Cytochrome P4501A (CYP1A) induction in rat and man by the benzodioxino derivative, fluparoxan

Article abstract of DOI:10.1080/004982597240668

1. Fluparoxan is an α₂-adrenoceptor antagonist that has a relatively planar, tricyclic structure and was considered a potential substrate and inducer of cytochrome P4501A (CYP1A) enzymes. 2. Structure-activity analysis indicated some potential for CYP1A interaction, although its greater log P and molecular depth, compared with many CYP1A inducers, suggested fluparoxan would be a weak ligand for the aryl hydrocarbon (Ah) receptor and only a weak inducer. 3. In vitro, fluparoxan showed little affinity for the CYP1A enzymes. The compound was not metabolized by human CYP1A1 or 1A2 heterologously expressed in yeast and its rate of metabolism in rat and human microsomes was unaffected by the addition of the 1A inhibitor α-naphthoflavone. Furthermore K(i)'s for fluparoxan against EROD activity were > 4000-fold higher than those of α-naphthoflavone. 4. In vivo, however, fluparoxan did show some capacity for CYP1A induction. In rat, hepatic EROD activity increased approximately 40-fold with seven once-daily oral doses of fluparoxan (50 mg/kg, solution), and immunoblotting studies confirmed induction of CYP1A2, though not of 1A1. In man, administration of 11 twice-daily oral doses of fluparoxan (8 mg tablet) produced some reduction in plasma levels of orally administered phenacetin and in the ratio of phenacetin AUC/urinary paracetamol, consistent with increased O-deethylation.

Full text of DOI:10.1080/004982597240668

xenobiotica , 1997, vol. 27, no. 2, 159±173
Cytochrome P4501A(CYP1A) induction in rat and
man by the benzodioxino derivative, ¯ uparoxan
A. P. BERESFORD*, W. J. ELLIS, J. AYRTON,
M. A. JOHNSON ‹ and D. F. V. LEW IS ‹
Bioanalysis and Metabolism Division, ‹ Clinical Pharmacology Department,
GlaxoWellcome Research and Development, Ware SG12 0DP, UK
‹ Molecular Toxicology Group, School of Biological Sciences, University of Surrey,
Guildford GU2 5XH, UK
Received 11 June 1996
1
. Fluparoxan is an a -adrenoceptor antagonist that has a relatively planar, tricyclic
#
structure and was considered a potential substrate and inducer of cytochrome P4501A
CYP1A) enzymes.
. Structure±activ ity analysis indicated some potential for CYP1A interaction, al-
(
2
though its greater log P and molecular depth, compared with many CYP1A inducers,
suggested ¯ uparoxan would be a weak ligand for the aryl hydrocarbon (Ah ) receptor and
only a weak inducer.
3
. In vitro, ¯ uparoxan showed little a nity for the CYP1A enzymes. The compound
was not metabolized by human CYP1A1 or 1A2 heterologously expressed in yeast and its
rate of metabolism in rat and human microsomes was unaŒected by the addition of the 1A
inhibitor a-naphtho¯ avone. Furthermore, K ’s for ¯ uparoxan against EROD activity were
i
" 4
000- fold higher than those of a-naphtho¯ avone.
4
. In vivo , however, ¯ uparoxan did show some capacity for CYP1A induction. In rat,
hepatic EROD activity increased approximately 40- fold with seven once- daily oral doses of
uparoxan (50 mg}kg, solution), and immunoblotting studies con®rmed induction of
CYP1A2, though not of 1A1. In man, administration of 11 twice- daily oral doses of
uparoxan (8 mg tablet) produced some reduction in plasma levels of orally administered
¯
¯
phenacetin and in the ratio of phenacetin AUC }urinary paracetamol, consistent with
increased O-deethylation.
Introduction
In order to develop safe and eŒective medicines, an awareness of the potential
interactions between a new drug substance and metabolic enzymes is extremely
important. The inhibition or induction of enzymes following drug administration
can aŒect the circulating levels of endogenous compounds and dietary constituents
as well as other xenobiotics ; with potentially calamitous results (Tarru s et al. 1987a).
!
Of particular importance in this area is the cytochrome P450 superfamily of
haem- thiolate proteins (Parke 1990), which represents a multiplicity of both
constitutive and inducible enzymes, capable of metabolising a broad range of
substrates (Nebert and Gonzalez 1987).
Historically, our understanding of P450 induction (Okey 1990) has largely
developed through studies on carcinogenic polycyclic aromatic hydrocarbons
(
PAHs) such as benzo(a)pyrene and 3-methylcholanthr ene (3- MC) and aromatic
amines such as 2- acetylamino¯ uorene, with the major PAH- induced cytochromes
having been established as the P4501A (CYP1A) subfamily (Nebert et al. 1989).
This subfamily (CYP1A) consists of two members, 1A1 and 1A2, (Nelson et al. 1996)
*
Author for correspondence.
049±8254 }97 $12±00 ’ 1997 Taylor & Francis Ltd
0

