JPET #203356 1. Title page Title: Metabolism and pathways for

JPET #203356

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1. Title page

Title: Metabolism and pathways for denitration of organic nitrates in the human liver

Authors: Mirco Govoni, Paola Tocchetti and Jon O Lundberg

Laboratories of origin: Department of Physiology and Pharmacology, Karolinska

Institute, Nanna Svartz väg 2, S-177 76 Stockholm, Sweden (M.G., J.O.L) and NicOx

S.A., Les Taissounières - 1681 Route des Dolines - Sophia Antipolis Cedex, France

(M.G., P.T.)

JPET Fast Forward. Published on April 17, 2013 as DOI:10.1124/jpet.113.203356

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 17, 2013 as DOI: 10.1124/jpet.113.203356

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2. Running title page

a) Running title: Metabolism of organic nitrates in the human liver

b) Corresponding author: Department of Physiology and Pharmacology, Karolinska

Institute, Nanna Svartz väg 2, S-177 76 Stockholm, Sweden and NicOx S.A., Les

Taissounières - 1681 Route des Dolines - Sophia Antipolis Cedex, France; Telephone:

+39 3398657744, Fax: +46 8 332278, email: [email protected]

c) Number of text pages: 32; Number of tables: 0; Figures: 9; References: 36; Number of words in the Abstract: 250; Number of words in the Introduction: 747; Number of words in the Discussion: 1234; d) Nonstandard abbreviations:

CYP, cytochrome P-450; CYP1A2, cytochrome P-450 1A2; GST, glutathione S-

transferase; GTN, glyceryl trinitrate; HCT 1026, [1,1’-biphenyl]-4-acetic acid,2-fluoro-

a-methyl,4-(nitrooxy)butyl ester; HCT 1027, [1,1’-biphenyl]-4-acetic acid,2-fluoro-a-

methyl,4-hydroxybutyl ester; ISMN, isosorbide mononitrate; NCX 2057, 3-(4-hydroxy-

3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester; NCX 2059, 3-(4-hydroxy-

3-methoxyphenyl)-2-propenoic acid 4-hydroxybutyl ester; NO, nitric oxide; NO2-,

nitrite; NO3-, nitrate; NOBA, (nitrooxy)butyl alcohol; NOC-5, 3-(aminopropyl)-1-

hydroxy-3-isopropyl-2-oxo-1-triazene; NOx, (NO3- + NO2

-); RSNO, S-nitrosothiols;

sGC, soluble guanylate cyclase;

e) Recommended section: Metabolism, Transport, and Pharmacogenomics


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Abstract

Liver first-pass metabolism differs considerably among organic nitrates but little

information exists on the mechanism of denitration of these compounds in hepatic

tissue. The metabolism of nitrooxybutyl-esters of flurbiprofen and ferulic-acid, a class

of organic nitrates with potential therapeutic implication in variety of different

conditions, was investigated in comparison with glyceryl trinitrate (GTN) in human

liver by a multiple approach, using a spontaneous metabolism-independent NO donor

(NOC-5) as a reference tool. Nitrooxybutyl-esters were rapidly and quantitatively

metabolized to their respective parent compounds and the organic nitrate moiety

nitrooxybutyl-alcohol (NOBA). Differently from GTN which was rapidly and

completely metabolized to nitrite, NOBA was slowly metabolized to nitrate. In contrast

to the spontaneous NO donor NOC-5, NOBA and GTN did not generate detectable NO

and failed to suppress the activity of cytochromeP450; an enzyme known to be inhibited

by NO. The direct identification of NOBA following liver metabolism targets this

compound as the functional organic nitrate metabolite of nitrooxybutyl-esters.

Moreover the investigation of the pathways for denitration of NOBA and GTN suggests

that organic nitrates are not primarily metabolized to NO in the liver but to different

extents of nitrite or nitrate depending from their different chemical structure. It follows

that cytochromeP450-dependent metabolism of concomitant drugs is not likely to be

affected by oral co-administration of organic nitrates. However the first-pass may

differently affect the pharmacological profile of organic nitrates in connection with the

different extent of denitration and the distinct bioactive species generated and exported

from the liver (nitrate or nitrite).


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Introduction

Organic nitrates have been harnessed therapeutically since 1879 when glyceryl trinitrate

(GTN) was first described as a remedy for angina pectoris (Murrell, 1879). GTN, acting

through the liberation of nitric oxide (NO) in vascular tissue, is still widely used in the

treatment of coronary artery disease and congestive heart failure (Parker and Parker,

1998). This compound is rapidly and efficiently absorbed from the mouth leading to a

rapid onset of action that is suitable for the treatment of acute angina attacks. After

absorption from the mouth or the skin GTN is rapidly denitrated by hepatic metabolism

(systemic t1/2 ~ 1min) (Needleman et al., 1976) to its dinitrate form (GDN) which has a

higher systemic half-life (t1/2 ~ 2h) (Needleman et al., 1976) but a lower potency than

GTN (Münzel et al., 2005). When GTN is given by oral route it undergoes extensive

liver first-pass metabolism (Needleman et al., 1972) leaving its dinitrate form to

circulate systemically with little or no parent compound appearing in blood (Needleman

et al., 1976l; Yu et al., 1988). For this reason GTN is administered orally only for

prophylaxis purposes due to its slower onset of action and higher doses required to

reach therapeutic efficacy in comparison to sublingual or transdermal administration.

Efforts to increase its duration of action and prophylaxis have led to the synthesis of

other organic nitrates, of which the most important is isosorbide mononitrate (ISMN).

