 F20101203_AACDWM dravid_p_Page_08.pro
a89b3b162604139186d0f2f359997865
8626fd53aaed8ac7dc70686d3c45344292457dc0
5360 F20101203_AACDRP dravid_p_Page_02.jp2
d9a9ad06006714d2547a6388f5e31b41
f7f6ca08d9490778c7a8d5a337de0604815c918f
6046 F20101203_AACEDJ dravid_p_Page_05thm.jpg
868846c93fc13b1c1b457c936bb95c9f
1aa3c1196fc71b0e7e661423d77e40be22cfb060
23925 F20101203_AACDWN dravid_p_Page_09.pro
eb0674b7b29e8ecea3d1dec90f49a702
3881d848289de62776b420b555aa13149f4d2900
7286 F20101203_AACDRQ dravid_p_Page_03.jp2
fa27778e7aa7dc9a4eceaa36f951967b
ed0fd8882eeac25d11543a68539ac8e269939fc9
4895 F20101203_AACEDK dravid_p_Page_06thm.jpg
b1fa393c34faafec53013f875cf97ee9
7eb2aa3f7638daa1026ec47efb1f3b2e6cec6ed8
5906 F20101203_AACDWO dravid_p_Page_10.pro
d53cfc97d2300dfc8aaa6086fbff3512
c27ef0eff70ef1a0c00cae155118b142d78a4ea0
69479 F20101203_AACDRR dravid_p_Page_04.jp2
889568d4759d8dc12bfe7435dad4401f
a0850f7ac909c6da6f983bab5df7adb4b62172f1
25259 F20101203_AACEDL dravid_p_Page_13.QC.jpg
366c67a3c078f2a8a5a902d3ea9505a5
38ffc82eabe99c5389f018d883b8c829d4bd7bca
42101 F20101203_AACDWP dravid_p_Page_11.pro
8bc5db361da79b1390f58cc6fef9c712
fba1624d50536e417c9622f3880f2a8192bcf59e
1051964 F20101203_AACDRS dravid_p_Page_05.jp2
13a985efb6b15e434edcfea05d499333
d2542d3480ea83aa3c47c067d2a0bf8874f01e2a
19493 F20101203_AACEDM dravid_p_Page_22.QC.jpg
0ff9e1751c0f97f219e01f5bf8e60869
ddb446817663844fdd20826b63e46947d909e7d1
24561 F20101203_AACEDN dravid_p_Page_26.QC.jpg
31809009d815c93f1331884e15c09419
417efededd62f9906a3e89148206afff19bbc5bb
52254 F20101203_AACDWQ dravid_p_Page_13.pro
075a51a97ac4f6a1d364506d96c07fcc
f120403eb34dae71caac6a5d93c473fdaf656d46
1051982 F20101203_AACDRT dravid_p_Page_06.jp2
009a27224094a1efca4b1a1bc68cb3ed
64c90c76d4c14e99faa5b3f68518de88df36102b
97948 F20101203_AACEDO UFE0021832_00001.xml FULL
d53bbc4a7469a1b8dbe099feb1190244
cb039847c0c875a41783b37312504cf8cf309b1c
754240 F20101203_AACDRU dravid_p_Page_07.jp2
03965ec6670c86aa0f767afac19f0557
e6938c4b4d4c19f302442e43426ceed7945cf685
52757 F20101203_AACDWR dravid_p_Page_15.pro
9f5ee2ede25c3cb17adef9d2587a74cf
652cc58c6b9acb6579457140773dd4fd5ccab1b2
7406 F20101203_AACEDP dravid_p_Page_01.QC.jpg
9f55e3ff0fd28441567a7c89a42a118b
c76870f5965576049eab5b3d5df553c1665140a4
1051965 F20101203_AACDRV dravid_p_Page_08.jp2
2ade0766c165ccb24c8bcf0af5cbb32b
1ff2cb427e30e9cc7f89d8d2265aa8157b64c136
48812 F20101203_AACDWS dravid_p_Page_16.pro
8169d6a0d40e17c4fc64ffcd72fc5572
a7a9bf5062faa76a5975691b36daf759ee566c3c
2354 F20101203_AACEDQ dravid_p_Page_01thm.jpg
15e08f2d740e120aac3128eee223387d
