INTESTINAL ABSORPTION AND AVAILABILITY: A VASCULAR PERFUSION STUDY OF THE RAT SMALL INTESTINE

WITH BENZOIC ACID

Diem Huyen Ton Nu Quy Cong

A thesis subrnitted in conformity with the requirements for the degree of Master of Science Graduate department of Pharmaceutical Sciences

University of Toronto

O Copyright by Diem Huyen Ton Nu Quy Cong (2000)

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Intestinal absorption and availability:

A vascular perfusion study of the rat srnail intestine with benzoic acid

Diem Cong (M. Sc.). ZOO0

Department of Pharmaceutical Sciences

University of Toronto

ABSTFUCT

The overall systemic bioavailability of dmgs is influenced by the intestine. a

tissue capable of absorption. metabolisni and secretion (exsorption). To date. modeling of

these concomitant processes in the intestine is scarce. and there is no existing approach

that can describe the well noted observation of route-dependent intestinal metabolism - a

greater extent of intestinal metabolism for oral over intravenous dosing. The purpose of

the current work is to inter-relate carrier-mediated absorption. metabolism. and

çxsorption in the overall absorption ofdrugs with use of a recirculating in situ rat srnail

intestine preparation. Benzoic acid (BA) was chosen for study since the substrate is

known ro undergo uptake. metabolism and emux by the intestine - uptake is mediated by

the H*lrnonocarboxylic acid transporter 1 (MCTI). rnetabolism is by glycine conjugation.

and exsorption is also by MCTI. bletabolism was. however. absent in the present

systemic and oral studies. but luminal secretion of BA was noted. Upon introduction of

the dose (0.166 to 3.68 prnol in 0.4 ml physiological saline solution or 0.4 to 9.2 mM) at

the duodenal end to the entire intestine. absorption of BA was rapid and almost complete

(> 95% dose) after 2 h of perfusion. A lack of dose-dependency of BA uptake was

observed. This was attributed to a large reserve length for BA absorption. The notion

was confirmed when extents of BA absorption From closed intestinal segments of shoner

lengths (12 or 20 cm) remained high (95 to 96% dose), albeit the extent was slightly

lower for the ileum (92% dose). Heterogeneity in the absorption rate constant was

observed arnong segments. denoting that the activity of MCTl was highest in the

jejunum. then duodenum. and was lowest for the ileum.

The developrnent of a physiologically-based model Further addressed the manner

in which absorptive. exsorptive and metabolic events are inter-related. For better

illustration of the metabolic effects. previously published data on morphine

elucuronidation in the vascularly pefised rat intestine preparation were utilized. A C

traditional. physiologically-based model (TM) which regarded the intestine as a single

cornpartment. with the intestinal blood in its entirety perfusing the tissue. was adequate in

describing the influence of transport. metabolism. flow. drug partitioning characteristics

and rlimination by other organs on intestinal clearance. intestinal availability. and

systemic bioavailability . However. a Segregated-Flow iModel (S FM) which viewed only

a portion of the intestinal tlow perfusing the enterocyte layer. where metabolism and

absorption are present. was more supenor in describing the metabolic data of morphine.

The flow characteristics of the SFM are consistent with the physiology of the intestine

and O ffered a sound explanation of route-dependent intestinal metabolism.

ACKNOWLEDGMENTS

Firstiy, I would like to extend my gratitude and appreciation to my supervisor. Dr. K. S . Pang for her patience and dance throughout my undergraduate and graduate studies. The completion of this project would not have k e n possible without her constant support and invaluable counsei.

I would also like to thank my cornmittee advisors, Dr. Bendaym and Dr. Piquette-Miller For thtir encouragement and geat advice.

Deepest thanks need to be expressed to those I've had the honour of working with over the years: Dr. W Geng, Dr. M Doherty, Dr. R Tirona. Mr. E Tan. Mr. N Abu-Zahn Miss E Cheng, Miss S Sanghera, Mr. F Baker and Mrs. L Tao. Their thoughtfulness. unwavering support and humour made my Me as a graduate student an enjoyable experience. 1 WU never forget the rnany laughs we shared over lunch and '80s days. God bless you al1 and good luck in your future endeavours.

Words fail to describe hou; grateful I am to have great hiends like Miss Y . W Lee and Miss J Chen. You two have stood by me through all hardships and have given me the confidence to believe in myself. Thank you for everything - for al1 the laughter. al1 the tears and late, coffee-drinking nights.

To the most qnerous and loving penon, Mr. M Erpelo - I thank God everyday for sending you into my hfe. Your love and support have given me the suength to becorne a better person. You have shown me the meaning of contentment.

Thanks are also due to Mr. and Mrs. Erpelo, for opening their hearts and home to me. You both have been like my second parents - providing me with love. understanding and hot meals. Manming salamat!

Last but not least, 1 want to extend my appreciation to my wonderful brothrr and to the world's greatest parents. Thank you so much for inspiring me to fulfill my drems and for always believing in me.

1 wish to also acknowledge my appreciation of the financiai support from the OntCario Graduate Scholarship and University of Toronto ûpen Fellowship. offered by the school of graduate ssnidies.

TABLE OF CONTENTS

PAGE

Title ........................................................................................... i . .

Abstract ......................................................................................... 11

............................................................................. Acknowledgments iv

.............................................................................. Table of Contents v

..................................................................... Abbreviations and Terms ix

................................................................................... List of Tables xiv

.................................................................................. List of Figures w

Chapters

.................................................................. 1 . General f ntroduction 1

7 .............................................................. 1.1 The Small Intestine - 7 1.1.1 Structure of the small intestine ....................................... -.

1.1.2 Circulation of the small intestine ..................................... 7 1 .1.3 Physiological functions of the small intestine ...................... 9

1.1.3.1 Absorption ...................................................... 9 ....................................................... 1.1 . 3.2 Exsorption 10

9 9 1 . 1 . Metabolism ................................................... 1 1 3 ........................................................... 1 . 4 Motility 1-

1 -2 Modes of Transport ............................................................... 13 1 2 . 1 Passive diffusion ......................................................... 13 1 2 . 2 Carriermediated transport ............................................. 17 1 . 2 Pore ..................................................................... 18 1.2.4 Pinocytosis and phagocytosis .......................................... 18

1.3 Factors Affecting Intestinal Drug Absorption ................................ 19 1.3.1 Bloodflow ............................................................... 19 1 .3.2 Physicochemical properties of substrate ............................. 19 1.3.3 Absorptive camen ...................................................... 21 1 . 3 Effluxproteins ........................................................... 27 I 3.5 Metabolic systems ...................................................... 29 1.3.6 Gastrointestinal motility ................................................ 30

1.3.7 Food ....................................................................... 31 ........................ 1.3.8 Segmenta1 heterogeneity of intestinal events 32

......................................... 1.4 Techniques to study intestinal events 36 ......................................................... 1.4.1 In v i ~ o methods 36

1.4.1.1 Ussingcharnber .................................................. 36 ............................................... 1.4.1.2 Membrane vesicles 37

........................................................ 1.4.1 -3 Everted sac 37 .......................................................... 1 .4 . 1.4 Cell lines 38

............................................. 1 .-l . 1 -5 Tissue homogenates 40 . . ......................................................... 1 A.2 In siru pertusion 40 ......................................................... 1 A.3 In vivo methods 41 . ............................................... 1.4.. 1 Luminal pertusion II

1 . 4.3 2 Portacaval Transposition ....................................... 43 ....................................... 1.4.4 Immunohistochemical methods 13

1.5 Physiological view of dmg absorption ....................................... 44

....... 1 -6 Approac hes for examination of various issues of dnig absorption 46 ...................................... 1.6.1 Benzoic acid as mode1 substrate 46

.................................... 1 . 6. 1.1 Metabolism of benzoic acid 46 ................................................... 1 .6 . 1.2 Protein binding 49

..................................... 1.6. 1.3 Absorption o f benzoic acid 49

............................................................ 1.7 Statement of Problem 50 1.7.1 bfethodofstudy ......................................................... 51 1.7.1 Development of comprehensive physiological mode1 .............. 51

S tatement of Purpose of Investigation .............................................. 53

2.1 Objectives ......................................................................... 54 2.2 Specific airns .................................................................... 55 2.3 Hypothesis testing ........................................................... 55

3 A New Physiologically Based . Segregated-Flow Mode1 to Explain ........................................... Route-dependent Intestinal Metabolism 56

3.1 Abstract ....................................................................... 57

3 . 3 Introduction.. .................................................................... 58

-v . 2.3 Theoretical ...................................................................... 60

3.3.1 Traditional mode1 (TM) ................................................ 61 * - 3.3.2 Segregated-flow model (SFM) ....................................... 63

3.4 Methods ........................................................................... 63 ................................ 3.41 Mass-bdanced and theorist equations 63

3.4.2 Simulation ............................................................ 66 ........................ 3 -43 Fitting of morphine data to the TM and SFM 69

3.43.1 Mass-balanced equations for TM and SFM of morphine ... 77

3.5 Results ............................................................................. 75 ..................................................... 3.5.1 Analflical solutions 75 - - 2 Simulation ................................................................ 76

3.1 2 . 1 Effects of intestinal metabolism and secretion on CLi, Fvs and FI at constant Fab, (0.667. with ka and kg as 1 and 0.5 min') .............................. 76

........... . 3.5.2.2 Effect of Cl ,,, ka and kg on F,, when CL, = 0 77 3.5.2.3 Effect of CL, and k, on Fsys when CL,, = O and

I kg = 0.5 min' ..................................................... 82 3.5.2.4 Effect of CLoth,,. CL, and CLd on metaboiism

with constant k, (0.05 min") ................................... 82 .................. 3 5.3 Application of the SFM: fitting of morphine data 86

........................................................................ 3.6 Discussion 88

. . . . 3.7 Statement O t Signi ticance ................................................. 95

4 . Preferential Absorption of Benzoic Acid by Jejunum of The In Situ Rat Small Intestine Preparation ...................................................... 97

.................................................. 4.1 Abstract ...................... ,., 98

4.2 Introduction.. ..... .. .......................................................... 99

4.3 Matenal and methods ........................................................... 101 ................................................ .............. 4.3.1 Materials ,., 101

4.3 2 Intestinal perfusion ..................................................... 101 4.3 2.1 Pemision apparatus and perfusate ............................ 101 1.3 2.2 S ystemic and intralurninal dosing ............................. 103

4.3 -3 Analyticd Procedures ................................................ IO5 4.3 3 Preparation of samples for HPLC injection .................. IO5 4.3.3 2 HPLC assay of unlabeled benzoate and hippuric acid ....... 105 4 3 . 3 Radioactivity in plasma . luminal fluid

and intestinal tissue .............................................. 107 4.3.3.4 n-Octanol and buffer partition of benzoic acid ............... 108 4.3 .3.5 n-Octanol and buffer partition of acetaminophen ............ 108

............................................................................. 4.4 Results 108 4.4.1 Intestinal Viability ....................................................... 108 4.4.2 Systemic administration of benzoic acid ............................. 109

vii

4.4.3 Intraduodend administration of benzoic acid to . . ...................................................... the entire intestine 110 4.44 Absorption of tracer dose of benzoic acid by various

closed segments of the rat small intestine - duodenurn . . . jejunum or ileum ........................................................ 1 1 1

4 . 5 n-Octanol and buffer partition of benzoic acid and acetaminophen ...................................................... 119

. . 4.3 Discussion ........................................................................ 120

4.6 Statement of Significance ...................................................... 126

5 . Discussion and Conclusions ......................................................... 127

5.1 Summary of Bndings .......................................................... 128 5.2 General discussion and significance .......................................... 129

........................................................................ 5.3 Conclusion 132

References .......................................................................................

Copyright Release .............................................................................. 163

viii

ABBREVIATIONS AND TERMS

ATP

AUC

BA

BBMV

Surface area avdable for absorption

Amount of h g in the enterocyte layer of the intestinal mucosa

Amount of metabolite in the enterocyte layer of the intestinal mucosa

Amount of h g in the mucosal blood to enterocyte compartment

Arnount of dmg in the intestinal blood

Arnount of dmg in the intestinal tissue

Arnount of metabolite in the intestinal tissue

Arnount of h g in the intestinal lumen

Amount of dmg in the reservoir

Arnount of dmg in the serosa and other non-eliminating intestinal structures

Arnount of drug in the serosal blood

Amount remaining to be absorbed

Adenosine triphosphate

Area under the concentration-time curve

Benzoic acid

Brush-border membrane vesicle

Colonic carcinoma cells

Concentration of ionized species

Concentration of unionized species

Influx inainsic clearance of morphine from blood cornpanment to tissue cornpartment

Efflux htrinsic clearance of morphine-3B-glucuronide from tissue compartment to blood compamnent

Intesthai gIucuronidation intrînsic clearance of morphine

Intrinsic luminal degradation clearance of morphine

CoA

Efflux intrinsic clearance of morphine from tissue compartment to blood c o m p m e n t

Secretory intrinsic clearance of morphine

Absorption inuinsic clearance of morphine

Inhinsic deconjugation clearance of morphine-3B-glucuronide

Secretory intrinsic clearance of morphine-3P-glucuronide

Efflux inirinsic clearance of morphine-3Pglucuronide from tissue cornpartment to blood cornpartment

Intrinsic clearance for morphine absorption

Inninsic clearance for morphine-3P-glucuronide absorption

Influx intrinsic clearance from blood cornparunent to enteroc yte compartment

Efflw inninsic clearance fiom enterocyte compartment to blood cornpartment

Mux intrinsic clearance from blood cornpartment to serosal compartment

EMux intrinsic clearance from serosal cornpanment to blood cornpartment

Luminal glucuronidation intrinsic cleannce for morphine

Intrinsic clearance for luminal degradation

Intestinal hydrolytic intnnsic cleamnce for morphine

Intestinal clearance

Clearance by other organs parallel to the intestine

Me tabolic intrinsic clearance of intestine

Secretory intrinsic clearance of intestine

Secretory inninsic clearance of intestine for morphine-3P- glucuronide

Total body or systemic clearance

DNP-SG

GLUT 1

HPLC

Cytochrome P-450 enzymes

Diffusion coefficient

Concentration gradient across the membrane

Diffusion rate of molecules at the site of absorption with respect to time

2,4-dinitrophenyl-S-glutathione

Thickness of the membrane

Concentration ratio of unionized to ionized species

Fraction of intestinal blood perfusing the enterocyte layer

Fraction absorbed

Intestinal avdability

S ystemic bioavailability

Glucose transporter 1

Glucose transporter 2

Glucose transporter 5

Kippuric acid

Kigh performance liquid chrornat~~mphy

intestinal bile acid transporter

Rate of flux per unit area

Rux of dnig across the membrane

Absorption rate constant

Luminal degradation constant

Michaelis-Menten constant

Morphine

Amount of morphine in the enterocyte layer of the intestinal mucosa

Arnount of morphine in the mucosal blood to the enterocyte layer

Amount of morphine in the luminal exudate

Arnount of morphine in the intestinal lumen

P&

PEPT 1

Amount of morphine in the reservoir

Arnount of morphine in the serosa and other non-eliminating intestinal structures

Amount of morphine in the serosal blood

Amount of morphine-3f3-glucuronide in the enterocyte layer of the intestinal mucosa

Arnount of morphine-3B-glucuronide in the mucosal blood to the enterocyte layer

Amount of morphine-3 P-glucuronide in the luminal exudate

Amount of morphine-3P-glucuronide in the intestinal lumen

Amount of morphine-3P-glucuronide in the serosa and othrr non- eiiminating intestinal structures

Amount of morphine-3B-glucuronide in the serosal blood

Monocarboxy late transporter 1

Human multidnig resistance gene 1

Rat multidrug resistmce gene la/b

Multidmg resistance-associated protein 2

Sodium-taurocholate cotransport polypeptide

Organic cation transporter

Apparent partition coefficient

True partition coefficient

Permeability coefficient

Effective permeability coefficient

Oligopeptide transporter 1

P-glycoprotein

Acid dissociation constant

xii

SFM

SGLT 1

TLC

UGT

Permeabiiity coefficient of the membrane

Blood 8aw to the enterocyte layer of the intestinal rnucosa

Total blood flow to the intestine

Blood flow to the serosal layer

Segregated-flow mode1

Sodium-glucose transporter 1

Sulfotransferase

Thin layer chromatognphy

Traditional mode1

UDP-glucosyltransferase

Volume of enterocyte layer

Volume of mucosal blood to the enterocyte layer

Volume of intestinal tissue

Volume of reservoir cornpartment

Volume of the serosal layer

Volume of serosal blood

Maximum transport velocity

LIST OF TABLES

Chapter 1

1 - 1 Intestinal carriers responsible for transport of endogenous .......................................................... and exogenous substrates.. 12

C hapter 3

3-1 Input parameters used for simulations of intestinal clearance and bioavailability according to the TM and SFM ................................ 67

3-2 Analytical solutions for intestinal clearance. areas and availabilities ............ based on the TM when rnetabolism occurs only within the tissue.. 68

3-3 Assigned and fitted parameters for simultaneous fitting of systemic and intraduodenal data of morphine and M3G from the

................. recirculating, perfused rat small intestine to the TM and SFM. 57

Chapter 4

4-1 Percent recovery of unlabeled and labeled benzoic acid From perfusate. ....... lwninal fluid and tissue when BA was administered into the reservoir.. 1 10

4-2 Extents of recovery of various intraluminally delivered doses of benzoic acid fiom perfisate. luminal fluid and tissue.. ........................... 1 13

4-3 Summary of volume recoveries. extents of absorption. luminal recoveries and absorption rate constants obtained after injection of tracer doses of ['4~]benzoic acid into various intestinal segments.. ........ 1 I 5

4-4 The tme octanol-water partition coefficient of benzoic acid.. ................... 120

xiv

LlST OF FIGURES

PAGE

Chapter 1

1 - I The four layers of small intestinal wall.. .......................................... 4

1-3 Schematic representation of artenal. venous and lymphatic circulation of the villus.. ............................................................. 7

.............................. 1-4 Proposed membrane topology of the MCT family.. 26

I-5 Glycine conjugation of benzoic acid.. .............................................. 47

Chapter 3

- 1 Schernatic representations of TM and SFM for intestinal ...... absorption, metabolism m d secretion of substrates given orally or i.v.. 62

3-2 Models for the TM and SFM describing the metabolism of morphine ta morphine-3p-glucuronide ( M X ) in the recirculating. perfused rat liver preparation.. ...................................................... 70

- 3 Simulated rffects of secretory intrinsic clearance and metabolic clearance on intestinal clearance for the TM and SFM.. ........................ 78

3-4 Simulated effects of secretory intrinsic clearance and metabolic clearance on intestinal availability for the TM and SFM ........................ 79

- 5 Cornparison of the ratios of intestinal clearance. systemic availability and intestinal availability simulated for the TM and SFM when the secretory intrinsic clearance and metabolic intrinsic clearance were altered.. .................................................... 80

3-6 Effects of the intestinal absorption rate constant and intestinal secretory intnnsic clearance on systemic bioavailability according

.................................................................... to the TM and SFM 81

3-7 Effects of intestinal absorption rate constant and intestinal metabolic intrinsic clearance on systemic bioavailability according O e TM d S M . . ............................................................... 84

3-8 Effect of transmembrane clearance (CLd) and rernoval by other organs (CLoaen) on intestinal metabolism when intestinal secretion and luminal loss are non-existent according to the TM and SFM.. ............. 85

3-9 Fitting of the SFM to data on the metabolism of morphine to M3G.. ......... .93

3-10 Comparison of residuals ofcornputer fits for the TM and SFM for orally and intravenously delivered morphine.. ..................................... 94

4- 1 Schematic illustration of the in situ pemised rat intestinal preparation.. ....... 1 02

4 - The disappearance of unlabeled and labeled benzoic acid in reservoir pefisate when BA dissolved in the perfusate was delivered into the recirculating perfused rat small intestine preparation.. ........................... 109

4-3 Absorption of ['"~]benzoic acid by the perfused rat small intestine when tracer doses in saline were delivered directly into the duodenum and exited at the ileocecd valve ..................................................... 1 12

4-4 Comparison of the influence of varying doses of benzoic acid on the extents of absorption by the entire intestine and on the absorption rate constant.. ........................................................................... 1 14

4-5 Absorption of lurninally delivered ['"~]benzoic acid by the equal lengths (1 2 cm) of duodenum and jejunum segments.. ........................... 1 17

4-6 Absorption of luminally delivered ['"~]benzoic acid by the equal lengths (20 cm) ofjejunum and ileum segments ................................... 1 18

4-7 pH-Dependence of octanol-buffer partitioning of beruoic acid.. ................ 1 19

xvi

CHAPTER 1

GERNERAL INTRODUCTION

1.1 THE SMALL INTESTINE

The small intestine is a vital tissue regulating the absorption of endogenous

compounds and exogenous substrates following oral ingestion. tt acts as the first

substantial barrier. restncting the entry of dmgs into the body. The tissue is noted not

only for its absorptive (Tsuji and Tamai. 1996) but also metabolic (Koster et al.. 1995;

llett et al.. 1990) and exsorptive (Arimori and Nakano. 1998) activities. Since the

intestine is positioned anatomically in series with the liver. it plays an important role in

regulating the tlow of substrates to the liver. the distal vital first-pass organ. and

consequently. contributes signi ficantly to the overall bioavailability of onlly

administered substrates.

1.1.1 Structure of the Small Intestine

The small intestine is a continuous tubular structure that is divided into three

structurally and functionally different segments: the duodenum. jejunum and the ileum.

Although the structure changes as do the functions along the length of the small intestine.

the changes are gradua1 and there is no sharp boundary behveen segments. The

duodrnum is the first. shortest. widest and least mobile segment of the small intestine.

beginning at the pyloris and ending at the ligament of Trietz. The segment is held to the

posterior wall of the abdomen by connective tissue. Two major ducts empty into the

duodenum: the common bile duct which delivers bile fiom the liver and the pancreatic

duct that brîngs pancreatic juice (Creamer, L 974).

The jejunum and ileum make up the last two segments of the small intestine. with

roughly two-fias of the small intestine being the jejunurn and three-fifis being the

ileum. These two intestinal segments fom a continuous, non-convoluted tube, with a

lack of valves. sphincters. or ducts opening into them. Although the jejunum and ileum

do not receive secretion From other organs. numerous secretory glands exist within the

w l l s of these segments. The ileum empties its contents into the large intestine at the

cecum: the two are separated by the ileocecal valve. The jejunum is wider. thicker and

more vascular than the ileum; whereas the aggregate lyrnph nodules (Peyer's patches) in

the ileum are more numerous and larger than those found in the jejunum. In addition. the

mucosal area per cm of the lower ileum is five tirnes less than that of the jejunurn (Magee

and Dalley. 1986).

The three regions of the small intestine share a common histological pattern.

Their ~valls. abutting out towards the lumen. are composed of the mucosa. the

submucosa. the muscle layers. and the serosa (Creamer. 1974: Thomas. 1988: Shiner.

1995) (Fig LI). The serosa is an extension of the peritoneurn and consists of a single

layer of tlattened mesothelial cells overlying some loose connective tissues. The

muscularis has an outer longitudinal layer and inner circular layer of muscle whose

tùnction is peristalsis. The submucosa is composed of a network of loose connective

tissue rich in small blood vessels. lymphatics. and nerve plexus. The mucosa has three

cornponents: a superficial lining of epithelitun. the lamina propria and the muscularis

mucosa. The epithelium. the outermost layer of the mucosa facing the lumen of the

intestine. consists of a single layer of colurnnar epithelid cells known as enterocytes.

