Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N 3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations

Article abstract of DOI:10.1124/jpet.105.097170

Thromboxane (TX) A₂, prostacyclin (PGI₂), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA₂, implemented synthesis of PGI₂, and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N¹-(4-trans-nitrooxycyclohexyl)-N ³-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC₅₀, 7.9 and 17.1 μM, respectively), an effect abolished by 10 μM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC₅₀, 9.8 μM) and TXB₂ formation (-71% at 10 μM), and its potency increased in the presence of aortic rings (EC₅₀, 1.4 μM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA₂ formation, reduced by 2NTX-99 (10-40 μM): contraction, -28 and -47%, TXA₂ formation, -37 and -75.4%, respectively, with concomitant increase in PGI₂. 2NTX-99 (20-40 μM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 ± 6.7 and -96 ± 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 ± 10 and -79.2 ± 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA₂ receptor isophorm α receptor, 2NTX-99 did not compete with the ligand, [³H]SQ29,548 ([ ³H][1S-[1α,2β(5Z),3β,4α]]-7-[3-[[2- (phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5- heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA₂ production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/μg/ml and a t_1/2 of 6 h were calculated, with a C_max value of 31.8 ± 8.2 μg/ml. An excellent correlation between plasma concentrations and TXA₂ inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential. Copyright

Full text of DOI:10.1124/jpet.105.097170

0022-3565/06/3172-830–837$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
JPET 317:830–837, 2006
Vol. 317, No. 2
97170/3096215
Printed in U.S.A.
Pharmacological Characterization of 2NTX-99 [4-Methoxy-N1-
(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-
benzenedicarboxamide], a Potential Antiatherothrombotic
Agent with Antithromboxane and Nitric Oxide Donor Activity in
Platelet and Vascular Preparations
Carola Buccellati, Angelo Sala, Giuseppe Rossoni, Vale´ rie Capra, G. Enrico Rovati,
Antonio Di Gennaro, Giancarlo Folco, Susanna Colli, and Cesare Casagrande
Department of Pharmacological Sciences, School of Pharmacy, University of Milano, Milano, Italy
Received November 11, 2005; accepted January 5, 2006
ABSTRACT
Thromboxane (TX) A2, prostacyclin (PGI2), and nitric oxide (NO) increase in PGI2. 2NTX-99 (20–40 ␮M) inhibited U46619-
regulate platelet function and interaction with the vessel wall. induced aggregation in rabbit platelet-rich plasma (PRP)
Inhibition of TXA2, implemented synthesis of PGI2, and supply of (Ϫ74 Ϯ 6.7 and Ϫ96 Ϯ 2.4%, respectively) and inhibited col-
exogenous NO may afford therapeutic benefit. 2NTX-99 [4-me- lagen-induced aggregation in human PRP (Ϫ48.2 Ϯ 10 and
thoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinyl-
Ϫ79.2 Ϯ 6%), whereas ozagrel was ineffective. In human em-
methyl)-1,3-benzenedicarboxamide], a new chemical entity re- bryonic kidney 293 cells transfected with the TXA2 receptor
lated to picotamide, showed antithromboxane activity and NO isophorm ␣ receptor, 2NTX-99 did not compete with the ligand,
donor properties. 2NTX-99 relaxed rabbit aortic rings precon- [3H]SQ29,548 ([3H][1S-[1␣,2␤(5Z),3␤,4␣]]-7-[3-[[2-(phe-
tracted with norepinephrine or U46619 (9,11-dideoxy-9␣,11␣- nylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-
methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC50, 7.9 and hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate ac-
17.1 ␮M, respectively), an effect abolished by 10 ␮M 1H- cumulation. After oral administration (50–250 mg/kg), 2NTX-99
(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 in- inhibited TXA2 production in rat clotting blood (Ϫ71 and
hibited arachidonic acid (AA)-induced washed platelet aggre- Ϫ91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of
gation (EC50, 9.8 ␮M) and TXB2 formation (Ϫ71% at 10 ␮M), 149.5 h/␮g/ml and a t1/2 of 6 h were calculated, with a Cmax
and its potency increased in the presence of aortic rings (EC50
,
value of 31.8 Ϯ 8.2 ␮g/ml. An excellent correlation between
1.4 ␮M). In whole rabbit aorta incubated with homologous plasma concentrations and TXA2 inhibition occurs. 2NTX-99
platelets, AA caused contraction and TXA2 formation, reduced controls platelet function and vessel wall interaction by multi-
by 2NTX-99 (10–40 ␮M): contraction, Ϫ28 and Ϫ47%, TXA2 factorial mechanisms and possesses therapeutic potential.
