Utility of hepatocytes to model species differences in the metabolism of loxtidine and to predict pharmacokinetic parameters in rat, dog and man

Article abstract of DOI:10.1080/004982599238650

1. The metabolism of loxtidine (1-methyl-5-[3-[3-[(1-piperidinyl) methyl] phenoxy] propyl] amino-1H-1,2,4-triazole-3-methanol) was studied in freshly isolated rat, dog and human hepatocytes. Metabolism in vitro was comparable with previously available in vivo data in all three species with the marked species differences observed in vivo being reproduced in the hepatocyte model. 2. The major route for the metabolism of loxtidine by rat hepatocytes was N-dealkylation to form the propionic acid and hydroxymethyl triazole metabolites. A minor metabolic route was the oxidation of loxtidine to a carboxylic acid metabolite. The major route of metabolism for loxtidine in dog hepatocytes was glucuronidation with oxidation to the carboxylic acid metabolite being of minor importance. Incubation of loxtidine with human hepatocytes resulted in the drug remaining largely unchanged but with the carboxylic acid metabolite being produced in minor amounts. 3. In vitro studies were undertaken with rat, dog and human hepatocytes to determine the Michaelis-Menten parameters V(max) and K(m) for the sum of all the metabolic pathways. These kinetic parameters were used to calculate the intrinsic clearance of loxtidine. Using appropriate scaling factors, the predicted in vivo hepatic clearance was then calculated. The predicted intrinsic clearances were 51.4 ± 12.4, 8.0 ± 0.8 and 1.0 ± 0.6 ml/min/kg for rat, dog and human hepatocytes respectively. These data were then used to calculate hepatic clearances of 24.5, 3.1 and 0.2 ml/min/kg for rat, dog and man respectively. 4. In vivo hepatic and intrinsic clearances for loxtidine were determined in rat, dog and human volunteers. The hepatic clearances of loxtidine were 26.6, 6.6 and 0.4 ml/min/kg in rat, dog and man respectively and intrinsic clearances were 58.5, 18.6 and 2.0 ml/min/kg in rat, dog and man respectively. 5. The present studies demonstrate that the hepatocyte model may be a valuable in vitro tool for predicting both qualitative and quantitative aspects of the metabolism of a drug in animals and man at an early stage of the drug development process.

Full text of DOI:10.1080/004982599238650

xenobiotica , 1999, vol . 29, no. 3, 253±268
Utility of hepatocytes to model species diåerences in
the m etabolism of loxtidine and to predict
pharm acokinetic param eters in rat, dog and m an
M. K. BAYLISS‹*, J. A. BELL‹, W. N. JENNER‹, G. R. PARK
and K. WILSONŒ
‹
Division of Bioanalysis & Drug Metabolism, Glaxo Wellcome Research and
Development Ltd, Ware SG12 0DP, UK
Œ Department of Biosciences, University of Hertfordshire, Hat®eld AL10 9AB, UK

Department of Anaesthesia, Addenbrookes Hospital, Cambridge CB2 2QQ, UK
Received 27 June 1998
. The metabolism of loxtidine (1-methyl-5-[3-[3-[(1-piperidinyl) methyl] phenoxy]
1
propyl] amino-1H-1,2,4-triazole-3-methanol) was studied in freshly isolated rat, dog and
human hepatocytes. Metabolism in vitro was comparable with previously available in vivo
data in all three species with the marked species diåerences observed in vivo being
reproduced in the hepatocyte model.
2
. The major route for the metabolism of loxtidine by rat hepatocytes was N-
dealkylation to form the propionic acid and hydroxymethyl triazole metabolites. A minor
metabolic route was the oxidation of loxtidine to a carboxylic acid metabolite. The major
route of metabolism for loxtidine in dog hepatocytes was glucuronidation with oxidation to
the carboxylic acid metabolite being of minor importance. Incubation of loxtidine with
human hepatocytes resulted in the drug remaining largely unchanged but with the
carboxylic acid metabolite being produced in minor amounts.
3
. In vitro studies were undertaken with rat, dog and human hepatocytes to determine
the Michaelis-Menten parameters Vmax and Km for the sum of all the metabolic pathways.
These kinetic parameters were used to calculate the intrinsic clearance of loxtidine. Using
appropriate scaling factors, the predicted in vivo hepatic clearance was then calculated. The
predicted intrinsic clearances were 51.4‰ 12.4, 8.0‰ 0.8 and 1.0‰ 0.6 ml}min}kg for rat,
dog and human hepatocytes respectively. These data were then used to calculate hepatic
clearances of 24.5, 3.1 and 0.2 ml}min}kg for rat, dog and man respectively.
4
. In vivo hepatic and intrinsic clearances for loxtidine were determined in rat, dog and
human volunteers. The hepatic clearances of loxtidine were 26.6, 6.6 and 0.4 ml}min}kg in
rat, dog and man respectively and intrinsic clearances were 58.5, 18.6 and 2.0 ml}min}kg
in rat, dog and man respectively.
5
. The present studies demonstrate that the hepatocyte model may be a valuable in vitro
tool for predicting both qualitative and quantitative aspects of the metabolism of a drug in
animals and man at an early stage of the drug development process.
Introduction
Information on the metabolism of candidate drugs is important in understanding
potential species diåerences in the pharmacological and toxicological properties
exhibited by a new chemical entity. The recognition of the limitations in
extrapolating animal data to man has increased the pressure for early information on
the behaviour of candidate drugs in man. In recent years isolated hepatocytes have
been used extensively to study biotransformation reactions and for preliminary
studies on the metabolism of a new chemical entity. Such studies have demonstrated
that isolated hepatocytes have clear advantages compared with subcellular fractions
in the prediction of both routes and rates of metabolism (Begue et al. 1983). The use
of the hepatocyte system as a predictive model for drug metabolism in diåerent
*
Author for correspondence.
Xenobiotica ISSN 0049±8254 print}ISSN 1366±5928 online ’ 1999 Taylor & Francis Ltd
http:}}www.tandf.co.uk}JNLS}xen.htm http:}}www.taylorandfrancis.com}JNLS}xen.htm

