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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1070
SEEKI�G THE SOFT SPOTS
Gabriele Cruciani1, Riccardo Vianello
2
1University of Perugia, Italy;
2Molecular Discovery, UK.
Metabolic stability is crucial in allowing drugs to reach therapeutic concentrations, and so
highly labile compounds are often filtered out early in the discovery process using metabolic
clearance assays. However, such assays do not provide information on the ‘soft spots’ on the
compound that undergoes rapid metabolism, leaving rational design to overcome this issue.
An in silico procedure called MetaSite has now been developed in order to predict soft spot
locations in drug candidates. The procedure can also be coupled with mass spectrometry (MS)
data from high-throughout clearance assays, so that the identification of soft spots can be
obtained more rapidly and at an earlier stage in the drug discovery process.
Data on metabolism mediated by cytochrome P450 (CYP) enzymes, the major contributors to
drug metabolism, are typically obtained using high-throughput microsomal assays early in the
compound optimization process. Linking experimental data with MetaSite could considerably
increase the likelihood of focusing optimization efforts most appropriately to improve the
metabolic stability of compounds. Numerous papers demonstrate that, by incorporating this
procedure in early drug discovery, more than 85% of soft spots were correctly identified.
Furthermore, gathering information in this way on structure–metabolism relationships could
help develop in silico models that not only identify soft spots with high success rates, but also
suggest chemical strategies for stabilization.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1071
SCALI�G DATA FROM RECOMBI�A�T CYTOCHROME TO HUMA� LIVER
MICROSOMES: IMPACT OF SCALI�G FACTORS A�D EXPRESSIO� SYSTEMS
Patrice Dehanne, Olivier Barberan
Aureus Pharma, 174 Quai de Jemmapes 75010 Paris, France.
Drug-drug interactions (DDIs) can lead to severe side effects and drug toxicities and have
resulted in refusal of approval, and withdrawal of drugs from the market. Many DDIs are due
to the co-administration of another drug that can alter the drug metabolism, in particular by
inhibition of cytochromes P450 (CYPs). Recombinant CYPs P450 systems are routinely used
in the industry to determine the intrinsic clearances of a drug candidate by each isozyme..
Anticipating potential issues through the identification of P450 isozymes concerned with the
metabolism of a drug candidate at an early stage of development is a great challenge for the
pharmaceutical industry. To answer this question, Aureus Pharma has developed a tool, DDI
Predict®
, based on a Knowledge database AurSCOPE ADME/DDI®
containing reliable
published data on pharmacokinetics and metabolism. This tool provides a fast and accurate
risk assessment of DDIs between a new chemical entity and drugs which are on the market or
have been withdrawn. Various approaches have been proposed to extrapolate data from
recombinant enzymes to human liver microsomes: abundance, RAF (Relative Activity
Factor), ISEF (Inter System Extrapolation Factor), all integrated in the DDI Predict®
application.
Cytochrome hepatic abundance was determined by a literature analysis. The scaling factors
RAF and ISEF were calculated using recombinant Vmax and human liver microsomes data
from AurSCOPE ADME/DDI®
database using the FDA probes substrates. The first step of
this study was to determine the relative contribution of each CYPs involved in the metabolism
of selected drugs using four different expression systems (lymphoblastoid, baculovirus, E.
coli, yeast) according to the approaches mentioned above. In the second part, we compared
clearance obtained from recombinant systems with values published in the literature.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1072
I�HIBITIO� OF HUMA� CYTOCHROME P450 E�ZYME (CYP) A�D THE
PREDICTIO� OF DRUG-DRUG I�TERACTIO�S (DDI): HA�DLI�G DIFFERE�T
I� VITRO-I� VIVO EXTRAPOLATIO� (IVIVE) TOOLS
�icoletta Pons, Fiorenza Bonomo, Paolo Rossato GlaxoSmithKline S.p.A., Medicine Research Center, Preclinical Development Drug
Metabolism and Pharmacokinetics Department, Verona, Italy.
The prediction of drug-drug interactions (DDIs) using in vitro enzyme kinetic data has been
and still is an area of increasing advances. This has proven to be a valuable endeavour
because DDIs remain an important issue in clinical practice and in the discovery and
development of new drugs. The earlier the potential for DDIs can be identified in new
chemical entities, the greater is the likelihood that this property can be removed through
improved design of the molecule. Also, for those compounds already undergoing clinical
trials, in vitro DDI data and their extrapolation can be important in the design of adequate and
appropriate clinical DDI studies. With increased understanding of the interactions between
drugs and drug-metabolizing enzymes a mechanistic approach to predict DDIs can be taken.
Several in vitro assays and modelling approaches have been developed by the pharmaceutical
companies in recent years in order to study new chemical entities in terms of DDI potential,
regarding particularly: 1) CYP inhibition, classified simplistically as reversible (mostly
competitive) and quasi-irreversible (mechanism based inactivation-MBI), and 2) CYP
induction.
In this presentation the use of in vitro IC50 parameter for reversible inhibitors and inactivation
kinetic parameters (Kinact and KI) for mechanism-based inactivators of human CYP3A4
enzyme has been examined, using human CYP selective activity markers in pooled human
liver microsomes [1]. These data have been combined with other parameters (systemic Cmax,
estimated hepatic inlet Cmax, fraction unbound, in vivo P450 enzyme degradation rate
constants and fraction of the drug cleared by the inhibited enzyme) to predict in vivo
increases in exposure of co-administered drugs. Finally, the predictions have been compared
with in vivo DDIs observed in clinical studies reported in the scientific literature using
reported equations [2]. In general, the use of unbound hepatic inlet concentration Cmax of
inhibitor has yielded the most accurate predictions of DDI. The predictions of DDIs have
been carried out using commercially available software Simcyp®
, population based ADME
simulator [3,4].
1. Grime KH et al. (2009) Eur J Pharm Sci 36:175
2. Obach RS et al. (2006) Drug Metab Disp 35:246
3. Jamei M et al. (2009) Expert Opin Drug Metab Toxicol 5:211
4. Rostami-Hodjegan A et al. (2007) Nat Rev Drug Discov 6:140
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1073
DY�AMIC CULTURE I� MULTI COMPARTME�T BIOREACTOR
UPREGULATES CYTOCHROME EXPRESSIO� I� HUMA� HEPATOCYTES
Bruna Vinci1, Cedric Duret
2, Patrick Maurel
2, Arti Ahluwalia
1
1Centro Interdipartimentale di Ricerca “E. Piaggio”, University of Pisa, Italy;
2Inserm U632
/ UM-I EA3768, Hepatic Physiopathology, Montpellier, France.
In vitro liver models for toxicity testing suffer from a number of drawbacks, including short
term viability, and phenotypic changes mainly associated with huge drops in P450 expression
of hepatocytes. This has been generally attributed to the fact that the complexity of the
physiological environment is not replicated in petri dishes or microplates. In fact, all cells are
exquisitely sensitive to their micro environment which is rich with cues from other cells, and
from mechanical stimuli due to flow, perfusion and movement. Current methods for
investigating cellular responses in vitro are inadequate in this sense, since the complex
interplay of mechanical and biochemical factors is in fact absent.
To address these issues we have developed a ‘system on a plate’ modular multicompartmental
bioreactor (MCB) array which enables microwell protocols to be transferred directly to the
bioreactor modules, without redesign of cell culture experiments. The new system offers
mechanical stimuli from flow and biochemical stimuli from cells placed in connected
modules, and can be used for assessing the human hepatotoxicity potential of drugs, or for
pharmacological or pharmacokinetic studies.
Human hepatocytes were cultured in the MCB system, by connecting eight modules in series,
and seeding cells in the long term medium described in [1], three weeks after isolation. The
cells were subject to a flow rate of 180 µl/min for up to 7 days, and gene expression of a large
number of enzymes were quantified with respect to freshly isolated hepatocytes from the
same liver sample, as well as cells in control (multiwell) conditions.
The results show that (as compared with the static controls) most P450 enzymes (CYP 3A4,
2B6, 2C9, 1A2, 3A7,1A1) are upregulated in the MCB, as are several phase II enzymes,
reaching about the same levels found in freshly isolated hepatocytes.
1. Ferrini JB et al. (1997) Chemico-Biol Inter 107:31
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1074
N
O
O
Ar1O
CH3
O
Ar2
H3CO
H3CO
OCH3H3CO
H3CO
OCH3
Ar1, Ar2 =
A B
H3CO
H3CO
OCH3 H3CO
H3CO
OCH3
C D
MULTI DRUG RESISTA�CE A�D CELL TRA�SPORT SYSTEMS: HISTORICAL
�OTES A�D PERSPECTIVES
Giampietro Sgaragli
Dipartimento di 9euroscienze, Università degli Studi di Siena, via A. Moro 2, 53100 Siena.
Many tumor cells become resistant to commonly used cytotoxic drugs due to the
overexpression of ATP-binding cassette (ABC) transporters. Pgp (MDR-1, ABCB1), MRP-1
(ABCC1) and BCRP (ABCG2) have been demonstrated to pump out of the cell a wide
selection of anticancer drugs thus rendering them ineffective [1]. Several generations of
inhibitors of these transport systems have been examined so far in preclinical and clinical
settings in the hope of overcoming multi drug resistance (MDR) of cancer cells. None of these
compounds, however, has gained access into the clinical practice. The main problems with
these drugs are poor specificity, low potency and interference with physiological functions
which gives rise to drug interactions [2]. In the last few years my Colleagues and I have
investigated several organic compounds as MDR reverters (e.g. 3,5-dibenzoyl-1,4-
dihydropyridines, taxuspines…).