formation, Ϫ37 and Ϫ75.4%, respectively, with concomitant
Aspirin, a nonreversible inhibitor of platelet cyclooxygenase approaches to the modulation of platelet function by preventing
(COX)-1, has been the mainstay of antiplatelet therapy for over
20 years (Antithrombotic Trialist Collaboration, 2002). Novel
platelet secretion and/or aggregation, their adhesion to the ves-
sel wall, or progression of thrombus development (Bhatt and
Topol, 2003) have been addressed in experimental and clinical
studies. Until now, the only novel drug suitable for chronic
therapy, which in some trials appeared superior to aspirin in
preventing cardiovascular accidents (CAPRIE, 1996), is clopi-
dogrel, an inhibitor of platelet P2Y12 receptor.
This work was supported by European Community Grant LSHM-CT-2004-
005033 (to G.F.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.097170.
ABBREVIATIONS: COX, cyclooxygenase; PG, prostacyclin; TX, thromboxane; NCX-4016, 2-acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester;
2NTX-99, 4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide; NO, nitric oxide; PRP, platelet-rich plas-
ma; AA, arachidonic acid; OKY-046, ozagrel; EIA, enzyme immunoassay; U46619, 9,11-dideoxy-9␣,11␣-methanoepoxy-prosta-5Z,13E-dien-1-oic
acid; NE, norepinephrine; GTN, glyceryl trinitrate; DMEM, Dulbecco’s modified Eagle’s medium; TP␣, thromboxane A2 receptor isophorm ␣;
SQ29,548, [3H][1S-[1␣,2␤(5Z),3␤,4␣]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]-methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid; IP, ino-
sitol phosphate; HPLC, high-performance liquid chromatography; CV, coefficient(s) of variation; ISMN, isosorbide mononitrate; ODQ, 1H-
(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PGH2, prostaglandin H cyclic endoperoxide.
830

2NTX-99, an Antithromboxane and NO Donor
831
Significant advancement in our understanding of the role
of platelets in the atherothrombotic process (Bhatt and
Topol, 2003) supports the concept that therapeutic efficacy
may be improved by a combined action on platelet activation
and interaction with the vascular wall. Indeed, several stud-
ies have shown that combined treatment with clopidogrel
and aspirin in acute coronary syndromes (Mehta et al., 2001)
or with dipyridamole and aspirin in the prevention of stroke
(Forbes, 1998), offer advantage over single treatment. How-
ever, concern about lack of response in subsets of patients
and about development of resistance during chronic treat-
ment (Gurbel and Bliden, 2003; Eikelboom and Hankey,
2004) stresses the need of novel therapeutic approaches
(Bhatt and Topol, 2003).
Different prostanoids originate from the common endoper-
oxide precursor PGH2, which is further metabolized by spe-
cific enzymes according to a strict cellular specificity, yield-
ing, e.g., mostly TXA2 in platelets, PGI2 in endothelial cells,
etc. (Maclouf et al., 1998). However, PGH2 can be also made
available extracellularly for further paracrine conversion to
bioactive eicosanoids. The inhibition of a given enzymatic
pathway within a defined cell facilitates an intercellular
shunt of PGH2 toward an alternative pathway, e.g., in plate-
let-endothelial cell coincubates, PGI2 synthesis is enhanced
when thromboxane synthase is inhibited (Nowak and
FitzGerald, 1989).