2
54
M. K. Bayliss et al.
species remains an active area of research. Good in vitro-in vivo correlations of
metabolic patterns have been reported using both isolated animal and human
hepatocytes (Le Bigot et al. 1987, Oldham et al. 1990, Lavrijsen et al. 1992, Sandker
et al. 1994, Pahernik et al. 1995) and studies reporting the failure of hepatocytes to
predict metabolism have been few (Humpel et al. 1989). However, there have been
few reports on the comparative metabolism of a single substrate in hepatocytes from
the three species central to drug development, namely rat, dog and man (Chenery
et al. 1987, Ekins et al. 1996, Lave et al. 1996a, b).
Recently, there has been considerable interest in the development of methods
predicting the clearance of drugs in vivo from experiments in vitro (Houston 1994,
Lave et al. 1996a, Houston and Carlile 1997, Iwatsubo et al. 1997a). Successful
predictive methods could lead to signi®cant savings in animals, time and cost during
the drug development process. Microsomal (Carlile et al. 1997, Iwatsubo et al.
1
997b, Sanwald-Ducray and Dow 1997) and hepatocyte (Oldham et al. 1986, Singh
et al. 1991, Lave et al. 1995, 1996a, b, 1997, Carlile et al. 1997) systems have already
been used successfully to predict quantitative hepatic elimination in vivo for some
drugs.
Loxtidine hemisuccinate (1-methyl-5-[3-[3-[(1-piperidinyl) methyl] phenoxy]
propyl] amino-1H-1,2,4-triazole-3-methanol) (®gure 1) is a potent, long-acting
histamine H -receptor antagonist (Brittain and Jack 1983). In laboratory animals
#
and human volunteers loxtidine undergoes metabolism by several routes (®gure 1)
and shows signi®cant species diåerences (Bell et al. 1983). The primary route of
metabolism is N-dealkylation in the rat and glucuronidation in the dog. In contrast
with animal species, loxtidine is eliminated predominantly by renal clearance in man
although a single minor oxidative metabolite has been detected in urine. No
evidence for the presence of a glucuronide metabolite was observed.
The observed species diåerences in metabolism make loxtidine an excellent
model for the prediction of xenobiotic metabolism and pharmacokinetics in vivo. In
the present studies, attempts have been made to model the metabolism of loxtidine
and predict hepatic clearance in vivo from the data generated in isolated hepatocytes
from rat, dog and man.
M aterials and m ethods
Chemicals
"
% C-Loxtidine hemisuccinate (speci®c activity 0.06 MBq}mg, radiochemical purity " 98 %) (for the
position of "% C-radiolabel, see ®gure 1) was prepared by the Radioisotope Laboratory, Process Research
Department, Glaxo Wellcome Research and Development (Ware, UK). Non-radiolabelled loxtidine
"
hemisuccinate (AH23844A, batch no. C215}153}1, purity
99 % as determined by nmr) used for
animal pharmacokinetic studies was supplied by Pharmaceutical Research, Glaxo Wellcome Research
and Development (Ware, UK).
Authentic non-radiolabelled metabolites of loxtidine : AH24160, GR30450 and AH25227 (®gure 1)
were synthesized by the Chemical Research Department, Glaxo Wellcome Research and Development
Limited, (Ware, UK). b-Glucuronidase (EC 3.2.1.31) and aryl sulphatase (EC 3.1.6.1, glucuronidase
free) were purchased from Sigma Chemical Co. (Poole, UK), collagenase (EC 3.4.24.3) was from
Boehringer Mannheim (Lewes, UK). Williams’ Medium E (WME) without phenol red, l-glutamine and
trypan blue were supplied by Flow Laboratories (Rickmansworth, UK). All other chemicals and solvents
were of Analar‡ grade or equivalent.
Preparation of isolated hepatocytes
Adult male Allen and Hanbury in bred random hooded rats (229‰ 27 g) and adult male or female
beagle dogs (10.8‰ 2.3 kg) were obtained from Biosciences Support Group (Glaxo Wellcome Research
and Development Ltd, Ware, UK). Livers were removed from rats anaesthetized with either halothane
or en¯urane, and from dogs that had been sacri®ced by an overdose of pentobarbitone sodium. Human

The metabolism and intrinsic clearance of loxtidine in vitro
255
Figure 1. Metabolic pathways of loxtidine (1-methyl-5-[3-[3-[(1-pipe ridinyl) methyl] phenoxy]
propyl] amino-1H-1,2,4-triazole-3-methanol) in rat, dog and man (Bell et al. 1983, Jenner et al.
1
984). *Position of the radiolabel.
liver samples were obtained from patients undergoing hepatectomy or from non-transplanted donor
samples with approval from the district Clinical Review Board.
Hepatocytes were isolated as described previously (Bayliss et al. 1994) using the biopsy perfusion
technique of Oldham et al. (1985). For the isolation of dog and human hepatocytes it was necessary to
double the units of collagenase activity used in the preparation of rat hepatocytes. Hepatocytes were
thrice washed in HEPES buåer containing bovine serum albumin (1 % w}v) and re-suspended in WME
without phenol red containing l-glutamine (4 mm) at a cell density of 23 10’ hepatocytes}ml. Viability
was determined by trypan blue exclusion and by lactate dehydrogenase (LDH) (EC 1.1.1.27) leakage
(
Jauregui et al. 1981). P450 content of solubilized hepatocytes was determined by the method of Omura
and Sato (1964a, b). Hepatocyte suspensions were sedimented by centrifugation (50 g for 4 min). The
supernatant was removed and the pellet dissolved in Renex 690 solubilizing buåer. The buåer was as
described by Warner et al. (1978) except that Renex 690 was substituted for Emulgen 911. Protein
content was determined by the method of Lowry et al. (1951).
Incubation procedures
"
% C-Loxtidine (in methanolic solution) was placed in polyethylene incubation vials to give ®nal
concentrations of 0.5, 1, 5, 10, 25, 50, 100 lm for rat, 0.5, 1, 5, 10, 25, 50, 100, 150 lm for dog and 1, 2.5,
, 10 lm for human hepatocyte incubations (2 ml incubation volume). A stream of nitrogen gas removed
5

2
56
M. K. Bayliss et al.
the solvent. Control (non-viable) hepatocytes were obtained by boiling for 10 min prior to cooling and
addition to vials containing "% C-loxtidine (0.5, 1, 10 and 100 lm). Metabolic and kinetic studies
commenced with the addition of hepatocyte suspensions (2 ml; 23 10’ hepatocytes}ml in WME
containing glutamine) which had been preincubated at 37 C for up to 30 min. All vials were incubated
at 37 C in a shaking water bath (C 70 oscillations}min) and were purged with water-saturated mixture
Ê
Ê
of oxygen and carbon dioxide (95 : 5 % v}v). Aliquots (200 ll) of the incubations were withdrawn from
each vial at 0, 0.25, 0.5, 1.0, 2.0 and 3.0 h after commencement and rapidly frozen in liquid nitrogen and
stored at –20 C.
Hepatocyte proteins were precipitated using 2 vols acetone and sedimented by centrifugation
Ê
(
(
12 000 g for 10 min). The supernatant was removed and reduced to dryness using a sample concentrator
Savant SpeedVac SVC 100, Stratech Scienti®c, Luton, UK). Concentrated residues were resuspended
in methanol (20 ll) and analysed by thin layer chromatography (tlc).
The presence of glucuronide}sulphate conjugates was assessed using speci®c hydrolysis. b-
Glucuronidase}sulphatase (b-glucuronidase free) (15 mg}ml), saccharo-1,4-lactone (10 mg}ml;
a
speci®c inhibitor of b-glucuronidase), phenolphthalein glucuronide and phenolphthalein sulphate
(
5 mg}ml) were dissolved in 0.05 m sodium acetate buåer (pH 5). Incubations with hydrolysing enzymes
were carried out for C 18 h at 37 C with rat and dog hepatocyte incubations containing "% C-loxtidine.
Ê
Incubations were terminated with 2 vols acetone and samples processed as previously described. The
activity of deconjugating enzymes was validated using the test substrates phenolphthalein glucuronide
and sulphate.
Analytical methodology
Quanti®cation of radioactivity. Diluted stock "% C-loxtidine solution (100 ll), aliquots of rat, dog and
human hepatocyte incubations (10±100 ll), hepatocyte protein precipitates and acetone washings of
precipitates (0.5 ml) were added to plastic scintillation vials containing Pico-Fluor 30 liquid scintillation
cocktail (8 ml, Packard Instrument Co.) and their radioactive content determined in a Tracor Analytic
Mark III (Model 6882) liquid scintillation system (Tracor Analytic, IL, USA). The external standard
channel ratio method was used for the determination of counting eæcacy, all counts being corrected
accordingly.
Thin layer chromatography. Samples of "% C-loxtidine solution (1±2 ll), non-radiolabelled loxtidine (1 mg
base}ml in methanol ; 10 ll), authentic loxtidine metabolites AH24610, GR30450 and AH25227
(
1 mg}ml in methanol, 10 ll) methanolic extracts of hepatocyte incubations (20 ll) and methanolic
extracts of hydrolysed rat and dog hepatocyte incubations (20 ll) were applied to 0.25 mm silica gel G60
F254 prelayered plates (E. Merck, Darmstadt, Germany).
Thin layer plates were developed in glass tanks equilibrated at ambient temperature with the
following solvent system : ethyl acetate :propan-2-ol:water:S.G. 0.88 ammonia (50 :30 :16 : 4 by vol.).
Radioactive areas on the thin layer chromatography plates were located and integrated by use of an
Isomess IM 3000 linear analyser (Raytest, Sheæeld, UK) linked to an Apple IIe data system.
Autoradiograms of tlc plates were prepared by placing the plates in contact with Hyper®lm b-max X-
ray ®lm (Amersham Intl, Amersham, UK) for a minimum of 24 h. The ®lms were developed and ®xed
according to standard procedures. Reference samples of non-radiolabelled loxtidine and authentic
metabolites were visualized on the tlc plates under UV light (254 nm).
Binding of "% C-loxtidine to hepatocyte protein
The non-speci®c binding of "% C-loxtidine or "% C-labelled metabolites of loxtidine to rat, dog and
human hepatocytes was investigated by centrifugation techniques. Rat, dog and human hepatocyte
suspensions at time zero and 3 h after incubation with "% C-loxtidine (1, 10 and 100 lm) were centrifuged
at 4 C for 5 min (12 000g) and the supernatant removed. The cell pellet was then washed with WME and
Ê
solubilized. The radioactive content of the supernatant, pellet wash and the solubilized cell pellet were
determined by radioassay to assess recovery of radiolabelled material.
Determination of enzyme kinetic constants. Enzyme kinetic parameters were determined by measuring the
rate of disappearance of "% C-loxtidine in rat, dog and human hepatocyte incubations. Estimates of
apparent Michaelis-Menten kinetic constants Km and Vmax for the metabolism of loxtidine in rat and dog
hepatocyte incubations were obtained initially by graphical analysis. The initial kinetic parameters
obtained were used as ®rst estimates in an iterative programme based on nonlinear least-squares
regression analysis. The actual curve ®tting procedure used was the Nelder-Mead Simplex method
(
O’Neill 1971) to calculate apparent kinetic parameters Km and Vmax in the general equation:
Vmax [S]
vo
=
Km 1[S]