Figure 1-1. The fou layers of s d intestioal wd. Ci& fol& are evident in mucosai layer of the intestine (Magee and Daiiey, 1986).

The iremendous absorptive power of the small intestine is due to severai unique

macroscopic and microscopie, physioiogicai featrms. The Iimiinal surface is not smooth.

Rather, the mucosa and submucosal layers are arranged as circuiar folds, known as plicae

circulares that project directly into the lumen (Fig 1-2). The folds are large and

numemus throughout the duodenum and the proximd half of the jejunum, and these

dirninish considerably both in size and quantity towanis the mid-ileum. The plicue

circufmes, however, are almoa absent in the terminal ileum (Magee and Ddey, 1986).

These folds increase the absorptive d a c e area of the intestine by as much as a factor of

3 (Sheehy and Floch, 1964). The mucosai surface is M e r Iined with fïnger-iike

projections known as vilii (Fig 1-Z), which M e r increase the luminal d a c e area

another eightfold (Sheehy and Floch, 1964). Not untike the nephrom of the kidney, these

v a constitute the fiinctiond units of the srnall intestine. They are h e d with enterocytes

and are endowed with a intricate circulatory system. The villus height and density

decrease from the duodenum to ileum Oomas, 1988; Shiner, 1995). The smaii

intestines of the commonly studied laboratory rodents lack the plicae circulares. having

only the villus and microvillus modifications.

At the base of the villus is the crypt (Fig 1-2), which is linked to the villus by a

network of connective tissue. The epithelial cells of the crypt resemble those that cover

the villi but they are immature and undifferentiated. The harsh luminal environment and

constant movement of the luminal fluid necessitate proliferation of the intestinal

epithelial cells in order to maintain integrity of the intestine. The cells divide in the crypt

and thrn migrate to the surface of the villus. fiom which they c m b r shed into the

intestinal lumen. The total lifespan of these cells is approximatel y 3-6 days (Magee and

Dalley. 1986).

The most distinctive feature of the apical or bmsh border membrane of the

intestine is the presence of a third set of projections. the microvilli (Fig 1-2). Thesr very

minute. parailel cylindrical extensions of the luminal cells are most effective in

increasing the absorptive surface area of the small intestine (Brown. 1962). The structure

of the membrane of the microvillus is based on the fluid mosaic mode1 of the ceII

membrane. where the barrier is believed to be a lipid bilayer with an array of enzymes

and transporters. The outer surface of the microvillus membrane. the glycocalyx. is rich

in carbohydrates (glycoproteins) and these sugar residues entrap water and rnucin

immediately adjacent to the bnish border membrane and constitute an unstirred water

layer (ho. 1965; Shiner. 1995).

Interspersed between the absorptive cells of the epitheliurn are goblet cells. which

are responsible for the secretion of mucus. The mucus lubricates and protects the

intestinal surface from the surroundhg environment. Once secreted fiom the goblet tells'

mucus laya can pose as a rate-limiting b e e r in absorption of some (Mimmddi and

bscnrhalcr, 1980; Wilanan et al., 1993) but not ail substrates ( W m e and V d q m ,

F i -2. Structure of the mucosa of the s m d intestine. h order of in- detail, the morphoiogy of (A) the circular folds; (B) the viE of the mucus membrane; (C) the microvilfi is show11 above ( C a s p q , 1987).

1.1.2 Circulation of the S m d Intestine

The blwd (500 to 1100 d m i n for human; 7 to 8 d m i n for rat) (Davies and

Morris, 1993) that courses through the srnail intestinaj tissue empties immediateiy into

the portal vein to enter the liver. incoming blood to the srnail intestine enters via the

superior mesenteric artery by way of an arching artcrial system (Parks and Jacobsen,

1987) (Fig 1-3) and disaibutes to the vafious layers of the intestine. In dogs,

approlamately three-fourths of the total resting intestinal blood flow is dispersal to the

mucosa and the remainder goes to the submucosa, muscdaris, and serosa (Bond and

Levitt, 1979; Bond et al., 1979). It has a h bem demonstrated that approximately 60 to

70% of the intestinal blood flow is disiributcd to the epithelial mucosal cells of cats and

rats (Biber et cil., 1973; Gore and Bohlai, 1977). It had been suggested that because only

part of the intestinai blood 'flow miches the mucosa, the mucosal blood flow and not the

total inîestùd blood flow should be used to reiaîe intPctinaI clearance and extraciion to

et al.

Figure 1-3. Schematic representation of arterial, venous and lymphatic circulation of the villus. (Casp.. L 987)

The blood flow in each region of the small intestine as well as in each layer of the

intestinal wall is related directly to the metabolic demands and to the fùnctional activity

within the region. During absorption. blood flow to the villi and adjacent regions of the

submucosa is greatly increased. whereas blood flow in the muscle layers increases with

motor activity (Bynum and Jacobsen. 1971). Afier a meal. blood flow to the intestine

increases by 30 to 130% of basal flow and as a result. hepatic blood fiow is also increased

(Bond and Levitt. 1979). A greater blood Bow translates to a lower transit tirne in either

organ. resuliing in reduction in extraction of the substrate by the first-pass organs

(Melander el al.. 1 977: Melandrr and McLean. 1983: Olanoff et al.. 1 986). Other factors

such as the types of food. neurohormonal and local regulatory mechanisms and exercise

c m further influence the blood flow rate to the intestine (Bond er al.. 1979).

The arterioles that supply the villi are branched into many small capillaries at the

tips of these luminal projections. The blood leaving the capillaries then drains into a

venule system (Fig 1-3). Because of the close proximity between the merioles and

venules in the villi. it is possible that some small molecules can diffuse out of the

ascending arterioles directly into the adjacent descending vendes without ever being

carried in the blood to the tip of the villus where the majonty of the intestinal drug-

metabolizing enzymes are Iocated (Kolars et al.. 1992). The arteriole-venule or

countercurrent exchange is probably accomplished mainly via simple diffusion created by

existing concentration gradients and the effect of this exchange may Vary arnong

substrates of d i f f e ~ g physicochemical properties (Bond et al.. 1977). The system of

countercurrent exchange in the villi is well noted in humans and animais (Hallback er al.,

1978; Parks and Jacobson. 1982) and tends to decrease the entry of the substrates from

the circulation into enterocytes. The presence of this countercurrent system is an

important feature of intestinal dmg metabolism and should be considered in

pharmacolcinetic models of intestinal metabolism of dmgs (Minchin and Ilett. 1982):

however. to date. the effect of the countercurrent exchange on intestinal absorption and

metabolism of dmgs has yet to be examined in detail.

1.1.3 Physiological Functions of the Small Intestine

1.1.3.1 Absorption

The srnall intestine, with its impressive surface area. is regarded as the most

important tissue for the absorption of Ruids. micronutrients (vitamins. minerals).

macronutrients (lipids. carbohyàrates. proteins) and dmgs from the gut lumen into the

circulation. To reach the circulation. molecules must first cross the apical membrane

facing the lumen then the basolateral membrane into blood. The intestinal bamer. similar

to other cellular membranes. consists of a lipid bilayer with glycoproteins dispersed

throughout. The lipoidal nature of the membrane restricts the movement of hydrophilic

substrates, whereas proteins regulate the transport of small size compounds by forming

pores or channels through the membrane.

Dissolved substrates present in the lumen can cross the epithelium via the

paracellular andor transcellular pathway(s). Paracellular transport which describes the

movement of the substrates around the enterocytes is a passive process and is restricted

by the tight junctions that exist between epitheliai cells. Functional studies show an easy

passage of small hydrophilic molecules, but there is a sharp cut off at molecular size of 8

A for movement across the jejunurn, and 3 A for uptake by the ileum (Fordtran et al.,

1 965).

Transcellular transport or the movement of luminal content through the epithelial

cells can be either passive or active. Passive transport is driven by a concentration or

electrochemical gradient across the membrane. The passive diffusion of a substrate

across the intestinal membrane is restricted by lipid solubility. which in turn is modulated

by the degree of ionization of the drug. The uptake of ionized or water-soluble

compounds is prevented by the lipoidal nature of the membrane. Transport of lipid

soluble chemicais. on the other hand, may be hindered by the unstirred water layer.

In addition to passive difision. the transport of certain substances is facilitated by

the presence of carriers selective towards specific molecules. Many such carriers are

located on the intestinal brush border and basolateral membranes. Dmgs that mimic

endogenous compounds. thus, may be recognized by existing membrane transporters for

effective intestinal uptake. The carriers form complexes with the substrates and render a

more rapid permeation across the membrane than that of the free substances.

Dissociation of the complexes on the other side of the membrane releases the free

substances into the cell. This form of facilitative difhsion is a saturable process. If there

is a requirernent of energy expenditure, the process is termed active transport. Needless

to say. carrier-mediated transport can be inhibited by substrates that compete for the

absorptive sites on the transport proteins.

Transport at the apical membrane of the small intestine has been demonstrated to

be bidirectional (Thiebaut et al., 1987; Hsing eî al., 1992; Karlson et al., 1993; Tsuji et

al.. 1996). There exists an increasing number of examples of molecules that are absorbed

and then secreted back into the intestinal lumen, resulting in lower overdl

bioavailabilities. Absorption and efflux may be mediated by different transport proteins.

Distinct efflu. transporters found on the brush-border include the P-glycoprotein (Pgp)

and the multidrug resistance-associated protein 7 (MRP2).

1.1.3.3 Metabolism

In addition to uptake and efflux functions. the smail intestine also possesses

metabolic activities. Intestinal metabolism. occuning either in the lumen or within the

rnterocytes. serves to limit the amount of h g entenng the circulation. thereby resulting

in subtherapeutic outcomes. On the other hand. metabolites generated by the intestine

c m be toxic. leading to undesirable effects. or may be active or more active than the

parent dmg. rendering their own therapeutic effects. The latter case serves as the basis

for pro-drug therapy.

Luminal metabolism is. in part. attributed to gastric and pancreatic secretions of

enzymes such as gastrin (Bynurn et al.. 1971). hydrolases (Imondi er al.. 1969).

pancreatin. trypsin. esterases. and alkaline phosphatases (Weiser. 1973) into the intestinal

lumen. Pancreatin and trypsin can deacetylate drugs (Trenholm et al.. 1969: Scaloni et

al.. 1992) whereas esterases affect the hydrolysis of various ester moieties of drugs such

as penicillins (Kabins et al.. 1966; Knott-Huruiker et al.. 1982: Valls et al.. 1984:

Branger and Goullet. 1987).

The presence of microorganisms adhered to the epitheliai surface also contributes

significantiy to the luminal biotransformation of substrates (Scheline. 1968: Smith. 1978;

IIen et al. 1990). The intestinal microflora are primarily derived from the environment

by mouth. The concentrations of these organisms tend to increase toward the distal end

of the intestine (Floch e t al., 1970). Bactena located in the jejunum are aerobic (e.g.

streptococci, staphylococci and lactobacilli) while those in the colon are mostly

composed of anerobes (e.g. bacteroides and bifidobactena) (Gorbach et al.. 1967b: Plaut

et al.. 1967). The ileum is a transitional zone and a rnixed flora is found.

Biotransformation of dmgs by luminal bactena is diverse and is influenced by factors

such as age and dietary habits (Gorbach et ai., 1967a). The propulsive motility of the

intestine. which is responsible for continually cleansing the tract. can also limit the

proli feration of microorganisms. thereby reducing the extent of luminal mctabo lism.

Concomitant antibiotic therapy which lowers the population of microorganisms decreases

or prevents biotransformation (Sandler et al.. 1969; Ilett et al.. 1990).

Numerous metabolic reactions. including phase 1 and phase 2. have been shown

to take place within the intestinal wall. Many of these reactions are similar to those

mediated by drug-metabolizing enzymes in liver. albeit at lower levels. The rate of

rnqmatic metabolism in the intestinal tissue is dependent on the concentration of

enzymes within the enterocytes and the intracellular residence time of the. Therefore. it

is important to determine the contents of various intestinal enzymes and their localization

to mess the relative contribution of intestinal metaboiism to overall dmg bioavailability.

The small intestine is not a stationary tissue, but rather. participates in the overall

motility of the gastrointestinal tract. There are two types of intestinal movements -

propulsive and mixing (Macagno and Christensen. 1981). The propulsive motion or

peristalsis carries the substrates down the intestinal tract and therefore. determines the

residence tirne of dmgs in the lumen. The intestinal transit rate is important since it

dictates the time for dmg release, dissolution, and absorption fiom an oral dosage form.

In addition. intestinal motility affects the extent of absorption by moderating the

residence time for which the luminal content dwells at specific surfaces/carrien. Mixing

movements of the srnall intestine are a result of contractions that divide a given region

into various segments. Such contractions chum the intestinal contents with luminal

secretions and bring substrates back and forth into contact with the epithelial surface.

thereby provide a large. effective absorptive area.

1.2 lMODES OF TRANSPORT

1.2,l Passive Diffusion

As mentioned earlier. the majority of lipid-soluble molecules permeate the

intestinal lipid membrane by passive difhsion. The driving force for passive diffusion is

the concentration gradient of the substrate across the membrane. and the rate of transport

increases proportionally with concentration. This rate of dnig penetration cm be

mathematically described based on Fick's first law of diffusion. which is s h o w below:

where dn/dt represents the difhsion rate of or the change in number of molecules at the

site of absorption with respect to tirne. D is the dif is ion coefficient. A is the surface

area AC is the concentration gradient across the membrane. and k x is the thickness of the

membrane.

Fick's law can then be simplified to describe flux or rate of change per unit area J.:

- D A C J = = P A C

where P is the permeability coefficient (or D/AC. the diffusion coefficient per unit

thic kness).

The tluv for a drug that partitions into the membrane (J,) with a partitionhg ratio

n (the ratio of concentration of substrate in the membrane to concentration in watet) is:

where Pm is the permeability coefficient (rrD/du) for the membrane.

It cm be concluded fiom the above equations that drue absorption is directly

dependent upon the membrane surface area available for diffusion and is affected by

intestinal motility and diseases since these rnodulate the area of absorption. One can then

undentand the reason for a more rapid and effective absorption in the small intestine

compared to the stomach. Furthermore, the equation indicates that the greater the

octanol/aqueous partition coefficient. x. the more rapid the rate of absorption of the

substrate.

The degree of ionization of a substrate is controlled by the luminal pH and the

pK, (acid dissociation constant) of the dnig and can be predicted by the Hendenon-

Hasselbach equation:

For acids:

pH = pKa + log ionized unionized

For bases:

pH = pKa + log unionized

C ionized

where Cionized and Cunionized denote the concentrations for the ionized and unionized

species. respectively .

-4ccording to the classic pH partition hypothesis. only unionized nonpolar dmg

penetrates the membrane. and at equilibrium. the concentrations of the unionized species

are equal on both sides. However. contrary to the pH-partition theory. recent studies

have shown that the ionized forms of drug moiecules can also permeate ce11 membranes.

although to a rnuch iesser cxtent than the unionized forms (Iseki rr ni.. 1992: Ottiger and

Wunderli-Allenspach. 1997: Avdeef et al.. 1998: Palm er al.. 1999). Ionized substrates

are postulated to cross membranes via the paracellular pathway and it has been

demonstrated that the paracellular route through the intestinal epithelium is more

permeable to cationic than either neutral or anionic drug molecules (Adson et al.. 1994:

Karlsson e t d.. 1994). Hence, the transport of the unionized f o m of weak acids across

the rat srna11 intestinal tissue was found to be 10. 000-fold more rapid than the transport

of the ionized form (Tai and Jackson. 1981). The selectivity of the intestinal membrane

for the unionized form of weak bases was. however. considerably smaller. approximateiy

500-fold (Tai and Jackson. 1982). The lower epithelial selectivity observed for the

unionized f o m of bases compared with acids may be a result of the higher paracellular

permeability of intestinal epithelia for cationic and not anionic dmg rnolecules.

Therefore. it is important to consider the intestinal permeability of the ionized forms of

drug molecules with high degrees of ionization, especially for cationic species, in

predicting intestinal drug absorption from in vitro permeability studies. Moreover. the

diffusion coefficient (D) may be different for the ionized and unionized forms of the

compound.

The concentration ratio of unionized vs ionized foms of a substrate (f or

Cunionixd/cbnized) is influenced by pH according to the following relationship:

For weak bases:

For weak acids:

f = 1

1 O (pH - pKa )

(1-71

Hence. the apparent partition coefficient. or the ratio of concentration of unionized drug

in organic phase to the concentration of unionized and ionized drug in aqueous phase

(na,, or P,,) is influenced by pH. generally increasing as the value o f f increases. It must

then be recognized that the rneasured C,,,,,i/C,,.,, in partitioning studies is an apparent

R. The true n,, or Pu,, value that describes the ratio of drug concentration in the octanol

vs water phase for the unionized species. on the other hand. is not affected by the pH

(Ishizaki et al.. 1997). The relationship between Po,, and Pa,, can be descnbed as:

and is universal for weak bases and acids.

1.2.2 Carrier-Mediated Transport

Two types of carrier-mediated transport. facilitated difision and active transport.

are involved in the intestinal absorption of endogenous and exogenous compounds.

Facilitative difision is passive. involving the transport of substrate dong a concentration

gradient without expenditure of energy. However. active transport involves movement of

substrate against a concentration gradient. The maintenance of this gradient requires

rnetabolic energy. Active transport can therefore be impeded by metabolic inhibitors.

Carier-mediated movement is substrate-specific and can be inhibited by substrates that

compete for the sarne binding site on the transport proteins. Transport is limited by the

capacity of the carrier and consequently. the rate of uptake c m reach a transport

maximum or Ci,. At low doses. the percentage of dmg absorbed increases linearly with

dose. But as the dose increases, the absorption percentage decreases as a result of

saturation of the transport mechanism. decreasing in absorption efficirncy. This

nonlinear relationship between absorption and concentration for carrier-mediated

substrates is similar to Michaelis-Menten saturable kinetics of enzyme systems:

- - vm, [ S I uptake K , + [ S I

where Km and Vm',, represent the binding affinity (concentration t e m ) and capacity of the

carrier protein (number of moles per unit tirne), respectively. and [SI is the concentration

of the substrate.

A unique feature of carrier systerns is the ability for some proteins to

simultaneously CO-transport molecules. In certain instances. two substrates are

transported in opposite directions by an exchanger or antiporter (e.g. N~+-K+ pump.

~ a * / r antiporter). On the other hand. there are cases where the two molecules are CO-

transported together in the sarne direction or symports (eg. N~~ID--glucose. Nar/arnino

ac id).

1.2.3 Pore

several ma l1 hydmphilic molecules (e-g. Luea. mehnol. fornunide) have Seen

demonstrated to penetrate the membrane with ease and faster than would be expected by

their octanol/water partition coefficients. It was suggested that the intestinal membrane.

although lipid in nature. is not continuous but is interrupted by srnaIl water-filled

channels or pores. As a result. small lipid-insoluble molecules can pass through these

aqueous pores while lipid-soluble molecules readily traverse the lipid reg ions of the

membrane. The effective pore radius is estimated to be 7-8.5 A and 3-3.8 in the human

jejunurn and ileum. respectively (Fordtran et al.. 1 965).

1.2.4 Pinocytosis and P hagocytosis

Large molecules of molecular weight over 900 daltons are genenlly not

transported through the membrane but are taken up by cells by means of endocytosis

(also called phagocytosis). Substances absorbed in this manner normally include

proteins. tetanus toxin. various antigens and drugs that are tightly bound to plasma

proteins. A sirnilar process for the uptake of liquid fiom the extracellular fluid. on the

other hand. is known as pinocytosis. By forming small invaginations in the membrane

(dso known as vesicles) the cell can engulf the material fiom the immediate

environment. The vesicles then transport the matenal into the ce11 where the substrate is

released.

1.3 FACTORS AFFECTING INTESTINAL DRUG ABSORPTION

1.3.1 Blood Flow

Blood periùsing the intestinal tissue continually removes and delivers substrates

from the enterocytes to the rest of the body. As a resdt. this rapid removal of dmg by the

ckcullition provides 3 "sink" condition for the Luiidirectiond flow of &mbed materia!.

The properties of the dosage form. in particular the dissolution rate, or the inherent

membrane permeability of the substrate are ofien the limiting factors for absorption.

However. the rate limiting step for the uptake of substrates with high membrane

permeability is perfusion. Since the membrane and the unstirred water layer offer linle or

no resistance. movement of these drugs into the circulation is dependent on the rate of

blood flow carrying the material away from the site of absorption (Doluisio et ai.. 1969:

Crouthamel et al.. 1970: W i ~ e . 1970: Wime and Remischovsky. 1970). In addition.

blood flow plays an important role in the absorption of compounds by an active

mechanism. a process which requires an expenditure of energy. If the blood tlow to the

absorptive site is reduced. oxygen delivery to the intestinal surface is also lowered and

thereby. reducing the absorptive capability of the carrier systems ( W i ~ e . 1973).

1.3.2 Physicochemical Properties of Substrate

The physicochemical properties of substrates play an important role in

detennining the extent of uptake across the lipid membrane. The ability of a dmg to

cross the intestinal bmier depends to a large extent upon its molecular size and shape,

and its solubility in aqueous and lipid phases (Higuchi et al.. 198 1: Pauleni et al.. 1997).

The passive difision of water-soluble h g s into the epithelial cells largely depends on

their molecular sizes. The 3-8 A width of the membrane aqueous channels restricts the

passage of molecules of rnolecular weights larger than 150-200. The extents of passive

permeation of lipid-soluble drugs, on the other hand. depend on oil/water

(membranehuffer) partition coefficient and the acid dissociation constant (pK,). Drugs

with higher oihvater partition coefficient will be absorbed more rapidly across the tissue

banier. However. there is an optimum value for the oiVwater partition coefficient and

ease of membrane transport (Wils et al.. 1994: Yodoya et ai.. 1994) since the unstirred

water layer lining the surface sugar coat of the luminal membrane restricts movement of

drugs with very high lipid solubility (Chiou, 1994). Because most drugs are weak acids

or weak bases. the pH of the luminal content and more specifically. the acidic

microclimate (pH 6.6 - 6.9) of the brush border surface (Said et al.. 1986) piays an

important role in determining the degree of ionization of the dmg. Weak acids are

mainly ionized in the intestine whereas bases are more nonionized at pH 6. i'ccording to

the pH partition hypothesis. only the nonionized species will be absorbed and thus. weak

bases are beaer absorbed in the intestine than weak acids due to their lower degree of

ionization,

The chemicai and structural stability of dmgs in the acidic environment of the

gastrointestinal tract (ranging fiom pH of 1 in the stomach to pH of 6.7 in the terminai

ileum and colon) can affect the amount of the intact substrate available for absorption.

Dmgs with ester or amide moieties can undergo acid hydrolysis in the stomach and thus.

lirniting the amount of substrate that reaches the small intestine for absorption (Boggiano

and Gleeson, 1976; Blaha et al., 1976). Such h g instability cm be improved through

special formulation techniques such as entetic coating. It is important to note that slow

release of the h g from this enteric coat. however. can lead to slow or incomplete

absorption.

1.3.3 Absorptive Carriers

Uptake carrier systems (Table 1-1) located in the intestinal brush border and

hasolatenl membranes serve to enhance the nverall evtent ahsorption of dn~gs that wodd

othenvise exhibit poor movement across the intestinal barrier (Tsuji and Tamai. 1996).