1
60
A . P. Beresford et al.
Figure 1. Comparative structures of benzodioxino derivatives and the potent CYP1A inducer, 2, 3,7,8-
tetrachlorodibe nzo-p-dioxin (TCDD). (a) Fluparoxan (GR50360), (b) GR37459, (c) TCDD.
having approximately 70 % nucleotide and amino acid sequence homologies
(
Kimura et al. 1984, Nelson and Strobel 1987, Fujii- Kuriyama et al. 1992) and both
are inducible via the Ah receptor (Nebert et al. 1993, Hankinson 1995).
Whilst there is not necessarily a direct correlation between the potency of a
polycyclic hydrocarbon as a CYP1A inducer and its carcinogenicity (Conney 1967),
it is clear that many of the features which confer Ah receptor a nity upon a molecule
are also those which render them potential mutagens }carcinogens (Ioannides and
Parke 1993). Consequently, computational studies designed to try and predict the
toxicity of such compounds are equally predictive of their Ah receptor binding
a nity (Ioannides et al. 1994, Lewis et al. 1994). Extensive molecular modelling
studies on a series of dioxins have shown that their potential Ah receptor binding
capability can be predicted (QSAR correlations up to r ¯ 0±95) using molecular
orbital calculated electronic parameters (e.g. frontier orbital energies) combined
with a number of structural descriptors such as log P, area}depth# and length }width
ratios (Lewis 1996).
Fluparoxan
Fluparoxan, 5- ¯ uoro-2,3,3a,9a- tetrahydro- 1H-[1,4]- benzodioxino-[2,3- c] pyr-
role (®gure 1a), is a potent a -adrenoceptor antagonist (Halliday et al. 1988), which
#
has been shown to inhibit the central a -adrenoceptors in man (Johnson et al. 1995)
#
and was developed as a potential anti-depressant. The compound progressed
satisfactorily through in vitro mutagenicity testing and animal toxicity testing to
clinical trials in man by 1988 (Gristwood 1990). However, development was halted
in 1994 when the compound failed to show a clear clinical advantage over existing
therapies.
Metabolic and pharmacokinetic studies (unpublished data) showed ¯ uparoxan
to be well absorbed following oral dosing in all species tested. Clearance was largely
metabolic, with both oral and intravenous doses being excreted mainly via the urine
("
90 % of administered dose), chie¯ y as phase II metabolites (sulphamic acid and
carbamoyl glucuronide conjugates). The compound was well tolerated on repeat

Cytochrome P4501A induction by ¯ uparoxan
161
oral administration to rat (% 200 mg}kg}day) and dog (% 80 mg}kg}day) for 12
months, although there was some reduction in weight gain at high doses and some
evidence of increased liver weight in rat, but not in dog.
As an increase in liver weight could be indicative of changes in metabolic
enzymes, investigations were carried out to assess the potential of ¯ uparoxan to
induce cytochromes P450, and particularly, in view of the relatively planar nature of
the molecule, those of the CYP1A family. Indeed, a degree of structural similarity
to the dioxins is apparent if the dichloro analogue of ¯ uparoxan, GR37459 (®gure
1
b) is compared with the potent CYP1A inhibitor 2,3,7,8- tetrachlorodibenzo-p-
dioxin (TCDD ; ®gure 1c). This paper describes the calculation of the Ah receptor
binding potential and CYP1A induction potential of ¯ uparoxan and a series of in
vitro interaction studies performed using rat and human liver microsomes, and
human CYP1A isozymes heterologously expressed in yeast. Also described are in
vivo studies carried out to monitor possible CYP1A induction by ¯ uparoxan in rat,
by measurement of microsomal ethoxyresoru®n- O-deethylation (EROD) and
immunoblotting, and a subsequent investigation in man, using phenacetin as the
probe substrate (Conney et al. 1976, Pantuck et al. 1979).
Materials and methods
Reagents
Fluparoxan (GR50360) and its dichloro analogue, GR37459 (6,7- dichloro-2,3,3a,9a-tetrahydro- 1H-
1,4]- benzodioxine- [2,3- c] pyrrole), were synthesized (as their hydrochloride salts) at Glaxo Research
[
and Development (Greenford, UK). Phosphate- buŒered saline (PBS, 100 mm, pH 7±4), NADPH
(
chemically reduced) and type H- 2 glucuronidase }sulphatase (crude solution from Helix pomatia) were
obtained from Sigma Chemicals (Poole, UK). Coomassie protein assay reagent was obtained from Pierce
and Warriner (Chester, UK). Dimethyl sulphoxide (DMSO) and all other solvents and reagents used
were Analar grade or equivalent.
Computational analysis
The potential Ah receptor binding capabilities (PEC ) of ¯ uparoxan and GR37459 were calculated
&
!
from a previously derived (Lewis 1996) quantitative structure±activ ity relationship (QSAR) based on a
series of structurally related 2,3- dichlorodibenzo-p-dioxins (DCDDs), and compared with TCDD. The
PEC equates to the negative logarith of the drug concentration required to produce 50 % displacement
&!
of TCDD from the Ah receptor. Sybyl software (Tripos Associates, St Louis, MO, USA) was used for
the generation of molecular orbital-calculated electronic structural descriptors, and HazardExpert
(
Compudrug, Budapest, Hungary) for calculation of log P via the method of Rekker et al. (1993).
A QSAR expression was also derived for CYP1A induction potential, i.e. the anticipated fold increase
in CYP1A activity per unit dose :
log CYP1A induction ¯ 2±67®0±15 CR ®0±40 l}w­0±23 log P,
where CR is the COMPACT radius ¯ o(a}d#®15)#­(DE®7)#, l}w (length }width) ¯ ratio of molecular
length and width for the minimum energy geometries, a}d# (area}depth #) ¯ ratio of molecular area and
square of molecular depth for the minimum energy geometries, and DE ¯ energy diŒerence between
highest occupied and lowest unoccupied molecular orbitals. This relationship gives a 0±99 correlation
with measured CYP1A induction (increased metabolic activity normalized for mmol }kg dose) for a series
of diverse chemicals using the data reported by Ioannides and Parke (1993).
Microsomal metabolism and inhibition studies
In vitro metabolism and inhibition studies were performed using both rat and human liver
microsomes. The latter were produced from tissue acquired as surgical waste from donor livers prepared
for paediatric transplant and were considered essentially normal. Samples from one male (H24, age 16,
previous treatment with antibiotics, cause of death meningitis) and one female donor (H26, age 18,
previous treatment with antibiotics, dopam ine and noradrenalin, cause of death subarachnoid
haemorrhage) were used. Tissue homogenates were centrifuged at 9000 g to remove cell debris and the
supernatants further centrifuged (100 000 g) to produce microsomal pellets, which were resuspend ed in
PBS containing 20 % (v}v) glycerol. In addition, microsomes containing the individual human CYP1A1