1914fc2d4a949f01b217559442a926788401e17f
58258 F20101203_AACDRW dravid_p_Page_09.jp2
9b6a6e5cc6e641c45640d786ae107f2b
50dc390c8fd661f8cb1d0e9a3ede789922cc50d3
52700 F20101203_AACDWT dravid_p_Page_17.pro
8bd513c47d4bcc8973b859f5e7dff842
703efd375579e912aab19422ee6d1088ad813fe7
1332 F20101203_AACEDR dravid_p_Page_02thm.jpg
e1f66bc4a0b59d3739d90527a9314157
33534ad624bde4c716b1f1b3ca34233dac4e0e64
14040 F20101203_AACDRX dravid_p_Page_10.jp2
f9b5b692bacd7cef1d7cff0dd6f79fc7
79de4b5383a389ab0c4b028bdb8af9ae0a00decd
52032 F20101203_AACDWU dravid_p_Page_18.pro
4eda265ee0b8e6bbcde122596281b4de
56ca68ce1a851e45dc01431312069bbf6e05da1a
1427 F20101203_AACEDS dravid_p_Page_03thm.jpg
036349adb61add1cb9984e06b56110e6
5e05bf070bce63ba736782ee40526111c02a9317
53246 F20101203_AACDWV dravid_p_Page_19.pro
0020854a1343c93e5f136c2cb2ad03a3
c603cec51fb86352d8adaf33a95b98ba700bf825
16307 F20101203_AACEDT dravid_p_Page_04.QC.jpg
c12fe593ea9d9d3aa1bc7d3b9fd34772
a9a4fb8caf9d9bd86222164cb8c4ef4487a37988
2257 F20101203_AACDPA dravid_p_Page_49.txt
23ba48116ecc4607265b2befcac777c7
b8b37f8de334418d28b924dab009f55a3f3befdb
95656 F20101203_AACDRY dravid_p_Page_11.jp2
3f370b7632ea0c972c5af14378026b07
f4fa38659f276685ad6872b8ba1d319450af61c8
56668 F20101203_AACDWW dravid_p_Page_20.pro
9435e15a8289af69f033c8673be3a77a
18ee98dc1e01461056d14fdc5914900d6df27932
4739 F20101203_AACEDU dravid_p_Page_04thm.jpg
d5203a5fb84044ee58385d60e8093451
8f9eee062427bda0e02bd60f1c851b4d6f638ae7
79312 F20101203_AACDPB dravid_p_Page_38.jpg
7c8e6bb7d8d934b3f991f28cdd7e6eaf
3ef2c39d28bb1e6afc87ea681a3c19edc6286e3a
26225 F20101203_AACDRZ dravid_p_Page_12.jp2
059d7b8bd492fa79cdc35d96f3239413
67baf5761d91129507b724a1b442efd2dddb264e
30813 F20101203_AACDWX dravid_p_Page_21.pro
725b7077f7132b01103713edaf31986e
653eda51c92ba11a464317b5a49061d44ff3da2b
2854 F20101203_AACEDV dravid_p_Page_07thm.jpg
5c73fd7cf749e38d80e3ec5acb57336c
83526c70755b5c84c7bce90310d06cfe88ecbf25
75816 F20101203_AACDPC UFE0021832_00001.mets
12550e76fce0bf792a3c5c84df6d09d7
1c4d9b9fc31ea4bf7bc7a1233dae938077bfad56
38475 F20101203_AACDWY dravid_p_Page_22.pro
b677fc4f5c1d3171d7ea74ed2c34cb8b
a3d1da475b0b34716bd05b69d0422e95403f0cf2
18136 F20101203_AACEDW dravid_p_Page_08.QC.jpg
8fc0594d5e93751ad0ea78d27726e6dd
35fe2566f0e6bed1695bb7bdc58b70252cf1b0f8
F20101203_AACDUA dravid_p_Page_02.tif
b1cf88cfdbdac47d9140907ffdbe5036
9c5416df964d06d4bf6fdef6adeaa8ee5a3ad349
54306 F20101203_AACDWZ dravid_p_Page_23.pro
c0a7c66374e412e2006e41bd63b5e00a
63358569a240d67d822b51af344ce9848c47bc7e