One such group of intestinal transporters is known for amino acids. There are at least four

distinctive arnino acid carriers and each is structurally restrictive (Ganapathy et al..

1994). Several amino acid analogs. e.g. gabapentin (an antiepileptic agent) (Stewart et

al.. 1993). a-methyldopa (Hu er al.. 1989: Amidon et al.. 1986: Hu and Borchardt. 1990).

and baclofen (Cercos-Fortea et al.. 1995) are absorbed from the small intestine by the

transporter for large. neutrai amino acids. Other brush-border arnino acid transport

systems include those responsible for the movement of basic. acidic and dipolar a-amino

acids. Several basolateral transport systems are also available for the rnovement of arnino

acids. The intestinal amino acid uptake by certain carriers is dependent on a sodium

gradient while transport by other systems is sodium independent.

A sodium-driven bile acid transporter (IBAT) has been shown to be responsible for

the reabsorption of biliarily excreted bile acids that enten the intestine at the duodenum

(Wilson. 1981). The cDNA for this carrier has been cloned and is 63% similar in

homology to that of the rat liver bile a c i d - ~ a transporter (NTCP or sodium-taurocholate

cotransport polypeptide) (Wong et al., 1994). Whereras the liver NTCP rnay participate

in hepatic clearance of organic anion metabolites (e.g. estrone-3-sulfate) and xenobiotics.

IBAT displays n m w substrate selectivity, specific for bile acids (Craddock et al.. 1998).

22

Table 1-1. Intestinal carriers responsible for transport of endogenous and exogenous substrates.

- -. -- -

Transport Carrier

Oligopeptide (PEPT 1)

Monocarbo'rylic acid (MCT 1 )

Phosphate

Bile Acid ([BAT)

Glucose (SGLT 1 )

Nucleoside

EFFLUX

P-glycoprotein (Pgp)

Organic cation (OCT)

MuItidrug resistance- associated protein 2

( W 2 )

In tes tinal Location

Brush-border and basolateral membranes

Brush-border membrane

Brush border membrane in

rnautre epithelial cells

Basolateral membrane in

undifferentiated crypt cells

Brus h- border membrane

Brush- border membrane

B rush- border and basolateral mem bnnes

Brush-border membrane

Brush-border membrane

Brush-border membrane

Brush-border membrane

Co-TransportlEnergy Dependence

H-- and Na--driven

Na--driven

Na--dependent or Nac-independent

Na*-driven

Examples of Substrates

baclotèn taurine a-methy [dopa

ACE inhibitors klactam antibiotics

Pravastatin benzoic acid lactate nicotinic acid salicy lic acid

forscarnet fosfomycin

taurocholate

D-g 1 ucose D- fruc tose D-galactose

Zidovudine S tavudine

cyc losporin- A digoxin

antihistamines antiarrhythm ics

glutathione conjugates

B-Lactam antibiotics such as penicillin and cephalosporins (Okano et al.. 1986;

Tsuji et al.. 1987; Dantzig and Bergin, 1 99O), angiotensin-converting enzyme inhibitors

(ACEi) such as lisinopril. captopil and enalapril (Amidon and Lee. 1994). and bestatin.

an anticancer agent (Tomita et al.. 1990). are reported to be transported by the brush

border membrane proton-dnven oligopeptide transporter 1 (PepT 1 ). PepTl has been

cloned fiom rabbit (Fei et (il.. 1994). rat (Miyamoto ei al. 1996) and human small

intestines (Liang et al.. 1995). with overlapping amino acid sequence homology among

species. Studies of complementary RNA (CRNA) of rabbit and human small intestinal

PepTl expressed in ,Yenoplis Iaevis oocytes revealed that the transport of

[ ' " ~ ] ~ l ~ c ~ l s a r c o s i n e was enhanced in the presence of an inwardly directed H--gradient

(Fei et al.. 1994: Liang et al.. 1995). The human dipeptide transporter transfected into

chinese hamster ovary cells also demonstrated a pH sensitivity (Covitz et ul.. 1996).

Studies on PepTl-mediated transport in oocytes revealed that small peptides containing

either neutral. basic or acidic amino acids. but not peptides larger than tetrapeptides. are

substrates (Fei et al.. 1995). The existence of a proton-coupled oligopeptide transporter

other than PepTl on the basolateral membrane has also been suggested (Dyer et al..

1990: Thwaites et al.. 1993 ; Inui et al., 1992).

The uptake of phosphate in the small intestine has also been s h o w to be carrier-

mediated in the isolated brush-border membrane vesicles prepared fiom rat (Berner et al..

1976) and human (Borowitz and Ghishan, 1989) intestines. Uptake of phosphate by rat

membrane vesicles was stimulated in the presence of an inwardly directed sodium ion

gradient and was also aEected by extravesicdar pH. The inhibition of the sodium-

dependent intestinal absorption of foscamet, an antiviral h g , by phosphate in rabbits,

mice and hurnans indicates the presence of a phosphate carrier system (Tsuji and Tamai.

1989: Swam and Tukker. 1989).

There are also carrier systems on the brush-border membrane responsible for the

movement of rnonosaccharides. The active transport of D-glucose and D-galactose.

energized by the electrochemical gradient of sodium ion. is rnediated by the membrane

protein. SGLTl (Hediger et al.. 1987; Hediger and Rhoads, 1994; Lee et al.. 1994). The

sodium-independent absorption of D-fi-uctose, however. is facilitated by the carrier.

GLUTj (Rand et ai.. 1993: Miyamoto et al.. 1994). Once absorbed into the enterocytes.

the monosaccharides are then transported by a Facilitative transporter. ULUTî or

GLUTI. across the basolateral membrane into the blood (Thorens tir al., 1990).

The absorption of nucleosides by the small intestine is mediated by sodium-

dependent transporters located on the brush-border membrane. The transporters are

either purine-selective. pyrimidine-selective or sçlective for both. Many antiviral and

anticancer drugs. for example. zidovudine and stavudine. are nucleoside analogs. These

have been shown to be uansported across the brush-border membrane by the nucleoside

carrier (Hu. 1 993: Waclawski and Sinko. 1996).

The intestinal absorption of weak organic acids has often been observed to be

much greater than that predicted by the simple pH-partition theory. Although intestinal

absorption by passive diffusion occurs, studies involving lactic acid (Tiruppathi et al.,

1988): acetic acid (Tsuji et al., 1990; Ogihara et al., 1996), salicylic acid (Takanaga et

al.. 1994), bemoic acid (Tsuji et ai.. 1994; Tamai et al., 19991, nicotinic acid

(Simanjuntak et al.. 1990) and pravastatin (Tamai et al.. 1995a) demonstrated

involvement of carrier-mediated transport across the brush-border membrane. The

intestinal flux of these organic acids has been s h o w to be regulated by a proton-driven

carrier-system (Tamai et al.. 199%). A family of such rnamrnalian H'lmonocarboxylate

transporters. MCTs. has been identified. The possible participation, albeit minor. of a

bicarbonate/anion exchanger in monocarboxylic acid uptake has also been suggested

(Simanjuntak er al.. 199 1 : Takanaga et al.. 1996; Yabuuchi et al.. 1998).

Nine different LLCT isoforms have been reported. but the MCTl and MCTZ are

regarded as major carriers (Halestrap and Price. 1999). The arnino acid sequences of

MCTl and MCT2 are 60% identical (Garcia c l al.. 1995). MCTs are ubiquitously

expressed throughout the body: skeletal muscle (MCTI . MCTJ). heart (MCTI. MCT2).

brain (MCTI. MCT2). testis (MCT?). kidneys (MCTI. MCT2. MCT4. MCTS). liver

(MCTî) and retina (MCT1. MCT2. MCT3 and MCT4) (Garcia et al.. 1995: Yoon er al..

1997: Jackson et al.. 1997: Broer et al.. 1997: Pnce et al.. 1998: Gerhart et al.. 1998;

Wilson et al.. 1998). Only MCTl has been identified in the intestinal tissue. The

ubiquitous expression of the monocarboxylate transporters is probably due to the need of

mammalian cells to transport lactic acid across the plasma membrane. either as an end

product that must leave the cell. or as a substrate that must enter the ce11 for respiration or

eluconeogenesis (Bonen et d.. 1998; Halestrap and Price, 1999). C

The membrane topology is predicted to be similar for al1 MCT isoforms. each

consisting of 12 transmembrane domains (TMs) (Fig. 1-4). The N- and C- termini are

located in the cytoplasm. TMs 6 and 7 are separated by a large hydrophilic loop. The

MCT farnily members exhibit the greatest sequence conservation in the putative

transrnernbrane regions and the shorter loop regions between them (Poole et al.. 1996;

Halestrap and Price, 1999). It has been proposed that the N-terminus is important for

energy (m coupling

nibstrate specincity

whereas the C-terminus may be important for the detemination of

(Donovan and Jennings, 1986; Kim e t al., 1992; Poole and

Halestrap, 1993; Garcia et al., 19941; Saier. 1994; Carpenter aad Halestrap, 1994; Baker

et al., 1998; Price et al., 1998; Rahman et al.. 1999).

Figure 1-4. Roposed membrane topology of the MCT family. The sequence shown is that of human MCTl. (Halestrap and Price, 1999)

MCTl was kst identified in Chinese hamster ovary (CHO) cells (Garcia et al.,

1994b). The subsequent expression of this transporter in Xenopus Zaevis oocytes has

ailowed for more detailed characterization of substrate specificity and tissue localization

(Broer et al., 1997 & 1998). MCTl from human, rat and mouse have now been cloned

and share about 95% sequence identity with that of CHO cells (Garcia et al., 1994;

Jackson et al., 1995; Takanaga et al., 1995: Carpenter et al., 1996; Koehler Stec et ai.,

1998). Tamai et al. (1999) recently confïrmed using immunohistochemical analysis that

the MCTl protein is present in the m a i l intestine, particularly in the duodenum and

jejunum. The MCT's are found on the basolateral membrane of immature crypt cells but

are shifted to the apical membrane in mature epithelial cells.

1.3.4. Enlux Proteins

Intestinal exsorption is known to decrease the overall bioavailability of oral

subs~ates (Leu md Huang, 1995; Lown ei al.. !99?: Arimc?ri and Nakanc 1998).

However. the repetitive process of extrusion and reabsorption of certain substrates c m

prolong the intracellular residence time. A portion of the excreted xenobiotics can be

reabsorbed into the enteroctyes and thus. be re-exposed to metabolizing enzymes leading

to an apparent high intestinal metabolism of the substrates. Most of the intestinal efflux

activities have been anributed to P-glycoprotein (Pgp) (Table 1-1). a 170 D a protein

found on the brush border membrane (Thiebault et al.. 1987). The protein. most notably

identified for its ability to confer multidrug resistance in mammalian tumour cells. is

encoded by hurnan MDRl and rodent mdrlah genes (Chin et ul.. 1989: Hsu et d.. 1989:

Higgins. 1992). The main substrates of this intestinal transporter are organic cations.

Inhibition of the release of [3~daunomycin fiom brush border membrane vesicles and of

the release of rhodarnine 123 from everted rat small intestine by P-glycoprotein substrates

diltiazem. colchicine. and verapamil demonstrated the involvement of Pgp in intestinal

secretion (Hsing et al.. 1992). The basolateral-to-apical fluxes of cyclosporin A

(Augustijns et al., 1993). celiprolol (Karlson et al.. 1993). vinblastine and docetaxel

(Hunter et al., 1993) in Caco-2 cells were also demonstrated to be limited by Pgp

inhibitors. Quinidine (Emi et al.. 1997) and veraparnil (Saitoh and Aungst 1995). well

known inhibitors of Pgp, have been also noted to be substrates of this efflux purnp. Leu

and Huang (1995) showed that the addition of C219. a monoclonal antîbody of the P-

glycoprotein. reduces the effluv of etoposide, an anticancer dmg. from the everted rat

small intestine. More direct evidence for a role of intestinal p-glycoprotein in limiting

h g absorption was derived from Nt vivo studies using paclitavel (Têuol) in mdrl a (4)

and mdr 1 a (+/+) mice (Spareboom et al., 1 997). The plasma AUC of Tavol was found to

be higher in the mdrla knockout mice than the mdrla (+/+) mice afier both i.v. and oral

administrations. In addition. the oral bioavailability in mdr 1 a (4) mice was significantly

greater than that in the rndrla (+/+) following oral dosing. Fecal escretion of the dmg

was also reduced in the knockout mice as compared to their counterparts. suggesting that

P-glycoprotein played a role in limiting Taxol absorption by rxcreting the drug into the

lumen.

Another cation transporter responsible for the secretion of organic cations was

also found on the brush border membrane of the intestine that facilitates the secretion of

organic cations. Dmgs from a wide array of clinical classes. including antihistamines.

skeletal muscle relaxants. and antiarrhythmics. are organic cations. The transport of

these substrates is regulated by the guanidine/proton antiporter (OCT) (Zhang et al..

1998: Koepsell. 1998).

Analogous to Pgp for the transport of cations, the rnultidnig resistance-associated

protein farnily (MRP). in particular MRPî, is known for its role in the exsorption of

organic anions such as glutathione and glucuronide conjugates. MRP? or cMOAT

(candicular multiple organic anion transporter) was first found to be responsible for

biliary excretion of several anions across the canalicular membrane (Oude Elfennk et al..

1995; Kusuhara et al.. 1998). The protein was also expressed in intestinal tissues. as

noted by Nothern blot anaiysis (Pauiusma eî ai., 1996; Ito et al., 1997; Kool e t al.. 1997).

Gotoh et al. (2000) showed that intestinal secretion of a glutathione conjugate. 2*4-

dinitrophenyl-S-glutathione (DNP-SG), was markedly reduced in EHBR rats whose

MRP2 is hereditarily defective compared to Sprague Dawley rats. suggesting that the

intestinal secretion of this conjugate is largely mediated by MRP?. A reduction in

intestinal clearance of DNP-SG in EHBR rats was also seen in everted sac studies. Using

brush border membrane vesicles from Caco-2 cells expressing MW?. Hirohashi et al.

(2000) showed that bimane glutathione. a substrate of MRP2 and not MRP3. a basohteral

membrane transporter, is excreted predominantly in the apical direction.

1.3.5 Metabolic Systems

Intestinal metabolism, similar to efflux activities. reduces the biavailability of

absorbed substrates (Gibaldi et al.. 1971 : Doherty and Pang. 1997: Lin et al.. 1999). In

addition to luminai bacteria and pancreatic secretions. the cytochromes P-450 in the

intestinal wall are responsible for the biotransformation of the majority of drugs and other

foreign compounds. The average total cytochrome P-450 content in human intestine was

found to be much lower than that in the liver, the major metabolic organ (Peters and

Kremers. 1989: Shimada et ut.. 1994). The metabolic activities of CYP3A. the dominant

P-450 cytochrome in the human intestine. for midazolarn (Thummel et al.. 1995).

tacrolimus (Lampen er al.. 1995) and (+)-bufùralol (Prueksaritanont et d.. 1995) was

demonstrated to be higher in the liver compared to the intestine. Other P-450 isoforms

such as CYP [Al. CYP2B 1/2. C Y P X CYP2D6. and CYP3A2 in rats have also been

detected in rat intestine (de Waziers et al., 1990). Like other dmg-metabolizing enzymes,

the activity of these intestinal cytochrome P-450s can be induced or inhibited. Oral

availability was decreased when cyclosporine was CO-administered with rifampin, a

CYP3A4 inducer (Hebert et al.,

inhibitors, erythromycin (Gupta et

1993). whereas, CO-administration with CYP3A4

al., 1988) and ketoconazole (Gomez el al.. 1995)

resulted in increased cyclosporine bioavailability. The Iocalization of CYP3AJ to the

columnar absorptive epitheiial cells of the villi and not in the goblet cells or epithelial

cells of the crypts in the human srna11 intestine was demonstrated by immunoreactivity

studies using CYP3A4 monoclonal antibody (Murray et al.. 1 988). A similar differential

distribution of intestinal P-450 was observed in rats: the P-450 content at the villus tips

was 10-fold greater than that at the crypt (Hoensch et al.. 1976).

In addition to the cytochrome P45Os. intestinal phase I I biotransformation

enzymes such as UDP glucuronyltransferases (UGT's) and sulfotransferases (ST's)

which are responsible for the conjugation of many dmgs in the intestine are also present

(Pacifici et al., 1988; Cappiello et al., 1989. 199 1 : Krishna and Klotz. 1997). Sirnilar to

the P-450'~. the activities of these enzymes were lower than those of the liver (Koster et

al., 1986: Cappiello et al.. 1991). Differential distribution of UGT activities dong the

villi of the rat intestine was observed. with greater concentrations of these in the villus

cells than in crypt cells (Dubey and Singh, 1988).

1 J.6 Gastrointestinal Motility

Because the intestine is the major site of absorption of most dmgs, any factor that

delays or hastens the movement of dmg from the stomach to the srnall intestine will

influence the rate and/or completeness of absorption. The retention of dnig in the

stomach can increase the percentage of a dose absorbed through the gastric mucosa or

c m induce a greater extent of degradation of acid labile substrates. Some drugs can

affect the rate and extent of absorption of other drugs by influencing the gastnc emptying

time (Nimmo. 19?6). For example, metoclopramide. a cholinergie agent. increases the

rate of gastric emptying, and hence results in earlier and higher peak concentrations of

dmgs which are rapidly absorbed from the upper small intestine (Eisner. 1968; Howells

et al.. 1971; Gothoni et al.. 1972a: Manninen et al.. 1973). Opioid analgesics (e.g.

codeine. morphine) (Burks. 1973: Persson. 197 1) and anticholinergic drugs (e.2. atropine.

loperamide) (Gothoni et al.. 1972a; Mackerer et al.. 1976). on the other hand. have the

reverse effect on gastric emptying rate, resulting in slower absorption and lower peak

dmg concentrations due to delayed gastric emptying.

The rate of intestinal motility dictates the contact tirne of luminal content to the

absorptive surface area and transport carriers and affects the residence time of substrates

and their absorption in the small intestine. Certain drugs can alter the rate of intestinal

motility: castor oil and other cathartics increase intestinal peristalsis and might decrease

the cornpleteness of absorption of dmgs while opioid analgesics and anticholinergic

agents decrease motility (Seeman and Kalant. 1989). Intestinal peristalsis is most

important for drugs that are slow- or sustained-release or that are enteric coated since

ereater intestinal rnotility results in Iess time for dissolution or release of dmg and for C

complete absorption. The transport rate of substrates with high lipid solubility is not

significantly affected by peristalsis since there is an excess area for uptake (Higucchi er

al.. 1981).

1.3.7 Food

Food can affect the efficiency of intestinal absorption by altering the rate and

extent of absorption of substrates. Food, especially one that is high in fat content

reduces the gastric emptying (Hunt and Knox, 1968: Nimmo, 1976), whose rate is

particularly important for compounds that are unstable in acidic environment and for

dosage forms designed to be released drug slowly. In addition. food increases the

viscosity of the luminal fluids and reduces the rate of dnig dissolution and dmg difision

to the absorbing membrane. Transport of certain substrates can also be decreased due to

their binding to specific food particles or their reaction with gastrointestinal tluids

secreted in response to the presence of food. A well-recognized binding interaction

between food and drug is tetracycline. The absorption of tetracycline is reduced by the

formation of water-insoluble and nonabsorbable complexes with iron (Neuvonen et d.

1970; Gothoni et al.. 1970), calcium sdts in dairy products and other cations such as

magnesium and aluminum (Bane rjee and Chakrabarti. 1976: Poiger and Schlatter. 1979;

Lambs et al.. 1988; Jung et a[.. 1997). Food. especially fat. delays the gastric emptying

rate (Nimrno. 1976; Hunt and Knox. 1968). resulting in decreased rate of absorption of

certain drugs.

1.3.8 Segmental Heterogeneity of Intestinal Events

intestinal absorption. metabolism and secretion demonstrate not only vertical. villi

to crypts. but also horizontal (segmental) localization. .4n understanding of the

distribution of the various intestinal events as well as their relative contributions along

the length of the intestine is important in order to make accurate predictions of intestinal

availability and consequently. overall bioavailability of a substrate. Ungell et al. (1 998)

used excised segments fiom rat jejunum. ileum and colon to determine the regional

permeability patterns for passively transported compounds with different

physicochemical properties. The permeability of hydrophilic dmgs was ranked as

jejunum > ileum > colon whereas that of hydrophobie compounds was the reverse. A

similar regional permeability pattern for hydrophilic substrates was observed by Jezyk et

al. (1 992) usine rabbit intestinal segments. Unlike the observation made by Ungell et al..

Jezyk found that the permeability of lipophilic compounds progressively increased fiom

the duodenurn to the colon. The permeability of leuprolide. an agonist of the Iutenizing

hormone-releasing hormone (LH-RH) receptor. in various isolated segments of rabbit

intestine also demonstrated regional differences - colon > ileum >> jejunum. The

absorption rate constant of leuprolide. determined in intestinal loop studies in

anesthetized rats. revealed the same pattern as region permeability seen in the isolated

segment studies (Zheng et ai.. 1999).

Narawane et al. (1993) observed that hydrophilic P-blockers. atrnolol. sotalol and

rnoderately lipophilic metoprolol penetrated al1 intestinal (duodenum. jejunum. ileum.

ascending and descending colon, and rectum) segments equall y wel 1. whereas timolo 1.

propanolol, levobunolol and betaxolol were better absorbed fiom the large than from the

small intestinal segments. The intestinal passive uptake of acetaminophen. a neutral

compound, was greatest in the proximal segment (Pang et al.. 1986) of the rat small

intestine perfusion. The absorption of griseofùlvin (Gramatté. 1996) and antipyrine

(Raoof et al., 1998) were found to be similar in the upper and lower small intestine. A

similar regional absorptive pattem was seen for carbovir. a carbocyclic nucleoside

analogue (Soria and Zimmerman. 1994). The reasons for a lack of similarities in the

observations of regional pemeability of passively diffised dmgs have not been hlly

clarified. However. it is surmised that the surface area is of paramount importance.

A regional pattern of carrier-mediated transport of D-glucose and L-leucine by

glucose- and amino acid carriers. respectively. was observed by Ungell et aL(1997). The

ranking was: jejunum < ileum < colon. The transport of salicylic acid. possibly by the

W/monocarboxylate carrier. however. was observed to be similar among al1 three

segments (Ungell et al.. 1997). Differential segmenta1 expression of another carrier

system. PepT1. which is responsible for the transport of peptides was more abundant in

the proximal (duodenum and jejunum) versus the distal intestine (Fei et al.. 1994). The

segmental expression of the protein. however. may not necessarily retlect the function of

the protein, as observed by Marino et al. (1996). who found that similar uptake of SQ-

29852. a specific probe of the dipeptide transporter dong the length of the srnail intestine

and colon albeit differences in segmental expression of the protein was observed. Most

recently. Tamai et al. (1999) demonstrated that the intensity of epithelial

E-I+/monocarboxylate transporter 1 (MCTI) expression was stronger in the proximal

regions of the rat small intestine. Segmental differences in the absorptive function of this

transporter has yet to be investigated.

Thummel et al. (1997) reported that CYP3A4 expression varies dong the length

of the small intestine. Media.. values of 3 1.23 and 17 pmol/mg microsomal protein were

measured in human duodenum. distal jejunum and distal ileum. respectively.