The metabolism and intrinsic clearance of loxtidine in vitro
257
Intrinsic clearance (Clint ) for loxtidine in rat and dog hepatocytes was determined from the ratio of the
maximum velocity (Vmax ) and the Michaelis-Menten aænity constant (Km) as described by the
relationship :
Vmax
Clint
=
Km
The rate of metabolism of loxtidine by human hepatocytes was signi®cantly slower than by animal
hepatocytes and as such required an approach using integrated forms of the Michaelis-Menten equation:
Co Vmax .t (Co –Ct)
ln
=
–
(1)
(2)
Ct
Km
Km
t
1
Km ln (C }C )
o
t
=
1
[
Co –Ct Vmax Vmax Co –Ct
where Co is the initial concentration and C is concentration at time t.
t
Equation 1 gives a straight line plot when Co ’ Km , the slope of which gives the intrinsic clearance.
Equation 2 yields estimates of Km, Vmax and intrinsic clearance (Chenery et al. 1987).
Scaling factors similar to those described by Houston (1994) were used to extrapolate in vitro Clint to
in vivo Clint . The ®gures used for rat, dog and human hepatocyte number}g of liver, liver and body weight
are detailed in table 1.
Hepatic clearance (Cl ) was calculated from in vitro data using the `well stirred ’ liver model as
described by the following relationship (Morgan and Smallwood 1990):
H
Q(fu –Clint
)
ClH
=
.
(
Q 1f Cl )
u int
Dose preparation and in vivo animal experimentation. Non-radiolabelled loxtidine hemisuccinate, 15.5 or
32.8 mg was dissolved in 5 or 20 ml isotonic saline for intravenous administration to either rat or dog.
2
Concentrations of dosing solutions were, therefore, 2.65 mg and 9.97 mg}ml expressed as loxtidine base.
The dose volume of the administered drug was 2 and 0.5 ml}kg bodyweight for rat and dog respectively.
Hooded rat (272‰ 33 g) and beagle dog (11.9‰ 2.1 kg) were denied access to either Laboratory Animal
Diet or SDS Diet A (Special Diet Services, Witham, UK) but were allowed water ad libitum for C 8 h
prior to the administration of loxtidine. During this time predose urine and blood samples were obtained
from both rat and dog. Animals received a single intravenous target dose of 5 mg loxtidine}kg by
administration into either the lateral tail vein of the rat or the cephalic vein in the foreleg of the dog. The
range of doses administered to rat (n = 6) and dog (n = 2) were 5.25 –5.37 and 4.9 –5.1 mg}kg
bodyweight respectively.
All animals were housed individually in stainless steel metabolism cages designed for the separate
collection of urine and faeces and were maintained in thermostatically controlled environments with a 12-
h light}dark cycle. Water was made available ad libitum throughout the experimental period, as was
animal diet from 2 h post-dose. Urine was collected from rats (n = 2) and dogs (n = 2) for 0±24 h post-
dose in containers cooled by solid carbon dioxide.
Samples of blood (100 ll) were taken from the tail vein of rats (n = 4) at 0, 2, 10, 20, 30, 45 min, 1,
2
6
, 3, 4, 6, 8, 12 and 24 h post-dose and placed into heparinized microtainers and centrifuged at 3000 g for
min to separate plasma from red cells.
Samples of blood (5 ml) were taken from the jugular vein of the dog at 0, 5, 15, 30, 45 min, 1, 2, 3, 4,
, 8, 12 and 24 h post-dose. The blood was placed in heparinized containers (Becton-Dickinson and Co.,
6
Rutherford, NJ, USA) and centrifuged at 1700 g for 15 min at 4
ÊC and the plasma removed. All samples
were stored at
–20Ê
C until analysed.
Human volunteer studies. Human pharmacokinetic data were obtained from four healthy male volunteers
aged 25±37 years (76.2‰ 7.95 kg) who had received varying doses of loxtidine. Each subject received an
intravenous bolus dose (0.025±0.5 mg}kg) of loxtidine. One volunteer received two separate intravenous
doses of loxtidine (0.25 and 0.5 mg}kg) separated by 48 h. Blood samples were taken at 1, 2, 3, 4, 5, 6 and
2
4 h post-dose and the plasma was separated by centrifugation and then analysed by radioimmunoassay.
Urine was collected for 24 h.
Radioimmunoassay. A radioimmunoassay for loxtidine with a detection limit of 4 ng}ml (Harrison et al.,
1
983) was used. Some of the metabolites of loxtidine cross-react in the assay system ; therefore, the parent
drug was selectively extracted from plasma or urine prior to assay. Samples were adjusted to pH 10 with
.2 m sodium borate buåer and extracted with water saturated dichloromethane. The phases were
0