The search for TXA2 inhibitors has targeted thrombox-
ane synthase or thromboxane receptors but has also devel-
oped “dual” inhibitors that combine inhibition of TXA2
formation with antagonism of the receptor-mediated ac-
tions of PGH2, of any residual TXA2, and of the isopros-
tanoid 8-epi-PGF2a (Dogne et al., 2000). In addition, shunt-
ing of PGH2 toward PGI2 may take place when, e.g.,
platelets interact with the vascular wall. Among dual in-
hibitors, only picotamide (Fig. 1) (Gresele et al., 1989) has
found clinical application in peripheral arterial disease in
diabetic patients (Modesti, 1995; Coto et al., 1998; Neri
Serneri et al., 2004).
An impaired production of nitric oxide (NO) by damaged
endothelium is considered one of the key factors in the de-
velopment of atherosclerosis and thrombotic events (Napoli
and Ignarro, 2001; Walford and Loscalzo, 2003). Potential
benefit of NO supply is suggested by the preventive action of
the NO-releasing derivative of aspirin, NCX-4016 (Napoli et
al., 2002), in murine models of atherosclerosis and restenosis,
an effect not shared by aspirin alone.
Structural modifications of picotamide, although pre-
serving its antithromboxane activity, allowed the insertion
of an NO donor moiety, leading to the synthesis of com-
pound 2NTX-99 (Fig. 1). 2NTX-99 is a new molecular en-
tity that targets three powerful regulators of platelet and
vascular function, i.e., TXA2, PGI2, and NO. TXA2 pro-
motes platelet activation and increases vascular tone and
neointima proliferation, whereas both PGI2 and NO, per se
or in synergy, counteract the biological actions of TXA2
(Moncada et al., 1991; Maclouf et al., 1998). In this article,
we report the pharmacological profile of 2NTX-99 on plate-
let and vascular preparations, as well as results from a
preliminary kinetic study following oral administration
to rats.
Fig. 1. Chemical structures of Picotamide, 2NTX-99, and 2NTX-101.
Materials and Methods
Synthesis of 2NTX-99 (Fig. 1)
The synthesis (U.S. Patent 6,525,078) started from the regioselec-
tive mono-amination of the dimethyl ester of 4-methoxy-1,2-ben-
zenedicarboxylic acid with 3-pyridynilmethylamine, yielding the
3-N-pyridinylmethylamide 1-ester. The corresponding acid, obtained
by alkaline hydrolysis, was activated with carbonyldiimidazole and
allowed to react with trans-4-hydroxycyclohexylamine to give the
isomerically pure N1,N3-disubstituted amide 2NTX-101 (Fig. 1). Its
esterification with nitric acid-acetic anhydride led to compound
2NTX-99, showing m.p. 153 to 154°C; its structure was confirmed by
1H NMR in dimethyl sulfoxide-d6.
Pharmacology
Blood Collection and Aorta Isolation. The use of experimental
animals adhered to the European Community guidelines. New Zea-
land male rabbits (Harlan Italy, Milan, Italy) weighing 2 to 2.5 kg
were anesthetized (Zoletil 20, 1 ml/kg), the left carotid was isolated
and cannulated for blood collection in 200 mM EDTA (1:40, v/v), and
rabbits were sacrificed by complete bleeding. Part of the thoracic
tract was cut into 2- to 3-mm-wide transverse rings, whereas a 3- to
4-cm thoracic-abdominal segment was isolated for contractility
studies.