ISMN has similar pharmacological actions as GTN but it is longer acting and it

undergoes low hepatic metabolism after oral administration (Abshagen, 1992).

Furthermore, the growing realization that nitrates may represent new therapeutic agents

in different areas led to the development of organic nitrates containing adjunct

pharmacophores to introduce biological properties beyond those of the parent

compound (Hodosan et al., 1969; Keeble and Moore, 2002). In particular,


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nitrooxybutyl-ester derivatives is a novel class of hybrid organic nitrates consisting of a

(-ONO2) structural unit connected via a butyl linker to the parent compound by a

carboxyl-ester bond.

While the capacity of organic nitrates to liberate NO in vascular tissue is quite well

established (Münzel et al., 2005; Kleschyov et al., 2003; Mulsch et al.1995), the exact

mechanism of denitration is still a matter of debate. Moreover, whether organic nitrates

are at all capable of generating NO in extravascular tissues, as for example in the liver is

still controversial. Some studies have provided evidence for NO generation from GTN

incubated in subcellular liver fractions (Servent et al., 1989; Kozlov et al., 2003); but a

generalized concept (not supported by any data though) is that the liver cannot

metabolize GTN to NO. Indeed, it is well established that GTN undergoes extensive

hepatic metabolism causing loss of vascular bioactivity (Needleman et al., 1972) but

this does not necessarily imply that the liver is not capable of acute NO generation from

GTN. A better understanding of how organic nitrates are metabolized in the liver is

important when characterizing novel organic nitrates that are under development. As an

example, NO has been reported to be directly and indirectly involved in the inactivation

of cytochrome P450 (Khatsenko et al., 1993; Wink et al., 1993; Stadler et al., 1994)

suggesting that liver CYP-dependent drug metabolism may be drastically affected by

oral co-administration of organic nitrates undergoing first pass metabolism.

Nitrooxybutyl-ester derivatives of anti-inflammatory and/or anti-oxidant agents as

flurbiprofen and ferulic acid is a novel class of organic nitrates with potential

therapeutic implication in variety of different conditions (Keeble and Moore, 2002;

Scatena et al., 2004) as neurodegenerative diseases (Wenk et al., 2002; Wenk et al.,

2004, Gasparini et al., 2005, Prosperi et al., 2004), inflammation and nociception


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(Ronchetti et al., 2009), learning and memory (Boultadakis et al., 2010), airway

inflammation and relaxation (Larsson et al., 2007; Larsson et al., 2009), osteoporosis

and bone resorption (Idris et al., 2004). Despite extensive pharmacological

characterization of these compounds, their metabolism and pathways of denitration

remain unknown. In particular, being targeted mainly for chronic diseases these

molecules are generally designed to be administered by oral route and an evaluation of

their liver metabolism is therefore critical.

In the present study we investigated the metabolism of nitrooxybutyl-ester compounds

in comparison with GTN and ISMN in the human liver by a multiple approach that

includes i) a chemiluminescent nitrite/nitrate/RSNO metabolic profile assessment ii) a

test of NO mediated inhibition of liver cytochrome P450 1A2 (CYP1A2) activity iii) a

direct electrochemical detection of NO and iv) the use of a spontaneous metabolism-

independent NO donor (NOC-5) as a reference tool.


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Materials and Methods

Biological material and chemicals

Human liver microsomes, cytosol and mitochondria were purchased from Tebu-bio,

Magenta, Italy, as pools of different mixed gender donors. HPLC-grade organic

solvents were purchased from Carlo Erba reagents (Milan, Italy). HPLC-grade water

was prepared with a Milli-Q water purification system. HCT 1026, [(1,1’-biphenyl)-4-

acetic acid,2-fluoro-α-methyl,4-(nitrooxy)butyl ester], HCT 1027, [1,1’-biphenyl]-4-

acetic acid,2-fluoro-α-methyl,4-hydroxybutyl ester, NCX 2057, 3-(4-hydroxy-3-

methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester and NCX 2059, 3-(4-

hydroxy-3-methoxyphenyl)-2-propenoic acid 4-hydroxybutyl ester were provided by

NicOx SA (Sophia-Antipolis, France). GTN and NOBA were kindly provided by

Dipharma S.p.A. (Milan, Italy). All other chemicals were purchased from Sigma-

Aldrich (Milan, Italy).

Optimization of the conditions for the evaluation of the inhibitory potential of a

selected test drug on CYP1A2

Phenacetin was used as a probe substrate. This compound is selectively converted to

acetaminophen by the microsomal enzyme CYP1A2. The optimization of the conditions

for evaluation of the inhibitory potential of drugs on microsomal CYP1A2 was

conducted according to the FDA guidance for industries; drug interaction studies, study

design, data analysis and implications for dosing and labeling (2006). Briefly, the

kinetics of acetaminophen formation from phenacetin incubated in human liver

microsomes was tested at 3 different protein concentrations (chosen in order to avoid

enzymatic saturation) for 5 different substrate concentrations. The experimental


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Michaelis-Menten constant values Km and Vmax were calculated by Lineweaver-Burk

equation for each protein concentration tested.

Inhibition experiments were further performed i) at the lowest microsomal protein

concentration tested (0.5 mg/ml) ii) incubating the substrate at the experimental

calculated Km value (50 µM) iii) incubating the substrate for a period of time in which

the formation of acetaminophen is linear and does not exceed the 30% of substrate

depletion (30 min).