2
58
M. K. Bayliss et al.
separated and the dichloromethane reduced to dryness. The residue was re-suspended in 0.5 m
phosphate buåer (pH 7.4). Rat, dog or human plasma samples, rat and dog urine (100 ll), loxtidine
standards and quality control samples were analysed as described by Harrison et al. (1983).
Protein binding studies. Whole blood was obtained from rat, dog and human volunteers by venepuncture.
Blood was immediately transferred to heparinized tubes and the plasma separated by centrifugation
(
1700 g for 15 min at 4 C). All experimentation was performed using freshly prepared plasma. Dialysis
Ê
was performed at 37 C for C 6 h in two-chamber dialysis cells using Spectrapor 2 membrane tubing (mw
cut-oå 12 000±14 000; Spectrum Medical Industries, Los Angeles, CA, USA). Dialysis cells were rotated
Ê
throughout the 6 h. Duplicate determinations were carried out at each drug concentration. "% C-Loxtidine
was placed in buåer for one determination and in plasma for the second and then dialysed against control
plasma or buåer respectively. After dialysis the radioactive content of the plasma and buåer was
determined by radioassay and the percentage protein binding determined using the following equation
Bound 1Free(x) –Free (y)
3
100%,
Bound 1Free(x)
where (x) is the radioactivity in the plasma compartment and (y) the radioactivity present in the buåer
compartment.
Pharmacokinetic analysis. Pharmacokinetic parameters of loxtidine in rat (n = 3), dog (n = 2) and human
volunteers (n = 5) were determined on each of the individual loxtidine plasma pro®les after intravenous
administration. The maximum plasma concentration (Cmax ) after intravenous administration was
determined by data inspection and extrapolation back to time zero. The area under the plasma
concentration time curves (AUC!-t and AUC , where t is the last data point), plasma half-life (t ),
"
#
!-
¢
elimination rate constant (k ) and volume of distribution (V) were calculated using SIPHAR (SIMED,
el
Creteil, France). The plasma AUC was calculated using the linear trapezoidal rule. Pharmacokinetic
parameters such as total plasma clearance, renal clearance, hepatic clearance and intrinsic clearance were
determined in each species from the dose administered, the AUC!-¢, urinary concentrations of drug and
in vitro plasma protein binding using standard relationships (Labaune 1989).
Results
Viability of hepatocyte preparations
"%
C-Loxtidine did not appear to have any signi®cant eåect on cell viability. The
viability of rat, dog and human hepatocytes as determined by trypan blue exclusion
was initially 86.8‰ 3.4 % (n = 12), 90.2‰ 4.4 % (n = 12) and 87.9‰ 2.7 % (n = 6)
respectively. After 3 h incubation at 37 C in the presence and absence of "% C-
Ê
loxtidine (100 lm ) the viability decreased to 71.8‰ 7.3 and 71.9‰ 3.6 % in rat
hepatocytes (n = 12) ; 70.9‰ 3.2 % and 72.5‰ 3.7 % in dog hepatocytes (n = 6), and
7
3.7‰ 4.9 and 71.7‰ 2.9 % in human hepatocytes (n = 3). Cytochrome P450 con-
centrations in rat, dog and human hepatocytes were 0.33‰ 0.04, 0.13‰ 0.03 and
’
0
1
.06‰ 0.02 nmol}10 cells respectively. Protein concentrations were 1.39‰ 0.16,
’
.05‰ 0.13 and 1.61‰ 0.36 mg}10 cells for rat, dog and human hepatocytes
respectively.
Recovery of radioactivity from hepatocyte incubates
The extraction eæciency of radiolabelled material from rat (n = 3), dog (n = 3)
and human (n = 2) hepatocyte incubations was 92‰ 2, 98‰ 2 and 98 % for each
species respectively. Less than 1 % of the total radioactivity in the incubations from
any species remained in the precipitate. The remainder of the radiolabelled material
(
up to 6 %) was associated with the precipitate washings indicating a full recovery of
radiolabelled material. These data indicate that very little if any non-speci®c binding
of loxtidine or metabolites occurs.

The metabolism and intrinsic clearance of loxtidine in vitro
259
Figure 2. Correlation of loxtidine metabolites formed in rat hepatocytes (1 lm and 100 lm)
incubated for 3 h and rat in vivo following intravenous administration (5 and 50 mg}kg) of
loxtidine (rat in vivo data from Bell et al. 1983).
Stability of "% C-loxtidine in isolated hepatocyte suspensions
No break down of "% C-loxtidine occurred in control (non-viable) or 3 h
incubations with either rat, dog (0.5 and 100 lm) or human (1 and 10 lm) hepatocytes
as determined by tlc (data not shown).
Rat hepatocyte incubations
The major radioactive metabolite observed at all concentrations was the
propionic acid metabolite (GR30450) which comprised 61.3‰ 3.6 and 19.1‰ 8.6 %
of the radioactivity in the 3 h incubation when the initial concentrations of "% C-
loxtidine were 1 and 100 lm respectively (n = 6) (®gure 2). The carboxylic acid
metabolite (AH24610) of loxtidine was detected in all rat incubations at all
concentrations studied. The carboxylic acid represented 27.4‰ 2.9 and 12.1‰ 3.0 %
of the radioactivity present after 3 h incubation with 1 and 100 lm "% C-loxtidine
respectively (®gure 2). Metabolites that chromatographed with a similar Rf to
!
loxtidine glucuronide represented 9.7 % of the radioactivity in rat hepatocyte
incubations. Trace amounts (up to 3 % of radioactivity present) of the phenolic
metabolite (AH25227), were also observed. The metabolism of "% C-loxtidine (1 and
1
00 lm) was completely inhibited in the presence of SKF525A (100 lm ) suggesting
that the metabolism of loxtidine in rat hepatocytes is mediated via the cytochrome(s)
P450.
Dog hepatocyte incubations
In dog hepatocyte incubations, the major radioactive metabolite con®rmed by
enzyme hydrolysis was the glucuronide conjugate of loxtidine. The aglycone formed
after hydrolysis of the dog hepatocyte co-chromatographed with "% C-loxtidine.
Incubation of the dog hepatocyte suspension containing the metabolite in the
presence of saccharo-1,4-lactone, a speci®c inhibitor of b-glucuronidase, resulted
in the major metabolite remaining unhydrolysed. This metabolite represented
3
6.1‰ 11.1 and 28.1‰ 9.1 % of the radioactivity in the 3-h incubations when the

2
60
M. K. Bayliss et al.
Figure 3. Correlation of loxtidine metabolites formed in dog and human hepatocytes (1 lm)
incubated for 3 h and dog and human volunteers following intravenous administration of loxtidine
(
in vivo data from Bell et al. 1983, Jenner et al. 1984).
initial concentrations of "% C-loxtidine were 1 and 100 lm respectively (®gure 3). The
other radioactive products were oxidative metabolites comprising up to 6 % of the
total. The major oxidative metabolite was the carboxylic acid metabolite, AH24610.
The ratio of metabolites did not change over the concentration range used.
Human hepatocyte incubations
Only one metabolite was detected in human hepatocyte incubations that co-
chromatographed with the authentic standard of the carboxylic acid (AH24610)
metabolite. This metabolite was observed at all concentrations of "% C-loxtidine
studied representing 5.1‰ 2.0, 4.7‰ 1.8 and 4.1‰ 1.8 % of the radioactivity on the tlc
plate. No evidence for the formation of the other metabolites of "% C-loxtidine seen
in animal species was detected in any of the human hepatocyte incubations (®gure 3).
Enyme kinetic analysis of loxtidine metabolism
Initial velocity data determined experimentally were modelled using mono-
phasic-®tting procedures. Computer generated Lineweaver-Burke transformations
of the experimentally determined initial velocities for rat and dog hepatocytes were
used to visualize the data. The mean apparent Km and Vmax for the metabolism of
"
% C-loxtidine by rat hepatocytes were 9.9‰ 4.0 lm and 0.114‰ 0.032 nmol min 10’
}
}
cells respectively. The apparent kinetic constants for the metabolism of "% C-
loxtidine in dog hepatocytes were 441‰ 257 lm and 0.512 ‰ 0.341 nmol}min}10’
cells respectively. Using the appropriate scaling factors, which included hepato-
cytes}g tissue, liver and body weight, ®nal means for the Cl of loxtidine by rat and
int
dog hepatocytes calculated from in vitro data were 51.4‰ 12.4 and 8.0‰ 0.8 ml}
min}kg respectively (table 1).
The slow rate of metabolism of "% C-loxtidine by human hepatocytes and the
limited data points did not allow for a full evaluation of apparent Michaelis-Menten
parameters. Therefore, an integrated form of the Michaelis-Menten equation was
used to calculate Clint as described in the methods. The Clint of loxtidine by human
hepatocytes was determined as 1.0‰ 0.6 ml}min}kg (table 1) when using the
appropriate scaling factors.