Recently, among novel transporter-dependent MDR inhibitors, four geometrical isomers
containing the 9,9-bis(cyclohexanol)amine scaffold esterified with two different aryl acids
(Ar1 = 3,4,5-trimethoxy-benzoyloxy; Ar2 = 3-(3,4,5-trimethoxyphenyl)acryloyloxy) were
obtained. At sub-uM concentrations they enhanced both nuclear pirarubicin retention and
doxorubicin cytotoxicity in doxorubicin-resistant leukaemia K562 cells, 1d exhibiting the
highest activity [3]. When evaluated for their Pgp inhibiting activity, by measuring cyto-
fluorimetrically the retention of Pgp substrate rhodamine 123 (R123) in MDR1-gene
transfected mouse T-lymphoma L5178 cells, the most potent were 1d and 1c the other two
showing IC50 values two-order of magnitude higher. The incomplete reversion upon repetitive
cell washings of the inhibition of Pgp caused by 1d prompted us to investigate the role of the
double bond between C2 and C3 of Ar2.
Sigla Compound Ar1 Ar2
DM407 1a (cis/trans) A B
DM410 1b (trans/trans) A B
MC185 1c (cis/cis) A B
MC176 1d (trans/cis) A B
MC252 2a (cis trans) A C
MC247 2b (trans/trans) A C
MC250 2c (cis/cis) A C
MC251 2d (trans/cis) A C
MC257 3a (cis/trans) A D
MC258 3b (trans/trans) A D
MC259 3c (cis/cis) A D
MC260 3d (trans/cis) A D
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1075
By substituting in Ar2 a 3-(3,4,5-trimethoxyphenyl)propionyloxy or a 3-(3,4,5-
trimethoxyphenyl)propioloyloxy radical, two groups of four isomers characterized by single
and triple bond, respectively, between C2 and C3 were obtained. Whilst the single-bond
isomers were equiactive, both 2d and 2c showed IC50 values two-order of magnitude higher
than the double-bond analogues 1c and 1d. On the contrary, the triple-bond isomers behaved
like the double-bond compounds, the most potent being 3d and 3c, the other two showing
IC50 values two-order of magnitude higher. Analysis of the 1c and 3d inhibition curves
showed both a high-affinity and a low-affinity constant, thus suggesting the existence of two
inhibitor-binding sites in the recognition domain of Pgp. The persistence of inhibition of Pgp-
mediated R123 efflux by the most active compounds showed itself to be intermediate between
that caused by cyclosporine A (Pgp substrate) and GF120918 (non Pgp substrate). When the
R123 concentration used to load cells was increased, there was a leftward shift of the
concentration-inhibition curve with all compounds examined, with the exception of 3d where
a rightward shift was evident. These findings indicate that both isomeric geometry and
limitation of Ar2 conformational freedom are crucial for Pgp inhibition by the 9,9-
bis(cyclohexanol)amine esters. Moreover, a positive modulation of the Pgp inhibition was
caused by R123 itself with most of the compounds considered here. In conclusion, 9,9-
bis(cyclohexanol)amine esters were the most potent Pgp inhibitors so far investigated in this
laboratory. They appear useful probes for studying Pgp and other transport systems associated
to MDR in cancer cells and very promising leads for the development of safe and effective
MDR reverters.
(This work was financed by Fondazione MPS, PAR and MIUR)
1. Fusi F et al. (2006) Curr Drug Targets 7:949
2. O’Connor R (2007) Anticancer Res 27:1267
3. Martelli C et al. (2009) J Med Chem 52:807
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1076
BIOMARKERS OF BIOTRA�SFORMATIO�, OXIDATIVE STRESS A�D
GE�OTOXICITY I� MOSQUITO FISH EXPOSED TO PW
Ilaria Caliani1, Maria Ferraro
2, Silvia Casini
1, Gabriele Mori
1, Silvia Maltese
1, Letizia
Marsili1, Maria Cristina Fossi
1
1Department of Environmental Sciences, University of Siena, Via Mattioli, 4 53100 Siena,
Italy; 2E9I S.p.A. Exploration & Production Division, Unita’ AMTE, Via Emilia 1, San
Donato Milanese, Italy.
Produced water (PW) is a complex mixture containing residual hydrocarbons, trace elements,
naturally occurring radioactive material and potentially toxic chemicals. The aim of this study
was to compare the CYP1A enzymatic activity in vitro to levels of PAH bile metabolites, and
to evaluate the PW toxicity in mosquito fish by examining, in addition, phase II enzymes,
oxidative stress and genotoxicity biomarkers. This research is part of the ENI S.p.a. project
entitled “Advanced Processes for Oily Water Treatment”. The test organism, mosquito fish
(Gambusia affinis), was divided into male and female groups and exposed for 8 and 30 days
to PWs from an Italian on-shore oil plant and from an Italian off-shore gas platform.
Specimens were also exposed to water and sediment from an Italian ship canal (Navicelli
Channel). The induction of phase I (EROD, BPMO activity) and phase II (GST activity)
biotransformation systems, FACs, LPO, antioxidants enzymes (GPX, GR, CAT), and a non-
enzymatic antioxidant (glutathione—GSH) were investigated. DNA damage was evaluated in
erythrocytes by single cell gel electrophoresis (Comet assay). The experimental groups
showed significantly higher EROD and BPMO activity compared with the control group
during the exposure to PWs from gas and oil installations. A decrease of CYP activities was
observed, in comparison with control, during the exposure to water and sediment from ship
canal, probably due to high concentration of lipophilic contaminants. The CYP1A responses
were also compared with the other biomarkers; in particular, positive Spearman correlations
were observed between EROD (or BPMO) activity and PAH metabolites in bile and Comet
assay.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1077
CYTOCHROME P450 I�DUCTIO� I� CRUSTACEA�S (BAR�ACLES A�D
CRABS) AS A DIAG�OSTIC TOOL I� THE ECOTOXICOLOGICAL
ASSESSME�T OF OFFSHORE GAS EXTRACTIO�: A REVIEW OF TWO
MO�ITORI�G STUDIES
Gabriele Mori1, Silvia Casini
1, Letizia Marsili
1, Ilaria Caliani
1, Silvia Maltese
1, Maria
Ferraro2, Cristina Fossi
1
1Department of Environmental Science, University of Siena;
2E9I S.p.A. E & P Division,
AMTE Dept., Via Emilia 1, San Donato Milanese, Milan, Italy.
The present work constitutes part of two more extended research projects called “Biomare” and
“Biodiversity” developed by the Department of Environmental Science at the University of Siena and
supported by ENI-Exploration and Production. Since the 1960’s Italian gas offshore activity is
predominantly on the northern sector of the Adriatic sea, where about 30 installations are operative.
During the year 2008 the total amount of natural gas production averaged 191,000 barrels oil
equivalent per day (boe/day) and represented approximately 70% of hydrocarbon production in Italy.
Natural gas fields located in the Adriatic Sea collectively accounted for 54%. Produced water is the
largest wastewater stream in the oil and gas exploration and production processes. Toxicological
effects of produced water on living organisms may be due to absorption of water soluble components
through their surface epithelia (e.g. body surface and gills) and/or to oral ingestion and digestion of
particulate material. Several studies were carried out on the toxicological effects of produced waters
on marine fauna such as fish and invertebrates. The majority of the work published on this aspect
concernes bivalve molluscs, particularly mussels, but there is very little information available on other
marine invertebrates like lower order crustaceans, a large, diverse and complex group of animals. The
aim of this work was to propose a suite of biomarkers (Benzopyrene monoxygenase activity (BPMO),
NAD(P)H Reductases activity, Acetylcholinesterase activity (AChE), Porphyrins, Antioxidant
enzymes), as well as levels of PAHs and heavy metals as potential diagnostic tools. We have
employed these diagnostic tools in the ecotoxicological monitoring of extractive offshore activities,
applying them to marine invertebrates, proposed here as new bioindicators, namely the barnacles
Balanus perforatus and Balanus trigonus and the crab Liocarcinus depurator. Previous studies carried
out by this Department demonstrated that barnacles Balanus perforatus and Balanus trigonus and the
crab Liocarcinus depurator are the most common species of crustaceans identified in macrobenthic
assemblages along the piles of offshore installations. In order to validate the use of crustaceans in
ecotoxicological monitoring of offshore activities, the experimental work was carried out in two
phases: first in the laboratory, then in the field. During phase 1, B. perforatus and B. trigonus were
experimentally exposed to water containing increasing doses of gasoline (a liquid mix of hydrocarbons
extracted with gas, and produced water) to check the sensitivity of selected bioindicators and identify
the most suitable biomarkers for each experimental design. Field studies followed in order to validate
the use of the multi trial biomarkers approach in a real environmental situation. Specimens of B.
perforatus, B. trigonus and L. depurator were collected along the piles of 4 different platforms in two
different areas: north Adriatic and Ionian sea. Laboratory studies showed high sensitivity of response
in barnacles for all biomarkers tested, particularly MFO and esterases, and high bioaccumulation
capacity. Field results showed differences in biomarker responses and PAH levels between the two
sites for barnacles and crabs. Moreover a different sensitivity of response between the two species of
barnacles was found. In conclusion, this study provided innovative results for the use of the selected
bioindicators and for the responses generated after chronic and acute exposure to petroleum
derivatives.