4827 F20101203_AACEDX dravid_p_Page_08thm.jpg
c36913ed2c2877d37076e2e2685bba79
54d6e1abe03ea639c8c5a13061d1de59d41a1c18
F20101203_AACDUB dravid_p_Page_03.tif
33cd21dca87d7b18af679dfaa0f12459
7f03631d73dcbe05a18812ddc6cbd7a352a47ee0
1839 F20101203_AACEDY dravid_p_Page_10thm.jpg
9a7216b273bf045e26bc8a147b3769a7
ce22a67e384a19189cd12067af5dea54a35ad1e6
25696 F20101203_AACEBA dravid_p_Page_27.QC.jpg
72b324ad30bfb439aed6316a5055acb1
b6e9c4f231116c8c169d6d655739f18be48b6f26
F20101203_AACDUC dravid_p_Page_05.tif
da7f4e535cde4fc4356ac7f549ac4e2f
f753612605bb69c56f4f97cfe34e639e69875c70
24618 F20101203_AACDPF dravid_p_Page_01.jpg
7f2f0755c3b01b7c7026f7cdb3255438
3dc31ec5170fb1dd0a53761eb03f109f8b42cfc9
7067 F20101203_AACEDZ dravid_p_Page_12.QC.jpg
163bac8c350b96ff569e6983d3771d57
c2536b1bb873a7179216c70712934875b85d03aa
2080 F20101203_AACDZA dravid_p_Page_17.txt
52cdd48ccbbea2d913fce6e21edae3c4
b7422d7e183cf3bba8e9ae68e1352b9a09d81226
6314 F20101203_AACEBB dravid_p_Page_37thm.jpg
6f3380adb198aa6ad18af5e9d0f08502
7bd61dd3eb2a032bd385c568a9eaa95845656a28
F20101203_AACDUD dravid_p_Page_06.tif
2766887253a8bfea872621caf1b66bf7
66e979c7a571ca80c8831befc8a233943e8a6a7a
9968 F20101203_AACDPG dravid_p_Page_02.jpg
b5b4934b434a5fbb5cde52aa86f77caa
0fc7e7577f954c09db63102e4a9173d1abc28c27
2052 F20101203_AACDZB dravid_p_Page_18.txt
aebfef912eda1c8d5b7665e111f31fa7
9cfab6a9a26a8d7fc55188b6e36de97c2bf6fd04
25940 F20101203_AACEBC dravid_p_Page_62.QC.jpg
d19d9a79799acab2d10d80bdff4f555c
86265c3c5ce740cb0aafe8b3856109c140fd687b
F20101203_AACDUE dravid_p_Page_07.tif
44f7af0787afb9b62dc533b0acbcdacf
7d16a55a6300102b1e568059d2c7d99ab962e099
11229 F20101203_AACDPH dravid_p_Page_03.jpg
84559226d017911f11e7dde242ea5386
5601ae009332c5518d4c662aebefda8ff2d0d9fa
2121 F20101203_AACDZC dravid_p_Page_19.txt
bf1935dcbf91e9c9fc6c357eb728394a
8d99b93486f64d4c9a2f094544b73616b79b0663
10862 F20101203_AACEBD dravid_p_Page_31.QC.jpg
28b9a0a738ebb7c1ee5bf2e8ff7da452
f736d62dd096371a1d8cd12c3ea08fdeb9c9c7b7
F20101203_AACDUF dravid_p_Page_08.tif
f6235359c28e44b411b6bf080ec0a4c7
ed73a4de760240ce4b8fd40d1d3faa03067c9f44
49780 F20101203_AACDPI dravid_p_Page_04.jpg
f8ba15fbf634b2f3eec013c9e7c9523d
c6e8d2b236579a2bf660a4340ee5a89cc45b8166
2218 F20101203_AACDZD dravid_p_Page_20.txt
5fa35f584ba1c36df3983a8480272484
b4710309cf5d241b0db5a72ff4d8794447b94f82
7059 F20101203_AACEBE dravid_p_Page_59thm.jpg
60684bc73e4cc8fcda6d7bac63c4c875
d20221a3eb33666e9f08c9d37f9b8ee5ca2582b0
F20101203_AACDUG dravid_p_Page_09.tif
830516e2eae98fc4a33d743f3fe2b438
1cda9e68890f2298f0910efcd1bcf60791963016