1
62
A . P. Beresford et al.
and 1A2 isozymes, heterologously expressed in Saccharomyces cerevisiae (Beresford et al. 1996), were
obtained by courtesy of the University of She eld, Department of Medicine and Pharmacology.
Microsomal protein contents were determined using Coomassie reagent (Bradford 1976) as directed by
the suppliers and cytochrome P450 content was determined by CO diŒerence spectroscopy (Omura and
Sato 1964).
Microsomal incubations (% 10 mg protein in PBS) were performed, in duplicate, at 37 °C in 1 ml
total volumes containing NADPH at an initial concentration of 1 mm. Metabolism of ¯ uparoxan (initial
concentrations 15 or 50 lm), was monitored using tritium- labelled drug (prepared as a 1 mm solution in
PBS). Aliquots of incubate were mixed with equal volumes of 0±4 m perchloric acid solution, centrifuged
to remove precipitated protein and the supernatant analysed by hplc using a radiochemical detector
(
(
(
Berthold LB506). The eŒect of a-naphtho¯ avone (ANF), a potent inhibitor of CYP1A activity
Diamond and Gelboin 1969), on ¯ uparoxan metabolism was determined following pre- incubation
2 min) with the inhibitor (10 ll in DM SO) prior to addition of the substrate.
For comparative purposes, microsomal ethoxyresoru®n- O-deethylation (EROD) activity and the
eŒect of ANF on this activity were similarly determined for an initial substrate (7-ethoxyresoru®n)
concentration of 15 lm (5 ll in DMSO). EROD measurements were based on the method of Burke and
Mayer (1974), with detection of the ¯ uorescent product (resoru®n) at excitation and emission wavelengths
of 522 and 588 nm respectively.
EROD activities were also measured using 7- ethoxyresoru®n at an initial concentration of 5 lm. For
determination of K ’s, ¯ uparoxan and ANF were added in 5±10 ll DM SO and pre- incubated for 2 min
i
prior to addition of substrate.
Induction studies in rat
Male, random -bred, Sprague- Dawley hooded rats, age 12 weeks, weight range 240±280 g, were
supplied by Glaxo Research and Development Animal Breeding Unit and oral doses of ¯ uparoxan were
administered by gavage as solutions (5 ml}kg) in distilled water.
Animals were given seven once- daily oral doses of ¯ uparoxan (0±1, 5±0 or 50 mg free base}kg) or water
only and sacri®ced by cervical dislocation, approximately 24 h after the ®nal dose, for production of
microsomes.
Microsomal EROD was determined as a measure of CYP1A (PAH- inducible) activity and, for
comparison, aminopyrine N -demethylation (APND), as a traditional measure of phenobarbitone-
inducible activity (CYP2B), was measured by trapping liberated formaldehyde with semicarbazide and
measuring the resultant semicarbazone colourimetrical ly at 415 nm (Nash 1953).
In order to provide an estimate of bioavailability in these experiments, the ¯ uparoxan plasma
concentrations following 50 mg}kg oral doses were compared with those from an earlier pharmacokinetic
experiment, carried out in support of toxicology studies, in which male rats were given single intravenous
doses of ¯ uparoxan at 5 mg }kg in physiological saline (2 ml}kg). Blood samples were obtained by vena
caval exsanguination (three animals per time- point) under ether anaesthesia, centrifuged to yield plasma
and the drug concentrations determined by hplc with UV detection (Beresford et al. 1992).
Immunoblotting. Microsomal proteins (50 lg) from the livers of vehicle and ¯ uparoxan treated rats were
separated on 8±5 % SDS- polyacrylamide gels (1±5-mm thick) using the method of Laemmli (1970).
Following Western blotting of the separated proteins onto nitrocellulose ®lters (Model TE22 Transfer
Unit, Hoefer Scienti®c Instrumen ts, Newcastle, UK) the CYP1A proteins were incubated with speci®c
rat 1A1 (Sesardic et al. 1990b) and 1A2 (Edwards et al. 1993) rabbit antibodies, kindly supplied by the
Royal Postgraduate Medical School, London. Visualization was achieved following incubation with
alkaline phospha tase-linked goat anti-rabbit second antibody and reaction with 5-bromo- 4-chloro- 3-
indolyl phospha te and nitroblue tetrazolium.
Comparisons were made with hepatic microsomes from additional rats pretreated with intraperitoneal
doses of either b-naphtho¯ avone (80 mg}kg}day for 4 days), isosafrole (150 mg}kg}day for 4 days) or 3-
MC (80 mg }kg single dose) in corn oil.
Induction study in man
The eŒects of ¯ uparoxan on the pharmacokinetics of phenacetin were monitored in 24 healthy male
volunteers (aged 18±40 years) using a placebo-controlled, double- blind, cross- over study. The study was
approved by the Ethical Review Committee of Glaxo Group Research and of Northwick Park Hospital,
and was conducted in accordance with the provisions of the Declaration of Helsinki and revisions.
Subjects received a full explanation of the nature and purposes of the investigation and provided their
written informed consent for participation in the study.
Following a run- in period of 21 days, during which time they were not permitted caŒeine- containing
beverages or barbecued food, each volunteer received 11 oral doses of either ¯ uparoxan (8 mg tablet) or
placebo, twice- daily. Approximately 24 h before dose 1 and 24 h after dose 11 of each treatment period,
a single oral dose of phenacetin was administered (900 mg suspended in 50 g hydroxypropyl-
methylcellulose with water to a total volume of 250 ml).