These results suggest that barnacles and crabs could be used in future biomonitoring programs to
evaluate both the effects of petroleum derived contamination, and the level of contamination itself.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1078
CYP1A A�D CYP3A GE�E EXPRESSIO� A�D CATALYTIC ACTIVITIES I� RED
MULLET MULLUS BARBATUS AS SE�SITIVE I�DICATORS OF PETROLEUM
HYDROCARBO� CO�TAMI�ATIO�: A FIELD STUDY I� FRO�T OF A� OIL
REFI�ERY
C. Della Torre1, I. Corsi
1, G. Guerrini
1, M.P. Tomasino
1, G. Perra
1, F. �ardi
2, F. Frati
2,
S. Focardi1
1Department of Environmental Sciences “G. Sarfatti”, via Mattioli, 4, University of Siena,
53100 Siena, Italy; 2Department of Evolutionary Biology, via Aldo Moro 2, University of
Siena, 53100, Siena, Italy
The aim of the present study was to investigate the response to petroleum hydrocarbon
contamination of CYP1A and CYP3A genes and related enzyme activities in the liver of the
red mullet Mullus barbatus.
In this field study specimens of red mullet were collected in the fall of 2007, from three sites
along a gradient of petroleum contamination from an oil refinery.
The expression of CYP1A and CYP3A genes was investigated in liver, by quantitative Real-
time PCR (based on a partial sequencing of the two genes), and enzymatic activities were
measured by assaying EROD, BROD, B(a)PMO and BFCOD. The 16 most toxic PAHs were
also measured in liver as an indication of petroleum contamination. Gene expression and
enzymatic activities profiles were strictly related to the spatial arrangement of sampling sites,
with highest expression/activities detected in specimens collected in front of the oil refinery.
A significant increase of CYP1A and CYP3A expressions, as well as EROD, B(a)PMO and
BFCOD activities was observed, compared to specimens from the reference site. A similar,
but less marked, trend was observed also for BROD activity. REST analysis indicated a
significant and comparable up-regulation of CYP1A and CYP3A, related to the proximity of
the oil refinery site. A strong positive correlation (r = 0.87) was found between CYP1A and
CYP3A activities. CYP1A and EROD activity were slightly correlated (r = 0.43), while no
correlation was observed between CYP3A and BFCOD activity.
Overall, our results suggest that red mullet inhabiting a petroleum contaminated site display
physiological modifications, that are detectable and measurable by using CYP1A and CYP3A
at both transcriptional and post-transcriptional level.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1079
CYPs, TRA�SPORTERS, RECEPTORS A�D A�TIOXIDA�T E�ZYMES I�
VARIOUS BRAI� REGIO�S OF PIG
Emilia Vaccaro1, Francesco Rossignolo
1, Roberto Tolando
1, Annalisa �annelli
2, Paolo
Rossato1, Mario Pellegatti
1, Vincenzo Longo
2, Pier Giovanni Gervasi
2
1GlaxoSmithKline S.p.A., Medicine Research Center, Preclinical Development Drug
Metabolism and Pharmacokinetics Department, Verona, Italy; 2Istituto di Fisiologia Clinica,
C9R, via Moruzzi 1, 56100 Pisa, Italy.
The basal expression of the cytochrome P450s (CYP) enzymes (CYP1A1, 1A2, 1B1, 2S1, 2B22,
3A22, 3A29, 3A46), nuclear receptors (AhR, PXR, CAR, Nrf2), transporters (MDR1, MRP1, MRP2,
LRP) and antioxidant enzymes (GST, catalase, superoxide dismutase, GSSG-reductase, GSH
peroxidase) was evaluated by Real-Time or traditional PCR in different pig brain regions (cortex,
cerebellum, midbrain, hippocampus) and in blood-brain interfaces (meninges and brain microvessels).
In addition, the effects of the typical drug metabolizing enzymes “inducers”, β-naphthoflavone (βNF)
and rifampicin (RIF), on the expression of the above listed genes was evaluated in the same brain
regions.
The following enzymatic activities were also determined in the same brain regions: ethoxyresorufin O-
deethylase (EROD, marker for CYP1A1), methoxyresorufin O-deethylase (MEROD, marker
CYP1A2), 7-benzyloxyquinoline O-debenzylase (marker for CYP3As), anthraldehyde oxidase
(marker for CYP2Bs), GST, catalase, superoxide dismutase, GSSG-reductase, GSH-peroxidase.
The mRNAs for AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at
different extent, in all the brain regions, while CYP2S1 mRNA was detected only in midbrain. In the
blood-brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the
hepatic one and even higher for CYP1B1 and Nrf2. The mRNA levels of the individual CYP3As,
CYP2B22, CAR and PXR in various cerebral regions and capillaries, were about or below 10% of the
corresponding hepatic mRNA values, whereas in meninges, the levels of CYP2B22, CAR and PXR
transcripts were similar to those observed in liver. The basal expression of MRP1, MRP2 and LRP
mRNAs, in the cerebral regions, were below 45% of those in liver, while the level of MDR1 mRNA
was comparable to that of liver. Notably, in capillaries and even more in meninges, the basal mRNA
expressions of all these transporters were comparable or higher than in liver.
βNF treatment caused induction of CYP1A1 and 1B1 mRNA levels in various CNS areas (but not of
AhR, CYP1A2 and Nrf2 mRNA); notably, CYP1A1 mRNA was increased about 300-fold in the
microvessels. After βNF treatment the enzymatic activity of EROD increased in microsomes but not in
mitochondria of all the CNS areas. No corresponding increase in microsomal MEROD activity was
observed. However, in the control animals, the mitochondrial EROD activities were comparable (in
midbrain and meninges) or higher (in cortex, cerebellum and hippocampus) than the microsomal ones,
suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities
of GST and antioxidant enzymes were found to be evenly distributed in all CNS tissues, only
marginally affected by βNF treatment and lower than the corresponding hepatic activities.
After RIF treatment, CYPs and transporters mRNA expression in brain regions did not substantially
change. The only exceptions were: CYP3A22 and 3A29 (cortex and hippocampus) and CYP2B22
(meninges). Unexpectedly, , an increased enzymatic activity was observed only for CYP3A, but only
in capillaries and not in all the other brain regions tested, unlike the liver.
The data obtained demonstrate that all the analysed genes are expressed in the tested pig brain regions,
even though some notable differences exist with respect to the liver. The high expression of metabolic
enzymes and transporters found in blood-brain interfaces could represent a very important defence of
the CNS toward toxins. Furthermore, a notable difference between brain and liver was observed in the
response to xenobiotics of AhR- and PXR-regulated enzymes. AhR-regulated CYPs positively
respond to induction with some high degree of response in blood-brain barrier interfaces. Whereas,
PXR-regulated enzymes, despite the presence of PXR and CAR, were resistant to induction, indicating
that the regulation of these proteins is more complex in brain than in liver.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1080
RAT BRAI� CYTOCHROME-P450 ACTIVITIES
�icola Pasquale Caradonna1, Giulia Franco
2, Ugo Zanelli
1, Goran Westerberg
1,
Massimo Valoti2
1Siena Biotech S.p.A. via del Petriccio e Belriguardo 35, Siena, Italy;
2Department of
9eurosciences, Pharmacology Section, University of Siena, Via Aldo Moro 2, Siena, Italy.
Although the overall cytochrome P450 (CYP) level in the brain is approximately 0.5-2% of
that in liver microsomes [1], it can play an important role in therapeutic and side-effects
responses of centrally acting drugs [2]. Furthermore the CYPs present in the various brain
regions differ in concentrations and isoform distribution. The objective of the current study
was to investigate the metabolism of CYP marker substrates (testosterone, dextromethorphan,
fluoxetine and bufuralol) in rat brain microsome preparations [3], comparing brain and liver
metabolism of these compounds.
UPLC/MS-TOF system allowed high resolution of isomer metabolites like testosterone
hydroxylates, while accurate mass (0.01Da window) allowed the detection of different types
of transformations with low background noise, relatively high sensitivity and wide dynamic
range. Dextromethorphan O- and N-dealkylation, fluoxetine demethylation and bufuralol
hydroxylation activities were all observed with brain microsomes, but the formation rate of
metabolites was about 3 orders of magnitude lower than the activity observed in liver.
However, our testosterone data showed a different metabolite pattern respect to those
observed in liver: with rat brain microsomes testosterone metabolism mainly produced the 2ß-
, 6α- and 16ß-hydroxytestosterone instead of the 2α-, 6ß- and 16α-hydroxytestosterone found
with liver microsomes. Moreover, gender differences in testosterone brain metabolism were
observed: 2ß- and 6α- hydroxytestosterone formation were more abundant in male than in
female brain microsomal preparations, 2α- and 16α-hydroxytestosterone were found only in
incubations with male rat brain microsomes.
Chlorpromazine was selected as model compound since differences were found between brain
and liver in the formation of chlorpromazine sulphoxide, nor-chlorpromazine and 7-
hydroxychlorpromazine. Further investigation of kinetic parameters both in liver and brain
microsomes will be presented. Our data highlight a qualitatively different CYP-dependent
metabolism of xenobiotics in the brain compared to the liver and this could contribute to
unpredictable therapeutic and/or toxicological effects of CNS drugs. This work was supported by the European Union. FP6 PRIORITY LSH-2005-2.2.0-8: Small-ligand libraries:
improved tools for exploration and prospective anti-tumour therapy. DePPICT Project (Designing Therapeutic
Protein-Protein Inhibitors for Brain Cancer Treatments) Contract number: LSHC-CT-2007- 037834
(http://www.deppict.eu/home.jsp)
1. Miksys SL et al. (2002) J Psychiatry Neurosci 27:406
2. Meyer RP et al. (2007) Curr Drug Metab 8:297
3. Dragoni S et al. (2003) J Neurochem 86:1174
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1081
A LO�G-LASTI�G DRIFT I� THE DRUG METABOLISI�G FIELD
Pier Giovanni Gervasi
Istituto di Fisiologia Clinica, C9R, Via Moruzzi 1, 56100, Pisa, Italy.