Liver subcellular fractions incubations

In vitro incubations were performed in reconstituted human liver homogenate

(microsomes 0.5 mg·ml−1, mitochondria 0.65 mg·ml−1, cytosol 2.05 mg·ml−1) (Kozlov et

al., 2003) or in the single subcellular fraction at the same individual protein

concentration. The following cofactor mixtures were used for all incubations: NADPH

2 mM, NAD+ 0.5 mM, NADH 0.5 mM, reduced glutathione (GSH) 2.5 mM.

Reconstituted human liver homogenate (microsomes + mitochondria + cytosol) or

individual subcellular fractions and cofactors were incubated at the final working

concentration in Na-phosphate buffer pH 7.4 and kept under slight shaking at 37°C

during the experiments. Drugs (HCT 1026, NCX 2057, NOC-5, GTN, ISMN, NOBA)

were dissolved in acetonitrile or water and added to human liver fractions at a final

solvent concentration ≤ 0.5% v/v.

Since diazeniumdiolates like NOC-5 release 2 moles of NO per mole of parent

compound (Fitzhugh and Keefer, 2000), for comparison purposes the compounds were

incubated under equinormal conditions meaning a concentration of GTN, ISMN or

NOBA double that of NOC-5 ([GTN] = [ISMN] = [NOBA] = 2[NOC-5]).


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Incubation for HCT 1026 and NCX 2057 metabolic assessment: the experiment was

started adding the compound (HCT 1026 or NCX 2057 at a final concentration of 250

µM) to reconstituted homogenate. At fixed time points (0, 1, 5, 7, 10, 15, 20, 30, 45, 60,

90, 120, 180, 240 min) 50 µl of the incubation mixture was removed and deproteinized

with 100µl of methanol for HPLC-ENO20 NO2-, NO3

- and NOBA analysis. The

remaining 200 µl of incubation mixture was removed at the same time, deproteinized

with 400 µl of acetonitrile + phosphoric acid 1% (v/v) and processed for HPLC/UV

HCT 1026 or NCX 2057 metabolite profiling.

Incubations for nitrite (NO2-) and nitrate (NO3

-) assessment: the experiment was

started adding the compound (HCT 1026, NCX 2057, NOC-5 GTN, ISMN or NOBA at

a final concentration of 250-500µM) to reconstituted homogenate or single fractions. At

fixed time points, 50 µl of incubation mixture was removed, deproteinized with 100 µl

of methanol and submitted for HPLC-VIS NO2- and NO3

- analysis.

Incubations for CYP1A2 inhibition and nitrosothiol (RSNO) assessment: For each

incubation furafylline was used as standard control (reference CYP1A2 inhibitor). After

thirty minutes of incubation in reconstituted homogenate with the drug (NOC-5, GTN,

ISMN, NOBA; concentration range 1-1000 µM), phenacetin (dissolved in acetonitrile)

was added at the final concentration of 50 µM (solvent final concentration ≤ 0.5% v/v).

After one hour of incubation 200 µl of the incubation mixture was spiked with 20 µl of

internal standard (ketoprofen 5 mM) and deproteinized with 400 µl of acidified

acetonitrile (acetonitrile + formic acid 0,1% (v/v)) for HPLC-MS/MS analysis. At the

same time 600 µl of the incubation mixture was spiked with EDTA (final concentration

2 mM) and N-ethylmaleimide (final concentration 5 mM) and processed for RSNO

chemiluminescence analysis.


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Incubations for NO assessment: the electrochemical sensor was immersed in 4 ml of

reconstituted human liver homogenate under continuous stirring. The experiment was

started by adding the compound (NOC-5 at a final concentration of 250 µM and GTN,

ISMN or NOBA at a final concentration of 500 µM) and the NO signal was recorded

continuously over 1 hour.

Assay for HCT 1026 and NCX 2057 metabolite profiling

The metabolic profile of HCT 1026 and NCX 2057 and their respective metabolites

was carried out by liquid chromatography analysis on an Agilent 1100 system

equipped with a UV/Visible diode array programmable detector. Separations were

achieved by reverse phase elution with a Synergi Hydro-RP 80 Å column (150 x 4.6

mm i.d., particle size 4 µm) equipped with an AQ-C18 precolumn (4 x 3 mm i.d.)

maintained at 25°C. The mobile phase, acetonitrile (solvent A) and sodium

phosphate buffer 25 mM pH 2 (solvent B), was delivered at a flow rate of 1 ml·min-1.

HCT 1026 and its metabolites were detected at λ = 246 nm with the following

gradient composition: 0 min A 20%, 1-8 min A 80%, 9-11 min A 20%. Under these

conditions the retentions times for HCT 1026, its direct denitrated derivative HCT

1027, and flurbiprofen were 6.8; 4.7; 4.2 minutes, respectively.

NCX 2057 and its metabolites were detected at λ = 325 nm with the following

gradient composition: 0 min A 20%, 4-8 min A 80%, 10-12 min A 20%. Under these

conditions the retentions times for NCX 2057, its direct denitrated derivative NCX

2059 and ferulic acid were 6.1, 4.6 and 4.1 minutes, respectively.

NCX 2057, HCT 1026 and their metabolites were chromatographically separated and

characterized on the basis of the typical UV fingerprint of the pure compounds.