Immunohistochemical studies revealed iocalization of CYP l A 1 in rat duodenum. with

undetectable levels in the jejunum or ileum (de Wazien et al., 1990). As with the

expression of the metabolic P-450~' the distribution of the UDP-glucosyl~ansferases

(UGT's) is also not *mifonn dong the length of the intestine. The bilirubin UGT activity

decreased significantly h m duodenum to ileum. whereas the UGT activity towards 4-

nitrophenol was roughly similar in human duodenum, jejunum and ileum (Peters et al.,

199 1). Segmentai differences in the metabolic esterase and ketone reductase activities

also exist (Narawane et al., 1993). These activities were found to be, on average, greater

in the small intestine than in the large intestinal segments.

Many have demonstrated that both the expresion of Pgp and its secretory function

are not uniformly disvibuted in the intestine. When the content of mRNA expression of

Pgp was studied over the total length of the human gastrointestinal tract. Fricker et ol.

(1996) found that there \vas a progressive increase of mRNA levels fiom the stomach to

the colon. with low levels in the stomach. intermediate levels in the jejunum. and high

levels in the colon. Fojo et al. (1987) also observed a higher level of MDRI mRNA in

the colon than the jejunurn. Unlike its mRNA expression. the efflux Function of Pgp was

concentrated in the jejunum. with some activity in the duodenum and ileum (Saitoh and

Aungst. 1995). The reciprocal protein concentration gradients of P-450 and Pgp

expression dong the length of the intestine retlect a very effective defensr system that

protects the body against toxic xenobiotics.

The expression and functional activity of the glutathione conjugate efflux carrier.

MRP?. has also been shown to be regionally localized. Nothem blot analysis indicated

significant expression of bfRP2 in the small intestine. with greatest concentration of

mRNA expression in the jejunurn. followed by duodenum and ileum. and very little in the

colon (Gotoh et al.. 2000). The secretory function of the carrier towards DNP-SG was

greatest jejunurn. as predicted by mRNA expression; however, excretion was higher in

the ileum than in the duodenum - a functional observation different from rnRNA

expression. Moreover. Peng et al. (1999) found that the basolateral membrane

expression of the multidrug resistance-associated proteins. MRP's. increased dong the

length. with duodenurn > jejunurn > ileum.

1.4 TECHNIQUES TO STUDY INTESTINAL EVENTS

Various methods are available for the evaluation of the different factors (e-g.

absorptive and eMuv carriers and metabolic systems) that affect to bioavailability. In

vitro. cellular. perfusion, in vivo and imrnunohistochemical techniques may be used for

the study of drug absorption and e.xhibit various inherent advantages and disadvantages.

U , l In C'itro Methods

1.4.1.1 Ussing Chamber

The Ussing charnber is a simple and quick procedure that has been widely utilized

to study intestinal drug permeabilities (Chissone et al.. 1990: Karbach and Rummel. 1990

& 1998: Yodoya eî al.. 1994: Lampen et al.. 1998). Desired intestinal segments are

excised fiom the body. mounted on holders and exposed to substrates in solutions. This

technique allows for the study of directional movement of substrates. from the apical to

serosal or from serosal to apical sides. The Ussing charnber can be used to assess not

only site specific absorption but also rnetabolism (Rogers er al.. 1987: Smith et al.. 1988;

Tjia et al.. 199 1 : Lampen et al.. 1996 & 1998).

The advantage of the Ussing chamber is that the selective barrier hnction of the

tight junctions of the excised segments is not aitered fiom that of the intact intestine

(Artursson et al.. 1993). In addition, the transport characteristics of the membrane are

also preserved. This in vitro method, however, is not without drawbacks - the nanirai

architecture of the intestine is disturbed and the preservation of organ viability is limited

to 90 minutes. Measurements of transepithelid potential difference is necessary to

ensure viability (Soderholm et of., 1998). The excised intestinal segments are also

denervated of central nervous control, and absence of blood flow and intestinal motility

can result in differences in the thickness of the mucous layer. Consequently. ion and

water transport are affected in this in vitro model.

1.4.1.2 Membrane Vesicles

Mernbnne xsicles prepared from the bmsh border menbmc (BBM) for v r b u s

intestinal regions have been used to assess intestinal absorption upon the loading of dmgs

to the extravesicular fluid and observing the rate and extent of removal of the substrates

into vesicles (Ishizawa et 01.. 1990: Yuasa et al.. 1993; Langguth et cd.. 1994: Takanaga

et ai.. 1996: Kitagawa et al.. 1999: Piyapolningroj et al.. 1999). The contributions of

various driving forces. such as pH. inorganic ions. and membrane potential. in energizing

the carrier functions under physiological conditions could be assessed in the in vitro

BBM vesicles. The intestinal BBM studies. in some instances. rnay not adequately

reflect the whole intestinal absorption process since net absorption through the intestinal

mucosa is the outcorne of many complex factors other than the brush-border membrane.

including the basolatenl barrier. motility, intracellular binding and translocation.

1.4.1.3 Everted Sac

The everted intestinal sac has also been employed as a simple and flexible in vitro

method to study intestinal absorption (Barr and Riegelman. 1970: Osiecka er al.. 1986)

and metabolism (Yamamoto et al.. 199 1 : Takeda er al.. L 997: Bouer et al.. 1999). The

procedure can easily be rnodified to snidy segmental absorption. metabolism and

secretion by isolating and removing the desired regions. The removed segments are

everted by means of a glass rod inserted through the lumen and tied at both ends. The

exposed lumen is then inserted into buffers containing the dmg of interest. Serial

sampling of known volumes may be made from both the luminal and serosal fluids at

various time points to examine for metabolism and to formulate an uptake-time profile of

the parent dmg and/or metabolite(s). Although this method can be used as an initial

screening procedure For the study of extent of intestinal absorption and metabolism of

drugs. it lacks several physiological factors such as intestinal motility, blood flow and

neural innervations that c m affect the overall absorptive process seen in vivo. However.

a useful advantage of the everted intestinal sac procedure is the opponunity to examine

the vertical differentiation of the intestinal processes along the crypt-villus mis by

obtaining various intestinal cells along this avis (Traber er al.. 199 1 ).

As an alternative to the above in vitro methods. attempts have been made to

develop rnonolayers of intestinal epithelial cells (enterocytes) as a possible dmg

absorption model. The cultured cells exhibited a presence of microvilli. tight junctions.

complex Golgi complex and basement membrane, which are al1 characteristics of mature

bmsh-border enterocytes. These cells. however, were in fact undifferentiated small

intestinal crypt cells (Quaroni et al.. 1979). A more usehil ce11 line. Caco-2. has since

been cultured and widely used to study the transport of drugs (Ogihara. 1996; Trrao et

al.. 1996; Walle and Walle, 1998; Li et al., 1999). The Caco-2 cells. denved fiom colon

adenocarinoma exhibit well-developed rnicrovitli, polarized distribution of enzymes (e.g.

lactase and maltase-glucomylase in the villus cells; sucrase-isomaltase. aminopeptidase

and dipeptidylpeptidase IV in the both villus and crypt cells) and other properties of

mature epithelial ceus (Zweibaum et al.. 1983 & 1984; Grasset et al., 1984; Hauri et al.,

1985). When grown in cultures. these cells form domes (Grasset et al.. 1985: Mohrmann

et al.. 1986). reflecting the presence of tight junctions and active ion transport processes -

typical of normal, transporthg epithelium. It has been s h o w that some transport carriers

such as Pgp. PEPTl (Dantzig and Bergin. 1990; Dantzig et al.. 1992). GLUT1. GLUT3

and GLUT5 (Blais et al.. 1987) are expressed in the Caco-2 cells. Inorganic phosphates

( M o h r m m et al.. 1986). vitamin Bi? (Dix et al.. 1987) and bile acids (Hidalgo and

Borchardt. 1990) can also be transported by the colonic cells. The Caco-2 system. as

with any othrr in vitro model systems. is not a perfect model of intestinal epithelium. For

example. the cells do not produce mucus nor express significant levels of drug

metabolizing enzymes of the cytochromr Pd50 class or transporters. Attempts have

been made to express relevant intestinal caniers and metabolizing enzymes in the Caco-2

cells by molecular cloning strategies (Covitz et al.. 1996; Crespi et d.. 1996: Hochman et

al.. 2000). The advantage ofthis approach is that it allows for the insertion of intestinal

transporters or enzymes of known structures that are not easily found in more complex

systems. One drawback of the molecular cloning strategy is that the cloned genes are

usually under the regulation of viral promoters. Thus. the natural up- and down-

regulation of gene expression cannot be controlled in these systems.

Goblet ce11 clones have been established from the hurnan intestinal epithelial ce11

line HT29 (Huet el al.. 1987: Roumagnac and Laboisse, 1987; Maoret et al.. 1989). The

monolayers that are formed can secrete mucin molecules and produce a mucus layer that

covers the apical ce11 surface sirnilar to mature goblet cells in vivo (Phillips et al.. 1988).

Thus, cultured goblet cells would provide a useN h g absorption model Uicorporating

the extracellular mucus barrier, thereby permitting detailed studies on the barrier

properties of an intact human intestinal mucus layer (Wikman et al., 1993). Transport

data derived from goblet cells can serve to complement those frorn the mucus-fiee

absorption models such as Caco-2 cells.

Schulthess et al. (1996) performed a comparative study of sterol absorption using

the in vitro bnish border membrane methods such as vesicles (BBMV). enterocytes and

Ussing charnber. They observed that al1 these models are limited in their use because of

instability and degradation. This problem is most apparent in the BBMV and enterocyte

systems. None of the approaches examined was satisfactory in explaining intestinal lipid

absorption.

1.4.1.5 Tissue Homogenates

Intestinal tissue homogenates and subcellular fractions (commonly cystolic.

microsomal or S9. the 9000x g supernatant fractions) have also been utilized as simple

initial screens to determine the degree of intestinal drug metabolism (Del Villar er al..

1974: Koster and Noordhoek. 1983: Young and Mehendale. 1986: Flinois et (11.. 1992:

Adams and Rickert. 1995: Chiba et al.. 1997: Larnpen et al.. 1998: Jacobsen et al.. 1999;

Madani er al.. 1999). Moreover. homogenates from various regions of the intestine can

be obtained to examine segmental locaiization of metabolic systems. The main

disadvantage. however, is the inability to account for regional differences in enzyme

populations dong the length of the organ.

1.42 In Situ Perfusion

The vascularly perfûsed small intestine offers a physiologically-based method to

evaluate the effect of route of administration on the overall bioavailability of a substrate.

A h g c m be delivered to the intestine dissolved in the circulation or directly into the

lumen to mimic conditions of i.v. or oral administration. respectively. However. variable

luminal fluid flow rate and accumulation of fluid may result. There is also a lack of

neurohormonal control. This in situ preparation enables intestinal evrnts to be studied in

isolation of other physiological influences such as biliary secretion and enterohepatic

recirculation. while minimally disturbing the natunl architecture and function of the

smail intestine. This technique not only ailows for examination of rate and extent of

absorption but also for rnetabolisrn and secretion (Pang et al.. 1986: Hirayama rr al..

1989: Doheny and Pang, 2000). Furthemore. regional differentiation of intestinal events

can be demonstrated by isolating the intestinal segments. administering the dnig into a

specific region. and Following the disappearance of the substrate from the lumen and

appearance in the circulating pefisate over time. Moreover. the in situ organ perfusion

method can be extended to include both the small intestine and the liver to examine the

inter-relationship betwern thrse two important first-pas organs (Xu e l cd.. 1989:

Hirayama et al.. 1990: Chen and Pang, 1997).

1.4.3 In Vivo Methods

1 .4.3.1 Luminal perfusion

Most early intestinal perfusion studies in man involved open or semiopen systems

where a solution of the cornpound(s) of interest and a nonabsorbable marker is infused

h to the intestinal test segment (Jobin et ai.. 1985: Vidon et al.. 1985; Merfeld et al..

1986: Borgstrom et al.. 1990). The nonabsorbable marker. often radiolabeled

polyethylene glycol 4000 ("c-PEG 4000), is used to monitor the viability of the

preparation and to correct for changes in the outlet dnig concentrations due to fluid

absorption and secretion. Samples of the luminal perfusate are collected when the

pemision has passed through the segment. The amount of substrate absorbed by the

intestine is calculated by determining the d i k e n c e between the inlet and outlet

concentrations of the dmg. In addition. sampling of the venous blood can also be made

for mass balance considerations (Barr and Riegelman. 1970). The disadvantage of these

earlier perfusion methods is the possible contamination of the test segment with the

luminal contents from the proximal and distal intestinal regions. Moreover. the

techniques require higher than normal perfusion flow rates and the recovery of the

perfusion tluid is low and variable.

To prevent lurninal contamination of the test segment in Ni vivo perfusion studies.

a mutichmel tube with two inflatable balloons c m be used. A 10 cm jejunal segment is

created between the bailoons. enabling perfusion of a defined and closed region of the

jejunum (Knutson et al.. 1990: Lennera et d.. 1992). The balloons are filled with air

when the proximal balloon has passed the ligament of Trirtz in order to prevent the

leakage of contents into the isolated region. The muliple charnels connected to the tube

allow for the infusion and aspiration of the pemisate as well as for the administration of

marker substances and drainage. The markers often used are phenol red. an indicator of

leakage fkom the stomach into the test segment, and PEG-4000. a volume marker. In vivo

perfusions involving closed loops of various intestinal segments have also been

perfomed in rats (Barr and Riegelman. 1970). The loops were held together by tubings

that allow for sampling of the perfusate.

1.4.3.2 Poriacaval Transposition

The systemic availability of certain drugs can be low due to both intestinal and

hepatic removal of the first-pass effect. In order to study the relative contribution of

intestinal absorption and metabolism to the overall disposition of these substrates in vivo,

perfusion fiom the intestine to the liver is diverted in portacaval shunts: the portal

systemic circulation is allowed to drain directly into the vena cava so that the venous

r e t m from the stomach. small intestine and colon bypasses the liver (Gugler er al.. 1975:

Giacomini er al.. 1980). In these studies. orally adrninistered drugs enter the circulation

after crossing only the intestine. Despite its usefulness. this surgical manipulation is

Iimited to only laboratory animals and cannot be performed in humans.

The in vivo perfusions described above are al1 very useful in providing insight to

intestinal processing of drugs in man. These methods. however. are not practical for use

in rapid screening ofdrug absorption.

1.4.4 Immunohistochemical Methods

Immunohistochemical techniques are usehil to detect the location of expressed

proteins in the various intestinal segments obtained fiom the in vitro methods described

above. Exposure of the intestine to various antisense probes and antibodies and use of

fluorescence and radioactivity allows for the detection of mRNA and proteins.

respectively (Thiebault et al., 1987; Murray et al.. 1988; Rich et al.. 1989: Garcia et al.'

1994: Tamai et al.. 1999). The drawback to these studies is the lack of antibody

specificity or interference of background artifacts. These immunohistochemical studies

are very useful in eliciting data regarding segmental localization of mRNA expression or

proteins; however, they do not provide the important information of functiond activities

of these proteins. ffiowledge of protein localization combined with that of segmental

differentiation of h c t i o n activity is required to provide a complete picture of the

absorptive events.

1.5 PHYSIOLOGICAL VIEW OF DRUG ABSORPTION

intestinal absorption. metaholism and secretion of varimir substrates have Lieen

thoroughly examined over the years. However. only few studies have concentrated on

the relationship of al1 these processes in dnig absorption (Pang et al.. 1986; Doherty and

Pang. 2000). Efficient uptake of substrates across the intestinal brush-border membrane

by either passive or facilitated pathways serves to enhance the overall rxtent of

absorption of drugs. Intestinal exsorption and metabolic systems. in contrat. decrease

the ovenll bioavailability of oral substrates by limiting the extent of unchanged parent

h g that reaches the circulation (Gibaldi el al.. 1971: Terao et al.. 1996: Doherty and

Pang. 1997: Watkins. 1997: Lin e t al.. 1999: Hall er ni.. 1999). Since dmgs cm influence

one or more of the intestinal processes (Inui et al.. 1992; Saitoh et al.. 1996; Wacher et

al.. 1998: Doherty and Pang, 2000). it becomes necessary to understand the individual

and collective contributions of each to oral bioavailability.

Pharmacokinetic models c m be designed to incorporate various cellular processes

- absorption. metabolism and efflw - in a comprehensive rnanner to accurately predict

their overall contribution on substrate bioavailibility. The modeling and cornputer fitting

of intestinal absorption to date has been based on a simplistic view of the intestine, where

the organ is considered as a hornogenous cornpartment separated fiom the lumen

cornpartment by the apical membrane and fiom the circulation by the basolateral

membrane (see chapter 3, fig 3-1). The entire organ blood supply is believed to traverse

through the absorptive site (Stigsby and Krag, 1983; Choi et al.. 1995; Yu and Amidon.

1998: Ito er ai.. 1999). These models have been usehl in describing the epithelial

transport of various agents. However, many of the rnodels lack consideration of one or

more of the important variables that are involved in determining overall intestinal

clearance. Moreover. none with the exception of the mode1 proposed by Klippert and

Noordhoek (1985). has been able to predict intestinal route-dependent metabolism which

implies that biotransformation of substrates is greater during on1 than i.v. dosing. The

metabolism of the dnig in some instances occurs only during absorption but not upon

subsequent circulation through the intestinal tissue. Biotransformation in the former

instance is described as pre-absorptive whereas the latter is regarded as a post-absorptive

event. Metabolisrn of enalapril (Pang et al.. 1985). morphine (Doherty and Pang. 2000)

and acetaminophen (Pang et al.. 1986) was observed with intraluminal administration but

not with systemic administration to the vascularly perfused nt intestine preparation.

Wen et al. (1 999) also demonstrated such route-dependent metabolism for the conversion

of (-)6-aminocarbovir to (-) carbovir. The reason why some substrates undergo pre-

rather than post-absorptive metabolism by the intestine is presently unknown. It can

possibly be explained by the inaccessibility of drugs in the circulation to the enzymes

(either mammalian or bacterial) that are available for pre-absorptive metabolism.

Since uptake. metabolism and efflux are not uniformly distributed dong the

intestine it is also important to examine the segmental differentiation of these individual

events as well as the significance of their localization relative to one another on intestinal

availability. Although many techniques are available for the investigation of the handling

of substrates by the intestine, few are capable of concurrent exploration of the

interactions of dl three processes and their segmental distributions while maintaining the

physiological integrity and characteristics of the intestinal tissue.

1.6 APPROACHES FOR EXAMINATION OF VAFUOUS ISSUES OF DRUG

ABSORPTION

1.6.1 Benzoic Acid as Mode1 Substrate

Benzoic acid (molecular weight = 122.12 g/mol) is a weak monocarboxylic acid

(pKa = 4.2) that exists naturally in plant material. especially fruits and berries. and is

most commonly found as sodium benzoate in food preservatives and dyes. It has been

used in the past for the treatrnent of patients with non-ketotic hyperglycinaemia (Ziter et

al.. 1968: Baurngartner et al.. 1969). Doses o f 250-750 mg kg" of benzoate daily reduce

the concentrations of glycine in the cerebrospinal fluid to improve control of seizures in

these patients (Wolff et al.. 1986) while smaller doses of 100-200 mg kg*' per day reduce

plasma glycine concentrations (Baumgartner et al.. 1969). Sodium benzoate has also

been used to treat patients with hyperammonemia caused by a genetic defect in urea

metabolism (Batshaw et al.. 1982). In these patients benzoate is used to divert

ammonium nitrogen and be excreted as urinary hippurate nitrogen (Batshaw et al.. 1982).

1.6.1.1 Metobolism of Benzoic Acid

The metabolism of benzoic acid (BA) occurs via three different pathways: (1)

taurine conjugation. (2) glucuronidation with UDPGA to form benzoyl glucuronide. and

(3) glycine conjugation to give hippuric acid (Fig 1-5). BA biotransformation is highly

species-specific. wherein taurine conjugation takes place in southem flounder and

channel catfish and glucuronidation occurs in various marnmals (Bridges et al.. 1970).

Glycine conjugation is the major metabolic pathway in most organisms, including cats.

rats and humans (Amsel and Levy, 1969; Gatley and Sherratî, 1977).

8enzoic Acid

+ Benzoic Acid

Glycine 4 - Glycine

Hippunc Acid 1

I d Hippuric Acid

Figure 1-5. Glycine conjugation of benzoic acid (Gatley and Sherratt, 1976).

Hippurate formation has been shown to take place in various tissues - within the

mitochondrial matrix of the liver (Bridges et al.. 1970: Gatley. 1977), kidneys (Wan and

Riegelman. 1972: Poon and Pang, 1995), and intestine (Strahl and Barr. 197 1). In the

perfused rat liver. Chiba et al. (1994) demonstrated that the overall glycine conjugation is

a system of relatively low Km (12 FM. based on unbound benzoate) and moderately high

PA, (102 nrno~~min"*~- ' liver). Unchanged benzoate is excreted into both bile and urine

(Hirom el al.. 1976) whereas the metabolite. hippuric acid. primarily undergoes renal

tubular secretion and little biliary excretion (Hirom et al.. 1976: Chiba er cd.. 1994:

Yoshimura al.. 1998).

The formation of hippurate has been shown to be capacity limited. with the

availability of glycine being the rate-limiting step (Amsel et cd.. 1969: Beliveau and

Bnisilow. 1987). Gregus et a[. (1992) had found a capacity limitation of glycine

conjugation by demonstrating a graduai dose-dependent reduction of benzoate blood

clearance and diminuation in maximal benzoate blood levels and urinary clearance of

hippurate with increasing benzoate doses. They found that there were similar time

profiles benveen the concentration of benzoate and excretion rate of the glycine

metabolite d e r injection of benzoate. Moreover, the maximal rate of urinary excretion

of hippuric acid was several-fold larger when hippurate was administered over than that

after benzoate administration. These results suggest that the maximal rate of urinary

output of hippurate is due to capacity limited formation not renal excretion of the glycine

conjugate. Benzoate may deplete hepatic coenzyme A without afTecting ATP levels

during the formation of hippurate. Others have, however. suggested that glycine

conjugation of benzoate is limited by the activity of benzoyl CoA synthetase, the enzyme

responsible for the initial activation of the acid sait to its CoA thioester derivative (Gatley

and Shemtt, 1977: Killenberg and Webster, 1980).

1 h.1.2 Protein Bhding

Chiba el ni. (1994) found that the blood to plasma ratio for benzoate (0.004-

fir)OpM) appmvimated ( 1 - hematoctit). ruggesting that distribution of henzoate into red

ce11 is insignificant. They also reported nonlinear albumin binding for BA. with low

binding to a single class of site on albumin with association constant. KA = 8.37 x 10 3

W.

1.6.1.3 Absorption of Benioic Acid

The mechanism of benzoic acid transport across the intestinal epithelial cells was

first rxamined by Tsuji et al. (1994) in Caco-2 cells. Benzoic acid uptake was found to

be greater than that of mannitol. a reference indicator of the pancellular route of

transport. The results suggest that a transcellular route must be involved in BA transport.

The other existing observations point towards the possible existence of a carrier-mediated

transport system for benzoic acid in these cells, since (1) concenuation-dependence was

obsrrved. (2) energy-dependence. the significant reduction of permeability in the

presence of metabolic inhibitors. DNP and sodium azide, (3) temperature-dependence -

decreased transport with reduction of temperature fiom 37*C to 4°C. (4) inhibition of

permeability of ['"~lbenzoic acid by dabeled benzoic acid and by other

monocarboxylic acids. and (5) significant reduction of cellular uptake when the proteins

on the extemal surface of the cells were digested with a proteinase. papain or modified

with amho acid rnodi@ing reagents (Tsuji et al., 1990). BA transport was also shown to

be highly pH-dependent - the greater the extracellular proton concentration. the greater

the pemeability. This is in concert with a H'-dnven gradient. Utoguchi et al. (1997)

made similar observations regarding BA transport when rabbit oral mucosal epithelial

cells were used. They demonstrated concentration-. pH-. temperature- and energy-

dependence for BA transport. They also showed a reduction of BA permeability when

monocarbo~ylic and not dicarboxylic acids were added and when proteinases were used.