Platelet Preparation and Aggregation Studies. Rabbit blood
was anticoagulated with EDTA to obtain platelet-rich plasma (PRP)
and further centrifuged at 1800g for 20 min at 20°C to isolate
platelets. The pellet was carefully resuspended in Tyrode-G (2.5 mM
KCl, 1 mM MgCl2, 120 mM NaCl, 25 mM NaHCO3, 5 mM glucose,
and 0.25% w/v gelatin, pH 6.5) with EGTA (0.2 mM) and centrifuged
under the same conditions (Bossant et al., 1990); finally, the pellet
was resuspended in Tyrode-G-Ca-HEPES (Tyrode G ϩ 0.9 mM CaCl2
and 4.2 mM HEPES, pH 7.4), and the platelet count was adjusted to
450,000 cells/␮l.
Platelet aggregation was studied using the Born turbidimetric
technique in a dual-channel Elvi 840 aggregometer (Elvi Logos,

832
Buccellati et al.
Milan, Italy). Before aggregation experiments, the aortic rings were Nitrite concentration was determined using sodium nitrite as stan-
Ϫ
preincubated with 1 mM acetylsalicylic acid, to inhibit endothelial dard. Results are expressed as nanograms per milliliter of NO2
cell cyclooxygenase activity, for 30 min at room temperature and
then washed twice in Tyrode-G-Ca-HEPES.
.
Thromboxane Receptor Studies
Aliquots (250 ␮l) of washed platelets, in the presence or absence of
aortic rings, were preincubated for 2 min at 37°C under stirring and
further preincubated for 3 min with drugs or their vehicles before
challenge with arachidonic acid (AA) (1.5–3.0 ␮M). Six minutes after
challenge, aggregation was stopped by adding 10 ␮M indomethacin
and 7.6 mM EDTA. The platelet suspension was then centrifuged at
11,600g for 5 min at room temperature. The supernatant was divided
in two parts and kept at Ϫ20°C until enzyme immunoassay (EIA) of
AA metabolites. The extent of aggregation was quantified as the area
under the aggregation curve from 0 to 6 min and expressed as weight
of paper (milligrams) of uniform density.
Platelet aggregation induced by U46619 was determined in rabbit
PRP, obtained by centrifugation of citrated blood (trisodium citrate
final concentration 0.3% w/v, 150g, 15 min). Threshold aggregating
concentration of U46619 was used (1–3 ␮M), and percentage inhibi-
tion by different compounds was calculated by the reduction of the
aggregation amplitude 5 min after challenge.
Culture and Transfection of HEK293 Cells. HEK293 cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) sup-
plemented with 10% fetal bovine serum, 2 mM glutamine, 50
units/ml penicillin, 100 ␮g/ml streptomycin, and 20 mM HEPES
buffer, pH 7.4, at 37°C in a humidified atmosphere of 95% air and 5%
CO2. Transfection with human TP␣ construct was performed as
described previously (Capra et al., 2004).
Ligand Binding Assays. Receptor expression was monitored
48 h after the transfection. A mixed-type protocol together with
heterologous competition were performed as described previously
(Capra et al., 2003, 2004). In brief, confluent adherent cells in 250 ␮l
of serum-free DMEM, containing 0.2% (w/v) bovine serum albumin,
were assayed in the presence of 0.1 to 3 nM of the specific receptor
antagonist SQ29,548 (48 Ci/mmol), 0.01 to 10 ␮M of the homologous
unlabeled ligand, or 0.1 nM to 10 ␮M of the heterologous unlabeled
ligands (U46619, 2NTX-99). After 30 min of incubation at 25°C, cells
were washed with ice-cold phosphate-buffered saline containing
0.2% (w/v) bovine serum albumin and lysed in 0.5 N NaOH. Analysis
of binding data was performed by means of the LIGAND program
(Munson and Rodbard, 1980).