Cytochrome P4501A induction by ¯ uparoxan
163
Blood samples were taken and plasma separated for phenacetin analysis. The compound was extracted
from plasma using cyanopropyl solid phase extraction cartridges and quanti®ed by hplc using
a
Spherisorb C18 column (100¬4±6 mm), with a mobile phase of potassium dihydrogen orthoph osphate
(
0±1 m)±methanol (33 : 67 v}v), at a ¯ ow rate of 1 ml}min and detection by UV absorptio n at 254 nm.
Urine samples were collected for determination of total paracetamol content following hydrolysis of
possible conjugates with sulphatase and glucuronidase (2 h at 37 °C in 0±1 m, pH 5 acetate buŒer). The
hydrolysates were analysed by hplc using a Nucleosil C18 column (150¬4±6 mm) and a mobile phase
of phosphat e buŒer (0±5 m, pH 3±8)±methanol±ethyl acetate (1400 : 220 : 4 by vol.), running at 2 ml}min,
with detection pf paracetamol by UV absorption at 242 nm.
Results
Potential for CYP1A induction by ¯ uparoxan
As shown in table 1, using a QSAR expression derived for a series of structurally-
related DCDDs, the potential Ah receptor binding a nities (PEC &! ), calculated for
¯
uparoxan (3±459) and even for the dichloro analogue, GR37459 (4±293), are
signi®cantly lower than that of TCDD (7±679). Using the QSAR expression for
CYP1A induction capability, derived using a structurally diverse series of chemicals,
the low values (log scale) for the benzodioxino compounds (0±456 and 0±684),
emphasize their low induction potential compared with TCDD (2±503).
Microsomal metabolism of ¯ uparoxan
Fluparoxan was readily metabolized in rat liver microsomes (720 pmol}min }mg
protein in samples from control animals). The metabolism was NADPH- dependent
but largely unaŒected by the addition of ANF (®gure 2). Under the same initial
conditions (15 lm substrate, 50 lm inhibitor), in the same microsomes, ANF
reduced EROD activity by 81 % (from 91 to 17 pmol}min }mg protein). As shown
in table 2, the rate of ¯ uparoxan metabolism in diŒerent batches of rat microsomes
was not re¯ ected by the EROD activity present : pretreatment of rats with BNF
producing a 350- fold increase in EROD, but no increase in ¯ uparoxan turnover.
Hence, ¯ uparoxan does not appear to be a substrate for CYP1A isozymes in the rat.
Likewise, the compound did not appear to be a substrate for human CYP1A
isozymes ; showing no detectable metabolism with either CYP1A1 (10 pmol) or 1A2
(
20 pmol) heterologously expressed in yeast, despite good turnover
("
200
pmol}min }mg protein) in human liver microsomes (table 2). As with rat
microsomes, the addition of ANF at a concentration (10 lm), which reduced EROD
activity by almost 90 % (from 28 to 3±3 pmol}min }mg protein), did not aŒect the
metabolism of ¯ uparoxan by human liver microsomes.
Determination of K ’s against EROD activity (®gure 3) showed that ¯ uparoxan
i
has a low a nity for CYP1A, with values of 356 and 311 lm being obtained in
expressed human CYP1A1 and human liver microsomes respectively, compared
with 23 and 72 nm for ANF in the same samples, i.e. at least a 4000- fold diŒerence.
Induction of CYP1A in rat by ¯ uparoxan
Measurement of plasma ¯ uparoxan levels con®rmed good oral absorption in the
rat, with the AUC for a single 50 mg}kg dose (458 lg}ml h) being slightly in excess
of the dose-corrected AUC (449 lg}ml h) obtained for a single intravenous dose
(
5 mg}kg).
The administration of seven, once-daily high oral doses (50 mg}kg) did not