The transport of other monocarboxylic acids displayed the sarne properties as that

of BA. It has long been postulated that the ~'/monocarbox~lic acid transporter MCTl is

involved in their intestinal absorption. Recentiy. Tamai et al. ( 1 999) demonstrated that

BA transport was indeed mediated by MCTl with localization of this carrier in the

intestinal bmsh border membrane. Uptake of BA in MDA-MB23 1 cells transfected with

rat MCT 1 -cDNA showed a rapid. time- and concentration-dependent uptake. The Km

and F,,,, for MCT1-mediated BA uptake was tstimated as 3-05 r 0.38 m M and 168 2

14.3 nrnol min-' (mg protein) -'. respectively. Transport of ["CIBA in these cells was

also pH-dependent. increasing continuously fiom pH 7.5 to pH 5.5. The uptake in mock

or control cells which did not contain MCTI was low. There was. however. a very slight

pH-dependence shown in the control cells. Moreover. transport of BA by the MCTl

transfected cells appeared to be bidirectional. although efflux of BA was significantly

lower than influx.

1.7 STATEMENT OF PROBLEM

In order to Mly understand the contribution of the intestinal processing of h g s

to overall bioavailability. various intestinal events such as absorption. metabolism and

secretion. and their relative distribution dong the length of the intestine must be

considered (Lin et al.. 1999; Doherty and Pang, 2000). Since BA has been shown to be

absorbed. possibly metabolized and secreted by the intestine. the compound serves as an

ideal substrate to study the interplay of these processes and to relate their individual

contributions to the overall intestinal availability of BA. Moreover. the expression of the

cmier MCT l has been s h o w by immunohistochemical experiments with anti-MCT 1

antibodies to be dominant in the proximal intestine. Since the functional activity of

MCTl along the intestine has not yet been demonstrated. segmental absorption of BA can

also be investigated.

1.7.1 Method of Study

The in situ recirculating rat small intestine preparation is a simple and ideal

technique that can be utilized to examine the functional activity of MCT1 in the intact

intestine and for the study of segmentai localization of expression and functional activity

of the transporter. Regional localization of metabolic and exsorptive activities that

govern the overall intestinal clearance of benzoic acid can also be explored.

1.7.2 Development of Comprehensive Physiological Model

A physiologically-based pharmacokinetic model that describes the collective

influence of various intestinal processes on bioavailability and predicts the important

aspect of intestinal route-dependent metabolism will be developed. The model will

incorporate absorptive. metabolic and efflux processes as well as movement within the

lumen (gastrointestinal transit), and dmg partitionhg characteristics in a comprehensive

marner. In addition. the clearance of the intestine in relation to other organs will be

examined. This model should be compatible with observations on route-dependent

intestinal metabolism. Moreover, the distinct tissue layers of the intestine, their varying

fmctions and the differential blood perfusions to these tissue layers should be recognized

and included in the model. The distinct blood flow patterns to the various intestinal

tissue layers has been suggested as an explanation for the observation of intestinal route-

dependent metabolism (Klippert and Noordhoek, 1985).

CHAPTER 2

STATEMENT OF PURPOSE OF INVESTiGATION

2.1 OBJECTIVES

Intestinal route-dependent metabolism has been noted by many: however. this

process has not been examined in detail. Moreover. existing intestinal pharmacokinetic

models have failed to adequately predict the phrnomenon on route-dependent

metabolism. The first objective of this investigation is to develop a physiologically-

based mode1 that would comprehensively incorporate intestinal processes such as uptake.

metabolism and r f f l ~ v alone with cellular diffusion properties of substrate in order to

accurately predict the intestinal and systemic availability of an orally administered agent.

The second objective of this investigation is to thoroughly examine absorption.

metabolism and secretion in the intact intestine. Benzoic acid (BA) was chosen for study

with the in situ rat intestinal perfusion technique. BA is reported to undergo carrier-

mediated uptake at the apical membrane by MCTI. the monocarboxylic acid 1

transporter. It has also been reported that the transport of aryl carboxylic acids by MCTl

is bidirectional. The transporter has been cloned. expressed and localized to the bmsh-

border membrane of the duodenum and jejunum. However. the intestinal distribution of

MCTl activity for betuoic acid is unknown. Benzoic acid is metabolized to hippuric

acid by the intestinal tissue: however. the segmental localization of the enzyme activity is

unknown. The various intestinal processes within isolated segments can be exarnined and

compared in the vascularly perfùsed rat small intestine preparation.

2.2 SPECIFIC AIMS

I . To develop a comprehensive pharrnacokinetic mode1 that explains the

intestinal disposition of a substrate and predicts intestinal route-dependent

metabolism.

3 -. To determine the kinetic parameten for intestinal uptake. metabolism and

secretion of benzoic acid in the whote intestine and afier administration

into the duodenal. jejunal and ileal segments.

2.3 HYPOTHESIS TESTING

We will test hypotheses that:

1. Route-dependent intestinal metabolism is dur to segregated flow pathways

to metabolizing (enterocytes) and non-rnetabolizing (serosal) regions.

2. The overall absorption of benzoic acid diffen among segmenta1 regions of

the rat small intestine.

CHAPTER 3

A NEW PHYSIOLOGICALLY BASED, SEGREGATED-FLOW MODEL TO EXPLAIN ROUTE-DEPENDENT INTESTINAL METABOLISM

Diem Cong, Margaret Dohertyi. and K. Sandy Pang

Department of Pharmaceutical Sciences. Faculty of Phmacy (D.C.. M.D.. K.S.P. ) and Department of Pharmacology, Faculty of Medicine (K.S.P). University of Toronto.

Toronto, Ontario, Canada M5S 2S2

'Present address: Victoria College of Pharmacy. Monash University. Melbourne. Austrrilia

Dmg Metabolisrn and Disposition 28(2): 224235,2000.

Reprinted with permission of The Amencan Society of Phmacoiogy and Experimental Therapeutics. rights resemed.

3 . 1 . ABSTRACT

Processes of intestinal absorption, metabolism and secretion rnust be considered

simultaneously in viewing oral dmg bioavailability. Existing models often fail to predict

route-dependent intestinal metabolism, narnely little metabolism occurs after systemic

dosing but notable metabolism exists following o d dosing. A physiologically-based.

Segregated-Elow Mode1 (SFM) was developed to examine the influence of intestinal -

transport (absorption and exsorption), metabolism. flow, tissue panitioning chariicteristics

and elimination in other organs on intestinal clearance, intestinal availability and systrmic

bioavailability. For the S m . blood flow to intestine was effectively segregated for the

pemision of two regions, with 10% reaching an absorptive layer - the çnterocytes at the

villus tips of the mucosa where rnetabolic enzymes and the P-glycoprotein reside. and the

remaining 9 0 8 supplying the rest of the intestine (serosa and submricosa). a non-

absorptive layer. The traditional, physiologically-based mode1 (TM). which regards the

intestine as a single, homogeneous cornpartment with ail of the intestinal blood tlow

perfusing the tissue, was also examined for cornparison. The anaiytical solutions under

fust-order conditions were essentially identical for the SFM and TM. differing only in the

flow rate to the absorptive/removal region. The presence of other elirnination organs did

nor affect the intestinal clearance and bioavailability estimates, but reduced the % dose

rnetabolized by the intestine. For both models, intestinal availabiiity was inversely relûted

to the intrinsic clearances for intestinal metabolism and exsorption. and was additiondly

affected by both the rate constant for absorption and that denoting luminal loss when dmg

was exsorbed; however, the effect of secretion by Pgp became attenuated with rapid

absorption. The difference in flow between models imparted a substantial influence on the

intestinal clearance of flow-limited substrates, and the SFM predicted markedly higher

extents of intestinal metabolisrn for oral over intravenous dosing. Thus, the SFM provides

a physiologicd view of the intestine and explains the observation of route-dependent.

intestinal metabolism.

3 .2 . INTRODUCTION

Dmgs adrninistered o d l y must frst be absorbed, either passively or vit1 ffacliiated

transport, across the intestinal luminal membrane to reach the systemic circulation. Much

is known about the various intestinai transport proteins that participate in the uptake of

dnigs (Tsuji and Tamai, 1996; Lin et al., 1999). Additionally, the intestine possesses

metabolic enzymes. notably the conjugating enzymes -UDP-glucuronosyItransferases.

glutathione S-transferases - (Koster et al., 1985: Dubey and Singh. 1988: Ilen t.r d..

1990) and cytochrome P450 3A (Watkins et ni.. 1987: Peters and Kremers. 1989: Kolürs

et al., 1992: Lampen et al., 1995; Paine et al., 1996, 1997). In some instances.

metabolism by the intestine was noted only during absorption and not upon subsequent

circulation through the intestinal tissue. That intestinal metabolism is "route-dependent".

k ing p a t e r with oral than with intravenous dosing, was observed for acetaminophen

(Pang et ai., 1986), enalapril (Pang et al., 1985), and morphine (Doherty and Pang. 1000)

and for the conversion of the prodrug (-)6-aminocarbovir to (-)carbovir (Wen et (il.. 1999)

in the perfused rat s m d intestine preparation. The observation was repeated for the

oxidation of midazolam in man (Paine et al., 1996, 1997). Furthemore. a 170 kDa

protein, the P-glycoprotein (Pgp), has been identifed to be responsible for dmg ~ M U X

59

into the intestinal lumen (Thiebault et al., 1987; Hunter et al., 1990: Hsing et d.. 1992:

Saitoh and Aungst, 1995; Srnit et al., 1996). Intestinal metabolisrn and exsorption

effectively reduce the bioavailability of ordy administered agents (Gibaldi et al.. 197 1 :

Leu and Huang, 1995; Doherty and Pang, 1997; Lown et al.. 1997: kirnori and Nakano.

1998: Haii et ai., 1999; Lin sr al., 1999).

Despite the large body of information on intestinal exsorption and merabolism.

only a few models exist to correlate these physiological processes with the overall dmg

absorption or bioavailability (Barr and Riegelman, 1970: Crouthamel et al. . 1975: S t igs b y

and Krag, 1983: Nakashima et al., 1984: Choi et al., 1995: Yu and Amidon. 1998: Ito rt

ai., 1999). Although the rnodels would account for multiple-sitelregional absorption.

metabolism, secretion, or even diffusion within the tissue. few would forecast route-

dependent intestinal rnetabolism. An exception is the model proposed by Klippen and

Noordhoek ( 1985) that suggests shunting of intestinal blood for prediction of route-

dependent metabolism.

A physiologicaily-based %gregated-aow Mode1 (SFM) was developed to explin

route-dependent intestinal metabolism: the model encompassed differential b lood

perfusions to distinct tissue layea of the intestine. The properties of the mode1 were

investigated upon engendering intestinal blood flow, the intestinal metabolic. secretory and

intrinsic clearances, tissue partitioning characteristics (diffusion-limited vs. flow-limited

oans to distribution) of substrate, and presence of eliminatory pathways in paralle1 or,

predict the intestinal clearance and systemic availability. The segregated flows could be

rationalized since distinct blood flow patterns have been noted for various tissue layers of

the intestine - the mucosa, submucosa and muscularis - with each contributing to one of

three hnctions of the small intestine. absorption, secretion. and motility (Granger et cd..

1980). and the serosa that Lies infenor to the muscularis. The large surface area for

absorption is attnbuted to the villi and microvilli of the mucosa. and rnetabolizing enzymes

are located within enterocytes at the villus tip (Kolan et al., 1992: Lown et al.. 1997). I t

has been noted that the majority of "resting" intestinal blood flow. some 60% to 70% of

the intestinal fiow, is disuibuted to the mucosa-submucosa due to p a t e r metabolic

demand (Schurgers and de Blaey. 1984). with approximately 18% (MacFrnan and

Mailman. 1977), 5-7% (Mailman. 1978; Granger et ni., 1980) or IO-30% (Svanvik. 1973:

Micflikier et al., 1976) of the intestinal blood flow perfusing the enterocyte layer of the

villus tips where the majority of the absorptive. metabolic and P ~ J activitirs reside. Sincr

flow perfusing the site of elimination c m influence the disposai of dmgs and since there is

differing blood flow distributions to vaious tissue Iayers of the small intestine. it becomrs

important to view intestinal h g metabolism beyond what is ordinarily considered in

traditional. cornpartmentai or physiological models. in which the absorptive layer is

assumed to receive 100% of the total intestinal blood flow.

3 3 THEORETICAL

Two physiological models for the intestine were examined: the Traditional Mode1

(TM) (Fig. 3-1A) and the Segregated-Flow mode1 (SFM) (Fig. 3- 18). Removal by other

parallel eliminating organs exists. and the effective clearance is descnbed by CL,,,,,.

Cornmon features of the modeis include the inter-connec tion of the blood comparunent

(cenaal or reservoir cornpartment in this instance) to the intestinal tissue via the circulation.

Only fust-order transport and removai processes are considered, and for the sake of

simplicity, the dmg is assumed to be completely unbound.

3.3.1. Traditional Mode1 (TM)

The intestine is subdivided into the vascular (intestinai blood), cellular (tissue) and

luminal subcompartments (Fig. 3-1 A). The tiswe i ç wpplied with blocd frmn ~he

superior mesenteric mery with the flow rate. Q,; venous blood retums through the pond

vein to the reservoir. The exchange of substrate between the cellular and vascular

compartments is described by the intrinsic transport clearance terms CL,, and CL,. that

characterize. respectively, transport from intestinal blood into intestinal tissue and vice

versa. The rate constant for absorption of the subsuate across the luminai membrane is

denoted by 4, whereas luminal removal of the dmg, either by metabolism. fecd excretion.

andor gasuointestinal transit, is represented by rate constant kg. Once in the intestinal

tissue, the dmg undergoes biotransfomation, and is uansponed out into blood or cffluxrd

into lumen - processes that are descnbed by intrinsic clearance terms CL,. CL,,:. md

CL,, respectively (Doherty and Pang, 2000).

3.3.2. Segregated-Flow Model (SFM)

This model is an expansion of the physiological model normdiy developed for the

intestine, but it further recognizes the subtle demarcation of tissue layers and distributions

in blood supply. The notion of Bow-bypass of tissdar regions of the intestine was dso

recognized by Klippert and Noordhoek ( 1985). Dmg in the serosal blood compartment

equilibrates with tissue with the transfer clearances CL, and CL,,, whereas drug in the

mucosal blood-enterocyte blood compartment equilibrates with tissue with the tnnsfer

clearances. CL,, and CL,. The absorptive, metabolic and efflux activities within the villus

tips of the enterocyte compartment are denoted by the rate constant. k,. and the intrinsic

clearances, CL, and CL,, respectively (see Fig. 3- 1B).

3.4. METHODS

3.4.1 Mass- balanced and Theorist Equations

Mass-balanced equations were written for the traditional model (TM) and the Sm.

For emphasis of intestinal metabolism, secretion, and absorption. the system dcscribed

was sirnilar to that for the recirculating system of the perfused intestine prepmiion

(Doherty and Pang, 200).

Trcrditionui Model f TM1.

For the rate of change in amount of drug in the reservoir (cornpartment "R)

For the rate of change in arnount of drug in the intestinal blmd (compartment "int'b")

intb -- A, A inrb - Q I K - ( C ~ ~ ~ +el)- Ainr d t Y n ~ b + CLd2 int

For the rate of change in amount of drug and formation of metabolite { mi ) in the intestinal

tissue (cornpariment "int")

A intb dAint = k d l u r n e n - ( CLdZ i CL,,, + CLrn ) Ai"t + CLdl V . dt 'int intb

For the rate of change in amount of drug in the intestinal lumen (compartment "lumen")

dAîumen Aint dt = int - ( ka + kg ) A~umen

Se ~ r e ~ated- Flow Mode1 ($FM).

For the rate of change in arnount of h g in the reservoir (compartment "R")

For the rate of change in amount of drug and rate of formation of metabolite ( m i } in

enterocyte layer of mucosa (compamnent "en")

For the rate of change in amount of drug in the mucosai blood to enterocyte cornpanment

(compartment "en,b")

For the rate of change in amount drug in the serosal blood (compartrnent "s,b")

For the rate of change in arnount of cimg in the compartment comprising of the serosa and

other intestinal structures (compartment "s")

For the rate of change in amount of dmg in the intestinal lumen (compartrnent "lumen")

It is noteworthy that if Q, equals QI, the SFM simplifies to the TM.

The coefficients in the mas-balanced rate equations for dmg with the TM (Eqs.3- 1

to 3-4) and SFM (Eqs. 3-5 to 3-10) were represented as elcments in 4 .u 4 and 6 x 6

matrices, respectively. inversion of these matrices with the software Throt-istQ on a

Macintosh computer (Power Macintosh 9500/130) provided the analytical solutions for

areas under the arnount-time curves per unit N or PO dose. Multiplication of these to the

ratios of administered doses to reservoir volumes furnished areas under the curves (AUC).

With the assumption that c l e m c e is constant under first-order conditions. the dose-

corrected areas under the curves were used to estirnate model-independent panmeters (a)

the total body or systemic clearance (CLt) from Dosew/AUC,,, (b) the intestinal clearance

(CLJ or (CL, - CLohem), and (c) the systemic bioavailability (F,,,) or AUCkpJAUCRIv.

The hction of drug that ultimately reaches the systemic circulation, Fsys, is a product of

the fraction of drug that is absorbed across the intestinal membrane (F,,,) and that portion

that escapes intestinal metaboiisrn and exsorption (F,). Based on the calculated F,!, and the

definition of the fraction absorbed CF,,, the ratio of the absorption rate constant to the sum

of the absorption and luminal degradation rate constants or ka/(k,+k,)], intestinal

availability (F,) was calculated as F,JF,,.

3.4.2 Simulation

Values of the intestinal clearance and the systemic and intestinal availabilities were

either simdated with the equations (Eqs 3-1 to 3-10. with the progrnm. Scientist'.

Micromath, Utah) or calculated utilizing the solutions obtained for both the TM and the

SFM. Various values for the volume. flow, and transport and intrinsic clearances (Table

3-1) were placed into rows/columns of the Worksheet in Excel (Version 5.0 for

Macintosh, Microsoft, Seattle, WA) and substituted into the solved equations (sce Table 3-

2) for estimation of the various parameters. The overall intestinal flow mte was set as 8

rnl/min. Since fiteranire values for the blood flow to the absorptive enterocyte iqcr of the

mucosa Vary greatiy, ranging from 5 % to 30% (Svanvik, 1973: MacFerran and Mailman.

1977; Mailman, 1978; Granger et al., 1980), the average flow to this compartment was

assigned 10% of intestinal flow for the sake of simpiicity, and the remainine compartment

- the serosa and other intestinal structures - received the other 90% of flow: the volumes

were partitioned in the same fashion. Furthemore, simulation was perfomed with

transport clearances between blood and tissue compartments being identical for the TM

(CL, = CL,, = CL,) and for S M (CL, = CL,, = CL,= CL, = CL,). The value of CL,

was set either as 0.5 or 50 ml/min, since these represented conditions of dmgs of low

(diffusion-kted distribution) and high (flow-limited distribution) permeability.

respec tively.

Table 3-1 Input parameters used for simulations according to both the traditional mode1 (TM) and segregated-flow mode1 (SFM) on intestinal clearance and bioavailability.

Description Symbol Traditional Segregated-flow Mode1 Mode1

Oral dose (mas units) IV d o s (mus unit$

Cornpartment volumes (ml) Reservoir Intestinal tissue

Enterocyte layer Serosa and other tissues

Intestinal blood volume Enterocyte blood Serosai biood

Flow rate ( m h i n ) Intestinal blood

Dose, Dose,

Mucosa blood to enterocyte layer Serosa and other tissue blood

Q; " es Clearances (mumin)

Drug nanspon clearance Metaboiic intrinsic clearance

CL, CL,

Secretory intrinsic clearance CL:

Absorption rate constant (min-') ka

Luminal degradation rate constant (min") kg

LOO" 100"

.L

Assigned parameten b

Value estimated based on Harrison and Gibaldi (1 977) where 10 ml was used for a 360 g rat (including cecum and stomach) and the average intestinal weight = 3 g (ref. Doherty and Pang, 2000)

C

Vaiue associated with the designated 80w to the enterocytes (0.1 *Q,) d

Vaiue associated with the designated 80w to the serosai and other tissue layer (0.9*Q,) C

Parameten v&ed during simulations

The intestinal metabolic inûinsic clearance (CL,, ranging from O. 1 to 50 rnllmin).

the exsorption or secretory ineinsic clearance (CL,, ranging from O to 50 drnin) . and

values of the absorption rate constant (4, from 0.01 to 10 min-') were varied under a

nonchanging kg (0.5 min*') to study the influence of these factors on the m a under the

curve, clearance, and bioavailability estimates.

In order to assess the importance of intestinal exsorption by Pgp on drug

bioavailability, the metabolic cornponent was set to zero (CL, = O). The secretory intrinsic

clearance (CL,), the absorption rate constant (kJ, and the rate constant for gastrointestinal

transitnoss (kg = 0.01.0.5 or 10 min'') were varied for a substrate with CL, = 0.5 luid 50

d r n i n . Lastly, the extents of intestinal dmg metabolism following IV and PO dosing

were compared between the models. In these simulations. CL,, and k. L were set as zero

while CL,, CL ,,,, and CL, were varied.

3.4.3 Fitting of Morphine data to the TM and SFM

The utility of the SFM vs. the TM was appraised with the recent data of Doherty

and Pang (2000) in which morphine (Ml, a substrate which is absorbed. glucuronidated.

and secreted, was given both systernicaüy and intraduodenally to the recirculating.

vascularly pemised rat small intestine prepantion. The models (Fig. 3- 1 ) were extended

to describe not only the disposition of M but also for the formation of the metabolite.

morphine-3P-glucuronide (M3G) by the rat intestine preparation; in this instance CLolh,,

was set to zero (Fig. 3-2).

7 1

For TM, infludefflux of M into the intestinal tissue kom the blood is characterized by

the transport clearance parameter, CL1 and CL2. respectively (Fig. 3-2A). Once M enters

the intestinal tissue, it undergoes biotransformation to M3G with the intestinal metabolic

clearance, CL L 1, or is exsorbed across the luminal (denoted by membrane with the

secretory intrinsic clearance CL3. The absorption intrinsic clearance of iLI from the

intestinal lumen is denoted by CL4. and the luminal degradation clearance. CL 12. M3G.

once formed in the intestinal tissue. can either efflux out to the perfusate blood (CL 10) or

be excreted into the lumen (CL7), where there exists deconjugation of the glucuronide

metabolite (with CL5) and re-glucuronidation of M (with CL6). The influx and eMiu..

clearances for M3G across the basolateml membrane are denoted by CL9 and CL IO.

respectively . The data had been fitted to mass balance relationships previousl y develo ped

to describe events occurring during the traverse of M and M3G across the intestine. The

inainsic clearances for dmg and metabolite absorption and luminal degradation. CL-I. CL8.

and CL 12, respectively, become the correspondhg rate constants upon division by the

volume of the lumen, VIme,.

