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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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