The metabolism and intrinsic clearance of loxtidine in vitro
261

2
62
M. K. Bayliss et al.
Table 3. Calculation of clearances of loxtidine after intravenous administration to rat, dog and
man.
Fraction
unbound
(fu)
Liver blood
(%)
Cl (
")
ClR (# )
ClH ($)
ClInt (&)
(ml}min}kg)
¯ow (Q)
(ml}min)
P
Species
(ml}min}kg) (ml}min}kg) (ml}min}kg)
Rat
Dog
Man
35.1‰ 9.2
8.3
8.5
1.7
26.6
6.6
0.74
0.42
0.22
69
44
25
58.5
18.6
1.0‰ 0.2
0.5‰ 0.1
0.4‰ 0.1
2.0‰ 0.6
Dose
AUC
1
2
. ClP
=
after intravenous administration.
Amount of drug excreted unchanged in urine to t
.
.
AUC
±
t
!
3
4
5
. ClH = ClP –ClR .
. Data from Boxhenbaum (1980).
Q.ClH
. Clint
=
.
f u(Q± ClH )
Pharmacokinetic analysis
Pharmacokinetic analysis of loxtidine plasma concentrations was performed on
each individual data set obtained from rat, dog and man. The mean rat, dog and
human pharmacokinetic parameters are presented in table 2. The AUC for the
loxtidine plasma concentration-time curve in the rat accounted for C 91±93 % with
only 7±9 % extrapolated. For one dog, the extrapolation was only 9 % ; however, the
extrapolated AUC for the second dog was 34 %. This was due to an extrapolation
from the 4 h point to in®nity rather than from 8 h to in®nity. The human volunteer
study was designed to assess the eæcacy of loxtidine and as such the initial time point
was 1 h after intravenous administration. Although the ®rst available time point was
1
h after administration the data are suæcient to describe the distribution phase of
the intravenous plasma pro®le, and thus allow an accurate extrapolation and
estimation of Cmax . The contribution of the extrapolated AUC between 0 and 1 h in
human volunteers was 11±20 % of the total and was considered signi®cant. All
AUC-derived pharmacokinetic parameters in man were calculated using total AUC
extrapolated to time zero by the computer program.
The mean half-life of loxtidine was 0.9‰ 0.1, 2.7 (2.4±3.1) and 5.8‰ 1.0 h in rat,
dog and man respectively (table 2). Loxtidine had a moderate volume of distribution
(
2±3 l}kg) in rat and dog and a low volume of distribution (0.4±0.6 l}kg) being
approximately equivalent to total body water in man (table 2).
Total plasma clearance (Cl ) of loxtidine in rat, dog and man was 35.1‰ 9.2, 8.3
P
and 1.0‰ 0.2 ml}min}kg respectively. Renal clearance (Cl ) of loxtidine determined
R
from the amount of loxtidine excreted in urine during time t in rat, dog and man was
8
.5, 1.7 and 0.5‰ 0.1 ml}min}kg respectively (table 3). Thus, from the relationship
Cl = Cl 1Cl the hepatic clearance (Cl ) of loxtidine was determined as 26.6, 6.6
P
R
H
H
and 0.4‰ 0.1 ml}min}kg in rat, dog and man respectively (table 3). In the above
relationship, hepatic clearance was assumed to be the sum of metabolic and biliary
clearance. The biliary clearance of loxtidine was shown to be negligible in rat and
dog (Manchee and Bell, unpublished data); therefore in the present studies hepatic
clearance was taken to equal metabolic clearance. The apparent extraction ratio (ER)
of loxtidine (ER = Cl }Q, where Q is hepatic blood ¯ow) in vivo was therefore 0.38,
H

The metabolism and intrinsic clearance of loxtidine in vitro
263
Table 4. Comparison of model-dependent parameters for the hepatic elimination of loxtidine
derived in vitro and in vivo in rat, dog and man.
Species
Parameter
Rat
Dog
Man
Predicted in vitro
Clint (ml}min}kg)
ClH ( (ml}min}kg)
51.4
24.5
29.2
8.0
3.1
3.2
0.07
1.0
0.2
0.2
0.01
" )
(
# )
Cl
(ml}min}kg)
H
ER(
", $)
0.36
In vivo
ClH (ml}min}kg)
26.6
58.5
45.4
6.6
18.6
17.0
0.15
0.4
2.0
2.0
0.02
ClH ( (ml}min}kg)
" )
(
# )
Cl
(ml}min}kg)
H
ER(
$)
0.38
1
. Determined using the `well stirred ’ model of hepatic elimination.
. Determined using the `parallel tube ’ model of hepatic elimination.
. Extraction ratios were calculated using liver blood ¯ows of 69, 44 and 25 ml}min}kg for rat, dog and
2
3
man respectively (Boxenbaum 1980).
0
.15 and 0.02 in rat, dog and man respectively, suggesting that the extraction ratio
of loxtidine was moderate in rat but low in dog and man (table 4).
Binding of "% C-loxtidine to rat, dog and human plasma proteins
The non-speci®c binding of "% C-loxtidine to the dialysis equipment including
the dialysis membrane was 3±5 % of the initial radioactivity. Binding to rat plasma
proteins was similar at all drug concentrations studied (24±30 %). However, the
binding of "% C-loxtidine to both dog and human plasma proteins was concentration-
dependent, becoming saturated at the highest concentrations of "% C-loxtidine
"
%
studied (10 000 ng}ml). Thus, the binding of C-loxtidine to dog plasma proteins
was 54±59 % at the lower drug concentrations but was reduced to 43 % at the highest
drug concentration. The binding of "% C-loxtidine to human plasma proteins was
7
6±80 % at the lower drug concentrations and 63 % at the highest drug con-
centration. Only the protein bindings at the lowest concentrations of drug were used
in any subsequent calculation. The fraction of loxtidine unbound (f ) was, therefore,
u
0
.74, 0.42 and 0.22 in rat dog and human respectively (table 3).
Calculation of in vivo intrinsic clearance
Loxtidine is a low to intermediate extraction ratio compound in rat, dog and
man, therefore the `well-stirred ’ liver model (Morgan and Smallwood 1990) was
applied to calculate Clint from in vivo measurements of hepatic clearance (Cl ) using
H
the following relationship :
Q, ClH
Clint
=
,
f (Q –Cl )
u
H
where f is the unbound fraction of drug in plasma and Q is the liver blood ¯ow.
.
u
Hepatic blood ¯ow was calculated from the relationship Q = 0.05543 B! )*% , where
B = body weight (Boxenbaum 1980). Using the above model the in vivo intrinsic
clearances of loxtidine in rat, dog and human were 58.5, 18.6 and 2.0‰ 0.6 ml}
min}kg respectively (table 3).