During the last 35 years, I have been deeply involved in the drug metabolising system, first
working in the Institute of Mutagenesis and Differentiation and later in the Institute of
Clinical Physiology of the CNR. At the very beginning, I was asked to address the
bioactivation of vinyl cloride which was found to form a mutagenic and carcinogenic
epoxidic intermediate. Therefore, in addition to vinyl cloride, I, along with my collaborators,
went on to study the biotransformation process of many olefins (vinyl cyclohexene, isoprene,
butadiene, 1-hexene), polycyclic compounds such as benz(a)anthracene and drugs containing
double bonds such as sobrerol, adriamycin, anthraquinones, all potentially able to produce
reactive epoxides. To perform these studies, experiments with animals (mainly rodents), cell
cultures, subcellular fractions, and pure enzymes were carried out. In particular, for the use of
individual cytochrome P450 (CYP), I began, after a training in the Lab of F. Guengerich, a
laborious purification of the principal P450s from rats, namely CYP1A1, 2B1, 2C11 and 2E1,
at that time not yet commercially available. These enzymes proved very useful to examine the
oxidative metabolism of many drugs (supplied by industry) and solvents, such as formamides
and t-butyl ethers (a study supported by an EEC grant).
During these years, I came across, by chance, to the presence of the drug metabolising system
in the nasal mucosa of rat. Since then, the biotransformation potential of nasal tissues (mainly
olfactory) has been very widely investigated in many vertebrate species (from fish to man).
In the last fifteen years, the molecular biology approach was introduced in our lab; then
cloning, regulation and protein expression in heterologous systems (mainly E. coli) were
addressed to unveil the presence and function of novel CYPs. These techniques were largely
adopted to identify and monitor the expression of CYP genes (i.e. 1A1 and 3A) in fish as
pollution biomarkers. More recently, these techniques were used to investigate the specific
presence and regulation of CYPs, receptors (AhR, CAR, PXR, HNF4, PPAR) and
transporters (MDR1, etc) in many organs (liver, kidney, small intestine, heart, lung, nasal
tissues, coronary and brain regions) of pig, as a new animal model for humans.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1082
PROTECTIVE EFFECT OF LISOSA� G AGAI�ST OXIDATIVE STRESS
I�DUCED BY CISPLATI� I� RAT TISSUES
V. Longo1, I. Gentile
2, P. G. Gervasi
2, M. Pellegrini
3
1Istituto di Biologia e Biotecnologia Agraria, UOS-Pisa, C9R, Pisa;
2Istituto di Fisiologia
Clinica, C9R, Pisa; 3Agrisan SRL, Larciano (PT), Italy.
Reactive oxygen species from both endogenous and exogenous sources may be involved in
the etiology of human diseases such as cancer, coronary artery disease and rheumatoid
arthritis. Diets rich in fruits and vegetables are associated with a reduced risk for these
pathologies and this protection has often been attributed to the presence of antioxidant
components [1]. Cisplatin, cis-diaminedichloroplatinum (II), is one of the most frequently
used anti-neoplastic agents for various types of tumors. It has a potent anti-tumor action
against a wide range of malignancies, including solid tumors. Despite its clinical usefulness,
cisplatin treatment has been associated with several toxic side effects such as nephrotoxicity
[2]. It has been shown that several antioxidant agents extracted from plants and dietary
components can reduce some of the side effects of this compound without altering the effects
of chemotherapy [2]. In our laboratory it has been recently shown that Lisosan G, a powder of
grain registered to the Italian Minister of Health as an integrator, did not interfer with
cytochrome P450 and phase 2 enzymes, had antioxidant properties and protective effects
against carbon-tetrachloride-induced hepatotoxicity in rats [3]. The purpose of the present
study was to evaluate the toxicity of cisplatin by measuring biochemical and enzymatic
parameters in plasma, liver, kidney and testis and to investigate the possible protective effect
of Lisosan G.
The animals were divided in the following groups: i) normal diet (Control); ii) lisosan G diet
for 15 days; iii) normal diet and a single i.p. injection of 20 mg /Kg of cisplatin and sacrifice
after 4 days; iiii) lisosan G diet and, after 11 days, treatment of the rats by a single i.p.
injection of 20 mg/Kg of cisplatin, followed by their sacrifice 4 days later.
Results: The results showed a significant increase in lipid peroxidation, creatinine, urea and a
decrease of testosterone and cytochrome P450 content in cisplatin-treated rats compared to
control. Following cisplatin treatment an increased amount of hydrogen peroxide, used as a
measure of oxidative stress, was also observed and a 2-4 fold reduction of the CYP-dependent
activities compared to control. The group of animals fed with Lisosan G and treated with
cisplatin (Lis+cis) showed levels of urea and creatinine similar to those of the control group,
indicating that Lisosan G protects from the toxic effects of cisplatin. In addition, a significant
reduction of hydrogen peroxide and an increased CYP content were observed in the Lis+cis
group compared to the cisplatin group.
The present results showed therefore that lisosan G exerts a hepatoprotective effect against
cisplatin toxicity.
1. Zhou S et al. (2004) Life Sciences 74:935
2. Liao Y et al. (2008) Pharmacol Res 57:125
3. Longo V et al. (2007) Biotechnol Lett 29:1155
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1083
RAT I�TESTI�AL PRECISIO�-CUT SLICES AS A TOOL TO STUDY
XE�OBIOTIC I�TERACTIO�S WITH TRA�SPORT PROTEI�S
Stefania Dragoni, MariaPia Possidente, Giada Materozzi, Massimo Valoti
Department of 9eurosciences, University of Siena, via A.Moro 2, Siena, Italy.
The intestinal activity of drug-metabolizing enzymes (of both phase I and phase II) and of
drug transporters (phase III) have been recognized as a major physiological mechanism to
protect from toxic compounds and for regulating their availability. Several phase III proteins,
commonly known as ATP-binding cassette (ABC), play key roles in tissue defense by
transporting metabolic waste and toxic chemicals out of the cells [1]. To study xenobiotic
interactions with ATP-dependent transporters intact cell systems are required. The aim of the
present study was to set up an intestinal precision-cut slice technique to study the interaction
of ABC transporters (namely Multidrug resistance 1/P-glycoprotein, MDR1, and multidrug
resistance associated proteins, MRPs) with xenobiotics. Slices were prepared as described by
De Kanter et al.(2005) [2] and individually incubated in RPMI 1640 under 95% O2 5% CO2
atmosphere at 37°C in 12 wells plates in presence of 0.5 µM calceinAM. Various
concentration of the well known MDR or MRP inhibitors verapamil, indomethacyn and
glibenclamide were also added. The intracellular deesterification of calceinAM to the
fluorescent compound calcein was measured spectrofluorimetrically. The presence of
transport inhibitors increased the intracellular concentration of calcein in time-dependent
fashion and showed the optimum incubation time to be 30 minutes. Furthermore verapamil,
indomethacyn and glibenclamide promoted a concentration-dependent accumulation of
calcein (EC50 3.28x10-6
M, 145x10-4
M, 190x10-6
M, respectively). These data suggest that the
precision-cut intestinal slices are a reliable, simple, and fast system to evaluate xenobiotic
interactions with ABC transporters. Data in the literature indicate that this model is also
suitable to study phase I and phase II drug metabolism [3], suggesting that precision-cut slices
give the possibility to investigate phase I, II and III reactions, all involved in intestinal
detoxifying mechanisms.
1. Benet LZ et al. (1999) Control Release 62:25
2. De Kanter R et al. (2005) J Pharmacol Toxicol Methods 51:65
3. Van de Kerkhof EG et al. (2007) Curr Drug Metab 8:658
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1084
CHLORPYRIFOS BIOACTIVATIO� BY HUMA� SMALL I�TESTI�E
(DUODE�UM, ILEUM/JEJU�UM)
Claudia Leoni1, Maria Balduzzi
2, Franca Maria Buratti
1, Emanuela Testai
1
1Environment and Primary Prevention Department, Mechanisms of Toxicity Unit, Istituto
Superiore di Sanità, Viale Regina Elena, 299 – 00161- Rome, Italy; 2Section of Toxicology
and Biomedical Sciences, Biotechnologies, Agro-Industry and Health Protection Department,
E9EA CR Casaccia, 00123 Rome, Italy.
Clorpyrifos (CPF), one of the most widely used organophosphorothionate pesticide, can be detected as
residues in food and drinking water; therefore the oral route is the major route of exposure for the
general population.
A recent in vitro study with Caco-2/TC7 intestinal cells evidenced high level of CPF inside the cells
corresponding to about 80% of the nominal concentration tested (30, 50 and 250 µM), representative
of the concentrations attainable in the intestinal lumen after actual levels of human oral exposure [1].
Furthermore, studies on human small intestine have evidenced its contribution to the overall first-pass
metabolism of several drugs (e.g. cyclosporine, midazolam, and verapamil) absorbed via the
transcellular route [2]. Similarly to the liver, CYP3A (3A4 + 3A5) has been demonstrated to be the
most abundant P450 subfamily in the small intestine, accounting for about 80% of the total CYP
intestinal content, although with large interindividual variations. However, CYP2C9 (15%),
CYP2C19 (2.9%), CYP2J2 (1.4%) and CYP2D6 (1%) are also present [2].
The aim of this study was to evidence CPF bioactivation to the toxic metabolite oxon (CPFO) and to
the detoxification metabolite 3,5,6 trichloro-2-pyridinol (PYR) by human duodenum (HDM)
microsomes from 11 male and female individual donors; in 4 out of 11 donors, microsomes from
ileum/jejunum (HSM) were tested too. The HDM and HSM were characterized for their intestinal
metabolic competence by using testosterone (TST) hydroxylation as marker of CYP activity by a
HPLC method. Furthermore, the P450 content in HDM was studied by means of western blotting,
using selective anti–P450 primary antibody (polyclonal anti-CYP3A4, CYP3A5, CYP2C9, CYP2C19,
CYP2B6). An enhanced chemiluminescent detection system was used for band visualization. All blots
were scanned and quantified densitometrically using the software program Labimage 1D 2006.