1
64
A . P. Beresford et al.
Table 1. Comparison of physicochemical parameters used in assessing the potential Ah receptor
binding a nity (PEC and CYP1A induction potential of the benzodioxino derivatives,
uparoxan and GR37459, and the potent CYP1A inducer, 2,3,7,8- tetrachlorodibe nzo-dioxin
TCDD).
)
&!
¯
(
Parameter
COMPACT
radius
CYP1A induction
potential (log)
Compou nd
log P
a}d#
DE
l}w
PEC
&!
Fluparoxan
GR37459
TCDD
0±862
2±125
7±256
3±025
3±166
7±599
9±326
8±884
8±090
1±455
1±693
1±785
12±199
11±983
7±481
3±459
4±293
7±679
0±456
0±684
2±503
log P ¯ calculated logarithm of the octanol}water partition coe cient ; a}d# (area}depth#) ¯ ratio of
molecular area and square of molecular depth for the minimum energy geometries ; DE ¯ energy
diŒerence between highest occupied and lowest unoccupied molecular orbitals ; l}w (length }width) ¯
ratio of molecular length and width for the minimum energy geometries ; COMPACT radius ¯
o(a}d#®15)#­(DE®7)#.
These parameters were used to calculate the potential Ah receptor binding a nities (PEC ) and
&
!
CYP1A induction potential based on QSAR expressions derived for a diverse series of possible CYP1A
inducers (see Materials and methods for details). Both values are clearly lower for ¯ uparoxan than
TCDD, principally due to the much lower log P and reduced a}d# ratio of the former.
Figure 2. Metabolism of ¯ uparoxan by rat liver microsomes. Tritium- labelled ¯ uparoxan (15 lm) was
incubated (37 °C) for 10 min with rat hepatic microsomes (5 mg protein) (A) in the absence or (B)
in the presence of NADPH (1 mm) and (C) with NADPH and the CYP1A inhibitor a-
naptho¯ avone (50 lm). Chromatograms illustrate the pattern of radioactivity obtained on hplc (y-
axis units ¯ counts sÕ ") following precipitation of microsomal proteins with perchloric acid
solution (0±4 m). Microsomal metabolism of ¯ uparoxan (Rt C 16 min) is clearly NADPH
dependent, but is largely unaŒected by addition of the inhibitor a-naphtho¯ avone. Under the
same initials conditions (15 lm substrate, 1 mm NADPH) in the same microsomes, 50 lm
a-naphtho¯ avone reduced EROD activity by
" 80 % (data not shown).

Cytochrome P4501A induction by ¯ uparoxan
165
Table 2. Comparative microsomal metabolic data for ¯ uparoxan and 7-ethoxyresoru®n.
Metabolic rate (}min }mg protein)
7
-Ethoxyresoru®n
(
Fluparoxan
(pmol substrate
metabolized)
pmol product
formed)
Microsomes
Control rat
Bnf rat
Iso rat
3±8 (³0±6)
1320 (³33)
94 (³2)
118 (³4)
21 (³1)
720 (³143)
80 (³15)
227 (³41)
507 (³76)
368 (³29)
806 (³96)
*n.d.
Flu rat
Human (male, H24)
Human (female, H26)
CYP1A1
36 (³4)
37 (³3)
CYP1A2
1±4 (³0±3)
n.d.
!
*
n.d. ¯
15 pmol}min }mg protein.
The microsomal metabolism of 7-ethoxyresoru®n (initial conc. 5 lm) was determined by spec-
trophotometric measurem ent of the ¯ uorescent product (resoru®n), and ¯ uparoxan metabolism (initial
conc. 50 lm) determined by radiochromatographic monitoring of tritiated parent compound. Metabolic
rates were compared in hepatic microsom es from untreated, b-naphtho¯ avone (Bnf, 80 mg}kg}day¬4),
isosafrole (Iso, 150 mg }kg}day¬4) or ¯ uparoxan (Flu, 50 mg}kg}day¬7) treated rats (pooled samples
from three animals), in samples of human liver microsomes (H24 and H26) and in microsomes containing
human CYP1A1 (25 pmol}mg protein) or 1A2 (12 pmol}mg protein) isozymes heterologously expressed
in yeast.
Data represent the mean of triplicate determinations (³SD) and show no obvious correlation
between the rate of ¯ uparoxan metabolism and that for the recognized CYP1A substrate
7
-ethoxyresoru®n.
produce any marked change in the plasma pro®le of the drug ; with peak plasma
concentrations of 59±6 and 57±0 lg}ml at 0±5 h post-dose and the calculated
absorption and elimination half-lives being approximately 0±3 and 3±3 h respectively
on both occasions. As shown in ®gure 4, this same oral dosage regimen did not
!
produce any change in hepatic APND, but did result in a signi®cant (p
0±01)
increase in EROD. An increase in EROD activity was also seen at the lower doses
of 5±0 and 0±1 mg}kg, and it was subsequently found that a single dose of ¯ uparoxan
at 50 mg}kg produced a signi®cant increase in EROD activity (data not shown).
Immunoblotting of microsomes from the ¯ uparoxan treated rats showed a
progressive increase in the level of CYP1A2 protein with dose (0±1, 5±0 and
5
0 mg}kg), but without increasing 1A1. This selectivity is illustrated in ®gure 5 by
comparing high oral dose (50 mg}kg) ¯ uparoxan microsomes with those from rats
given intraperitoneal isosafrole, b-naphtho¯ avone or 3-methylcholanthr ene. At the
total protein loading used here (50 lg), there was no detectable CYP1A2 in
microsomes from vehicle dosed rats, but the band intensity from ¯ uparoxan dosed
animals was similar to that from isosafrole treated animals, which also showed a
similar level of EROD activity (around 100 pmol}min }mg). Similarly, CYP1A1
was undetectable in control microsomes, but, in contrast to 1A2, this remained
undetectable after dosing with either ¯ uparoxan or isosafrole. Based on the blotting
of puri®ed CYP1A proteins (data not shown), the limit of sensitivity for this
procedure is considered to be about 0±25 pmol. Hence, neither compound had
"
increased CYP1A1 concentrations
5 pmol}mg microsomal protein. The CYP1A1
isozyme was, however, readily detectable in microsomes following BNF and 3- MC
dosing ; the EROD activity being considerably higher in these microsomes
("
1000 pmol}min }mg protein).