Chromatographic peaks were processed for spectral purity characterization by


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statistical analysis for automated comparison of spectra (Agilent Chemstation

software). Only peaks showing spectral matching factors higher than 99% were

considered acceptable. Chromatograms were integrated with an Agilent Chemstation

software and area under the curve converted into concentration using reference

compounds,

Assays for NO2- and NO3

- and NOBA analysis

NO2- and NO3

- and NOBA concentrations in liver fractions were measured after

methanol precipitation of proteins (1:2 v/v) by using a dedicated HPLC system

(ENO-20; EiCom, Kyoto, Japan) coupled with and Agilent 1100 autosampler. The

method (Yamada and Nabeshima, 1997) is based on the separation of nitrite and

nitrate and NOBA by reverse-phase/ion exchange chromatography, followed by on-

line reduction of nitrate and NOBA to nitrite with cadmium and reduced copper.

Nitrite is then derivatized with the Griess reagent and the level of diazo compounds

is measured by a visible detector at 540 nm. The retentions times for nitrite, nitrate

and NOBA were 5.2, 8.6 and 29.5 minutes, respectively.

Gas phase chemiluminescence assay for RSNO

RSNOs were determined by gas-phase chemiluminescence with a NO analyzer

(Aerocrine AB, Sweden) after reductive cleavage and subsequent determination of the

NO released into the gas phase. The method has been described in detail elsewhere

(Lundberg and Govoni, 2004). NO signals were collected and reported as area under the

curve with Azur (v 3.0) chromatographic software (Datalys, Saint-Martin d'Héres,

France). RSNO were reduced to NO with a solution consisting of 45 mmol/l potassium

iodide (KI) and 10 mmol/l iodine (I2) in glacial acetic acid at 60°C and measurement

performed by direct sample injection (300 µl).


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Since the reducing solution is able to convert both nitrite and nitroso/nitrosyl

compounds to NO, RSNO were quantified by simple subtraction of the peak area of

sample aliquots pretreated with sulphanilamide + mercuric chloride at room temperature

for 30 min [10% (v/v) of a solution 5% sulfanilamide + 0.2% HgCl2 in 1 N HCl] from

that of sample aliquots treated only with sulfanilamide at room temperature for 15 min

[10% (v/v) of a solution 5% sulfanilamide in 1 N HCl]. Under these conditions, nitrite

reacts with sulfanilamide to form a stable diazonium ion that is not converted to any

appreciable extent to NO and HgCl2 selectively cleaves the S–NO bond (Feelisch et al.,

2002) without affecting peak shape or recovery of other detectable NO species. The

calibration curve was obtained with freshly prepared S-nitrosoglutathione.

HPLC-MS/MS analysis for CYP1A2 inhibition evaluation

The phenacetin O-deethylase activity of CYP1A2 was evaluated using liquid

chromatography. Analyses were carried out on an Agilent 1100 system coupled with a

triple quadrupole mass spectrometer (API 2000 LC-MS/MS system, Applied

Biosystems/MDS Sciex Toronto, Canada). Separations were achieved by reverse phase

elution with a Phenomenex Synergy Hydro-RP 80 Å (50 x 4 mm i.d., particle size 4

µm) equipped with a Phenomenex AQ-C18 precolumn (4 x 3 mm i.d.) maintained at

25°C. The mobile phase, water containing 0.1% formic acid (solvent A) and acetonitrile

(solvent B), was delivered at a flow rate of 0.35 ml·min-1 at the following gradient

composition: 0-1 min A 100%, 1.10-4.30 min A 0%, 4.40-15 min A 100%. Under these

conditions the retention times for acetaminophen, phenacetin and ketoprofen (internal

standard) were 3.87, 4.12 and 4.4 minutes, respectively.

The following mass spectrometer settings were used: turbo ionspray interface

maintained at 300°C; detection (positive ion mode) of the analytes using the multiple


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reaction monitoring (MRM) scan mode was performed with collision activated

dissociation gas (CAD) at 5 psi; curtain gas (CUR) at 20 psi; turbo ion spray voltage at

+5500 V; channel electron multiplier (CEM) at 2400 V; source gases (GS1 and GS2)

pressures at 40 and 50 psi, respectively; declustering potential (DP), focusing potential

(FP) and entrance potential (EP) were set at 31, 370 and 10.05 V, respectively;

experiments were performed using a dwell time of 50 ms. The spectra were acquired at

unit resolution.

The mass transitions [MRM] of acetaminophen, phenacetin and ketoprofen were m/z

152→110, m/z 180→138 and m/z 255→105, respectively. Chromatograms were

integrated with Analyst software (version 1.2 Applied Biosystem, CA, USA) and area

under the curve converted into concentration using reference compounds.

Assay for electrochemical detection of NO

The measurement of NO release was performed with the inNO nitric oxide

measurement system using the amiNO-700 sensor electrode (Innovative Instruments

Inc., U.S.A.). The output current was monitored and recorded on a Windows 98

compatible system using analysis software provided with the inNO system.

The sensor was calibrated by adding known volumes of standard nitrite solution to an

acidified solution in the presence of iodide ion according to the manufacturer's

specifications and settled for temperature compensation before the experiment.

Data analysis

NOx (NO3- + NO2

-), nitrite and nitrate values in the incubations were subtracted from

the basal (time 0). One-way analysis of variance was used to evaluate differences

between groups and it was followed by a Tukey post hoc test for comparisons between

multiple groups. Differences were considered significant at p < 0.05. IC50 values for


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enzyme inhibition were obtained by fitting to the sigmoid equation:Y= Y0 + [a /

1+(X/IC50)b] were Y = % inhibition and X = inhibitor concentration. Data were modeled

by nonweighted nonlinear regression analysis using SigmaPlot 8.02 (Systat Software,

Inc., Point Richmond, CA).


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Results

Liver metabolism of nitrooxybutyl-ester compounds.