2
64
M. K. Bayliss et al.
Discussion
The results of the present study show that in rat, dog and man the metabolites of
loxtidine produced by isolated hepatocytes closely parallel those produced in vivo.
Thus, the major route for the metabolism of loxtidine in rat hepatocytes was N-
dealkylation to form the propionic acid metabolite. This metabolic pathway has
been shown to be saturable in vivo (Bell et al. 1983) and saturation was also observed
in rat hepatocyte incubates in this study where the relative extent of N-dealkylation
was greatest at the lowest concentration of loxtidine studied. Other workers have
demonstrated saturation of metabolic pathways in rat hepatocytes. For example,
isolated rat hepatocytes have been used to reproduce the in vivo dose-dependant
metabolism of diphenylhydantoin (Inaba et al. 1975) and propranolol (McCormick
et al. 1988). In contrast with the rat, the major route of metabolism of loxtidine in
the dog in vivo is conjugation with glucuronic acid. This was also observed in dog
hepatocytes, the metabolite being con®rmed by enzyme hydrolysis. The metabolism
of loxtidine in the dog is independent of dose up to and including 50 mg loxtidine}kg
(
Bell et al. 1983) ; this ®nding was con®rmed in vitro by the results of this study
where the extent of metabolism remained constant through out the concentration
range examined. Administration of loxtidine to man has revealed that the drug is
eliminated predominately by renal clearance with minimal metabolic clearance. A
single metabolite has been identi®ed, the carboxylic acid (unpublished data).
During the present studies, loxtidine was incubated with hepatocytes isolated from
six diåerent human samples. In each case, loxtidine remained largely unchanged
with only one metabolite produced. The metabolite formed by human hepatocytes
co-chromatographed with an authentic standard of the carboxylic acid metabolite,
thus re¯ecting the in vivo situation.
The present studies have, therefore, shown that rat, dog and human hepatocytes
are a good quantitative model for the in vivo metabolism of loxtidine. The marked
species diåerences observed in vivo were reproduced in the hepatocyte model. Thus,
the results from the present studies add to the growing number of published data
demonstrating a good correlation between the drug metabolite pro®les detected in
hepatocyte preparations, and those observed in vivo (Green et al. 1986, Chenery et
al. 1987, Le Bigot et al. 1987, Chenery 1988, Guillouzo et al. 1988, McCormick et
al. 1988, Seddon et al. 1989, Fabre et al. 1990, Jajoo et al. 1990, Lacarelle et al. 1991,
Lavrijsen et al. 1992, Sandker et al. 1994, Pahernik et al. 1995). To our knowledge
loxtidine is at present unique in that other examples of in vitro-in vivo correlations
do not exhibit such marked species diåerences in metabolic pathways. Furthermore,
many other workers have failed to extend their studies to one of the major species
used in the pharmaceutical industry, namely the dog. Although no one animal model
represents an exact model for the metabolism of loxtidine in man, both rat and dog
form the loxtidine metabolite observed in man. In summary, the hepatocyte model
oåers a versatile system providing the opportunity to elucidate metabolic pathways
of new chemical entities in animals and man at an early stage of drug development
and may provide an opportunity for establishing the most appropriate toxicology
species.
The potential of the hepatocyte system for the prediction of drug metabolism
pathways has been established for a variety of chemical entities. However, interest
in the use of hepatocytes to predict quantitative pharmacokinetic parameters of drug
biotransformation has grown signi®cantly in recent years using rat (Oldham et al.
1
986, Chenery et al. 1987, Singh et al. 1991, Houston 1994, Ashforth et al. 1995,

The metabolism and intrinsic clearance of loxtidine in vitro
265
Hayes et al. 1995, Zomorodi et al. 1995, Carlile et al. 1997), or, less frequently,
human hepatocytes (Lave et al. 1995, 1996a, b, 1997). Only recently has there been
a greater interest in comparative studies. This has lead to a signi®cant increase in the
number of published studies where the in vivo intrinsic clearance of a new chemical
entity in rat, dog and man have been predicted from in vitro data (Chenery et al.
1
987, Lave et al. 1995, 1996a, b). In the present study intrinsic and the subsequent
hepatic clearance was determined for loxtidine using rat, dog and human hepatocyte
data. These in vitro data were con®rmed by studies in vivo in rat, dog and human
volunteers.
Under ®rst-order conditions, intrinsic clearance can be calculated from the ratio
Vmax }K . The extent of drug binding to cellular material including proteins can
m
in¯uence the apparent kinetic parameters by altering the eåective substrate
concentration at the drug metabolizing enzymes. Otherwise, the calculation of
intrinsic clearance data is independent of the various models of hepatic elimination
(
Morgan and Smallwood 1990). Since, the binding of loxtidine to rat, dog and
human hepatocyte proteins was negligible and therefore did not signi®cantly aåect
the apparent Michaelis-Menten parameters derived in vitro, the intrinsic clearance
was expressed directly as apparent Vmax }apparent K .
m
To compare Clint determined in vitro with in vivo data, the ratio V }Km was
max
scaled to give units of ml}min}kg. Scaling requires consideration of factors such as
numbers of hepatocytes per g liver, animal liver weight and body weight. It is the
uncertainty of these scaling factors, particularly the number of cells per g liver that
make the prediction of intrinsic and hepatic clearance from in vitro data precarious.
Houston (1994) has reviewed the use of scaling factors. Several workers have
discussed the appropriate methods to determine the number of hepatocytes per g rat
liver and Houston (1994) and Carlile and co-workers (1997) have reviewed the
approaches taken. The current studies used ®gures of 1203 10’, 2403 10’ and
1
203 10’ hepatocytes per g rat (Mizuma et al. 1982, Pang et al. 1985), dog and
human liver (Arias 1988). These data were con®rmed by relating the microsomal
protein content of 1 g liver, the nmol P450 per mg microsomal protein and the nmol
P450 per 10’ hepatocytes for each species.
Scaling of Clint for loxtidine gave 51.4, 8.0 and 1.0 ml}min}kg in rat, dog and
human hepatocytes. Using the ` well-stirred ’ liver model (Morgan and Smallwood
1
990) these Clint values provide ClH of 24.5, 3.1 and 0.2 ml}min}kg. These values
compare favourably with the Cl derived in vivo, 26.6, 6.6 and 0.4 ml}min}kg in rat,
H
dog and human (table 4) demonstrating the ability of the model system to predict in
vivo.
The predicted apparent hepatic ER (ER = Cl }Q) of loxtidine derived from in
H
vitro data was 0.36, 0.07 and 0.01 in rat, dog and man using blood ¯ows of 69, 44,
2
5 ml}min}kg respectively (Boxenbaum 1980). These data compare well with the
ER of 0.38, 0.15 and 0.02 determined from in vivo studies in rat, dog and man. ER
was low in dog and human hepatocytes, vindicating the use of the ` well-stirred’
model. However, the predicted apparent hepatic extraction ratio in rat (0.36) was
close to a value that is considered to be intermediate. Pharmacokinetic data were also
evaluated using the `parallel tube ’ liver model (table 4) (Morgan and Smallwood
1
990). The use of the `parallel tube ’ model resulted in little change in the ClH
determined for dog and human. A small diåerence was observed for the ClH
predicted for the rat when using the ` parallel tube ’ model (29.2 ml}min}kg)
compared with the `well-stirred’ model (24.5 ml}min}kg), suggesting that in the