1
66
A . P. Beresford et al.
Figure 3. EŒect of ¯ uparoxan addition on microsomal ethoxyresoru®n- O-deethylation (EROD).
Microsomes from human liver (0±25 mg protein) or Saccharomyces cerevisiae cells heterologously
expressing human CYP1A1 (10 pmol P450) were incubated with 7-ethoxyresoru®n following
pre- incubation (2 min at 37 °C) with ¯ uparoxan and NADPH (1 mm), and the formation
of resoru® n (V ¯ pmol}min) monitored over 5 min. The double reciprocal plots illustrate the
determination of K using three ethoxyresoru®n concentrations (+ , 0±2 ; _ , 1±0 ; E , 5±0 lm)
i
range of ¯ uparoxan concentrations. Each point represents the mean of triplicate
over
a
determinations. Comparative data are provided for the known CYP1A inhibitor a-
naphtho¯ avone, in the same microsomes at the same three substrate concentrations, and show an
approximately 4000- fold greater a nity for the enzymes than ¯ uparoxan.
Induction of CYP1A in man by ¯ uparoxan
Considerable variability in the plasma concentration}time pro®les for phenacetin
were seen between volunteers before and after both placebo and ¯ uparoxan
treatment, with four pretreatment and seven post-treatment sample sets containing
no detectable drug (!
20 ng}ml plasma). A non-parametric statistical method, as
described by Koch (1972), was therefore applied to the data. This indicated a
statistically signi®cant (p ¯ 0±040) reduction in maximum phenacetin concentration
following ¯ uparoxan treatment (median 369 ng}ml) compared with placebo (median
4
56 ng}ml) and a reduction in the area under the phenacetin plasma concentration}
time curve (from a median value of 441 to 407 h ng}ml) which approached statistical
signi®cance (p ¯ 0±070). An illustration of these changes is provided by the plots of
median plasma concentrations in ®gure 6. The suggestion that the changes were the
result of increased phenacetin metabolism, rather than decreased absorption, was
supported by a signi®cant decrease (p ¯ 0±025) in the ratio of phenacetin AUC }
paracetamol excreted in the urine following ¯ uparoxan treatment (from 2±9 to 1±9),
compared with placebo (3±2 both before and after treatment).

Cytochrome P4501A induction by ¯ uparoxan
167
Figure 4. EŒect of ¯ uparoxan administration on hepatic aminopyridine- N -demethylation (APND) and
ethoxyresoru®n- O-deethylation (EROD) in the rat. Animals (n ¯ 4 per treatment) were given
seven once- daily oral doses of ¯ uparoxan (0±1, 5±0 or 50 mg }kg) in water (or water only) and
hepatic microsom es prepared approximately 24 h after the ®nal dose. The histograms illustrate
the change (³SD) in microsomal APND and EROD rates compared with the mean for vehicle-
dosed controls and show little eŒect of the compound on APND but a signi®cant induction of
EROD.
Figure 5. Induction of rat hepatic CYP1A proteins by ¯ uparoxan. Hepatic microsomes were prepared
from rats given seven once- daily oral doses of ¯ uparoxan (0±1, 5±0 and 50 mg}kg) in water or water
only. Microsomal proteins (50 lg samples) were separated on SDS- polyacrylamide gels (Laemmli
1
1
970), transferred to nitrocellulose ®lters (Western blotting) and probed with either CYP1A1 or
A2 speci®c antibodies. Samples from three rats at each dose level were probed individually and
showed a progressiv e increase in CYP1A2 blot intensity with dose, but with CYP1A1 remaining
undetectable (!
0 25 pmol). The immunoblot shown compares microsomes pooled from the 3
±
high oral dose (50 mg}kg) ¯ uparoxan (Flu) rats with those given water only (Con) and animals
given intraperitoneal doses of the known CYP1A inducers, isosafrole (Iso), b-naphtho¯ avone
(
Bnf) and 3- methylcholanthrene (3Mc).