The chemical structures of HCT 1026, NCX 2057 and their metabolites arising from

biotransformation pathways involving hydrolytic cleavage of the ester functions in the

molecule are depicted in Fig. 1A and Fig. 1B, respectively.

HCT 1026 and NCX 2057 were rapidly and extensively metabolized at the carboxyl-

ester function. HCT 1026 and NCX 2057 rapidly disappeared (t1/2 = 7.03 ± 1.24, 9.15 ±

0.28 minutes, respectively) with a stoichiometric formation of flurbiprofen and ferulic

acid, respectively (Fig. 1A,B). At the same time the formation of NOBA and NOx

species coming from its metabolism, was consistent with the generation of flurbiprofen

and ferulic acid, respectively. In fact, the kinetics of NOBA generation from HCT 1026

and NCX 2057 overlapped the kinetics of flurbiprofen and ferulic acid, respectively,

over the first 20-30 minutes of incubation and thereafter declining consistent with the

slow metabolism of NOBA to NOx species (tmax = 30 and 45 minutes for HCT 1026 and

NCX 2057, respectively and Cmax = 70.78 ± 8.13 and 83.38 ± 3.06 % of incubated for

HCT 1026 and NCX 2057, respectively). The elimination of NOBA was consistent with

the generation of equimolar amount of NOx exclusively in the form of NO3- for both

compounds tested.

HCT 1026 was converted to flurbiprofen with V0 = - 19.32 ± 5.79 µM/min consistent

with V0 of flurbiprofen and NOBA formation of 18.63 ± 1.54 µM/min and 18.39 ± 3.05

µM/min , respectively, (- V0 HCT 1026 ≈ V0 Flurbiprofen ≈ V0 NOBA) (Fig. 1A). NCX 2057 was

converted to ferulic acid with V0 = - 13.87 ± 2.51 µM/min consistent with V0 of ferulic

acid and NOBA formation of 12.93 ± 0.15 µM/min and 11.30 ± 1.23 µM/min,


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respectively, (- V0 NCX 2057 ≈ V0 Ferulic Acid ≈ V0 NOBA) (Fig. 1B). HCT 1027 and NCX

2059 were not observed over the incubation time.

Incubations with the vehicle in liver homogenate or with the compounds alone in buffer

did not produce any increase in NOx concentrations.

The liver metabolism of other compounds belonging to the class of nitrooxybutyl-ester

derivatives was investigated and showed very similar metabolic properties to HCT 1026

and NCX 2057 (data not shown).

NOC-5, GTN, ISMN and NOBA liver metabolism

NOC-5 and GTN were rapidly and extensively metabolized to NOx species. The

kinetics of NOx generation from the two compounds overlapped over 90 min of

incubation with an almost identical V0 (13.0 ± 0.9 µM/min and 12.9 ± 0.4 µM/min for

NOC-5 and GTN, respectively) (Fig. 2). However, the generation of nitrite and nitrate

as individual species was different. While GTN generated nitrite as the main metabolite

(V0 NO2- = 13.1 ± 0.1 µM/min ≈ V0 NOx >> V0 NO3

- = 0.4±0.1 µM/min) (Fig. 3A),

NOC-5 generated both nitrite and nitrate with similar V0 (V0 NO2- = 7.3 ± 0.2 µM/min,

V0 NO3- = 5.7 ± 0.2 µM/min) (Fig. 3B).

NOBA incubated under the same experimental conditions displayed a slower rate of

metabolism to form NOx than GTN while NOx formation from ISMN was very low

(Fig. 4). An analysis of the individual species generated from NOBA also showed

differences in terms of metabolic products. In fact, NOBA led exclusively to the

generation of NO3- with a V0 NO3

- ≈ V0 NOx = 1.2±0.1 µM/min and a generation of NO3

-

70 µM and 460 µM after 60 and 240 minutes of incubation, respectively (Fig. 3C).

Incubations with the vehicle in liver homogenate or with the compounds alone in buffer

did not produce any increase in NOx concentrations.


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RSNO formation

Fig. 5A shows the generation of RSNO measured 1 hour after incubation of NOC-5 and

GTN at different concentrations. Although the amount of RSNO formed was much

lower in comparison to the initial concentration, NOC-5 showed a higher nitrosating

capacity than GTN,in particular at the highest concentrations tested, where RSNO levels

reached 9.8 ± 2.1µM for NOC-5 and 3.5 ± 0.3µM for GTN (Fig. 5A). Incubations with

NOBA or ISMN under the same experimental conditions did not produce any RSNOs,

nor did incubations with vehicle in liver homogenate or with the compounds alone in

buffer.

Inhibition of CYP1A2

The effects of GTN, ISMN, NOBA and NOC-5 on CYP1A2 activity are shown in Fig.

5B. The estimated IC50 values obtained showed that organic nitrates as ISMN, NOBA

and GTN did not significantly inhibit the phenacetin O-deethylase activity of CYP1A2.

In contrast, NOC-5 completely inhibited CYP1A2 activity at a concentration of 500µM

with an IC50 of 114.2 µM. Incubations were performed in homogenate (microsomes +

mitochondria + cytosol) to guarantee the presence of a complete pool of liver enzymes

and complete liver metabolic activity.

Direct measurement of NO formation

Generation of NO was assessed by an electrochemical sensor and large differences were

noted between NOC-5 and the organic nitrates GTN, ISMN and NOBA. The

spontaneous metabolism-independent NO donor NOC-5, showed a rapid generation of

NO peaking (tmax) at 6 min and reaching a maximum concentration (Cmax) of 4.15 µM

(Fig. 5C). In contrast, GTN, NOBA and ISMN did not show any NO increase over the


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study period (Fig. 5C). Lower concentrations of organic nitrates were also tested but

again, no formation of NO was detected (data not shown).