Metabolism of 1 mM TST evidenced a high variability in metabolite formation among tested HDM: 4
samples were endowed with substantial CYP3A4 activity (as evidenced by formation of 6β-OH TST,
a specific marker) and significant levels of androstendione (a marker for the 2B and/or 2C family),
while the other samples showed low or negligible levels of any metabolite formation. A similar trend
was observed with immunoblotting detection: at least one donor contained CYP3A4 and CYP2C9,
most samples were positive only for CYP3A4, but some of them were negative for all P450s tested.
Analogously, high variability was observed in CPF metabolism (50 -600 µM CPF, range of tested
concentrations) measured as CPFO formation, whereas PYR production was below the limit of
detection with all donors. Kinetic parameters related to CPFO formation were derived and compared
with the hepatic situation. The HSM samples, coming from the same donors showing the highest
CYP3A content and CPFO formation in HDM, showed no activity with either TST or CPF,
confirming that the vast majority of small intestine metabolic competence, associated with CYP3A
content, is confined in the proximal region (duodenum) and declines sharply toward the distal ileum.
In conclusion our data show the capability of human small intestine to bioactivate CPF at the site of
absorption, which can be relevant considering the major route of exposure for the general population.
In addition, since it has been reported that CPFO is a substrate for Pg-P protein, the transport of the
toxic metabolite in the lumen could limit the entering into the bloodstream, acting as an additional
detoxication mechanism.
1. Paine MF et al. (2006) Drug Met Disp 34:880
2. Tirelli V et al. (2007) Toxicol In Vitro 21:308
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1085
PARTIAL SEQUE�CI�G OF CYP1A A�D CYP3A CD�A I� RED MULLET
MULLUS BARBATUS (LI��EAUS, 1758) O�E OF THE WIDELY USED
BIOI�DICATOR SPECIES OF THE MEDITERRA�EA� SEA
M.P. Tomasino1, I. Corsi
1, C. Della Torre
1, F. �ardi
2, F. Frati
2, S. Focardi
1
1Department of Environmental Sciences “G. Sarfatti”, via Mattioli, 4, University of Siena,
53100 Siena, Italy; 2Department of Evolutionary Biology, via Aldo Moro 2, University of
Siena, 53100, Siena, Italy.
The cytochrome P450 system is one of the most informative marker of exposure to specific
classes of pollutants in fish species. At the transcript and biochemical level, induction of this
system indicates a physiological condition of stress associated to the exposure to organic
contaminants. Biochemical analysis are widely used in environmental biomonitoring
programs, whereas the molecular approaches based on gene expression are less common.
Nevertheless a molecular characterization of CYP genes is crucial for our understanding of
the cellular defence mechanisms towards pollutants in marine species. Therefore,
characterization and expression profiling of specific gene families such as CYP1A and
CYP3A, known to be involved in the response of selected fish species to aquatic pollutants,
could be extremely important in environmental studies. The red mullet (Mullus barbatus), is
one of the most suitable specie to be used as bioindicator in Mediterranean marine coastal
areas (UNEP, GESAMP), being a territorial benthic fish with well-known ecological and
physiological characteristics and pronounced sensitivity to pollution. Pollutant-induced CYP
enzymes activities, such as ethoxyresorufin-O-deethylase (EROD), has been widely used in
biomonitoring studies due to their sensitive response to pollution in the liver of red mullet.
However, to our knowledge, no data are currently available on genes encoding such enzymes
such as CYP1A as well as other CYP families in the liver of this species. The aim of the
present study is thus to indentify CYP1A and CYP3A genes in the liver of red mullets
collected from an Italian coastline environment. Primers were designed based on highly
conserved regions from known teleostean species. Partial sequences of CYP1A and CYP3A
genes were obtained, and functional domains as well as conserved regions have been
recognized. Substrate recognition sites (SRS) have been identified based on a comparison
with closely related fish species, displaying high similarity in CYP1A and a more pronounced
variation in CYP3A. Polymorfic sites have been detected in both genes, although it is not
possible at present to differentiate between inter-allelic differences or a recent gene
duplication. Biochemical confirmation of enzymatic activities (EROD and 7-benzyloxy-(4-
trifluoromethyl)-coumarin O-debenzylase; BFCOD) encoded by the two gene families was
also obtained. Detectable EROD and BFCOD activities confirm the expression of both
CYP1A and CYP3A genes, altough the association between BFCOD activity and CYP3A
expression needs further confirmation. The results of the present study provide novel
information on crucial genes involved in the response towards environmental pollutants in the
red mullet, and might represent a useful tool in the application of mRNA-based biomarkers to
ecotoxicological field studies.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1086
I�DUCTIO� OF CYP1A I� CARETTA CARETTA LYMPHOCYTES EXPOSED TO
PAHS A�D PBDES
Silvia Casini1, Letizia Marsili
1, Cristina Panti
2, Stefania Ancora
1, Silvia Maltese
1, Chiara
Caruso3, Ilaria Caliani
1, Maria Cristina Fossi
1
1Department of Environmental Sciences, University of Siena, Siena, Italy;
2Department of
Evolutionary Biology, University of Siena, Siena, Italy; 3Centro Recupero Tartarughe Marine
Talamone, Italy.
The Mediterranean population of the loggerhead turtle (C. caretta), a threatened species, may
be especially subjected to ecotoxicological hazard due to high levels of contaminants in this
sea, one reason being its long mean life expectancy. The need to develop sensitive non
destructive biomarkers to investigate the health status of this species has focused attention on
the possibilities offered by different non destructive biological materials. The aim of this
study was to investigate protein expression and induction of CYP1A in loggerhead turtle
lymphocytes. Lymphocytes (obtained from blood of turtles hospitalised in a recovery center
in Italy) were cultured using RPMI-based complete medium. Cultures were treated with two
doses of PAHs (benzo(a)pyrene and β-naphthoflavone) and two doses of a mix of 27 PBDEs.
Western blot was used to test for presence and induction of CYP1A in the different treatment
groups and to compare the induction potential of PAHs and PBDEs. CYP1A was detected in
all lymphocyte cultures with a molecular weight of about 59 kDa. Cultures treated with PAHs
showed clear induction of CYP1A, though no evident differences were found between the two
doses. Cultures treated with PBDEs showed induction of CYP1A that increased with dose and
was several fold higher than observed after PAH treatments. The results of this study are the
first evidence of induction of CYP1A by contaminants in loggerhead turtle lymphocytes, and
represent an important starting point for an in vivo application of this biomarker of exposure.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1087
MODULATIO� OF DRUG METABOLISI�G E�ZYMES (DMES) A�D RELATED
�UCLEAR RECEPTORS (�RS) GE�E EXPRESSIO� PROFILES I� THE LIVER
A�D EXTRA-HEPATIC TISSUES OF CATTLE I�DUCED WITH
PHE�OBARBITAL (PB)
Vanessa Zancanella1, Mery Giantin
1, Rosa Maria Lopparelli
1, Carlo �ebbia
2, Mauro
Dacasto1
1Dipartimento di Sanità pubblica, Patologia comparata e Igiene veterinaria, Università degli
Studi di Padova, viale dell’Università 16, 35020 Legnaro (Padova); 2
Dipartimento di
Patologia Animale, Università degli Studi di Torino, via Leonardo da Vinci 44, 10095
Grugliasco (Torino).
In mammals, PB induces hepatic and extra-hepatic DMEs by the activation of specific NRs
[1]. Although barbiturates pharmacological effects were discovered more then 40 years ago,
molecular mechanisms involved in PB induction have been only recently understood in
human and laboratory species [2]; furthermore, few data about PB effects upon DMEs and
NRs of veterinary species have been published [3-5]. In the present work, the transcriptional
effect of PB, administered at inductive dosage regimen, was investigated upon DMEs and
NRs in the liver and extra-hepatic tissues of cattle.
Seven male Friesian cattle (10 months old) were used; four of them (PHEN) received PB by
gavage (18 mg kg-1
body weight day-1
for 7 days), while the other three remained untreated
(CTRL). The experiment was performed according to the EC Directive 86/609 and the Italian
D.L. 27/01/1992, nr 116. Bovines were slaughtered the day after the suspension of PB
administration. Small aliquots of liver, duodenum, kidney, lung, testis, adrenal, and muscle
were collected, immediately snap frozen in liquid nitrogen, and stored at -80°C until use.
Firstly, the most valid reference genes for the comparative evaluation of data from different
tissues were identified, by using the NormFinder program [6] and four candidate genes were
considered (β-actin, ACTB; acidic ribosomal protein large P0, RPL0; beta-2-microglobulin,
β2M and peptidylprolyl isomerise A, PPIA). Thereafter, the transcriptional effect of PB upon
a set of target genes was measured by using a quantitative Real Time RT-PCR approach. The
target genes investigated were cytochromes P450 (CYPs) 2B22, 2C87, 2C31, 2C49 and
3A28; the constitutive androstane receptor (CAR), pregnane X receptor, retinoic X receptor-
alpha (RXRα); and, finally, glutathione S-transferase A1 (GSTA1), sulfotransferase 1A1 and
2A1 (SULT1A1 and SULT2A1, respectively).