1
68
A . P. Beresford et al.
Figure 6. EŒect of ¯ uparoxan administration on plasma phenacetin concentrations in man. Male
volunteers (n ¯ 24) were given single oral doses of phenacetin (900 mg suspensio n) at each end of
a treatment period comprising 11 oral doses of either ¯ uparoxan (8 mg tablet) or placebo, twice-
daily. The graphs represent the median phenacetin plasma concentrations from the 24 volunteers,
determined before and after placebo or ¯ uparoxan treatment. The plots are intended to illustrate
the overall decrease in
C
(p ¯ 0±040) and AUC (p ¯ 0±070) apparent due to ¯ uparoxan
treatment and no attempt has been made to show the wide inter- and intra- subject variability seen
before and after both treatments.
max
Discussion
The CYP1A enzymes have long been associated with the activation of chemical
mutagens and carcinogens and, more recently, linked with other potentially
procarcinogenic cellular events such as the activation of protein kinase c and
phosphorylation of EGF (reviewed in Beresford 1993, Ioannides and Parke 1993).
These eŒects have largely been attributable to highly lipophilic, planar, polycyclic
aromatic compounds and aromatic amines, which are often substrates for the
CYP1A subfamily as well as potent inducers of the enzymes via the Ah receptor.
Induction via this receptor results in an increase in both CYP1A1 and 1A2 activity
(
Nebert and Jones 1989) and it has proved possible to correlate the Ah receptor
binding a nity to the mutagenic }carcinogenic potential of such compounds (Ayrton
et al. 1990, Lewis et al. 1994). This approach may be of considerable value in future
if it would permit the calculation of potential mutagenicity before a compound has
even been synthesized.
Fluparoxan is a relatively planar aromatic amine, and, although the compound

Cytochrome P4501A induction by ¯ uparoxan
169
was not found to be a mutagen in cultured human peripheral lymphocytes and did
not cause gene mutation when administered to Chinese hamster ®broblasts in
culture (unpublished data), the potential Ah receptor binding a nity (PEC &! ) and
CYP1A induction capability were calculated. Using a QSAR expression generated
for a series of DCDDs, the PEC &! computed for ¯ uparoxan was extremely low
compared to a wide range of known CYP1A inducers (Lewis 1996). Indeed, even the
dichloro analogue of ¯ uparoxan, GR37459 (®gure 1b), which shows a considerable
structural similarity to the highly potent CYP1A inducer, TCDD (®gure 1c), shows
a low PEC &! (table 1). Likewise, the CYP1A induction potential (log scale),
calculated using a QSAR expression derived for a series of structurally diverse
chemicals, was extremely low for ¯ uparoxan and GR37459 (0±456 and 0±684
respectively), compared with TCDD (2±503) or, as previously calculated (Lewis
1
996), to 3-MC (2±371) or BNF (2±929). The low calculated values for ¯ uparoxan, in
part, result from the relatively low log P and are re¯ ected in the poor a nity for
the CYP1A isozymes apparent in the in vitro studies (table 2, ®gure 3). Despite this,
the data obtained in vivo in the rat (®gure 4) demonstrate that administration of
¯
uparoxan can produce hepatic CYP1A induction. If the assumption is made that
induction was maximal after the ®rst dose, then the actual increase in EROD activity
(
table 2), normalized for dose (mmol}kg), is much higher than anticipated for
¯
uparoxan (119), but is about 10- fold lower than for BNF in this study (1180).
Interestingly, the greater molecular ` depth ’ (compared to the TCDDs) imparted
to ¯ uparoxan by the saturated pyrrole ring, has previously been noted as a possible
descriptor for CYP1A2 versus 1A1 selectivity for aromatic amines (Lewis et al.
1
994) and immunoblotting of the rat microsomes indicated that induction by
uparoxan is CYP1A2 selective (®gure 5). This additional depth will also be a
¯
feature of the CYP1A2 selective methylenedioxyp henyl (MDP) compounds,
isosafrole and piperonyl butoxide (Adams et al. 1994), and may indicate that the
hydrocarbon- responsive element diŒers between the CYP1A1 and 1A2 genes : the
form of the resultant complex between the Ah receptor and diŒerent ligands
allowing diŒerential induction. For example, the tight binding of TCDD may
produce a conformational change compatible with both 1A1 and 1A2 genes, whilst
a weak, deeper ligand may only permit attachment to the 1A2 gene. Such
diŒerentiation might also apply for induction through post-transcriptional stabil-
isation of mRNA, which has been shown to play a greater role in CYP1A2 induction
than 1A1 induction (Pasco et al. 1988). In this respect, it is perhaps worth noting that
preliminary data (not shown) on the dichloro analogue of ¯ uparoxan, GR37459,
suggests that it is capable of inducing both CYP1A1 and 1A2.
In addition to the MDPs a number of tricyclic compounds, such as acen-
aphthylene and dibenzofuran, have also been shown to act as CYP1A2- selective
inducers. As this induction occurs in TCDD receptor- de®cient mice, it has been
proposed that the selectivity is via an Ah receptor- independent mechanism (Cook
and Hodgson 1985, Chaloupka et al. 1994). In the case of the MDPs, induction is a
two-phase process in which increased CYP1A2 protein synthesis follows an initial
inhibition of the enzyme by formation of MDP metabolite- P450 complexes (Ryu et
al. 1995), and a number of studies have suggested the presence of an autoregulatory
loop in which the presence of functioning CYP1A protein negatively controls its
own gene expression (Puga et al. 1990, RayChaudhuri et al. 1990, Jorgensen and
Antrup 1996). This may involve some, as yet unidenti®ed, endogenous substrate for
the enzymes, the concentration of which would rise in the presence of inhibitors and