Incubations with the vehicle in reconstituted homogenate or with compounds alone in

buffer did not produce any NO.

Metabolism of GTN in different liver compartments

Conversion of GTN to NOx. differed between the various liver compartments studied.

Among the fractions, the cytosol was able to metabolize GTN with the highest rate V0

cytosol = 7.8 ± 0.5 µM/min being more than 4 times higher than the metabolism in the

other fractions (V0 mitochondria = 1.0 ± 0.1 µM/min, V0 microsomes = 1.7 ± 0.1 µM/min) (Fig.

6A). No significant differences were observed when GTN was incubated in

homogenate (microsomes + mitochondria + cytosol) compared to incubations in the

cytosolic fraction (V0 Cytosol ≈ V0 microsomes+mitochondria+cytosol = 7.9 ± 0.6 µM/min) (Fig.

6A). Moreover the generation of nitrite in the first part of the pharmacokinetic profile

(0-10 min) in homogenate and cytosol were similar (Fig. 6B)

Although GTN was found to be stable in phosphate buffer (data not shown), it was

slightly unstable in the presence of cofactors. Incubations performed in presence of each

cofactor alone (NAD+, NADH, NADPH or GSH) showed that this instability is due to

GSH slowly reacting with GTN and generating NOx species (data not shown).


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Discussion

In a previous study (Govoni et al., 2006) we showed that nitrooxybutyl-ester derivatives

undergoes ubiquitous and fast esterase-mediated carboxyl-ester hydrolysis in rat liver

and plasma, yielding to the formation of the parent compound and an organic nitrate

moiety speculatively identified as the nitrooxybutyl-alcohol species (NOBA). The direct

and unequivocal identification of NOBA following metabolism of nitrooxybutyl-ester

(Fig. 1) has been made possible only in the current study with the development of a

novel chromatographic method based on the on line conversion of organic nitrates to

nitrite and subsequent derivatization with the griess reagent. It follows that NOBA is in

fact the active organic nitrate metabolite of nitrooxybutyl-esters and for these reasons it

has been used on its own to investigate the mechanism of denitration of this class of

compounds in human liver in comparison to GTN, ISMN and the spontaneous NO

donor NOC-5. The various organic nitrates displayed considerably different rates of

liver metabolism to NOx, with GTN being rapidly metabolized NOBA showing an

intermediate rate and ISMN being negligibly metabolized (Fig. 4). Notably, NOC-5 and

GTN generated identical quantities of NOx (Fig. 2). Assuming that the first metabolic

step of organic nitrates in the liver is associated with the release of NO which is

consequently oxidized to nitrite and nitrate, these data might have been taken as

evidence for GTN and NOC-5 having the same NO releasing kinetics in the liver.

However, when looking at the generation of NO2- and NO3

- separately (Fig. 3A,B) it

became evident that the mechanism of denitration is different from these two molecules.

While NOC-5 denitration is consistent with a direct generation of NO followed by

oxidation to both NO2- and NO3

-, GTN denitration led almost exclusively to the

generation of NO2- (Fig. 7). Notably, also the metabolic profile of NOBA was not


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consistent with a direct generation of NO leading exclusively to the generation of NO3-

species (Fig. 3C). In addition to the different NO2-/NO3

- metabolic profile, GTN, NOBA

and NOC-5 also showed different nitrosating capacity measured as S-nitrosothiol

generation. In fact, consistent with the direct generation of NO in turn forming the

potent nitrosating agent N2O3 (Fig. 7), S-nitrosothiols formation was higher for NOC-5

(Fig. 5A). A further indirect assessment of GTN and NOBA metabolism comes from

the experiments looking at the activity of CYP1A2, an enzyme potently inhibited by

NO (Stadler et al., 1994). NOC-5 was found to extensively inhibit CYP1A2 activity

while the different organic nitrates did not (Fig. 5B).

These results clearly suggest that organic nitrates share a common pathway of

denitration in the liver not primarily being metabolized to NO. Instead, depending on

their chemical structure, they are metabolized to nitrate or nitrite. This was further

demonstrated by measuring NO formation as shown in Fig. 5C. While NOC-5 directly

released NO, GTN and NOBA did not show any detectable NO formation.

Importantly, the denitration of GTN in liver homogenate is exclusively carried out by

the cytosolic fraction with a negligible contribution from the mitochondrial as well as

the microsomal compartments. This is clearly evidenced by the fact that the denitration

of GTN in the presence of a complete pool of liver enzymes (microsomes +

mitochondria + cytosol) was the same as when using only cytosolic enzymes (Fig. 6).

Interestingly it has been demonstrated previously (Kozlov et al., 2003) that the cytosolic

fraction of the liver is not capable of significative GTN bioactivation to NO. Taken

together these findings suggest that the absence of intrahepatic NO generation is

explained by the much higher rate of denitration carried out by the cytosolic enzymes

thereby consuming GTN before it reaches the other compartments.