2
66
M. K. Bayliss et al.
case of high extraction compounds the choice of the model to determine ClH may be
important. However, the hepatic extraction ratio predicted for loxtidine from data
in vitro using either model was not substantially diåerent.
In the absence of pharmacokinetic data in animals and man in vivo, the present
studies with isolated hepatocytes would have accurately predicted the species
diåerences in the hepatic clearance of loxtidine. These data would have indicated
that loxtidine has increased metabolic stability in man compared with animal species
because of the lower hepatic clearance value in man. Taken together, these data
suggest that relatively little loxtidine would be eliminated by hepatic metabolism in
man implying that the half-life of loxtidine may be increased when compared with
!
animal species, which is in fact the case. The half-life increases from
to 6 h in man.
1 h in the rat
This type of kinetic information produced from predictive in vitro systems early
in the development process could help to eliminate the possible pharmacokinetic
shortfalls some new chemical entities encounter when they are ®rst administered to
man during the later stages of drug development.
Acknow ledgem ents
We thank Dr Gary Manchee for providing some of the unpublished loxtidine
data in man and Mr Edmund Champness for helping in the preparation of the
®
gures.
References
Arias, I. M., 1988, In I. M. Arias, W. B. Jakoby, H. Popper, D. Scahacter and D. A. Shafritz (eds), The
Liver: Biology and Pathobiology (New York : Raven), pp. 3±10.
Ashforth , E. L., Carlile , D. J., Chenery , R. J. and Houston , J. B., 1995, Prediction of in vivo
disposition from in vitro systems : clearance of phenytoin and tolbutamide using rat hepatic
microsomal and hepatocyte data. Journal of Pharmacology and Experimental Therapeutics, 274,
7
61±766.
Bayliss , M. K., Bell , J. A., Wilson , K. and Park, G. R., 1994, 7-Ethoxycoumarin O-deethylase
kinetics in isolated rat, dog and human hepatocyte suspensions. Xenobiotica , 24, 231±241.
Bell , J. A., Bradbury, A., Jenner , W. N., Manchee , G. R. and Martin , L. E., 1983, The metabolism
of loxtidine in rat and dog. Biochemical Society Transactions, 11, 715±716.
Begue, J. M., Le Bigot , J. F., Guguen -Guillouzo , C., Kiechel , J. R. and Guillouzo , A., 1983,
Cultured human hepatocytes: a new model for drug metabolism studies. Biochemical Phar-
macology, 32, 1643±1646.
Brittain , R. T. and Jack, D., 1983, Histamine H -antagonists ± past, present and future. Journal of
#
Clinical Gastroenterology, 5, 71±79.
Boxenbaum , H., 1980, Interspecies variation in liver weight, hepatic blood ¯ow and antipyrene
clearance: extrapolation of data to benzodiazepines and phenytoin. Journal of Pharmacokinetics
and Biopharmaceutics, 8, 165±176.
Carlile , D. J., Zomorodi , K. and Houston , J. B., 1997, Scaling factors to relate drug metabolic
clearance in hepatic microsomes, isolated hepatocytes and intact liver. Drug Metabolism and
Disposition , 25, 903±911.
Chenery , R. J., 1988, The utility of hepatocytes in drug metabolism studies. In G. G. Gibson (ed.),
Progress in Drug Metabolism, vol. 11 (London : Taylor & Francis), pp. 217±265.
Chenery , R. J., Ayrton , A., Oldham , H. G., Standring , P., Seddon , T. and Kirby , R., 1987,
Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog guinea pig and man. Drug
Metabolism and Disposition, 15, 312±317.
Ekins , S., Williams , J. A., Murray, G. I., Burke, M. D., Marchant , N. C., Engeset , J. and
Hawksworth , G. M., 1996, Xenobiotic metabolism in rat, dog and human precision cut liver
slices, freshly isolated hepatocytes and vitri®ed precision cut liver slices. Drug Metabolism and
Disposition , 24, 990±995.

The metabolism and intrinsic clearance of loxtidine in vitro
267
Fabre, G., Combalbert , J., Berger, Y. and Cano, J.-P., 1990, Human hepatocytes as a key in vitro
model to improve pre clinical drug development. European Journal of Drug Metabolism and
Pharmacokinetics, 15, 165±171.
Green , C. E., Le Valley , S. E. and Tyson , C. A., 1986, Comparison of amphetamine metabolism using
isolated hepatocytes from ®ve species including human. Journal of Pharmacology and Experimental
Therapeutics, 237, 931±936.
Guillouzo , A., Begue, J.-M., Maurer, G. and Koch, P., 1988, Identi®cation of metabolic pathways of
pindolol and ¯uperlapine in adult human hepatocyte cultures. Xenobiotica , 18, 131±139.
Harrison , C., Jenner , W. N., Martin , L. E. and Young , S. N., 1983, Radioimmunoassay of loxtidine
and lamtidine. Biochemical Society Transactions, 11, 713±714.
Hayes, K. A., Brennan , B., Chenery , R. J. and Houston , J. B., 1995, In vivo disposition of caåeine
predicted from hepatic microsomal and hepatocyte data. Drug Metabolism and Disposition, 23,
3
49±353.
Houston , J. B., 1994, Commentary. Utility of in vitro drug metabolism data in predicting in vivo
metabolic clearance. Biochemical Pharmacology, 47, 1469±1479.
Houston , J. B. and Carlile , D. J., 1997, Incorporation of in vitro drug metabolism data into
physiologically based pharmacokinetic models. Toxicology in Vitro, 11, 473±478.
Humpel , M., Sostarek , D., Gieschen , H. and Labitzky , C., 1989, Studies on the biotransformation of
ionazolac, bromerguride, lisuride and terguride in laboratory animals and their hepatocytes.
Xenobiotica , 19, 361±377.
Inaba , T., Umeda , T., Mahon , W. A., Ho, J. and Jeejeebhoy , K. N., 1975, Isolated rat hepaotcytes as
a model to study drug metabolism : dose-dependent metabolism of diphenylhydantoin. Life
Sciences, 16, 1227±1232.
Iwatsubo , T., Hirota , N., Ooie , T, Suzuki, H., Shimada , N., Chiba , K., Ishizaki , T., Green , C. E.,
Tyson , C. A. and Sugiyama , Y., 1997a, Prediction of in vivo metabolism in the human liver from
in vitro metabolism data. Pharmacology Therapeutics, 73, 147±171.
Iwatsubo , T., Suzuki, H., Shimada , N., Chiba , K., Ishizaki , T., Green , C. E., Tyson , C. A., Yokoi ,
T., Kamataki , T and Sugiyama , Y., 1997b, Prediction of in vivo hepatic metabolic clearance of
YM796 from in vitro data by use of human liver microsomes and recombinant P-450 isozymes.
Journal of Pharmacology and Experimental Therapeutics, 282, 909±919.
Jajoo, J. K., Blair , I. A., Klunk , L. J. and Mayol, R. F., 1990, In vitro metabolism of the antianxiety
drug buspirone as a predictor of its metabolism in vivo. Xenobiotica , 20, 779±786.
Jauregui, H. O., Hayner , N. T., Driscoll , J. L., Williams -Holland , R., Lipsky , M. H. and
Galletti , P. M., 1981, Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes
±
freshly isolated cells, cell suspension, and primary monolayer cultures. In Vitro, 17, 1100±1111.
Jenner , W. N., Brown, I. L., Bradbury, A. and Manchee , G. R., 1984, The metabolism of loxtidine in
man (unpublished).
Labaune , J.-P., 1989, Interpretation of kinetic data. In M. H. Rubenstein (ed.), Handbook of
Pharmacokinetics : Toxicity Assessment of Chemicals (Chichester: Ellis Horwood), pp. 293±499.
Lacarelle , B., Marre, F., Durand, A., Davi , H. and Rahmani , R., 1991, Metabolism of minaprine in
human and animal hepatocytes and liver microsomes ± prediction of metabolism in vivo.
Xenobiotica , 21, 317±329.
Lave, T., Coassolo , P., Ubeaud, G., Brandt, B. B. R., Schmitt , C., Dupin , S., Jaeck, D. and Chou,
R. C., 1996b, Interspecies scaling of bosentan, a new endothelin receptor antagonist and
integration of in vitro data into allometric scaling. Pharmaceutical Research, 13, 97±101.
Lave, T., Dupin , S., Schmitt , M., Kapps , M., Meyer , J., Morgenroth , B., Chou, R. C., Jaeck, D. and
Coassolo , P., 1996a, Interspecies scaling of tolcapone,
a new inhibitor of catechol-O-
methyltransferase. Use of in vitro data from hepatocytes to predict metabolic clearance in animals
and humans. Xenobiotica , 26, 839±851.
Lave, T., Dupin , S., Schmitt , C., Valles , B., Ubeaud, G., Chou, R., Jaeck, D. and Coassolo , P., 1997,
The use of hepatocytes to select compounds based on their expected hepatic extraction ratios in
humans. Pharmaceutical Research, 14, 152±155.
Lave, T., Schmitt -Hoffmann , A. H., Coassolo , P., Valles , B., Ubeaud, G., Brandt, B. B. R. and
Chou, R. C., 1995, A new extrapolation method from animals to man : application to a metabolised
compound, mofarotene. Life Sciences, 56, 473±478.
Lavrijsen , K., Van Houdt, J., Van Dyck, D., Hendrickx , J., Bockx, M., Hurkmans , R.,
Meuldermans , W., Le Jeune , L., Lavwers , W. and Heykants , J., 1992, Comparative metabolism
of ¯unarizine in rats, dogs and man : an in vitro study with subcellular liver fractions and isolated
hepatocytes. Xenobiotica , 22, 815±816.
Le Bigot , J. F., Begue, J. M., Kiechel , J. R. and Guillouzo , A., 1987, Species diåerences in
metabolism of ketotifen in rat, rabbit and man : demonstration of similar pathways in vivo and in
cultured hepatocytes. Life Sciences, 40, 883±890.
Lowry, O. H., Rosebrough , N. J., Farr, A. L. and Randall , R. J., 1951, Protein measurement with
the Folin-Phenol reagent. Journal of Biological Chemistry, 193, 265±275.
McCormick , D. J., Fitzgerald , T. C. and McKillop , D., 1988, The metabolism of propranolol