88579 F20101203_AACDPJ dravid_p_Page_05.jpg
a76d4e4cda494e25bf9a0e9641a7dee3
46306e2d83a0a19229646b2b98053f9db3b25c51
1632 F20101203_AACDZE dravid_p_Page_21.txt
92c4420e2d7eb1bd07faa90d9973f34d
9d9453ea4b5d68e4b8bebd85f60e21e5aa43f28d
6884 F20101203_AACEBF dravid_p_Page_26thm.jpg
13f747610b627add6e05f9d58f54e36d
7e65a9b0cea4c36a0ae5010c7e415725b73ebf94
F20101203_AACDUH dravid_p_Page_10.tif
9dac7f360170d7563a8a69cf3c3fe745
2eafbfc389a65f4005193215edea040a3a739f77
75363 F20101203_AACDPK dravid_p_Page_06.jpg
29c7c943778baaebfbc5d932755d7954
64c24ff626ef4cd5824977a7c9765e4f36fcc3f3
1570 F20101203_AACDZF dravid_p_Page_22.txt
35b3e0b961e37ad661b495dcdf7c1fa4
0c6dcbf2650c119504c66b961c7e2588641cc2e7
6988 F20101203_AACEBG dravid_p_Page_43thm.jpg
cab988c44191a290d982c155e8599b38
e81b13bdc83a0500f421e2dec1e088fe8dd6612a
F20101203_AACDUI dravid_p_Page_11.tif
d70cbda57e85318fca075fc2a577e307
53003048530d827bca836a0d31372ec91425aaa6
32969 F20101203_AACDPL dravid_p_Page_07.jpg
ccd43d459c6712946aa7222dc90c9675
89db2afebfe882a8ee57ca22bae0da8322bde24c
2140 F20101203_AACDZG dravid_p_Page_24.txt
d8ce48310beeeada5bb09c0fb797e0e3
5aea7c14baa1dc99bc42bb2b654616676c9508c4
F20101203_AACDUJ dravid_p_Page_13.tif
0f31acb4a93066e49428846fae0b6e33
f7a9027729b6277d08e03a878adf1ea4dc345b49
64693 F20101203_AACDPM dravid_p_Page_08.jpg
0f77b0e2fea20e6d570bff6987b5c14a
cff5237061adf09a986d5e1bb50d47577f3f8afa
2057 F20101203_AACDZH dravid_p_Page_25.txt
75cb4e52fef36422d21fe242b17dd9a9
4d5850a515eb3bbeeb0d3a1685cf7957bb025512
4252 F20101203_AACEBH dravid_p_Page_45thm.jpg
2679d6d07793b06e9441a508ca954959
026e26d06d33d53a5816079f5780e3456f2cf72a
F20101203_AACDUK dravid_p_Page_14.tif
e563d38cf108ab09cdd9250281faf48c
cc597bce3f80f0b121885f1fa85a067c50d80975
42347 F20101203_AACDPN dravid_p_Page_09.jpg
19f22f010ba4418da50eb137465097c1
a7433b2e1c96753d13bff11e4a01d9f58ce76961
2002 F20101203_AACDZI dravid_p_Page_26.txt
b08efad1b64233e7ad7fd5aa92e94b81
f3f2857efeac1212bca05fbf2a943b2513e80929
2196 F20101203_AACEBI dravid_p_Page_55thm.jpg
882e4ea6293eefe512fb1c254db4bfe1
c5c9f21954a9e0b30cd4041ebeeb712ab3eabd05
F20101203_AACDUL dravid_p_Page_15.tif
be5c499f577c0c16baf66cea0b530178
d59128fb8dbb1fb1ddee6e344bbb2655af8b07f7
14916 F20101203_AACDPO dravid_p_Page_10.jpg
e00ae5f777dccd4566bb432515730d0f
ce889686632e70623fdb1391dbfc793aab864c92
1960 F20101203_AACDZJ dravid_p_Page_27.txt
421c8d2ee5b94cb535f22994295f4ddf
8dd151c2947476a91509916e49fcef8333b3f60d
26703 F20101203_AACEBJ dravid_p_Page_49.QC.jpg
465d3d84ca67d190da9c5432f839c855
e30952ea1d54f19f0e3f739fa02aebd7c2891767