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Recently it has been reported that the enzymatic metabolism of organic nitrates might

be concentration-dependent (Daiber et al., 2009) and therefore a careful evaluation of

the test concentrations used is needed when investigating the metabolism of these

compounds. GTN, ISMN and nitrooxybutylester-compounds can be taken by the oral

route in doses of 46 µmoles, 628 µmoles and 2,2 mmoles, respectively (Parker and

Parker, 1998; Fagerholm et al., 2005). These molecules are lipophilic and very rapidly

absorbed to reach the liver. GTN is almost completely metabolized during first passage

leaving the dinitrate metabolite GDN to circulate systemically (Needleman et al., 1976,

Yu et al., 1988). The tmax of intact detectable GTN in vivo in plasma after oral intake is

5 minute after administration (Yu et al., 1988). This suggests that the entire dose taken

is rapidly concentrated in the liver during first pass. Nitrooxybutylester-derivatives are

rapidly absorbed from the GI tract and their parent compound appears in the circulation

with almost complete bioavailability (Fagerholm et al., 2005). This suggests formation

of NOBA at relevant concentration in the liver. Following these considerations the mid

micromolar concentrations tested in the present work seem appropriate for analyzing the

metabolic pathway during first passage. These concentrations might however not be

suitable to assess the functional metabolism of organic nitrates after the first passage

since the concentrations of intact drug reaching the target tissues is orders of magnitude

lower.

NO donor compounds are known to inhibit CYP by forming nitrosyl-heme CYP

reversibly in competition with oxygen (Ignarro et al., 2002). Such decreased CYP

mediated activity might have implications for drug interactions when NO donors are

given in association with other drugs metabolized by CYPs. In this in vitro study

organic nitrates were not found to inhibit CYP1A2 (Fig. 5B) most likely due to lack of


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NO release (Fig. 5C). Translated in vivo these findings suggest that CYP-dependent

drug metabolism of concomitant drugs would not be affected by co-administration of

organic nitrates.

Another implication of the results of current study is that while the systemic

bioavailablity of organic nitrates might be decreased by its liver metabolism to nitrate or

nitrite this specific metabolic route may support another recently described NO

pathway. Nitrate can be converted in vivo to nitrite that is emerging as a stable

circulating NO pool and a variety of pathways have been described for the reduction of

nitrite to NO in blood and tissues (Lundberg et al., 2008; Lundberg et al., 2009). Such

NO formation is expected to be slower and more prolonged than direct release of NO

from most organic nitrates and can significantly contribute to the final therapeutic effect

of these molecules in connection to the extent and identity of species generated (nitrate

or nitrite) and exported from the liver.

In conclusion, our data suggests that organic nitrates are not primarily metabolized to

NO in the liver and their different chemical structures not only dramatically change the

extent of denitration in this organ but also the entity of the metabolic species generated.

ISMN undergoes negligible liver metabolism while GTN is rapidly and extensively

metabolized primarily to nitrite. Nitrooxybutyl-ester compounds on the other hand are

first rapidly metabolized to the organic nitrate moiety NOBA which is eventually

slowly metabolized to inorganic nitrate. If the lower extent of denitration of NOBA in

comparison to GTN was also reflected in vivo during first passage, NOBA might have

the potential to overcome the liver and reach (at least in part) the systemic circulation

allowing for bioactivation to NO in the vascular compartment or elsewhere where a

different enzymatic pool than in the liver exists. In summary the first-pass may


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differently affect the pharmacological profile of organic nitrates in connection with the

different extent of denitration and the distinct species generated and exported from the

liver (nitrate or nitrite).


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Authorship Contributions.

Participated in research design: Govoni, Tocchetti, Lundberg Conducted experiments: Govoni Contributed new reagents or analytic tools: Govoni, Lundberg Performed data analysis: Govoni Wrote or contributed to the writing of the manuscript: Govoni, Lundberg


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Footnotes.

a) This work was supported by NicOx S.A., Sophia Antipolis Cedex, France

c) Person to receive reprint requests:

Name: Mirco Govoni

Affiliation: Department of Physiology and Pharmacology, Karolinska Institute

Street address: Nanna Svartz väg 2

Postal code: S-177 76

City: Stockholm

State:Sweden

Telephone: +39 339 8657744

Fax: +46 8 332278

email: [email protected]

d) Authors name:

Mirco Govoni1,2, Paola Tocchetti2 and Jon O Lundberg1

1Department of Physiology and Pharmacology, Karolinska Institute, Nanna Svartz väg

2, S-177 76 Stockholm, Sweden

2NicOx S.A., Les Taissounières - 1681 Route des Dolines - Sophia Antipolis Cedex,

France


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Legends for figures.

Fig. 1. Chemical structures and metabolic profile of HCT 1026 (A), NCX 2057 (B) and

their metabolites arising from biotransformation pathways involving hydrolytic

cleavage of the carboxylic and nitric ester functions in reconstituted human liver

homogenate (cytosol + microsomes + mitochondria) with cofactors. HCT 1026 (A) and

NCX 2057 (B) were incubated at a final concentration of 250µM and their metabolic

profile followed over the incubation time. Filled up triangles (�) show the kinetic of

flurbiprofen and ferulic acid formation from HCT 1026 and NCX 2057 (�),

respectively, while the direct denitrated metabolites HCT 1029 or NCX 2059 were not

observed over the incubation time. Open squares represent the kinetics of NOBA

generation from HCT 1026 (A) and NCX 2057 (B) (�) and filled up diamonds the time

course of NOx species generated over the incubation time (�). NOx values are

subtracted from the basal (time 0). Data are presented as mean ± S.D., n ≥ 3.

Fig. 2. Extent of denitration of GTN and NOC-5 in reconstituted human liver

homogenate (microsomes + mitochondria + cytosol) with cofactors. GTN (�) and

NOC-5 (�) were incubated at equinormal conditions (500µM and 250µM for GTN and

NOC-5, respectively) and the profile of NOx species generated followed over the

incubation time. Values are subtracted from the basal (time 0). Data are presented as

mean ± S.D., n ≥ 3.