Two valid housekeeping genes (ACTB and RPL0) were identified among the chosen
candidates, thereby allowing a more precise comparison of transcriptional data obtained from
tissues richly endowed of DMEs (liver, duodenum, kidney, lung) with those showing from
low to very low level of expression (testis, adrenal, muscle).
Phenobarbital and other PB-like chemicals induce liver CYP2A, 2B (mostly), 2C, 3A and as
well as others DMEs, albeit to a lower extent; moreover, CAR plays a key role in such up-
regulations [7]. In cattle liver, a PB-dependent significant increase (p<0.01) of CYP2B22,
2C31, 2C87 and 3A28 mRNAs was recorded, while the expression of the CYP2C49 gene was
unaffected. These results agree with those previously reported in humans, rats and mice as
well as with post-translational data from cattle themselves [5,8]. On the contrary, NRs gene
expression profiles did not show differences among CTRL and PHEN groups. Phase II DMEs
did not respond univocally to PB: a significant increase (p<0.05) was observed for GSTA1;
by contrast, SULT1A1 gene expression was significantly (p<0.05) decreased, while that of
SULT2A1 was unaffected. The pattern of phase II gene expression now reported resembles
that described in rodents, where too PB induced GSTA1, but reduced in a dose-dependent
manner both SULT1 and SULT2 [9-10].
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1088
As regards the extra-hepatic sites of drug metabolism, statistically significant changes were
found only in the duodenum of PB-treated animals, where a general inhibitory trend was
observed; such a down-regulation was significant for CYP2C87 (p<0.05), RXRα (p<0.05)
and SULT1A1 (p<0.05). By contrast, CYP2C31 gene was not expressed at all. Altogether,
these results confirm that NRs are expressed also in cattle gastrointestinal tract [11], but
disagree with the transcriptional PB induction of CYPs observed in vitro and in vivo in human
and rat intestine [12]. Remarkable effects attributable to PB were never found in target gene
expression profiles in the kidney, lung, testis and adrenal of PHEN group, as reported in rats
and humans [13]; besides, some of them were poorly expressed or undetectable. Finally, a
significant inhibition of SULT1A1 gene (p<0.05) was noticed in muscle, where the increase
of CYP2B22 and CAR mRNAs was not significant.
Phenobarbital, at inducing dosage regimens, prompted different transcriptional effects upon
cattle liver and extra-hepatic DMEs and NRs mRNAs. These results partially disagree with
those reported in previous comparative studies. Confirmatory investigations are thereby
needed and are planned in our laboratory.
Acknowledgements. This study was supported by grants from Università degli Studi di Padova (60A08-8818/08
and 60A08-5793/09) to D.M.
1. Ohno M et al. (2009) Biol Pharm Bull 32:813
2. Handschin C et al. (2001) Mol Endocrinol 15:1571
3. Dupuy J et al. (2001) Can J Physiol Pharmacol 79:848
4. Kawalek JC et al. (2003) Am J Vet Res 64:1167
5. Cantiello M et al. (2006) J Vet Pharmacol Therap 26:109
6. Andersen J et al. (2004) Cancer Res 64:5245
7. Handschin C et al. (2003) Pharmacol Rev 55:649
8. Waxman DJ et al. (1992) Biochem J 281:577
9. Romero L et al. (2006) Mol Pharmacol 70:277
10. Runge-Morris M et al. (1998) Drug Metab Dispos 26:795
11. Mäkinen J et al. (2002) Mol Pharmacol 62:366
12. Martignoni M et al. (2004) Chem Biol Interact 151:1
13. Nannelli A et al. (2008) Toxicology 252:105
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1089
PRESE�CE A�D I�DUCIBILITY OF CYP2C ISOFORMS I� PIG AS A �EW
A�IMAL MODEL
Emanuela Puccinelli1, Roberto Fiorio
1, Vincenzo Longo
2
1Istituto di Fisiologia Clinica C9R, Pisa, Italy;
2Istituto di Biologia e Biotecnologie Agrarie
C9R, Pisa, Italy.
Cytochrome P450s (CYPs) play an important role in the oxidative metabolism of numerous
xenobiotics as well as endogenous compounds. In human, the CYP2C subfamily is one of the most
important, accounting for roughly 20% of total CYPs and being responsible for the metabolism of
about 30% of drugs [1].
In the last few years, the pig has been proposed as a new model for pharmacological and toxicological
studies, and its liver may be used as bioreactor for patients waiting for liver transplantation. However,
up to now, little information is available on its metabolic systems, and in particular on the expression
of its CYP enzymes.
In the present study we investigated by RT-PCR and by marker activity experiments the presence and
regulation of three recently cloned isoforms of the CYP2C subfamily (2C33, 2C42 and 2C49) in liver
and some extrahepatic tissues of pigs. The expression and activity of these CYP2Cs were evaluated in
pigs treated with three typical human CYP inducers: rifampin, phenobarbital and β-naphtoflavone,
inducers, respectively, of CYP3A4 (through PXR activation), CYP2B6 (through CAR activation) and
members of CYP1 family (through AhR activation).
Seventeen male castrated pigs of about 20-30 kg were utilized: seven pigs were used as controls, three
were treated with rifampin (40 mg/kg i.p. for 4 days), three were treated with β-naphtoflavone (30
mg/kg i.p. for 4 days) and four were treated with phenobarbital (20 mg/kg i.p. for 3 days). Total RNA,
extracted from liver, kidney and small intestine, was retrotranscripted and amplified by PCR using
specific primers. Hepatic and renal microsomes were also prepared in the standard way to investigate
the activity of CYP2Cs through reactions specifics for human CYP2C isoforms (paclitaxel
hydroxylase, tolbutamide hydroxylase, diclofenac 4'-hydroxylase, S-mephenytoin 4'-hydroxylase and
methoxytrifluoromethylcoumarin O-demethylase). Additionally, the hydroxylation of testosterone was
evaluated in liver and renal samples. Further inhibition studies are currently in progress in liver
microsomes with typical inhibitors of human CYP2Cs (quercetin, sulphaphenazole, ticlopidine).
The three CYP2C isoforms considered here were constitutively expressed in all porcine organs
examined and, in liver, their expressions were transcriptionally inducible by rifampin and
phenobarbital, but not by β-naphtoflavone. Furthermore, phenobarbital increased the transcript of
CYP2C42 and 2C49 genes in the kidney and the transcripts of all three CYP2C isoforms in the small
intestine. All the CYP2C isoforms were transcriptionally inducible by rifampin in the kidney. The
markers activities of rat CYP2Cs (methoxytrifluoromethylcoumarin O-demethylase and testosterone
2α- and 16α-hydroxylase) were increased by phenobarbital in liver microsomes. In agreement with
what has been reported for CYP2C9 in human hepatocyte experiments [2], treatments of pigs with
rifampin or phenobarbital increased the hepatic activity of tolbutamide hydroxylase and diclofenac 4'-
hydroxylase but, unlike human CYP2C9, these porcine activities were not inhibitable by
sulphaphenazole. Other differences between human and pig were also found in the metabolism of both
paclitaxel and S-mephenytoin. Rifampin and phenobarbital increased the oxidation of paclitaxel, but
6α-hydroxypaclitaxel, the product of human CYP2C8 catalysis, was not observed [3]. Lastly, the
typical oxidation product of S-mephenytoin by human CYP2C19, namely the 4'-hydroxy derivative,
was not detected with hepatic microsomes of control and rifampin-treated pigs, while it was observed
with phenobarbital-treated microsomes.
In conclusion, the results on the expression and activities of CYP2Cs presented in this study show
some differences between human and pig CYP2Cs, suggesting that pig might not be a suitable model
for the study of drugs metabolized in humans by this subfamily.
1. Skaanild MT et al. (2008) Basic Clin Pharmacol Toxicol 103:487
2. Madan A et al. (2002) Drug Metab Dispos 31:421
3. Vaclavikova R et al. (2004) Drug Metab Dispos 32:666
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1090
CYP3A4/P-GP SI�ERGISTIC ACTIVITY MODULATES THE ABSORPTIO� A�D
DELIVERY OF XE�OBIOTICS
Carmela Inglese, Maria Grazia Perrone, �icola Antonio Colabufo
Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via Orabona 4, 70125 Bari,
Italy.
P-gp is an efflux pump belonging to the ATP Binding Cassette (ABC) transporters and
localized in the apical membrane of the intestinal epithelial cells of small intestine, where it
modulates the absorption of drugs and xenobiotics [1].Cytochrome P-450 isoenzyme 3A4
(CYP3A4) is the most prominent oxidative enzyme localized in the endoplasmic reticulum of
intestinal epithelial cells of small intestine [1]. CYP3A4 and P-gp act together in a
cooperative manner controlling the intestinal absorption and a considerable overlap in the
substrates/inhibitors selectivity of the CYP3A4 enzyme and P-gp has been reported [2].
Several drugs are P-gp substrates so that they display poor intestinal absorption due to the P-
gp efflux activity [1]. An improvement of intestinal absorption could be obtained by
employing a P-gp inhibiting agent, so as to increase drug cell permeation. The ideal P-gp
inhibitors should block the pump, leaving the CYP3A4 metabolic activity unaltered;
unfortunately some P-gp inhibitors also reduce the activity of CYP3A4, increasing systemic
exposure to the drug and causing potential toxic effects [3].
Recently, we developed potent P-gp modulating agents, namely MC18 and MC266, as PET
probes [4] to recognize P-gp activity and expression in all biological barriers, such as Blood
Brain Barrier (BBB), Cerebro Spinal Fluid (CSF) and Gastro Intestinal (GI) tract, where P-gp
plays a critical role in the absorption mechanism [5]. In in vitro assays carried out in tumor
cell lines (Caco-2), MC18 and MC266 in co-administration with a cytotoxic drug, displayed
an increased cellular drug accumulation [6]. However no study has so far been carried out to
investigate their ability to interfer with CYP3A4 activity.