The SFM was employed for the shultaneous fitting of the data (Fig. XB). The

distinction of this mode1 from the TM lies in that only a fraction (fQ) of the intestinal

flow (QI) perfkes the enterocyte layer of the mucosa where both CYP3A and Pgp reside.

The remaining fiow of the intestine or (1- fQ)Qi perfuses the serosa and other structures.

73

If fa is unity, the SFM simplifies to the TM. In the SFM. substrate in the serosal blood

(s,b) and mucosal blood to the enterocyte layer (en,b) equilibrates with those in tissue:

these are descnbed by transport clearances for M (CLéI and CLa) and M3G (CLdl-\13C

and CLeMG). Conversion and secretion of M proceed with the intrinsic clearances of

CL, and CL,,,, respectively . The intrinsic clearances for dmg and metabolite absorption

and luminal degradation, CL,, CLawG, and CL,,, respectively. are related to the rate

constants. km k a M 3 ~ and k, by the volume of the lumen: intrinsic clearance = VI,,,, x rate

constant. The metabolite, M3G is secreted with an intrinsic clearance. CLseç.h13ü In the

lumen, hydrolysis of M3G is associated with the hydrolytic intrinsic clearance. CL!,

whereas M glucuronidation is denoted by the luminal glucuronidation intrinsic c l e m c e

CL,. Mass balance rate equations were M e r developed to describe events pertaining to

the metabolite, M3G.

3.4.3.1 Mass-balanced Equations for TM and SFM of ilforplrine.

For M and M3G in reservoir (R) cornpartment

For M and M3G in serosa and other non-mucosal tissue (s) cornpartment

dMs -= Ms,b CLdl-- - Ms

dt CL, -

vs,b vs

For M and M3G in enterocyte layer (en) in mucosal compartment

dMen -- M ~ u m e n - CL,-- Men Menb

dt (CL,,, + CLd2 f CL,) - + CLdi -

Vlumen ven Vcn.b

M3Gen M3Gcn.b - (CL se, M G + CL,. MG )- + CLdi. 413~ lumen vcn Ven.b

(3-16)

For M and M3G in serosal blood (s,b) compiutment

For M and M3G in blood to enterocyte layer (en.b) in mucosal compartment

For M and M3G in lumen (lumen) compartment

d W u m e n Men = CL,,,- - M ~ u m e n (CLg + CL, + c L ~ r r ) - + CLh M 3 G t u m n

dt Ven v~umen 'lumen (3-2 1 )

The amounts of M in exudate and lumen were summed to provide the total amount collected

in the sampling tube at 120 min. The same was done for M3G.

Data for M and the fomed M3G avex used for fitting (see Table 1 of Dohcny

and Pang, 2000). The effects of binding of M at tracer concentration were neglected sincr

binding was linear and constant and would not contribute to changes. Equivalent total

values of volume and flows were assigned. aithough the tlows and tissue volumes were

partitioned for the SFM, with 10% of the total volume assigned to the tissue and blood

volumes for the enterocyte region and the remaining 90% for the serosal tissue and blood

(see volumes and flows in Table 3-1). Due to published accounts on the lack of

deglucuronidation of M3G to M (Kenyon and Calabrese. L993) and absence of bI

glucuronidation to M3G in lumen in our systemic studies. CL5 and CL6 for the TM or

CLh and CL, for the SFM were set to zero. Fitting was performed with differential

equations for the SFM with

the Simplex method, then,

the program, Scientist. Initial estirnates were obtained with

Ieast square optirnization was pertbrmed on data ofter the

administration of trace doses of [)H]M alone (systemic and duodenal administration).

Vanous weighting schemes were employed to arrive at optimal fits: the weighting of

unity furnished the best fit.

3.5. RESULTS

3.5.1 Analytical solutions

Mathematical solutions for the AUCs of IV and PO administrations. obtained Crorn

inversion of the square matrices, were used to calculate the total and intestinal clearances.

and systemic and intestinal avdtibilities for both Ihe TM ünd SFM. when niernbrÿnt.

transport clearances were distinct (CL,, + CL,, and CL,, # CL, # CL, # CL,,) (Table 3-

2); these solutions readily provided simplified versions when the transport clearances were

equai (CL,, = CL,,, and CL,, = CL, = CL, = CL,). The solutions differed in the tlow

rate terms: QI for the TM and Q,, for SFM. The presence of other clearance (CL,,,,,, > 0 )

did not influence expressions for the intestinal clearance and systemic bioavailability.

solved for the firsst time when absorption, lurninal degradation. and intestind secretion and

metabolism are al1 present. The solutions were complex relations encompassing the ternis -

blood flow rate to the intestinai tissuelenterocyte layer. transport clearance. intestinal

metabolic intrinsic clearance, exsorption intrinsic clearance. and the lurnind degradation

(kg) and absorption (k,) rate constants, and CL,,,,. The AUC's were simplified when

CL,, was zero: AUC,, were the same for the TM and SFM aithough the AC'CR.,v

differed due to the flow terms- Q for the TM and Q, for SFM. as did CL,. F,,, and F,.

Interestingly, the transport clearances of dmg across the serosal membrane (CL, and CLJJ

and the serosal flow rate (Q) were absent in the solutions of the SFM. This is dur to the

role of the serosa serving oniy as a noneliminating, drug-distribution compartment (Fig. 3-

LB). Because of exsorption of h g , the absorption rate constant, and the luminal

degradation rate constant, kg, were present in the solutions of CL,, CL,, F,,,, and F,. In

absence of secretion by Pgp, 4 and k, these constants are absent in the equations for CL,,

CL,, and FI, except for AUC,, and F,, which are influenced by F,, (Table 3-2).

3.52 Simulations

3 S. 2.1 Effects of intestinal metabolism and secretion on CL&= and F L a

constant Fa.. (0.667. with t and k.. as 1 and O. 5 min"\

The intestinal clearance (CL,), systemic availability (F,,,) and intestinal availability

(F,) were found not to be influenced by the presence of other eliminatory pathways (CL,,,,,,

>O). CL, was affected directfy by both the intestinai secretory and metabolic intrinsic

clearances (Fig. 3-3). The magnitude of the intestinal clearance for any combination of

CL, (frorn O to 50 d m u i ) and CL, (from 0.1 to 50 ml/min) was greater for the TM (Figs.

3-3A and 3-3B. upper panel) than for the SFM (Figs. 3-3C and 3-3D. lower panel). As

expected, CL, increased with increasing CL, and CL,, and the increases were more

obvious for a highly permeable (flow-lirnited) substrate (transport intrinsic clearance = 50

rnümin, Figs. 3-3B and 3-3D). These changes were more gradua1 for the TM (Fig. 3 8 ).

but the changes were more abrupt for the SFM (Fig. 3-3D). By contnst. F, was modulated

by CL, and CL, in an inverse manner (Fig. 3-4, and the changes were more graduai for

drugs with high permeability (cf. Figs. 3-4B and 4D to Figs. 3-4A and 34C) and with the

TM. For dmgs with low permeability, values of FI decreased dramatically to almost a

constant value upon increasing the CL, and CL, from O to 10 d m i n ; further increases in

CL, and CL, were, however, ineffective in decreasing the value of F,, which was already

close to zero (Figs. 3-4A and 3 4 3 . The trends for F,,, were identicai to those for F,

inasmuch as Fa,, was constant due to the nonchanging k, and kg (data not shown: values

were lower because of the fraction, F,,).

Generd trends were identified with the simulations. The values of the intestinal

clearance (CLJ, systemic (F,,) and intestinal (F,) availabilities sirnulated with varying

values values of CL, and CL, for SFM were consistently lower against correspondin,

based on the TM. The ratios of the vdues for SFM to TM were al1 less than unity (Fig. 3-

5). The smallest ciifference between the two models existed when intestinal metabolism and

secretion were absent, Le. CL, = O and CL, = O; a p a t e r discrepancy was observed for

the flow-limited substrate (cf. Figs. 3-5B vs. 3-5A). An increase of either CL, or CL,,

from zero resulted in a dramatic disparity in panmeter values between the two models.

In absence of metabolism, secretion and absorption represented the processes

effecting the cycling of dnig between lumen and intestine. However. the overail

bioavailability depended not only on the vdues of CL,, k,, but k,. the "luminal

degradation" constant associated with gastrointestinal transit time or loss. When k, wüs set

to zero, CL, became zero regardless of the value of CL, because of drug re-absorption md

total lack of loss in the system (CL, and kg = O). High secretion tended to be offset with

rapid absorption (high k) when minird loss existed in the lumen (kg = 0.01 min"). and

the systemic availability tended to remah close to unity (data not shown). At increasing

values of kg (0.5 min*'), however. F,,, became attenuated (Fig. 3-6), and the trend

persisted with even higher kg (10 min') (data not shown).

Membrane-Limited (CLd = 0.5 mllmin)

Flow-Limited (CLd = 50 mllmin)

Figure 3-5. Coiiiparison 01' tIic ratios of intestinal clearüiicc (C'Li), systriiiic avüilabili ty ( FSy,) and intestinal üvüilabi lity (FI) siiiiulüted br ilie SFM üiid the trüdit ioiiül mode\ wlirii tlic secreiory iiitrinsic clearance (CL,,,) and iiietabolic iiitrinsic cleüraiice (Ci..,,,) wcre altrred. The absorption aiid luniiiial degradaiion constants, k, iind kg, were krpt coiistüiii iit 1 üiid 0.5 iiiiii". respectively.

3.5.2.3 m a n d k o&whenCL=Oand kh=0.5 min-'.

In absence of secretion (CL, = O), increasing the values of k, failed to alter

AUC,, or CL, (see Table 3-2) but increased values of F,,,, the singe ppanmeter changing

with &. The greatest changes existed for dmgs with low CL,; w hereas changes were more

gradual for the bigh perme~bility dnrgs (Fig. 3-7). Similar trends ..iere o b ~ e z i d at CL,, =

5 d m i n , albeit the values for FsYs were attenuated (data not shown). It was noted that

values of F,,, for the SFM were consistently smailer than those for the TM. md the ratios

of the values were aiways less than one.

3.5.2.4 Effect of Cl* CL and CL. on metabolism with constant k. i 0.05

min").

The simulation with Scientist according to the differential equations reveded

different extents in intestinal metaboiisrn between IV and PO doses for the SFbI and TM,

when values of CL,,,, CL, and CL, were varied in absence of secretion and luminal loss

(CL, and kg = O). When CL,, = O, intestinal metabolism accounted for 100% of the

administered N and PO doses regardless of the value of CL, for dmg sincr metabolism

was the ody route of removai (data not shown). With degradation or loss occumng within

the lumen (kg >O), however, the % dose metabolized by intestine could become greatrr for

the N over the PO dose due to incomplete absorption (F, < 1).

In the presence of altemate, paraiIel pathways, both models displayed "route-

dependent" metaboiism, with a greater extent of intestinal metabolism occurring with PO

than with IV dosing. However, the difference was much greater with the SFM. The S M

predicted that since there was slower intestinal flow rate (10% flow rate) to the enterocyte

layer, the absorbed drug tended to remain longer in the intestinal tissue due to the sluggish

flow, thereby dowing a greater extent of intestinal metabolism. The difference in flow for

the models led to a smaller intestinal clearance for the SFM. leading to much reduced

intestinal metabolism following IV dosing. Hence discrepancy in intestinal metabolism

between the PO and IV doses was greater with the SFM. and this trend was augmentrd at

low CL, (Fig. 3-8A vs. Fig. 3-8B). The same reasoning may be used to rxplain the

intestinal metabolism for the TM. The greater intestinal flow rate to the site of absorption

would effect the dispersal of the oraily absorbed drug rapidly into the systernic circulation.

thereby reducing the extent of intestinal metabolism. Moreover. due to the greater flow rate

to the absorptive and metabolisrn region of the intestine. CL, and intestinal metabolism

would be high with N dosing. For this reason. there was less discrepancy in intestinal

rnetabolism between the PO and N doses with the TM. Additional simulation with

increased values of predicted higher extents of intestinal metabolism.

Transmembrane Clearance, CLd (ml/min)

F@m 3-8 ERm of CLd and Q#S on intestbal rneabolûm when intesànal

~4~retion and luminal Ioss are non-existent (h, and kg = O) according

to the TM (A) and the SFM (BI.

3.5.3 Application of the SFM: fitting of morphine (M) data

The optimized parameters obtained from simultaneous fitthg of the systemic and

oral data of M and M3G to the TM and SFM are summarized in Table 3-3. Parameter

estimation for M was more reliable since the standard deviations (S.D.'s) of the estimates

were less than the values of the estimates. Expectedly, those for M3G were much less

reliable due to the very high S.D.'s of the estimates. This situation was not unique since

the metabolite was not given, and there were too many fitted parameters. Nonetheless.

least-square fitting was best with a weighting scheme of unity. and the resultant tïts

generally yielded good correlation with the data (Table 3-3. Fig. 3-9). The quality of the

fits was, however, better for the SFM. Mthough an adequate fit of the TM was obsenved

for intraduodenal data (Fig. 3-98). a systematic trend existed for the fit to the intravenous

data of M; M3G formation. though not detected in the system. was over-predicted (Fiy.

3-9A). The SFM furnished, in cornparison, supenor fits. as s h o w by the higher value

for the MSC (Mode1 Selection Cnterion), the slightly improved correlation coefficient.

the lower RSS or residual sum of square of residuals (Table 5-3). and increased

randomness in the residual plots (Fig. 3-10). An improved fit was observed with the

intravenous data since the serosai cornpartment effectively provided a distribution space

for M pig. 3-9A). The fitted value for the fraction of the intestinal fiow perfusing the

enterocyte layer (fq) was very low, representing only 2.4% of the total intestinal flow.

87

and was different f?om zero or unity. If fQ were unity, the SFM would degenerate to the

TM.

Table 3-3 Assigned and fitted parameters for simultaneous fitting of systemic and intraduodenal data of morphine and morphine 3Pglucuronide from the recirculating, vascularly perfused rat small intestine to the Traditional and Segregated-Flow models (Fig. 2).a

Traditional Model -

Parameters CL 1 (drnin) CL2 (mvmin) CL3 (drnin) CU(mI/min) CL5 (mvmin) CL6 ( d m i n ) CL7 ( d m i n ) CL8 (mYmin) CL9 (mvmin) CL 1 O ( m h i n ) CL1 1 ( d m i n ) CL 12 ( d m i n )

h b r C

Weighting i

M S C ~ RSS

Segregated-Flow Model

Fitted Values

' Data for intravenous (n=4) and intraduodenal (n=J) dosing of M were fitted simultaneously with mass balanced equations shown in the appendix for the SFM and compared to the fitted results of Doherty and Pang (2000) for the TM.

Assigned Calculated as CL4/(CL4+CL 12) or CLJ(CL,+CLGiT) Model Selection Criterion - the greater the number, the better the fit

3 .6 . DISCUSSION

The ovenll systemic availability of an oraüy administered substrate depends on the

outcome between intestinal absorption and elimination by first-pass ogans such as the

intestine, iiver, and iungs. Indeed, the importance of the intestine as an ingress organ in

regulating the net absorption of drugs into the portal circulation is weil recognized

(Rowland 1972; Doherty and Pang, 1997). However, unlike the attention given to the

examination of physiological variables influencing liver drug clearance (for revirw. see

Pang et al., 1998), removal processes such as metabolism and secretion (or exsorption)

and the physiological variables such as intestinal flow and gastrointestinal transit tirne on

intestine clearance and availability have not been Fully investigated.

Until now, modeling and cornputer fitting of dnig absorption have been based on a

=ensous simplistic view of the intestine, where the tissue is considered as a homo,

cornpartment separated from the lumen cornpartment by an apical membrane and from the

organ blood by a basolateral membrane. Although these cornpartmental models have been

applied to describe the intestinal absorption of various agents. the models lack

consideration of one or more of the processes that are critical in detemiinhg reliably the

overall clearance of the intestine. More specifically, the mode1 assumed by Barr and

Riegelman (1970) allowed for efflux and intacellular rnetabolism of ordly administered

dmgs but did not include the transfer constant from the blood cornpartment to the tissue.

Crouthamel et al. (L975), on the other hand, included the revenible transfer of drugs

berneen the tissue and blood compartments but both intestinal secretion and metabolisrn

were ignored in modeling of the pharmacokinetics of sulfaethidole. Transport processes.

such as the exchange fIom blood to tissue or the efflux fiom tissue to lumen. and intestinal

metabolic activities were absent in the kinetic models proposed by Choi et ni. (L995) and

Nakashima er al. (1984). Recently, Ito et al. (1999) introduced a theoretical

phannacokinetic mode1 to relate the influence of intestinal CYP3M metabolism. Pgp efflux

and intracellular diffusion on d m g absorption. Not unlike both of our TM 'and SFM. ho's

rnodel was able to predict the inverse relationship between bioavailability and metabolism

and/or efflux. However, the transport clearance term that describes the partitioning of dmg

from the circulation to the epithelial cells was absent, precluding the intestind accumulation

or exsorption of intravenously administered drugs. and transfer processes between the _out

lumen and epithelial ceiis were omitted in their defnition of absorption clearance. The

extended cornpartmental absorption and transit (CAT) rnodel developed by Yu and Amidon

( 1998) had simultaneously considered passive absorption. saturable absorption.

degndation, and transit kinetics in the smdl intestine. But processes such as lurninai and

intracellular metabolism and exsorption were excluded. The present rnodel is developed to

comprehensively illustrate the interaction between the effective flow to the intestine. the

absorption rate constant, intestinal enzymatic and secretory activities, and the intluence of

other clearances on systemic bioavailability. The SFM - based on the view that the

absorptive site of the intestine receives only a portion of the overall ogan blood flow. is in

theory, not dissidar to the bypass phenornenon proposed by KLippert and Noordhoek

(1985), with the exception that the flow rate to the intestinal tissue is conserved and h g

distributes into the nonabsorptive and nonelimuiatory layer of the serosa and submucosa.

90

A close scrutiny of the SFM and TM reveals notable differences because of the

different effective perfusion of the absorptive/metabolic/secretory layer. Theoretical

solutions for both the traditional and segregated-flow models differ only in the tlow tenns

(Q, vs. Q,) (see Table 3-2). Elimination within other parailel (non first-passl organs hils

to affect the intestinal clearance, as expected of the additivity of organ c lemces ammg

parailel, elimination pathways, and does not impact on bioavailability. The present

communication also uncovers that, for both the SFM and TM. CL, and F, are

directlylinversely related to the intestinal metabolic and exsorption intrinsic clearances (CL,,

and CL,) and blood flow to the absorptive layer (Figs. 3-3 and 34): the panmeters are

additionally affected by k, and kg when there is dnig exsorption (Table 3-2). Values for the

SFM are, however, consistentiy lower than those for the TM (Fig. 3-5).

The frequent question addressed on whether the role of Pgp on secretion is

overemphasized (Lin et al., 1999) cm now be answered. The exsorption of substrate from

the intestinal tissue to the lumen (CL,> O) exerts a direct influence on F,?, - the liirger the

exsorption clearance, the less the systetnic availability. Dmg secretion by Pgp. viewed best

in absence of metabolism and loss fiom lumen, reveals that indeed. secretion may be

obliterated when dmg absorption is rapid (Fig. 3-6). However, the concurrent absence of

secretion and metabolism (CL, = O; CL, = O) will result in a h a t i c increase in the

systernic (or intestinal) availability.

The difference in flow between the models also affects the extents of intestinal

metabolism. The condition was best shown when CL, and kg = O; a greater difference in

the extent of intestinal metaboikm is found between the PO and IV doses with the SFM

(see Fig. 3-8). According to the S M , the lowered flow rate pemising the enterocyte layer

renden lower values of intestinal clearance, since there is reduced dmg delivery to

intestinai erizymes or secretory sites. However. during orai absorption. the rntire oral1 y

administered dose must traverse the enterocyte layer before the substrate enters the

circulation. The consequence of the partial flow to the enterocyte cornpanment leads to

sluggish dispersai of dmg into the circulation and a longer transit time within the intestinal

tissue. The differential exposure with the site of administration results in different extents

of metabolism by intestinal enzymes a d exsorption. and contributes to the observation on

"route-dependent" metabolism (Klippert and Noordhork. 1985: Pang n d.. 1 985. 1986:

Wen et ni.. 1998). Intestinal meiabolism may then be viewed effectively as a single pre-

absorptive event, occumng predominantly dunng the absorption of the substrate across the

luminal membrane and is substantidly less upon recirculation of the cimg. It has bern noted

that fiow can also be a limiting factor of intestinal absorption since it affects the net

substrate flux from the lumen into the circulation and vice versa (Crouthamel er c d . . 1975:

Winne, 1978; Schurgers and de Blaey, 1984). However, the flow rate to the enterocyte

layer is now recognized as critical to intestinal clearance and bioavailability. Although the

nature of the change remains largely untested, the magnitude of this fiow is rxpected to be

of paramount importance to the initial absorptive flux and drug extraction as well as on

subsequent recirculation of the substrate.

Finally, the confirmatory evidence that the SFM is the better rxplanation of

intestinal metabolism is substantiated by the fit to the experimentd data of morphine.

Statisticaily, the fits of the SFM to data on route-dependent glucuronidation of morphine

92

in the vascularly perfused intestine preparation (data of Doherty and Pang. 1000) are

improved over those afforded by the TM (Table 3-3. Fig. 3-10). In particular. the fit of

the SFM to the N data of M was supenor since the distribution phase was better

descnbed by the SFM due to the presence of the serosal cornpartment acting as the

srorageidistribution companmem (Fig. 3-9A). The tissue panitioning ratio (vaiue of 8 )

for M for the SFM was more reasonable than the much higher value of 22 predicted for

the TM (CLYCLI or CLd2/CLdl), when levels of total ndioactivity in the tissue were

low (5 to 6% dose). Although there were notable levels of M3G accumulated in the

reservoir fier the intraduodenai dose. M3G was not detected after intravrnous

administration. The total level of M3G predicted by the SFM was lower than that by the

TM (6.6% for TM and 2% for the SFM).

Tirne (min)

F ~ R 3-9. fitting of the SFM (- -) to data on the metabolism of morphine (M) to morphine-3fbglucumnide (M3G). M was given intravenously (A) and intraduodenally (B) ta the recircdating perfwd rat tiver preparation (data of Doherty and Pang, 2CKlû). The SFM was more superior in descrihg the data compared to TM (- ) described by Doherty and Pang (2000). Note that M3G was not observed after the intravenous dosing of M althou* a trace amount of M3G was predicted io be formed according to the SEM (B), and thne-fold tbat was predicted with the TM (A).

( A ) IV-Morphine

Figure 3-10.

Predicted Amount in Reservoir (% dose)

Predicted Amount in Reservoir (% dose)

Cornparison of residuals of computer Bts for the TM and SFM for oraiiy (A) and intravenously (B) delivered morphine. There is no systematic pattern to the scatter of the residud plots for both routes of administration. A similar residud plot was observed for the fitting of i.v. data for both modeis. The residuais of the fitting of the p.o. dosing of morphine for the SFM was smaller (doser to zero) than those for the TM, indicating a better computer fit of the p.o. data for the SFM.

Currently, the intestine is regarded as a single compartment. The SFM is

physiologically sound and affords a plausible explmation of route-dependent metabolism.

Due to the many examples of route-dependent rnetabolisrn of the intestine. it is anticipated

that the proposed intestinal SFM may be important in future endeavors to accurately relate

Ni-viîro parameters with in-vivo physiological events on absorption and bioavailability.