1
70
A . P. Beresford et al.
subsequently increase CYP1A gene transcription and protein production (Beresford
993, Reick et al. 1994). With ¯ uparoxan, although the in vitro metabolic data
1
presented here (®gures 2 and 3, table 2) show little evidence that the compound is
either a substrate or inhibitor for the CYP1A enzymes, it is worth noting that after
oral dosing at 50 mg}kg, the maximum plasma concentrations in rats (around
6
1
0 lg}ml or 380 lm) do exceed the K ’s calculated for both CYP1A1 (356 lm) and
i
A2 (311 lm). Hence, it remains possible that CYP1A induction by ¯ uparoxan
could result from some negative feedback mechanism activated by a decrease in
enzyme activity ; though it is not clear why this should be CYP1A2 selective, unless
the K against 1A1 in rat is higher than that for the human isozyme.
i
In man, hepatic CYP1A2 activity appears to vary substantially between
individuals (Sesardic et al. 1988, Lucas et al. 1993), which makes the estimation of
induction due to drug administration di cult. In addition to genotypic variability,
phenotypic diŒerences due to the eŒects of dietary and environmental chemicals on
CYP1A2 (Conney et al. 1976, Pantuck et al. 1979) make it di cult to assess the
clinical relevance of any induction seen. This has been particularly well illustrated
by debates over the signi®cance of CYP1A induction caused by the gastric acid
suppressant, omeprazole (Petersen 1995). In the study performed herein, the
exposure of volunteers to phenacetin before and after ¯ uparoxan treatment was used
as an indicator of CYP1A2 activity and showed a decrease in exposure (as plasma
AUC ) following drug treatment. Unfortunately, considerable variability in the
plasma concentration}time pro®les for phenacetin were seen between volunteers
before and after both placebo and ¯ uparoxan treatment. This probably re¯ ects
inherent variability in the ®rst- pass metabolism of phenacetin, as well as diŒerences
in absorption rates, as demonstrated by Raa¯ aub and Dubach (1975). Whilst the
clearance of phenacetin is not due solely to CYP1A2 activity (Mineshita et al. 1986),
the enzyme is considered to be largely responsible for its O-deethylation to
paracetamol (Sesardic et al. 1988). Consequently the plasma phenacetin AUC and
the total urinary excretion of paracetamol were compared and the ratio of these
parameters did indicate a signi®cant increase in O-deethylation following ¯ uparoxan
treatment. However, in view of the variability seen between individuals and the
known eŒects of diet and environment on CYP1A2 previously mentioned, as for
omeprazole, the change was not considered to be of clinical concern.
Individual variability considered, CYP1A2 is the third major isoform present in
human liver (Shimada et al. 1994). In untreated laboratory rats, however, the
hepatic CYP1A2 content would appear to be negligible (Nedelcheva and Gut 1994).
Certainly the level of EROD in the control rat microsomes prepared in the current
work, was lower (at least 5-fold) than that of the human liver microsomes when
expressed per mg microsomal protein (table 2), and this activity in the rat may
mainly be due to CYP2D6 (Burke et al. 1994). The situation therefore exists that a
potentially major route for metabolism of a xenobiotic in man may be only a minor
route in the species most likely to be used for its metabolism and toxicity testing.
This would be particularly important if the metabolic product was an activated
chemical species.
Currently, inference of CYP1A involvement in metabolic pathways is made
using microsomes from rats dosed with inducers such as 3- MC, BNF and isosafrole,
in combination with selective CYP1A inhibitors such as ANF, furafylline (Sesardic
et al. 1990) or anti-CYP1A antibodies (Edwards et al. 1993). Of the inhibitors,
furafylline has proved to be the most selective CYP1A2 inhibitor and can be

Cytochrome P4501A induction by ¯ uparoxan
171
employed in vivo (Tarru s et al. 1987b), although it exhibits a considerable
!
interspecies diŒerence in potency (Sesardic et al. 1990a). The inducers listed above
are themselves potentially toxic, with only isosafrole being relatively selective for the
induction of rat hepatic 1A2 (Thomas et al. 1983) and, unfortunately, known to bind
to the enzyme which it induces (Ryan et al. 1980). Considerably more work would
be required to con®rm that ¯ uparoxan has minimal eŒects on other metabolic
enzymes (including phase II systems). However, its minimal interaction with the
CYP1A2 isozyme which it induces could make the compound a useful tool for in
vitro and in vivo studies on this isozyme or to investigate Ah receptor- independent
mechanisms of CYP1A induction.
Acknowledgements
We gratefully acknowledge the considerable eŒorts of Mrs D. Herriott of the
GlaxoWellcome BioMet Division in establishing the potential CYP1A2 selectivity
of ¯ uparoxan and we are indebted to Dr G. R. Park, MD, FRCA, Director of the
John Farman ICU, Addenbrooke’s NHS Trust, Cambridge, for the human liver
surgical waste tissue, which was provided to GlaxoWellcome with approval from the
district Ethical Review Board. We also thank Dr S. W. Ellis of the University
Department of Medicine and Pharmacology, Royal Hallamshire Hospital, She eld,
for his expertise and help in the provision of S . cerevisiae microsomes containing
heterologously expressed human CYP1A1 and 1A2 enzymes, and to Dr R. J.
Edwards of the Department of Clinical Pharmacology, Royal Postgraduate Medical,
Hammersmith Hospital, London, for provision of speci®c CYP1A1 and 1A2
antibodies.
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