Fig. 3. Metabolism of GTN (A), NOC-5 (B) and NOBA (C) in reconstituted human

liver homogenate (microsomes + mitochondria + cytosol) with cofactors. GTN, ISMN


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and NOC-5 were incubated at equinormal conditions (500µM for GTN and ISMN and

250µM for NOC-5) and the profile of NO2- and NO3

- species generated followed over

the incubation time. Values are subtracted from the basal (time 0). Data are presented as

mean ± S.D., n ≥ 3.

Fig. 4. Extent of denitration of organic nitrates in reconstituted human liver homogenate

(microsomes + mitochondria + cytosol) with cofactors. GTN (�), ISMN (�) and

NOBA (�) were incubated at a final concentration of 500 µM and the profile of NOx

species generated followed over the incubation time. Values are subtracted from the

basal (time 0). Data are presented as mean ± S.D., n ≥ 3.

Fig. 5. (A) RSNO generation from GTN and NOC-5 in reconstituted human liver

homogenate (microsomes + mitochondria + cytosol) with cofactors. GTN and NOC-5

were incubated at different concentrations ranging from 5µM to 1000µM and the

amount of RSNO species generated was measured after 1 hour of incubation.

Comparisons were made between groups incubated at equinormal concentrations

([GTN] = 2[NOC-5]). Data are presented as mean ± SEM, n ≥ 3. *, p < 0.05. Notably

NOBA and ISMN incubated in the same experimental conditions did not show any

RSNO increase and were therefore omitted from the present figure. (B) CYP1A2

inhibition in reconstituted human liver homogenate (microsomes + mitochondria +

cytosol) with cofactors. NOC-5 (�), GTN (�), ISMN (�) and NOBA (�) were

preincubated for 30 minutes at different concentrations (ranging from 1 to 500µM) and

the phenacetin O-deethylase activity of CYP1A2 measured 30 minutes after addition of

phenacetin. The CYP1A2 activity was considered 100% in presence of the vehicle. Data


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are presented as mean ± S.D., n ≥ 3. *, p < 0.05. (C) Direct measurement of NO by

electrochemical sensor in reconstituted human liver homogenate (microsomes +

mitochondria + cytosol) with cofactors. ISMN, NOBA, GTN and NOC-5 were

incubated at equinormal conditions (500µM for ISMN, NOBA, GTN and 250µM for

NOC-5) and the generation of NO followed over 1 hour of incubation.

Fig. 6. (A) Extent of denitration of GTN in the different liver compartments. GTN was

incubated at a final concentration of 250 µM and the profile of NOx species generated

followed over the incubation time in reconstituted human liver homogenate

(microsomes + mitochondria + cytosol) with cofactors (�), human liver cytosol with

cofactors (�), human liver microsomes with cofactors (�), human liver mitochondria

with cofactors (�), buffer + cofactors (�). (B) Nitrite formation in liver homogenate

(microsomes + mitochondria + cytosol) and cytosol in the first 10 min of incubation.

Values are subtracted from the basal (time 0). Data are presented as mean ± S.D., n ≥ 3.

Fig. 7. Metabolism of NOC-5, GTN and NOBA in human liver. While NOC-5 is

spontaneously converted to NO which in turn is metabolized in the liver to nitrite,

nitrate and nitrosothiols, the organic nitrates GTN and NOBA are directly metabolized

to nitrite and nitrate, respectively.


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0 50 100 150 200 2500

20

40

60

80

100

120

% o

f inc

ubat

ed

Time (min)

NCX2057 Ferulic Acid NOBA NOx

Fig. 1

A B HCT 1026

O

O

FO

N+O

O-FLURBIPROFEN

OH

O

F

+ HO

ON+

O

O-

NOBA

NCX 2057

HO

O

O

OO

N+

O

O-

FERULIC ACID HO

O

O

OH

+

HOO

N+

O

O-

NOBA

0 50 100 150 200 2500

20

40

60

80

100

120

% o

f inc

ubat

ed

Time (min)

HCT1026 Flurbiprofen NOBA NOx


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Fig. 2

0 10 20 30 40 50 60 70 80 900

50100150200250300350400450

GTNNOC-5

Time (min)

NO

x C

once

ntra

tion

(µM

)


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Fig. 3 A

B

0 50 100 150 200 2500

100

200

300

400

500

NitriteNitrate

GTN

Time(min)

Con

cent

ratio

n (µ

M)

0 50 100 150 200 2500

100

200

300

400

500

NitriteNitrate

NOC-5

Time (min)

Con

cent

ratio

n (µ

M)

C

0 50 100 150 200 2500

100

200

300

400

500

NitriteNitrate

NOBA

Time (min)

Con

cent

ratio

n (µ

M)


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Fig. 4

0 50 100 150 200 2500

100

200

300

400

500

GTNISMNNOBA

Time

NO

x C

once

ntra

tion

(µM

)


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Fig. 5

B

A

C


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Fig. 6


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GTN

2NO+O2→2NO2

NO2+NO → N2O3

N2O3 + H2O→ 2NO2-+2H+

N2O3+RSH→RSNO+NO2-+ H+

NO2-

NO

NO2- RSNO

NO+ Fe(II)O2→Fe(III) + NO3-

NO3-

2NO+O2→2NO2

NO2+NO→N2O3

N2O3+RSH→RSNO+NO2-+ H+

NOC-5

Fig. 7

NOBA

NO3-

Nitrooxybutyl-esters


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Documents

JPET #203356 1. Title page Title: Metabolism and pathways for