A useful ex vivo assay that permits to characterize at the same time the metabolism and the
intestinal absorption of drugs is the everted gut sac model [7]. This system is particularly
useful in the study of intestinal absorption of drugs that are subjected to P-gp activity and to
CYP3A4 metabolism. The metabolism data obtained with the everted gut sac system reflects
the interaction of substrates with the CYP3A4 enzyme inside the enterocytes, during their
passage through the cells [8]. MC18 will be tested in this model in the presence of a CYP3A4
substrate (midazolam or testosterone) to verify whether MC18 affects the enzyme activity of
CYP3A4. The identification by LC-MS of the most representative metabolites (1-OH-
midazolam and 6-β-hydroxytestosterone) both in and out of the biological compartments will
permit to establish the CYP3A4 inhibiting activity of the tested compound.
MC266 will be co-administrated with a known P-gp inhibitor unaffecting CYP3A4 activity
for evaluating the metabolic stability of this compound because it is a promising PET
radiotracer in epilepsy diagnosis. The results of synergic P-gp/CYP3A4 activity in gut sac
model will be reported.
1. Benet LZ et al. (2004) Int J Pharmaceutic 277:3
2. Cummins CL et al. (2002) J Pharmacol Exp Ther 300:1036
3. Rubin E et al. (2002) Clin Cancer Res 8:3710
4. van Waarde A et al. (2009) J Med Chem in press doi: 10.1021/jm900485a
5. Hammarlund-Udenaes M et al. (2009) Curr Top Med Chem 9:148
6. Colabufo NA et al. (2008) Bioorg Med Chem 16:362
7. Barthe L et al. (1998) Eur J Drug Metab Pharmacokinet 23:313
8. Arellano C et al. (2007) J Pharm Pharmaceutic Sci 10:26
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1091
DRUG METABOLIZI�G E�ZYME DIFFERE�CES AS A RISK FACTOR I�
CHILDRE�
Virginia Tzankova
Department of Pharmacology, Pharmacotherapy and Toxicology, Medical University of
Sofia, Faculty of Pharmacy, 2 Dunav Str, 1000 Sofia, Bulgary.
The mechanism of adverse drug reactions (ADR) in children have been often related to the
differences in patient response at the pharmacokinetic level. Drug metabolizing enzyme
systems and drug transporters have a particularly important role in the control of
pharmacokinetic properties of drugs. Pharmacogenetic variability contributes to the broad
range of drug responses observed in children. Important drug metabolizing enzyme genes
associated with drug response include those of phase I enzymes (cytochrome P450; CYP) and
of phase II enzymes. Genetic polymorphisms, responsible for clinically important interpatient
variability in drug response have been described for CYP2D6, CYP2C9, CYP2C19,
CYP3A4, CYP3A5, CYP3A7, glucuroniltranspherases and drug transporters.
In children, undetected genetic variations in drug metabolizing enzymes may lead to serious
morbidity and even mortality. Recent findings of genetic polymorphism and age connected
variations in drug metabolizing enzymes have increased awareness of the existence and the
possible prevention of unexpected undesirable drug responses. The major consequence of
pharmacogenetic polymorphism in drug metabolizing enzymes and transporters is
concentration dependent toxicity due to impaired drug clearance. The important effects of
gene polymorphism of the most important drug metabolizing isoforms of CYP as well as of
phase II enzymes will be discussed.
The role of pharmacogenetics in drug biotransformation will be reviewed with particular
emphasis to pediatrics drug therapy. Pharmacokinetics can differ widely in children due both
to immaturity of enzyme systems and/or differences in genes encoding drug metabolizing
enzymes. The finding that drug response can be influenced by the patient’s genetic profile has
offered great hope for realizing patient-specific targeted therapy, so as to decrease adverse
drug reactions due to a different response. Prevention requires clinical monitoring in pediatric
population.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1092
POLYMORPHISMS OF THE OMEGA CLASS GLUTATHIO�E TRA�SFERASE
GE�ES A�D ASTHMA: A POSSIBLE �EW ASSOCIATIO�
Sara Piacentini, Renato Polimanti, Maria Fuciarelli
Dipartimento di Biologia, Università degli Studi di Roma “Tor Vergata”, via della Ricerca
Scientifica 1, 00133 Roma.
Glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that
catalyze the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous
electrophilic compounds; these enzymes are critical in the protection of cells from reactive
oxygen species [1]. Human GSTs are divided into three main families: cytosolic,
mitochondrial and membrane-bound microsomal. The cytosolic family is further divided into
seven classes: Alpha, Mu, Omega, Pi, Sigma, Theta and Zeta. Unlike other GSTs, Glutathione
S-transferase Omega (GSTO) has an active site cysteine that is able to form a disulfide bond
with GSH and exhibits glutathione dependent thiol transferase and dehydroascorbate
reductase activities, reminiscent of thioredoxin and glutaredoxin enzymes [2]. Many of the
GST genes are polymorphic; interest has focused on whether certain allelic variants are
associated with various phenotypic expressions of disease. In humans, polymorphism in GST
genes has been associated with susceptibility to various diseases though some recent data
indicate that these genotypes modify the disease phenotype. Asthma, one of the most common
chronic diseases in modern societies, has like other diseases both genetic and environmental
components in its etiology. For the development of preventive strategies, it is important to
identify inherited and acquired host factors that modify the individual risks of developing
asthma. It has been suggested that inherited differences in the capacity to metabolize
environmental pollutants (as a result of genetic polymorphism in xenobiotic metabolizing
enzymes) modify an individual’s susceptibility to asthma. GST enzymes may play an
important role in the detoxification of environmental pollutants. Therefore, it has been
hypothesized that genetic alterations of GST enzymes may change the ability of the airways
to deal with toxic substances and influence airway inflammation and lung development [3]. In
humans, Omega class GST are coded by two genes, hGSTO1 and hGSTO2. Three
polymorphisms in hGSTO genes (hGSTO1*A140D, hGSTO1*E155del and
hGSTO2*N142D) have been identified in ethnic groups, although their relationship with
asthma has not yet been explored. Expression of GSTO1 is abundant in a wide range of
normal tissues [4]. The widespread distribution of GSTO1 suggests that it has important
biological functions: It is possible that the glutathione-dependent dehydroascorbate-reductase
and thiol-transferase activities of GSTO1 may be required for cellular processes [5]. Although
the physiological role of GSTO1 is not well understood, it is thought to participate in
intracellular homeostatic reactions: Reduced thiol-transferase activity may result in defective
protection against cellular oxidative stresses. Considerable GSTO2 expression has been
observed in liver, kidney, skeletal, muscle with lower expression in the heart [4].
Unfortunately the insolubility of GSTO2 has prevented its characterization and comparison
with GSTO1. The aim of this case-control study was to analyse the possible correlation
between polymorphism in hGSTO1 and hGSTO2 genes and asthma development in a sample
of individuals from Rome (Central Italy). DNA was extracted from buccal cells.
GSTO1*A140D and GSTO2*N142D polymorphisms were determined using a PCR-RFLP
method [6] while GSTO1E155del polymorphism was determined using the confronting two-
pair primer method [7]. The results presented here show that the allele frequencies for GSTO1
polymorphisms (GSTO1*A140D, GSTO1*E155del) were nearly equal in both the asthmatic
and in the control group . The Chi-square analysis established that there were no significant
differences in genotype distribution for GSTO1 polymorphisms between the asthmatics and
the controls.
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1093
The odds ratios of GSTO1 polymorphisms did not reach the level of statistical significance.
For GSTO2 gene polymorphism (GSTO2*N142D) allele frequencies in the control group
were nearly equal to those found in the asthmatic group. The Chi-square test for GSTO2
showed significant differences in genotype distribution between asthmatics and healthy
controls (p=0.009**). Subjects with the GSTO2 homozygous D142/D142 genotype have an
increased risk of having asthma when compared to control subjects (OR = 6.77; 95% CI:
1.42–32.37). In conclusion the work now presented demonstrates a potential association
between GST polymorphism and asthma. This finding is especially significant given that as
yet in the literature there have been no epidemiological studies on the GST Omega class and
asthma.
1. Strange RC et al. (2000) Toxicol Lett 112-113:357
2. Girardini J et al. (2002) Eur J Biochem 269:5512
3. Kabesh M (2006) Toxicol Lett 162:43
4. Whitbread AK et al. (2003) Pharmacogenetics 13:131
5. Board PG et al. (2000) J Biol Chem 275:24798
6. Takeshita H et al. (2009) Clin Exp Pharmacol Physiol 36:283
7. Fujihara J et al. (2007) Clin Chem Lab Med 45:621
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1094
EXPOSURE OF CD1 MICE I� UTERO TO RED WI�E OR ETHA�OL: A
PRELIMI�ARY STUDY O� LIVER A�D BRAI� CYP2E1 mR�A ESPRESSIO�
Susanna Vichi1, Mauro Ceccanti
2, Marco Fiore
3, Luigi Aloe
3, Rosanna Mancinelli
1,
Emanuela Testai1, Simonetta Gemma
1
1Dipartimento Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanità,
Roma; 2Università “La Sapienza”, Roma;
3Istituto di 9eurobiologia e Medicina Molecolare,
C9R, Roma.
