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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009



Gabriele Cruciani1, Riccardo Vianello


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




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


, 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®


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


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009





�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


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Bruna Vinci1, Cedric Duret

2, Patrick Maurel

2, Arti Ahluwalia


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009














Ar1, Ar2 =










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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Ilaria Caliani1, Maria Ferraro

2, Silvia Casini

1, Gabriele Mori

1, Silvia Maltese

1, Letizia

Marsili1, Maria Cristina Fossi


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


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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009






Gabriele Mori1, Silvia Casini

1, Letizia Marsili

1, Ilaria Caliani

1, Silvia Maltese

1, Maria

Ferraro2, Cristina Fossi


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


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009






C. Della Torre1, I. Corsi

1, G. Guerrini

1, M.P. Tomasino

1, G. Perra

1, F. �ardi

2, F. Frati


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Emilia Vaccaro1, Francesco Rossignolo

1, Roberto Tolando

1, Annalisa �annelli

2, Paolo

Rossato1, Mario Pellegatti

1, Vincenzo Longo

2, Pier Giovanni Gervasi


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


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009



�icola Pasquale Caradonna1, Giulia Franco

2, Ugo Zanelli

1, Goran Westerberg


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


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009



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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




V. Longo1, I. Gentile

2, P. G. Gervasi

2, M. Pellegrini


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Claudia Leoni1, Maria Balduzzi

2, Franca Maria Buratti

1, Emanuela Testai


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009





M.P. Tomasino1, I. Corsi

1, C. Della Torre

1, F. �ardi

2, F. Frati

2, S. Focardi


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Silvia Casini1, Letizia Marsili

1, Cristina Panti

2, Stefania Ancora

1, Silvia Maltese

1, Chiara

Caruso3, Ilaria Caliani

1, Maria Cristina Fossi


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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009






Vanessa Zancanella1, Mery Giantin

1, Rosa Maria Lopparelli

1, Carlo �ebbia

2, Mauro


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].

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Emanuela Puccinelli1, Roberto Fiorio

1, Vincenzo Longo


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Carmela Inglese, Maria Grazia Perrone, �icola Antonio Colabufo

Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via Orabona 4, 70125 Bari,


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




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


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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




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.

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009


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


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Susanna Vichi1, Mauro Ceccanti

2, Marco Fiore

3, Luigi Aloe

3, Rosanna Mancinelli


Emanuela Testai1, Simonetta Gemma


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Giada Materozzi, Giulia Franco, Stefania Dragoni, Maria Frosini, Massimo Valoti

Dipartimento di 9euroscienze, Università degli Studi di Siena, via A. Moro 2, 53100 Siena,


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

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Pharmacologyonline 3: 1070-1096 (2009) Abstracts CYP 2009




Mariapia Possidente1, Stefania Dragoni

1, Jose Luis Marco

2, Mercedes Unzeta

3, Massimo


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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