2
68
The metabolism and intrinsic clearance of loxtidine in vitro
(
InderalTM) and xamoterol (CorwinTM) by isolated rat hepatocytes: in vivo-in vitro correlations.
Xenobiotica , 18, 1401±1412.
Mizuma , T., Machida , M., Hayashi , M. and Awazu, S., 1982, Correlation of drug conjugative
metabolism rates between in vivo and in vitro : glucuronidation and sulphation of p-nitrophenol as
a model compound. Journal of Pharmacobiodynamics, 5, 811±817.
Morgan, D. J. and Smallwood , R. A., 1990, Clinical signi®cance of pharmacokinetics: models of
hepatic elimination. Clinical Pharmacokinetics, 18, 61±76.
Oldham , H. G., Chenery , R. J., Standring , P., Norman , S. J., Garton , A. and Ayrton , A. D., 1986,
Drug metabolism kinetic studies in a hepatocyte model. Food and Chemical Toxicology, 24,
7
61±762.
Oldham , H. G., Norman , S. J. and Chenery , R. J., 1985, Primary cultures of adult rat hepatocytes ±
a model for the toxicity of histamine H -receptor antagonists. Toxicology, 36, 215±229.
#
Oldham , H. G., Standring , P., Norman , S. J., Blake, T. J., Beattie , I., Cox, P. J. and Chenery , R.
J., 1990, Metabolism of temelastine (SKF93944) in hepatocytes from rat, dog, cynomolgus
monkey and man. Drug Metabolism and Disposition, 18, 146±152.
Omura , T. and Sato, R., 1964a, The carbon monoxide-binding pigment of liver microsomes. I.
Evidence for its hemoprotein nature. Journal of Biological Chemistry, 239, 2370±2378.
Omura , T. and Sato, R., 1964b, The carbon monoxide binding pigment of liver microsomes. II.
Solubilisation, puri®cation and properties. Journal of Biological Chemistry, 239, 2379±2385.
O’Neill , R., 1971, Function minimisation using a simplex procedure. Journal Royal Statistical Society
C, 20, 338±345.
Pahernik , S. A., Smihmid , J., Sauter, T., Schidberg , F. W. and Koebe , H. G., 1995, Metabolism of
pimobendan in long term human hepatocyte culture : in vivo-in vitro comparison. Xenobiotica , 25,
8
11±823.
Pang, K. S., Kong, P., Terrell , J. A. and Billings , R. E., 1985, Metabolism of acetaminophen and
phenacetin by isolated rat hepatocytes. Drug Metabolism and Disposition, 13, 42±50.
Sandker , G. W., Vos, R. M. E., Delbressine , L. P. C., Sloof , M. J. H., Meijer , D. K. F. and
Groothuis , G. M. M., 1994. Metabolism of three pharmacologically active drugs in isolated
human and rat hepatocytes: analysis of interspecies variability and comparison with metabolism
in vivo. Xenobiotica , 24, 143±155.
Sanwald-Ducray, P. and Dow, J., 1997, Prediction of the pharmacokinetic parameters of reduced-
dolasetron in man using in vitro-in vivo and interspecies allometric scaling. Xenobiotica , 27,
1
89±201.
Seddon , T., Michelle , I. and Chenery , R. J., 1989, Comparative drug metabolism of diazepam in
hepatocytes isolated from man, rat, monkey and dog. Biochemical Pharmacology, 38, 1657±1665.
Singh , K., Tripp , S. L., Dunton , A. W., Douglas , F. L. and Rakhit , A., 1991, Determination of in
vivo hepatic extraction ratio from in vitro metabolism by rat hepatocytes. Drug Metabolism and
Disposition , 19, 990±995.
Tsuru, M., Erickson , R. R. and Holtzman , J. L., 1982, The metabolism of phenytoin by isolated
hepatocytes and hepatic microsomes from male rats. Journal of Pharmacology and Experimental
Therapeutics, 222, 658±661.
Warner, M., La Marca, M. V. and Neims , A. H., 1978, Chromatographic and electrophoretic
heterogeneity of the cytochromes P450 solubilised from untreated rat liver. Drug Metabolism and
Disposition , 6, 353±362.
Watkins , J. B. and Klaassen , C. D., 1983, Chemically induced alteration of UDP-glucuronic acid
concentration in rat liver. Drug Metabolism and Disposition, 11, 37±40.
Zomorodi , K., Carlile , D. J. and Houston , J. B., 1995, Kinetics of diazepam metabolism in rat hepatic
microsomes and hepatocytes and their use in predicting in vivo hepatic clearance. Xenobiotica , 25,
9
07±916.