Alcohol exposure during pregnancy may result in children with Fetal Alcohol Syndrome (FAS), which
has been described as the simultaneous occurrence of several abnormalities in children, such as fetal
growth retardation, malformations, developmental defects, and/or spontaneous abortion (10% of
pregnant women are heavy drinkers in western countries) [1]. A better understanding of the risks
factors involved in the development of FAS and of other alcohol-related problems in children is
extremely important in order to plan appropriate prevention strategies. The contribution of genetic
factors as the presence of allelic variants for those enzymes involved in ethanol metabolism such as
cytochrome P4502E1 (CYP2E1) has been recently suggested by animal models and molecular
epidemiological studies [2]. Alcohol toxicity has to be addressed not only with respect to ethanol itself
but also to its metabolic products, that is acetaldehyde and reactive oxygen species (ROS) produced
during its biotransformation. Ethanol, once absorbed, is bioactivated to acetaldehyde by alcohol
dehydrogenase (ADH) and CYP2E1. Acetaldehyde is subsequently oxidized to the inactive acetate by
aldehyde dehydrogenase (ALDH). The oxidative stress produced by CYP2E1 during its catalytic
action contribute to the adverse effects of alcohol exposure, by promoting lipid peroxidation and
subsequent severe damages to DNA and proteins. After ingestion of moderate alcohol doses, CYP2E1
is responsible only for about 10% ethanol oxidation, but its contribution becomes relevant at high
alcohol doses, when ADH is saturated [1]. In addition, CYP2E1 is inducible up to 10 fold by ethanol
itself [3]. Under these conditions ethanol-related damages are more severe and this is also true in the
presence of low levels of antioxidants. The aim of this study was to evaluate alterations in CYP2E1
expression in F1 CD1 outbred mice due to pre- and post-natal chronic exposure to ethanol. Dams were
treated with ethanol solution (11% v/v) or with red wine at equivalent alcohol concentrations starting
from 60 days before pregnancy up to weaning of the offspring. Besides ethanol, red wine is known to
contain also several substances with putative antioxidant properties which may be able to protect
against the oxidative stress induced by ROS. Mice were housed under standardized conditions using a
12L:12D lighting regime. All animal experiments were conduced in strict accordance with Directive
86/609/ (EEC European Council, 1986). Pregnant females were divided in four groups: ethanol, red
wine, sucrose and water (n = 8 for each group). The ethanol group received, ad libitum, the ethanol
solution as only source of liquid. Animals treated with red wine received red wine ad libitum as only
source of liquid. The sucrose control group received a sucrose solution at equivalent caloric intake of
the ethanol group. A water group was used as a further control group. 8 pups were sacrificed from
each litter at different development ages (8 days, 5 weeks and 7 weeks), and their organs immediately
stored at -80°C. RNA extracted from the liver and cerebellum was retro-transcribed into cDNA and
analyzed by Real Time PCR using SYBR Green chemistry. No alteration of CYP2E1 expression in the
liver was observed in the sucrose and water control groups and no differences were found between
controls and mice exposed to ethanol at any time. A strong increase in the CYP2E1expression levels
compared to controls was observed in the red wine group of mice sacrificed 8 days after birth. The
difference was no longer present 5 and 7 weeks after birth. Preliminary experiments on CYP2E1
expression in CNS (cerebellum), the target system for FAS, seem to indicate that the trend is similar,
although the levels of expression are extremely low. Overall the prenatal exposure to ethanol per se
appeared not to significantly influence the CYP2E1 expression in mice, likely because ethanol
exposure from the mother is limited. However, a different effect on hepatic CYP2E1 expression was
found depending on the form in which alcohol was given (ethanol vs red wine). A possible
contribution of red wine components other than ethanol may explain the up-regulation of CYP2E1
experimentally observed in the red wine group, or alternatively, a different absorption kinetics may be
involved. 1. Gemma S et al. (2007) Neurosci Biobehav Rev 31:221
2. Warren KR et al. (2005) Birth Defects Res A Clin Mol Teratol 73:195
3. Lieber CS (2000) Annu Rev Nutr 20:395
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1095
PRECISIO�-CUT LIVER SLICES TO STUDY DRUG METABOLISM:
CYTOCHROME P450-DEPE�DE�T METABOLISM OF l-DEPRE�YL
Giada Materozzi, Giulia Franco, Stefania Dragoni, Maria Frosini, Massimo Valoti
Dipartimento di 9euroscienze, Università degli Studi di Siena, via A. Moro 2, 53100 Siena,
Italy.
Several in vitro systems are available for studying the metabolism and toxicity of novel
compounds in the liver. Although subcellular fractions, such as microsomes, and isolated
hepatocytes, have been most extensively used, precision-cut liver slices could be an
alternative and useful tool as it presents some significant advantages: no proteolytic enzymes
are necessary for preparation, and cell heterogeneity, cell-cell interactions, and multienzymes
system are maintained [1]. The aim of the present study was to set up the preparation of rat
liver slices in order to provide an improved tool for the study of xenobiotic metabolism.
Precision-cut rat liver slices were prepared from tissue core (8 mm) using a Krumdieck tissue
slicer filled with oxygenated, ice-cold Krebs-Henseleit buffer. After a pre-incubation period
of 30 min, slices were individually incubated in RPMI 1640 complete medium under
carbogen atmosphere and incubations were carried out at 37°C in 12 well plates with
continuous gentle shaking [2]. The slices retained good cell-viability for 48 h of incubation
(as measured by glutathione content, MTT test, and LDH leakage). These slices possessed
relatively stable metabolic functions, measured by the time-dependent metabolism of 7-
ethoxycoumarin, CYP dependent enzyme analysis, and Western blot. In a second part of the
studies, the kinetic analysis of the well-known drug l-deprenyl was performed both in the
precision-cut liver slices model and in microsomes. The formation of l-deprenyl metabolites
was determined by GLC [3] in the incubation medium. A preliminary assay of l-deprenyl N-
dealkylation by rat liver microsomes and rat precision-cut liver slices gave rise to the primary
metabolites l-methamphetamine and l-nordeprenyl: the amount of products formed increased
linearly with time up to 45 min and the initial rates were proportional to the amount of
microsomal protein added. The formation of low amounts of the secondary metabolite l-
amphetamine was also evident. In the precision-cut slices, the kinetic study of l-nordeprenyl
and l-methamphetamine formation showed atypical Michaelis-Menten kinetics. The same
analysis performed in microsomal fractions of rat liver revealed that both metabolites possess
a biphasic Michaelis-Menten kinetics. These data, together with the different Vmax values
obtained, could be the result of the complex multi-enzymatic system represented in the slices
that may interact with the ldeprenyl, modifying its concentration at the enzymatic site.
In conclusion, the precision-cut liver slices represent an in vitro system alternative to
microsomes for studying drug metabolism. Further studies are necessary to clarify the
differences between the two systems.
1. Lerche-Lagrand C et al. (2000) Toxicology 153:221
2. De Kanter R et al. (1999) Toxicology In Vitro 13:737
3. Dragoni S et al. (2003) Xenobiotica 33:181
Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009
1096
CYTOCHROME P-450-DEPE�DE�T METABOLISM OF �EWLY SY�THESIZED
MO�OAMI�O OXIDASE I�HIBITOR
Mariapia Possidente1, Stefania Dragoni
1, Jose Luis Marco
2, Mercedes Unzeta
3, Massimo
Valoti1
1Dipartimento di 9euroscienze, Università degli Studi di Siena, via A. Moro 2, 53100 Siena;
2Laboratorio de Radicales Libres y Quìmica Computacional, Madrid,
3Departamento de
Biologia Molecular, Universidad Autonoma de Barcellona, Spain.
The selective monoamine oxidase-B (MAO-B) inhibitor, l-deprenyl, is still used for treating
Parkinson’s patients. However, a disadvantage of its use lies in the formation of l-
amphetamine and l-methamphetamine. More recently new MAO-B inhibitors have been
designed which possess a propargylamino group. ASS94 was such a new compound
synthesized in the Laboratorio de Radicales Libres y Quìmica Computacional, CSIC, Spain
with the aim to inhibit MAO, but also to exhibit protective properties against
neurodegenerative diseases. The aim of this present work was to study ASS94 phase I
metabolism in human liver microsomes in order to study the influence of this drug on the
activities of the major cytochrome P-450 (CYP) and to exclude potential drug-drug
interaction. In fact, the presence of propargylamino moiety could represent a potential
molecular site to the formation of suicide substrates. Effects of ASS94 on human CYP 3A4
were assessed with use of selective substrate, [3-[3(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-
[4-(methylsulfonyl)- phenyl]furan-2-(5H)-one] (DFB) [1]. The interaction of DFB with
human liver microsomes was assessed by a photofluorimetric method, using an excitation
wavelength of 360 nm and emission at 460 nm. When human microsomes were incubated
with DFB in presence of different concentration of ASS94, concentration-inhibition curves
were obtained. ASS94 inhibition gave IC50 value of 27 µM. In a second set of experiments,
human liver microsomes were pre-incubated for 15 min with ASS94, at its IC50
concentration, in presence or absence of NADPH. After this incubation time, DFB was added
and activity measured. In all experiments, ASS94 inhibition was not dependent on pre-
incubation time and on the presence of NADPH in the medium. ASS94 reversible inhibition
of DFB metabolism was assessed by dilution experiments. Reaction mixtures (containing
microsomes, ASS94, NADPH, and appropriate substrates) were pre-incubated for 15 min and
then diluted 10 folds. The residual activity observed exhibited values close to those of control
conditions, suggesting that the inhibition was reversible. These data indicate that ASS94
inhibits DFB metabolism by human CYP 3A4 in a competitive manner, suggesting that the
MAO inhibitor is a substrate for this isoenzyme. Further experiments will be carried out in
order to clarify the structure of the metabolite(s) and the interaction with other CYP-isoforms.
This work was supported by COST Action D34/003
1. D’Elia P et al. (2009) Eur J Pharmacol 614:7
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