Moreover, this mode1 rnay be readily expanded to describe the physiological sebmental

divisions of the intestine - duodenum. jejunurn and ileum - and transport and merabolic or

secretory heterogeneity within these se,pents (Dubey and Singh. 1988: Paine er trl.. 1997:

Saitoh and Aungst. 1995; Fei et al., 1994; Aldini et al, 1996). With the development of

these kinds of models, predictions on the first-pass removal/metabolism and dnig-drug

interactions within the intestinal tissue would then be made accurately.

3.7. STATEMENT OF SIGNIFICANCE

In this chapter we demonstrated the usefulness of a new physiologically-based

mode1 (SFM) in predicting intestinal clearance and systemic bioavailability of oraily

administered substrates. Simulations showed that accunte interpretations of the intestinal

handling of drugs require an understanding of the overall contribution of rnany important

intestinal factors such as uptake, metaboiism (mamalim or bacterial). efflux. luminal

rnotility, membrane permeability characteristics of the substrate and differential blood

flows. The clearance of the drug by other paraiiel organs also contributes to the

interpretation of intestinal availability. The inclusion of the distinct blood flow rates to the

absorptive tissue layer, which inchdes the enterocytes, and the non-absorptive layer in the

SFM was sigrilfcant in ailowing for the observation of route-dependent intestinal

96

rnetabolism of morphine. The fit to the data, performed by Dr. Pang, showed the

superiority of the SFM.

CHAPTER 4

PREFERENTIAL ABSOWTION OF BENZOIC ACID BY JEJUNUM

OF THE INSITU PEWUSED M T SMALL INTESTINE PREPARATION

Diem Cong and K. Sandy Pang

Department of Pharmaceuticai Sciences. Faculty of P h m a c y (D.C.. K.S.P.) and Department of Pharmacology. Faculty of Medicine (K.S.P.). University of Toronto.

Toronto, Ontario. Canada MSS 2SZ

The recirculating in sitir perfused rat small intestine preparation was used to examine

the absorption of benzoic acid (BA), a substrate which is putatively transported by MCTI.

the rnonocarboxylic acid transporter 1. ïhere was lack of metabolism for both systemic and

intraluminal administrations. However. unchanged BA was recovered in luminal fluids of

intestinal segments not exposed to the drug. Absorption of varying BA doses (0.166 to 3.68

pmole in 0.4 ml physiological saline solution or 0.42 to 9.2 miM) which were introduced into

the proximal duodenum and exited the ileocecal end was almost cornplete p 9 5 % dose) at

2 h. with similar first-order absorption rate constants (ka or 0.0464 2 0.00 1 O min"). When

BA was injected into closed segments of much shorter lengths (13 or 20 cm). the absorbed

amounts remained high for the duodenum and jejunum (95 to 96% dose). albeit a slightly

lower extent (92% dose) existed for the ileum. suggesting a large reserve length for BA

absorption. Recovery of dose in the lumen of the injected segment was also similar (< 2%)

for the duodenum and jejunurn. but was higher for the ileum ( 5 % dose). Values of k, were

highest for jejunum (0.05 I92O.OOOl and O.Oj6J+O.OO 12 min". respectively. for 12 and 20

cm segments) and exceeded those for the duodenum (12 cm segment. 0.0442 t 0.00 1 1 min*')

and ileum (20 cm segment. 0.0380 2 0.0024 min") as injection sites. suggesting the

unevenness in absorption of BA among intestinal segments.

3.2. INTRODUCTION

The intestine is well recognized for its myriad of fùnctions - absorption (Kohn et al..

1965; Bodemar et al.. 1979). metabolism (Koster et al.. 1995: Paine et al.. 1996; Ilett et al..

1990) and exsorption (Augustijns et al.. 1993: Su and Huang. 19%). Intestinal transport is

due. in part. to the presence of various transport proteins (for review see Tsuji and Tamai.

1996: Lin et al.. 1999). and efflux activities have been attributed to the existence of the P-

glycoprotein (Pgp) (Thiebault et al.. 1987: Saitoh and Aungst. 1995) and the multidrug

resistance-associated protein 2 (MRP?) (Konig et cd.. 1999). The overall intestinal

availability of orally administered dmgs is highly dependent on the intimate dynamics of

these processes (Doherty and Pang. 1999: Lin et al.. 1999: Cong et al.. 1000).

With recent advancement in the expression cloning of intestinal transporters (Hediger

et tri.. 1987: Rand et al.. 1993: Saito et cri.. 1995; Hirohashi et al.. 2000). there is increased

interest in the examination of regional distribution of the camers. The location of apical

absorptive transporters is particularly important in relation to the exsorption carriers. since

these intluence the overall bioavaiiability (Gramatté and Richter, 1994: Homsy et d. 1995;

Lin et cd.. 1999). Among these transporters is the proton-driven monocarboxylate CO-

transporter 1 or MCTI. which is responsible for the flux of carboxylic acid substrates such

as acetic acid (Bugaut. 1987: Tsuji et al.. 1990), nicotinic acid (Simanjuntak et al.. 1990):

lactate (Timppathi et al.. l988), salicylic acid (Takanaga et al.. 1994), benzoic acid (Tsuji

et al.. 1994). pravastatin (Tamai et al., 1995a) and propionate (Harig et al.. 199 1 ) . The

farnily of MCT transporters is pK-dependent and ubiquitously expressed. and the MCTl

isoform is known primarily for the transport of aryl acids across the intestine. MCTl was

1 O0

first cloned from the intestine of the hamster (Garcia et al.. 1994: Covitz et al.. 1996). and

identified on the enterocytes and not the crypt cells of the duodenum and jejunum of the rat

(Tamai et ul. 1999). The segmenta1 absorptive h c t i o n of this transporter. however. has not

been previously described.

Benzoic acid (BA). a common preservative that is used clinically For the treatment

of inbom errors in urea synthesis (Batshaw et ai.. 1982: Barshop et ai.. 1989) is a substrate

of MCT1 (Tsuji et ai.. 1994: Tamai et al.. 1999). and is mainly metabolized to hippuric acid

(Gatley and Sherratt. 1977: Beliveau and Brusilow. 1987: Gregus ei cd.. 1992). In the

present investigation. vie employed BA for the study of segmental metabolism. rxsorption.

and transport by MCTl . For our studies. we utilized the in situ perhsed rat small intestine

preparation for direct assessrnent of net absorption. metabolism and secretion. The innate

circulatory patterns and cellular architecture of the small intestine are preserved in this

preparation such that processes such as absorption. mrtabolism and efflux of drug into the

circulation occur simultaneously. In addition. regional intestinal absorption OF orally

administered drugs c m be studied by the injection of dose into the physiologically relevant

segments - duodenurn. jejunum or ileum. The proper charactenzation of intestinal

absorptive. metabolic and exsorptive behaviour in the various intestinal segments will

undoubtedly result in an improved understanding of the interplay of the processes and

improved design of oral dmg delivery. The n-octano1:buffer partitioning coefficient of BA

was investigated. For cornparison, the n-octano1:buffer partitioning of acetaminophen, a

neutral compound whose transport is normally by passive di f ised was also examined.

4.3. MATERIAL AND METHODS

4.3.1 Materials

Unlabeled benzoic acid (BA) and its glycine conjugate. hippuric acid (HA) were

purchased from Sigma Chemical Co. (St. Louis. MO). [ ' 4 ~ ] B ~ (specific activity. 16

mCi/mmol) was obtained tiom New England Nuclear Co (Boston. MA). The radiochernical

purity of BA was >99%. as judged by HPLC. Al1 reagents used were of glass-distilled

HPLC grade or of the highest purity available.

4.3.2 Intestinal Perfusion

4.3.2.1 P erfusian apparatus and perfusate

A Two/Ten perfuser (MX International. Aurom CO). equipped with two reservoir

units. was used for perfusion of the rat small intestine preparation. Reservoir 1. containing

the blank pefisate. was used for equilibration (20 min) of the intestinal preparation prior to

the commencement of the experiment upon recirculation of perfusate (200 ml) fiom reservoir

2. The perfusate consisted of 20% of washed. freshly obtained bovine red blood cells (kind

gifi of Ryding Regency. Toronto. ON). 4% bovine serum albumin (Sigma Chemical, St.

Louis. MO), 300 mgdl glucose (50% dextrose. Abbott Laboratories Ltd., Montreal. QC) and

a complement of 20 amino acids in Krebs-Henseleit bicarbonate (KHB) solution. buffered

to pH 7.4 and oxygenated with carbogen (95% 04% CO,) and 0: (BOC Gases. Whitby.

ON)-

Male Sprague Dawley rats (300-400 g, Charles River, St. Constant. QC) were used

as intestine donors. These rats, housed in accordance to protocols set forth by the University

of Tomnto Animai Cornmittee and kept iinQ &cial Li@ on a 12: 12 h light-dark cycle,

anGsthcsia with sodium pcxxtobarbital ( j i t o d dosc of 50 rng/kg), surgcrp was

c o n d d as dtscribcd previously (Hira)ama et ol, 1989) v~g 4-1). The supahr

m t s d c artay (SMA) that scrvai as tk idet was ~~lnir lrr trr l by a b l d 18 gange

by a sphygmomanorncter (mode1 AB, Data Instruments, L a b g t o n , MA). The orrdcr. the

Raritan, NJ), with thc Op of the d e t e r fàüng thc vcnous drainage of the intestine S u o m a ~ r n c ~

\ h a r i v m

Smaü unnrme

K o w v t w L u n i n i l fluid

d

Schemaric illustration of the in situ perfused rat intcstinal preparation with flow rate of 8 ml/min. The nrpaior mesentcric artery and p o d canntiisnrri for innow and oudow, qectMly. Limiinal fluid was dowed to drain k i y ouî an O- made at the î i e o d end of the intestint (fOr &oie inmine d e s ) or at the end of the various segments (for s w shidies). S and P rcprcsent rhc sphygmomanomcter used to monitor artaial prrssurr thughout the crpcrimcat, and pafuson pimip. r c q e d ~ ~ i ~ -

Perfusate exiting the intestine was retumed to the reservoir for recirculation of the intestine

at the flow rate of 8 mumin. Following surgery. the intestinal preparation was stabilized For

20 min with recirculation of perfusate fiom reservoir 1. During this equilibration penod. an

opening was made near the ileocecal junction (for whole intestinal perfusions) or at the end

of non-injected intestinal segment(s) (for segmental studies) to allow for the outtlow of

mucus/chyme that would othenvise present blockage of luminal tlow during the experiment.

Outflow cannulae (PE 240) were made at various places dong the length of the intestine to

divert luminal ewdate into 12-ml polypropylene tubes for monitor of drug exsorption for

mass balance considerations. At the commencement of the study. perfusate fiom reservoir

7 (100 ml) \vas utilized for recirculation of the intestine preparation. A heating lamp was

usrd to maintain the temperature of the preparation at 37" C. The pH of was monitored and

adjusted to 7.4 by altering the inflow of gases (oxygen or carbogen) to the reservoir. The

hematocrit of the perfusate was determined before and &er each expenment by a hematocrit

centrifuge (Microhge B. Beckrnan Instruments. Pa10 Alto. CA).

4.3.2.2 Systemic and in truiuminal dosing

For systernic administration, BA in two concentrations (tracer [ I 4 C ] B ~ of 44 + 2.3

x 10' dpm/ml or 1.3 k 0.06 PM. and 432 t 13 @Q was mixed thoroughly in the pemisate

of reservoir 2. For studies which entailed the luminal administration of BA into the entire

intestine, the dose (1 19 to 3680 nrnole, containing 6.5 f 7.8 x 106 dpm), dissolved in 0.4

ml physiological saline solution, was injected via a L -mi tuberculin syringe directly into the

lumen of the duodenum at 2 cm below the pyloric sphincter. An outflow cannuia was made

1 04

at the proximity of the ileocecal end. For other segmental studies. a tracer dose of ['"CIBA

(5.3 t 2.9 x 106 dpm or 150 f 84 nmole) was injected into discrete. closed segment of the

duodenum. jejunurn or ileum under investigation. For these studies. the length of the small

intestine was traced by a piece of silk thread. and care was taken not to damage the intestinal

tissue or its blood supply. BA was then administered into one of the three intestinal

segments. In view of the shorier length of the duodenum. a 17 cm closed loop was chosen

for injection of the duodenum. and a similar length was used for the jejunurn for cornparison:

however a longer length of 20 cm was used for the closed segments of both jejunum and

ileum. Ligatures were placed proximally and distally of the intestinal segment (12 cm

duodenum. onginating close to the pyloric sphincter: jejunum - 12 or 20 cm segments: ileum

- 10 cm closed segment. -2 cm From the ileocecal end) for the creation of a closed loop so

as to entnp BA within the desired segment for absorption. Outflow cannulae were made at

the ends of the segments not receiving the dmg. Perfusate samples were taken from reservoir

2 at 0.2. 5. 10. 15. 30.45. 60. 75. 90. IO5 and 120 min after the recirculation of reservoir

perfusate containing BA (systemic studies) and afier the injection of BA to the lumen

(intmluminal studies). The total sarnpling volume accounted for less than 10% of the

original volume. At the conclusion of the experiment? the intestinal segments (injected or

noninjected) were cleared of their luminal contents and cleansed by hvo 1 ml saline washes.

The contents fiom the sarne segment were pooled. The intestine was isolated from the

carcass. gently rinsed, weighed and homogenized for analysis of radioactivity. The volume

of perfusate remaining in reservoir 2 was recorded and added to the volume of perfbate

sampled for volume and mars conservation considerations.

43.3 Analytical Procedures

1.3.3.1 Preparation of saniples for HPLC injection

Since BA and HA were not distnbuted into red blood cells (Poon and Pang. 1995.

Geng er ni.. 1999). plasma samples obtained by centrifugation of blood perfusate were used

for analyses. The HPLC procedure of Chiba et al. (1 994) was modified for the quantitation

of BA and possible metabolite. hippuric acid in the perfusate. The blood samples were

centrifuged to obtain plasma. To 350 pl of the plasma samples. 50 pl of the intemal

standard. rnethoxybenzoic acid (16 pg/ml solution in water) and 800 pl of acetonitde were

added. The samples were voneaed and centnfuged at 2700 rpm for I O min to precipitate

protein. The supernatant was then transferred to a new tube. dned under nitrogen and

reconstituted with 200 pl of mobile phase (0.5% acetic acid:acetonitrile: 90: 10 v/v). The

reconstituted sarnple was centnfùged again and 150 pl of the supernatant was used for

HPLC. Standards for calibration curves (varying amounts of unlabeled a d o r [''CIBA) were

processed under the same condition as that used for the quantitation of BA in the plasma

samples.

4.3.3.2 HPL C Assay of unlabeled benzoate and It ippicric acid

The chromatographie system (Shimah. Shirnadm Corporation. Kyoto. Japan)

consisted of two LC-IOAT purnps, SCL- I OA system controller. GT- 1 O4 degasser. FCV-

1 OAL low-pressure mixing chamber? SIL- 1 OA autoinjector and an SPD- 1 OA UV-Vis

1 O6

detector. The wavelength of the detector was set at 254 m. A reverse-phase Beckman

Ultrasphere column (0.46 x 25 cm i.d.: particle size. 5pM) and a Waters C ,, guard colurnn

(2.2 x 0.34 cm i.d.: particle size. 37-55 pM) were used for separation. The initial condition

of the mobile phase was 0.5% acetic acid (purnp A) and acetonitnle (pump B). 90:lO v/v. at

a flow rate of 1 mumin. At 10 min. a linear gradient was used to increase the acetonitnle to

27.5% for the next 2 min. Then at 15 min. the tlow rate was decreased to 0.9 rnl/min for

improved resolution of the peaks for BA and the intemal standard. The condition was

maintained for 5 min before reverting back to the initial conditions over a course of 2 min.

This \vas rnaintained for 5 min before the start of a washing period which involved

increasing the percent organic phase fiom 10 to 30. then 50% over a 2 min period. before

gradually retuming to the original condition over 6 min. The wash procedure was necessary

since continuous HPLC injections of plasma samples resulted in poor resolution of the peaks

afier several such injections. The total run tirne per injection was 40 min. The retention

times were: hippuric acid. 14 min: benzoic acid. 24 min: and rnethoxybenzoic acid (internal

standard). 26 min. Radiolabeled BA was collected into 20 ml scintillation tubes afier pre-

determining the collecting interval by characterizhg the radioelution of a representative

sample at 1 min intervals. Afier the addition of 5 ml of scintillation cocktail (Ready Safe.

Beckman instruments. Pa10 Alto. CA), the HPLC elutions were counted using a two-channel

liquid scintillation spectrophotometer (mode1 LS 580 1. Beckman Instruments. Pa10 Alto.

CA). Unlabeled BA was quantified by comparing the ratio of the area of BA to area of

internal standard against known concentrations of unlabeled BA and intemal standard in the

calibration cuve.

4.3.3.3 Radioactivity in plasma, tuminaifluid and intestinal tissue

In addition to HPLC. a thin layer chromatographic procedure (c horo form:

;yclohcsanc: acctic acid, SO:20:IO rhh and Silica Gcl GF 3 0 pin phtes. Aialtcch.

Newark. DE) was used to ven@ the presence or absence of ["CIHA in the system: however.

none was found in al1 the sarnples examined. Since metabolites were absent in the luminal

fluids and plasma. the total radioactivity of the sarnple was taken to represent ["CIBA.

Aliquots of plasma were assayed by liquid scintillation spectrophotometery (Beckrnan LS

580 1. Beckrnan Canada Mississauga, ON). The luminal fluid \vas analyzed for radioactivity

following an extraction procçss. Subsequent to centrifugation of the luminal contents for

removal of particdate matter. aliquots of the supernatant (q.s. to I ml) were added 5 ml of

aceronitrile and mixed thorou&ly. and 3 ml of the resultant solution was removed for liquid

scintillation counting. To account for recovery. known arnounts of [ I 4 C ] ~ ~ was added to

blank luminal tluid ( 1 ml) and subjected to the same procedure. The recovery was 73%.

Intestinal tissues were also analyzed for radioactivity. The weighed tissue was

reduced to fuie pieces. then homogenized (Ultra Tunav T25 homogenizer. Janke and Kunkel,

KA-Labortechnik. Staufen im Briesgau, Gemany) with 2 volumes of KHB. One milliliter

of the homogenate was then added 5 ml of acetonit.de and mixed thoroughly. and 3 ml of

the resultant solution were subjected to scintillation counting. To account for recovery.

known arnounts of ["CIBA were added to blank homogenized tissue (1 ml) and subjected

to the same procedure. The recovery of ["CIBA was 57%.

4.3.3.4 n-Octanul und bu ffer partition of benzoic acid.

Radiolabeled BA (approximately 100,000 dpm) was placed into glass tubes and dried

under ninogen. Buffers (1.95 ml at pH 1.1. 5.6.7 and 8) and 50 pl of a saturated solution

uT unlabçird BA wrrr added to dissoive the dried [!'C]BA. Equivoiiimrs ( 2 mi) of buEer

containing [''CIBA and n-octanol were then mixed. The tubes were rocked for 2 to 3 h with

an aliquot mixer. then left to equilibnte ovemight. A volume ( 1 . j ml) of n-octanol and 500

FI of the buffer (in triplicates) were removed for scintillation counting.

4.3.3.5 n-Octanui and buffer partition of ocetaminoplien.

Aliquots of 500 pl of unlabeled acetaminophen (1 mM) were added to buffers ( 1 .j

ml) of varying pHWs (1. 2. 5. 6. 7 and 8). Partition studies were carrird out in the same

manner as those described for benzoic acid. Acetaminophen in bufferin-octanol was

analyzed afier removal of25 pl of the sarnples into 175 pl of buffedn-octanol. Then 5 pl

was injected into a C,, pBondapak column. Separation was achieved using a mobile phase

consisting of 25% methanol-water at a flow rate of 0.7 ml/min with W detection

wavelength at 254 nm. The retention time of acetaminophen was 5.9 min.

4.4.1 Intestinal Viability

The viability of the vascularly perfùsed rat mal1 intestine preparation was similar to

that previously characterized in our laboratory (Hirayama et al.. 1989). There was good

recovery of the volume of the reservoir at the end of each study (94 f 2.1%) and pemision

pressure measured at the SMA was constant (54 k 17 mm Hg) during the perfusion study.

The hematocrit of the pefisate at the end of the perfusion study increased only by 9.5 +

1.9% of the original values. These values are indicative of sound viability of the intestinal

4.4.2 Systemic Administration of Benzoic Acid

Upon recirculation of BA at low (1.2 to 1.3 PM) and high (41 4 to 450 PM)

concentrations to the perfùsed intestine preparation. HA was not detected in either plasma

or luminal tluid. LrveIs of BA in perfusate remained high in the reservoir perfusate

(93 ~0 .8% for high dose and 94.3 = 1.2% for the tracer dose of ["CIBA. Fig 4-2) afier 7 h.

Time (min)

Figure 4-2. The disappearance of uniabeled and labeled benzoic acid in reservoir perfusate when BA dissolved in the perfusate was delivered into the recirculating perfùsed rat small intestine preparation. The glycine conjugate. hippuric acid was not detected in reservoir perfksate. The data was expressed as mean r SD.

Any loss of BA in the perfusate was almost cornpletely attributed to the appearance of BA

in lumen (4.5 = 0.8% For high dose and 3.5 r 1.5% for the tracer dose of ["CIBA) (Table 4-

1). After accounting for the partitionhg of BA into intestine tissue. recovery of dose was

virtually complete. and there was no statistical difference (p > 0.05) in dose recovery

between the tracer and high dose of BA (Table 4- I) .

Table 4-1. Lack of metabolism but presence of excretion of benzoic acid in the recirculating perfused rat intestine preparations when labeled ( 1.16 to 1.30 PM) or unlabeled (4 14 to 450 FM) doses of BA were administered into the

Recovery (% dose)

Reservoir pefisate

Luminal fluid

Tissue

Totalb

["CI Benzoic acid (n = 3)

Unla beled-benzoic acid (n '4)

Tuo metabolite (hippuric acid) was found in either the perfusate or luminal fluid b Surn of amounts of beruoic acid in perfusate and lumen 'Not measured

4.1.3 Intraduodenal Administration of Benzoic Acid to the Entire Intestine

M e r an intraduodenai injection of a tracer dose of BA (1 66 2 35 nrnole comprising

only of 5.8 2 1.2 x 106 dpm ['"C]benzoic acid), appearance of BA in the recirculating

perfusate was rapid (Fig. 4-3A). The extent of dnig absorption at the end of 2 h perfusion

was virtuaily complete (96.7 t 1.1 % dose; Table 4-2), with only a minor proportion of the

dose recovered fiom the lumen (1.8 t 0.08% dose). The apparent First order rate constant.

b, obtained upon plotting the amount remaining to be absorbed (ARA. was calculated by

subtracting the percentage of dose absorbed at various time points from that acquired at the

completion (last data points) of the experiment) vs time on semilogarîthmic paper (Gibaldi

and Perrier. 1982) (Fig UB). In addition. the kinetics of absorption of unlabeled benzoic

acid (doses of 0.166 to 3.65 pmol) were found to be unaltered (Fig. 4 4 ) . There was no

apparent change in the extent of BA absorption (Fig. U A . table 4-3: p > 0.05). Recovenes

of BA in reservoir perfusate (94.6 x 0.9%). lumen (2.5 r 0.9% dose) and intestine ( - 0.2%

dose) were sirnilar For the various doses. Upon performance of the ARA plots. the k,

remained independent of the BA dose (Fig. 4-4B). Again. no metabolite was found in the

perfusate or luminal fluid. Good recovery of the dose and perfusate volume was observed

(Table 4-2).