F20101203_AACDUM dravid_p_Page_16.tif
fd6513bc9ba29f2561173f8bd1fa333b
8b2fb641ffd32057c2df58a60bce0d638ae213dd
65755 F20101203_AACDPP dravid_p_Page_11.jpg
309ddfefe238cb9dc0fd49fc3d03301b
fed5a462e4aa07614f86e06770782698b7d38b84
2125 F20101203_AACDZK dravid_p_Page_28.txt
6db3be8bbbee1e41bc1691fb07b9ce04
2aab1633c54e5793dddee88bb74f9c2b3dc24367
26776 F20101203_AACEBK dravid_p_Page_14.QC.jpg
e5d667a04730d3edb779252dccfec54b
7a0c5d70559d1eab989eb34fe51421078210223c
F20101203_AACDUN dravid_p_Page_17.tif
c4fae52e570a465a72237e41b7f05231
9e3db75d00d0e46a1f7be911f0acdda6c490f100
21725 F20101203_AACDPQ dravid_p_Page_12.jpg
8aa50f32c58247949fa791a8c87b5ce7
a6c83d9c044fdec3fc38527386792902adab6032
F20101203_AACDZL dravid_p_Page_29.txt
9f1a1b98ace702f7306c273a34e6a2ec
a127d61f30c047e343a7ed5cd4a52a23ee30c6c2
7183 F20101203_AACEBL dravid_p_Page_49thm.jpg
14dbf3e62c881925dfc1d0bd28b1bf91
f61a4a5339bf6915fb2f4aef7d710cf0b80c2fea
F20101203_AACDUO dravid_p_Page_18.tif
9f1cd7b4fdc2e96135cb9e4d7b004451
ddd8dbac74b0a0f631264b07bde19a9ec906cfd6
76722 F20101203_AACDPR dravid_p_Page_13.jpg
b20c945c199c84f214277650ef57c5a9
69a05631381d3c0cb8a7280f2abb378258ba6627
2067 F20101203_AACDZM dravid_p_Page_30.txt
d4c8340becb5d57c9e5de74f0e72ae59
21c0a9a7588926f2b575757dc4ebeab84778e5f7
25763 F20101203_AACEBM dravid_p_Page_43.QC.jpg
5747d41099019216bf406e951208514e
0f70a2bf7a5cd20525c9ffa3d860aa54a977a91c
F20101203_AACDUP dravid_p_Page_19.tif
a5ff000b2ff9bf27ae3c42293e6c2d64
603e39f24d76b0647234accd7f904df2102f5cad
81274 F20101203_AACDPS dravid_p_Page_14.jpg
4b62c88fd2e378e8adbdf39673fea24b
ee84dbbf36451cecf2dbc51b0c7e612a036305d8
20281 F20101203_AACEBN dravid_p_Page_11.QC.jpg
5b28bd216e7542e6160ba2cb04ec1822
5886a68e7ba364aa3afa917a2fff7a01b2639f51
F20101203_AACDUQ dravid_p_Page_20.tif
a3cde04c82a199363fa211057c33a391
d077420e5b5a3d5d138ae45688e8538c6e6a58ec
79008 F20101203_AACDPT dravid_p_Page_15.jpg
376cd244dd70f76ffa14a5e314001530
73f83b9067896d0426301c23777cb6dc28cfaf82
824 F20101203_AACDZN dravid_p_Page_31.txt
bec68898f54b4b8617d5b3887154ea75
ef83045e03a2c890b94364bc4f4e674f0ce69f57
11569 F20101203_AACEBO dravid_p_Page_48.QC.jpg
1eac69f6f16556f4a03dea314eb428f7
74a9bd62f57e3d0a2b937586a359d0401c222ed5
F20101203_AACDUR dravid_p_Page_21.tif
5f46452f910cffdffbad2e2e00f380d4
3144e39bb0f83e29ddb24ff6f3f040063c6aee8c
71001 F20101203_AACDPU dravid_p_Page_16.jpg
674cdb13c2c7dabb6b4b06403243cc77
f6ac27462dbf894b233bc3d8480686dd3709e113
1542 F20101203_AACDZO dravid_p_Page_32.txt
b8e5af7f2550b882860c7462be3339f5
d722a1c4b4ece640cd589183a2d458e7aed40718