4.1.4 Absorption of Tracer Dose of Benzoic Acid by Various Closed Segments

of the Rat Small Intestine - Duodenum, Jejunum or Ileum

Inasmuch as the lack of dose-dependence in the kinetics of absorption of BA.

intrasegmental injection studies were conducted with tracer doses of ["CIBA. Linle

difference \vas found in the extents of absorption (Table 4-3), regardless of the segment and

the length of the closed loops for injection. The total radioactivities remaining at the closed

loop for injection were 2%. 1.3 to 1.7% and 4.5 % dose. respectively. for the duodenurn,

jejunum. and ileum. and were not difierent (p > 0.05, ANOVA) From that for tracer dosing

of ["CIBA to the entire intestine (Table 4-3).

Time (min)

Figure 4-3. Absorption of ["C]benzoic acid by the perfused nt small intestine when tracer doses in saline (1 19 to 214 nmole) were delivered directly into the duodenum and exited at the ileocecai valve. Reservoir perfusate simples were monitored at various time points and the mdioactivity recovered was expressed as percentage dose (A). (B) a semilog plot of the arnounts remaining to be absorbed versus the corresponding Mie points (ARA plot) was obtained. The regressed fine was restricted to data for the first 20 min and absorption was almost completed within 60 min. The Fust-order absorption rat coastant (kJ for the entire intestine was deierrnined by multiplying the dope of the ARA plot with constant, 2.303.

Absorption Rate Constant (min- ') Percent of Dose Absorbed

C I ,

lu - C

O -

There was a statistically significant difference in the absorption rate constants. k, for BA

absorption by the entire intestine. duodenum. jejunum and ileum segments (p > 0.05.

ANOVA). Recovery of radioactivity from lumen of the non-injection segments accounted

for less than 1 % dose at the end of 2 h. Again. only a minor amount of BA ( - 0.1% dose)

was detected in homogenized tissue. and the metabolite. HA. was absent in the system.

Upon a closer comparison of the extents of absorption of benzoic acid into the

recirculating blood perfusate. no difference was detected for the absorption of BA by the

duodenum and jejunum (, 12 cm and 20 closed loops. Fig. 4-SA: Table 4-3) regardless of the

lrngths of intestine used for study. However. the extent of benzoic acid absorbed by the

ileum was statistically lower (p < 0.005) than the jejunum of comparable length (20 cm

closed loops Fig. 4-6A). The MU plots revealed that the absorption rate constant. k.. of the

jejunum for BA was slightly greater than that of the duodenum (Fig. 4-jB), and that the k,

for the jejunum was greater than that for the ileum (Fig. 4-6B) when paired results from

comparable Iengths of closed-loops were compared (Table 4-3). Cornparison of the

absorption rate constants from the various segments (regardless of physiological lengths)

revealed the srnailest k, for the ileum. followed by the duodenurn. and a largest k, for the

jejunum. These differences resulted in a higher @ < 0.0005) amount of benzoic acid left in

the injection sekgment at the end of the 3 h perfusion for the ileum. However. no statistical

difference was observed in the percent of tracer doses secreted into various intestinal

segments. although it is noteworthy that a greater intra-animal variability existed in luminal

secretions for the ileum as the injection segment. Good recoveries of the doses and volumes

were again observed.

Figure

0 Duodenum (1 2 cm) O Jejunum (12 cm)

Duodenum (12 cm) O Jejunum (1 2 cm)

Time (min)

4.5. Absorption of luminaily delivered ['%]benzoic acid by the equal lengths ( l? cm) of duodenum and jejunum segments. No difference in the extents of absorption by the two regions (A); however, the jejunum dispiayed a greater absorption rate constant (kJ than the duodenum. as seen by the steeper slope of the ARA plot for the jejunum (B). * represents statisticaiiy simifïcant data.

lleum (20 cm) O Jejunum (20 cm)

Time (min)

Figure 4-6. Absorption of Iurninaily delivered ['"Clbenzoic acid by the equd lengths (20 cm) of jejimum and ileum segments. The jejunurn appeared to exhibit slightly greater extent of benzoic acid absorption at the end of 2 h perfusion (A), and a greater absorption rate constant, k,, than the ileum (B). * represents statistically significant data.

4.4.5 n-Octanol and Buffer Partition of Benzoic Acid and Acetaminophen

Partition studies of BA between n-octanol and buffer demonstrated a pH-dependence

and the preferential distribution of BA into the organic phase at low pH's. A plot of the

apparent partition coefficient (P,,) of BA us pH reveaied a sigrnoidal decrease of P,, with

increasing pH (Fig 4-7). By contrast. the n-octanolhuffer partition of acetarninophen. a

neutral and lipophilic compound. was. however. virtually pH-independent. S ince the

concentration ratio of ionized to unionized BA is 10 'Pt' -pK"' . the ratio of C ,,,,,,, to C ,,,,, is

1/( 1 O (PH With the values of P,,,. the true P,, was calculated according to Eq. 1 -8.The

value of P, estimated for BA \vas high and similar for al1 the pH's used (70 2 13). excepting

that at pH of 8 (Table 44) .

+ Acetaminophen .+- Benzoic acid

pH of buffet

Figure 4-7. pH-Dependence of octanol-buner partitionhg of benzoic acid. The partition of acetamiophen, a neutral compound, into n-octanol was. however, pH- insensitive.

120

Table 44. The true octanol-water partition coefficient of benzoic acid ( p y = 4.2) at varying pH's.

abtaincd fiom n-octanol and buffer partitioning studies b According to Eq. 1-7

'According to Eq. 1-8

4.5. DISCUSSION

The present perfùsion studies on rat intestine. designed to examine processes ot

intestinal transport. metabolism and secretion of benzoic acid. revealed that the entire dose

was recovered as unchanged BA in perfusate and lumen after systemic dosing (Table 4-1).

Among the intraiuminal studies. absorption of benzoic acid was rapid and almost complete

at the end of 2 h perfusion (Tables 4-2 and 4-3). Conjugated metabolites were again absent

in either lurninai tluid or perfûsate when BA was given intraluminally. These results differed

kom the observation of Strahl and Ban (1971) who observed intestinal glycine conjugation

121

of [''~Ibenzoic acid to ["C]hippuric acid. albeit small. in the in vitro rat intestinal slices and

rverted intestinal preparations. The small amount of HA fonned in the studies of Strahl and

Barr ( 197 1 ) tvas materially insignificant and would not affect the overall mass balance of the

system. By contnst. luminal secretion was more substantial. and together with the tissue and

pehsate contents of BA. accounted for the entire doses of BA administered.

The rapid and almost complete absorption of BA. a weak organic acid with p& of

1.19. may implicate the presence of a transporter. Indeed. Tamai er ol. ( 1999) demonstrated

a concentration- and pH-de pendent transport of benzoic acid by the proton-monocarboxy late

transporter 1. in MCT-1 transfected cells vs. mock cells. Immunohistochemical studies

reveaied that MCTl was present throughout the gastrointestinal tract. from the stomach to

the large intestine. In the small intestine. the transporter was localized in the villi. Moreover.

MCTl was f o n d on the brush border membrane of mature cells of the vilii and was

localized on the basolateral membrane of immature cypt cells. MCT1 -mediated transport

in enterocytes also appeared to be bidirectional. and the asymmetric and much lower efflux

of [''CIBA by the rat MCTl expressed in MDA-MB23 1 cells couid explain the low secretion

observed in the present intestine preparation.

The role of passive diffbsion in BA absorption mua also be appraised. Nomally. the

effective permeability (P,) is a parameter often used for the estimation of the rate and extent

of absorption (Lemerna et al., 1992; Amidon et al.. 1995). The parameter is dependent on

severd physiologicai characteristics of the intestinal tissue and the physicochernical

properties of the substrate, including lipophilicity, molecular size, hydrogen bonding

122

capacity and polar surface area (Winiwater et ai.. 1998). Lipophilicity. a major determinant

in predicting the extent of absorption. is often correlated with the partition coefficient. When

lipophilicity is viewed in terms of the true partition coeficient (P,,) which is pH-

independent. a large value (70 1 13) was observed For BA. The high value was quite

unexpected. but may possibly be due to inter-molecular hydrogen bonding. The apparent

partitioning (P,,) of BA into organic phase (octanol) vs aqueous (buffer) phase at pH 7 (the

sarne pH as that of the luminal fluid for the perfusion study) was. however. very low (0.13)

in comparison (Table 44). Yet. the rat smail intestine exhibited a relatively high first-order

absorption rate constant k, of 0.0464 r 0.0010 min-'. As a point of comparison.

acetaminophen. a neutral lipop hilic compound. was transported by passive diflbsion with a

higher k, of 0.224 z 0.041 min-' into the perfused rat small intestine (Pang et al.. 1986). The

higher k, of acetaminophen is undoubtedly due to its high apparent partitioning into n-

octanoI(- 2 or 15 times that of BA). which was relatively pH insensitive (Fig. 4-7). If the

absorption of BA were purely by passive difision. the low partitioning value of BA at the

pH of 7 in the lumen (O. 13) would have predicted a much lower k, than that observed. It is

likely that simple passive diffusion plays only a minor role in the intestinal uptake of BA.

It is thus s m i s e d that MCTl contributes significantly to the intestinal absorption of BA.

The involvement of the transporter MCTl should have displayed dose-dependent rate

constants and decreasing extents of absorption with increasing doses (1 66 to 19 13 nmole).

However. statistically indistinguishable k's (about 0.0467 min-') and similar extents of

absorption were observed. Although the concentration of the administered dose reached 5

133

mM. a value close to the Km of MCTl (Tamai et al.. 1999). the lack of concentration-

dependence in the uptake of BA could be explained by the rapid dilution of dose within the

lumen. as observed during the course of the study. This is because a large portion of the

intestine in vivo is reserve length. the length not utilized sincr absorption is already

completed. The entire intestine is capable of absorbing BA due to rapid absorption.

However. uptake is carried out by only a small section of the tissue while the remaining

segment(s) is not utilized in overall absorption (Ho et al.. 1983). Even though saturation in

drug absorption could have existed. the dmg is passed quickly dong the reserve length with

peristalsis and the drug will be absorbed sequentially. Hence. the overall absorption by the

intestine rnay appear to be dose-independent. In contrast. the in vitro uptake studies

involving MCTl (Tamai et t i f . . 1999) posed as a stagnant system in which the intluence of

peristalsis and reserve length was absent and would not affect transport of BA.

Heterogeneity of intestinal uptake of BA was observed (Table 4-3). Again. due to the

excess reserve length. the absorptive activity of the entire intestine was not a sum of those

of the individual segments for the perfused n t intestine. Rather. the absorption rate constant

for the jejunurn (20 cm) was highest. followed by that of the duodenum and ileum. The 12-

cm and the 20-cm lengths of the jejunum revealed the same extent of absorption as the

duodenum. The rate constant for this segment. however. was lower. albeit not significantly

than that of the longer jejunum length. The activity of the 20 cm jejunurn (0.05 19 t 0.000 1

min-') was significantiy greater than that of the ileum of equal length (0.0380 5 0.0024 min'

) Due to the excess reserve length, the k, should not be corrected for the lengths of the

intestine used for absorption.

Our observation of the unevenness in transport of benzoic acid among the segments.

particularly with greatest activity in the jejunum. cannot simply be explained by the

difference in surface area available for passive absorption between the segments. The

duodenum and jejunum possess the greatest surface area due to the concentration villi and

microvilli in that region. whereas the ileum has the least of the luminal projections (Magee

and Dalley. 1986). The greatest intestinal uptake of acetaminophen. a drug that most likely

enters by passive diffusion. by the first-third (duodenum and jejunum) of the intestinal

preparation over the second (jejunum plus proximal ileum) and third (ileum) segments (data

of Pang et cd.. 1 986) rnay retlect the segmenta1 di fferences in surface area. If the absorption

of BA were due to passive transport alone. similar duodenal and jejunal activities would have

been predicted. The absorption of benzoic acid. however. is highest in the jejunum.

Indeed. other examples on heterogeneity of intestinal transport have been

demonstrated. Expression of the proton-coupled oligopeptide transporter (PEPTI) was found

more abundant in the proximal intestine (duodenum and jejunum) (Fei et al.. 1994). despite

that the absorptive function among the various sites of the smail intestine (duodenum.

jejunum and ileum) and the colon towards the dipeptide SQ-29852. a specific probe of the

system. was not statistically different (Marino et al.. 1996). The carrier-mediated transport

of D-glucose and L-Leucine displayed a regional pattern of jejunurn < ileum < colon (Ungell

et ai., 1997). A greater absorption of atenolol by the jejunum was observed than for the

ileum (Narawane et al., 1993), altbough another report suggested that the net mucosal to

125

serosal absorption was the same in al1 intestinal segments (Fagerhom e l al.. 1997). The

absorption of griseofulvin (Gramatté, 1996) and carbovir (Soria and Zirnmerman. 1994) was

found to be same arnong al1 segments. For vempa.mil. net mucosal to serosal absorption was

ereater for the ileum than for the jejunum (Saitoh and Aungst. 1995). and this could be due C

to the presencr of other complicating factors such as the efflux pumps (Pgp) operating most

efficiently in the jejunum (Saitoh and Aungst. 1993). Gotoh et al. (2000) demonstrated

dominance in mRNA expression of MRP3 in the jejunum. followed by the duodenum and

ileum. with very linle in the colon. The excretion of the glutathione conjugate 2.4-

dinitrophenyl-S-glutathione (DNP-SG) by MRP? was greatest in the jejunum. as expected

by mRNA expression: however. excretion by ileum was greater than bby the duodenum

(Hirohashi et al.. 2000). a pattern that differed From the mRNA expression.

In summary. data fkom the present midy revealed that BA was not metabolized by the

n t small intestine. Rather. rapid and uneven absorption of BA rxisted among the segmenta1

regions. being highest in the jejunum and slightiy Iower in the ileum. The absorption of BA

was not explained by passive difision and implicated the role of MCTI. the

monocarboxyiate acid transporter 1. From the present studies. it was demonstrated that the

in siirr recirculating smail intestine preparation was a useful technique in providing

S o m a t i o n on dmg absorption, exsorption, and metaboiism in segmented regions of the

intestine. It is surrnised that future studies on mode1 substrates that display differential

absorption. metabolism and exsorption by the various segmental regions - duodenum.

jejunum and ileum - wouid allow for the integration of these events and the examination of

their overall influence on dmg bioavailability.

4.6. STATEMENT OF SIGNIFICANCE

The in situ perfusion \vas a useful technique that can be utilized for the examination

the segmenta! clbsorption. metabolism and cxsorption of kenzoic cicid in the innct smdl

intestine. Heterogeneity was indeed observed for the overall absorption of BA. with the

ereatest absorption O C C U ~ ~ ~ in the jejunum and the least was observed in the ileum. The C

elvcine conjugation of benzoic acid was not observed: however. BA was evcreted into L I

intestinal segments not exposed to dmg. The lack of dose-dependency on S and the

persistent high percent absorption among the segments suggest a large "reserve length" for

BA intestinal absorption.

DISCUSSION AND CONCLUSIONS

5.1. SUMMARY OF FINDINGS

In the present investigation. pharrnacokinetic modeling and the in sini perfused rat

intestinal preparation were utilized to examine the physiological variables of intestinal

availability and clearances and to study the intestinal absorption. metabolism and emux

of benzoic acid. The following observations were noted:

A. (1) Kinetic parameters that pertain to absorption. rnetabolism and efflux. drug

partitioning characteristics in cell/blood and clearances by other organs needed to

be considered for the assessment of intestinal clearance and availability and the

overall systemic bioavailability.

(3) The traditional physiological mode1 that viewed the intestine as a single

homogeneous tissue cornpartment which receives the intestinal blood flow in its

entirety was adequate in predicting the intestinal drug clearances and overall

bioavailability . But an improved prediction of drug absorption and metabolism

existrd with a segregated flow modei. Intestinal route-dependent metabolism - a

ereater extent of biotransformation following oral than i.v. dosing or during CI

subsequent circulation of the absorbed drug molecule through the intestinal

tissues - was accurately predicted by the SFM when differential intestinal blood

flows were considered.

(3) Both the SFM and TM described that intestinal availability was invenely

related to the inûinsic metabolic and exsorptive clearances. and luminal loss

(either by degradation or motility).

B. (1) Benzoic acid \vas not metabolized by the intestinal lumen or tissue.

(2) Benzoic acid uptake by the nt small intestine is rapid and complete. The

extent of absorption was 95 to 96% dose regardless of the length of the segment

(whole intestine. 12 or 20 cm segments) used for the absorption study. The

degree of absorption for the ileum was slightly lower than those for the jejunum

and duodenum.

(3) There was a great intestinal reserve length for absorption of benzoic acid.

Dose-dependency transport of benzoic acid was not observed due to the large

reserve length.

(4) Uptake of benzoic acid was heterogeneous dong the length of intestine. The

absorption of benzoic acid was greatest in the jejunum (absorption rate constant.

k, = 0.05 19 r 0.000 1 and 0.0564 2 0.00 12 min". respectively. for 12 and 20 cm

segment). followed by the duodenurn (ka = 0.0442 z 0.00 1 1 min". 12 cm

segment) and then the ileum (ka = 0.0380 = 0.0024 min-'. 20 cm segment).

5.2. GENERAL DISCUSSION AND SIGNIFICANCE

The concepts developed in the present investigation will enhance our

understanding of the overall intestinal handling of drugs. The SFM incorporated not only

transport and metabolic charactenstics of the intestine as well as the physicochemical

properties of the substrate. This development is an advancement in modeling and

cornputer fitting of drug absorption data since previous models were overly simplistic and

failed to include efflux. metabolism. intestinal transit kinetics and circulatory dynamics

within the intestine. The simulations based on the Segregated-Flow model described in

chapter 3 demonstrated the usefulness of this new model in accurately predicting

intestinal clearance and systernic bioavailability of orally administered drugs. The

inclusion of drug partitioning characteristics (CLd) as well as other intnnsic clearances in

the simulation study allowed for the examination of difierential influence of blood flow

on substrates with high vs low permeability (ie. flow-limited vs membrane-limited). The

SFM was able to predict the observed intestinal clearance estimates of morphine in the in

siru rat srna11 intestine preparation (Doherty and Pang. 3000). The better fit of the SFM

over that afforded by the traditional physiological mode1 (TM) substantiated the need to

consider importance of segmentai flow to the enterocyte layer on rates and extents of

intestinal mrtabolism and exsorption. The theory that intestinal enzymes are inaccessible

to drues in the circulation posed as a possible explanation for pre-absorptive vs post-

absorprive metabolism. According to the SFM. the lower Row rate perfusing the

rnterocyte layer resulted in reduced drug delivery in the circulation to the intestinal

enzymes and secretory carriers. However. during oral absorption. al1 drug must traverse

the enterocyte layer before their release to the circulation. The consequence of partial

How to the enterocyte cornpartment leads to sluggish dispersal of the drug into the

circulation and a longer transit time within the intestinal tissue. Ieadinç to greater

exposure to the rnetabolic enzymes during drug absorption. Since copious examples of

route-dependent intestinal metabolism exist. it is anticipated that the SFM would serve to

accurately relate in vitro parameters to in vivo physiological events on absorption and

bioavailability. However. further validation of the SFM is needed.

The SFM can be improved with the inclusion of parameters on intraceIlular

diffusion as descnbed by Ito et al. (1999). Indeed. drug diffusion through the cytoplasm

influences the rate and extent of h g . Intracellular diffusion c m be limited by organelle

binding - the greater the binding, the lower the di fision constant - and thus. the slower

the appearance of drug in the circulation. A greater intracellular binding can also affect

the extent of Free drug available for metabolism. The noted heterogeneity in absorption.

metabolism and/or effiux dong the length of the intestine for the SFM can be extended to

describe the physiological segments of the intestine. It is well recognized that luminal

metabolism mediated by microorganisms and gastrointestinal transit both lead to dmg

loss from the lumen. These are incorporated into the term k,. which could be funher

segregated into its two components. if necessary.

The hypothesis that the overall absorption of benzoic acid is di fferentiall y

localized dong the length of the intestine. as described in section 2.3. is true. In absence

of metabolism. heterogeneity in absorption and exsorption among various segments was

present. Should segmental rnetabolism exists. this added variable would be readily

incorporated into an expanded segregated-flow mode1 that divides the tissue into three

segments.

Although intestinal transport of benzoic acid was mediated by MCTI. saturation

\vas not observed in the perfusion studies even though the dose concentration exceeded

the Km. ïhe large reserve length of the intestine for BA absorption and luminal peristalsis

could apparently result in the observation of dose-independent absorption. If the

absorption of BA were purely by passive difision, the low octanol-buffer partitionkg

value of BA at the pH of 6 - 7 of the lumen would have provided absorption rate

constants that are lower than those observed. It is likely that simple diffusion across the

intestinal membrane is not the pnmary mode of uptake for benzoic acid. In order to

ascertain that the intestinal uptake of benzoic acid obsewed in our in situ preparation is

carrier-rnediated,

inhibition studies

investigations with much higher doses (3 K Km) at varying pH's and

need to be performed.

The absorption of benzoic acid in our intestinal studies was greatest in the

jejunum. followed by the duodenurn and ileum. respectively. Since the carrier-mediated

transport of benzoic acid is proton-driven (Tamai et al.. 1999). intestinal absorption of

benzoic acid in vivo. however. may be seen as greater in the duodenum than the other

segments. The duodenum. receiving the highly acidic content of the stomach. contains

the greatest concentration of protons. As the contents move down the intestinal length.

there is a gradua1 neutralization of the luminal content due to the secretion of bicarbonate.

As a result. special considerations need to be given to this pH effect when utilizing data

obtained from these isolated intestinal perfusion studies to predict in vivo observations.

especially when physiological factors such as gastric emptying and hormonal control play

significant roles in absorption.

Efflux of benzoic acid was obsewed following the systemic delivery. The

recovery of BA in the lumen after intraluminal dosing was found to be similar for al1

segments. albeit a slightly greater recovery was observed for the ileum. Luminal BA

recovery during intraluminal studies. however. was not conclusive evidence of exsorption

since the BA remaining in the lumen could be due to incomplete absorption not rfflux.

5.3. CONCLUSION

Simulations and computer fittings based on a new physiologically-based

segregated-flow mode1 (SFM) support the fim hypothesis stated in section 2.3. The view

of segregation of blood flow to the various tissue layers of the intestine is a plausible

explanation of route-dependent intestinal metabolism. The SFM is comprehensive and

incorporates the kinetic parameters on absorption. metabolism. e M m and drug

partitioning properties in a dynamic fashion for the prediction of intestinal availability of

oraily administered agents. The in situ studies with benzoic acid support our hypothesis

that the overdl absorption of the substrate is differentially localized along the length of

the intestine. The absorption of BA did indeed demonstrate heterogeneity. with the

greatest absorption occumng in the jejunm (jejunum > duodenum > ileum). The

glvcine conjugation of benzoic acid to hippunc acid was not observed. Emux appeared k +

to be sirnilar for al1 the segments. albeit a slightly greater extent existed for the ileum.

The heterogeneous localization of intestinal transporters For absorption needs to be

considered in the interpretation of overall intestinal clearance. An extension of the

current SFM to include segmental localization is possible and would represent an even

greater refinement of the current intestinal clearance model.

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