Total Inositol Phosphate Determination. The functional ac-
tivity of receptor was assessed 48 h after transfection by measuring
total inositol phosphate (IP) accumulation as described previously
(Habib et al., 1997; Capra et al., 2004). HEK293 cells were labeled
with 1 ␮Ci myo-[2-3H]inositol (17 Ci/mmol) for 24 h in serum-free,
inositol-free DMEM, containing 20 mM HEPES buffer, pH 7.4, and
0.5% w/v Albumax I. Cells were washed and incubated with 25 mM
LiCl for 10 min, pretreated with the indicated concentrations of
study compounds (SQ29,548, 2NTX-99), and then were incubated for
30 min with either vehicle or 1 ␮M of the agonist U46619. Cells were
then lysed and extracted with an anion exchange AG 1X-8 column
(Bio-Rad, Hercules, CA). Free inositol and glycerophosphoinositol
were washed with 40 mM ammonium formate/formic acid buffer, pH
5, and total IP was eluted with 4 ml of a 2 M ammonium formate/
formic acid buffer, pH 5.
2NTX-99 Oral and Intravenous Administration to Rats.
2NTX-99 was administered to anesthetized rats (thiopental sodium
salt, 50 mg/kg i.p.) both i.v. (25 mg/kg) and p.o. (50 or 250 mg/kg).
Blood was collected 30 min after i.v. 2NTX-99 infusion or 30 to 360
min and 18 h after p.o. administration. Samples were incubated for
30 min at 37°C to obtain serum or added with EDTA (10 ␮g/ml) and
Na-heparin (50 U/ml) to obtain plasma. Systemic blood pressure and
heart rate were determined by insertion into a carotid artery of a
Collagen-induced platelet aggregation was determined in PRP
from healthy donors as described previously (Tremoli et al., 1984).
For each subject, a collagen concentration (0.5–1.0 ␮g/ml) that in-
duced a 50 to 60% decrease of optical density within 5 min was
selected to test the effect of the drugs. Data are expressed as percent
inhibition of platelet aggregation.
Rabbit Aorta Contractility. Four aortic rings were set up for
isometric recording in oxygenated buffer (Krebs-Henseleit: 5 mM
KCl, 1 mM MgSO4 ϫ 7H2O, 119 mM NaCl, 1 mM KH2PO4, 25 mM
NaHCO3, 5 mM glucose, and 2.5 mM CaCl2) at 37°C. Responses to an
endothelium-dependent vasodilating agent such as acetylcholine
(1–3 ␮M) were tested following enhancement of vascular tone with a
submaximal (1 ␮M) concentration of NE (or alternatively with the
TXA2 analog, compound U46619, 10 nM) to verify endothelium in-
tegrity. Vessels that gave a relaxation lower than 50% were not used.
After wash of the preparations, the vascular tone was again in-
creased with the submaximal concentration of NE, and a concentra-
tion-response curve of 2NTX-99 (0.1–100 ␮M) was constructed. The
capacity of 2NTX-99 to produce tachyphylaxis was investigated by
exposing the aortic rings to a concentration of the compound that was
the highest possible given its solubility profile (300 ␮M, 3 h) or
appropriate dimethyl sulfoxide blank.
A vascular segment with the endothelial lining exposed was pre-
pared for isotonic contraction recording as described previously (Buc-
cellati et al., 2002). After testing of the functionally intact endothe-
lium as described above, the entire chamber volume was substituted
with the washed platelet suspension (2.5 ml, 450,000 cells/␮l), pre-
treated for 15 min with the drugs under test or with vehicle. AA (12
␮M) was added 15 min later to stimulate platelet TXA2 formation
and vascular contraction (30-min total platelet-drug incubation).
Aliquots of the incubation suspension (200 ␮l) were collected for EIA
quantitation 15 min after AA challenge.
PE-60 cannula connected to
a pressure transducer (HP-1280;
Hewlett-Packard, Waltham, MA).
To assess bleeding time, a small incision was applied longitudi-
nally between the median and lower dorsal portion of the tail (be-
tween 4 and 6 cm from the end of the tail), taking care to avoid the
artery. Blood from the wound was collected onto a filter paper every
30 s. Bleeding times were recorded as the interval between incision
Assay of TXA2, PGI2, and NO. The stable metabolites of TXA2
and PGI2 (TXB2 and 6-keto-PGF1␣, respectively) were evaluated by and bleeding arrest starting at 30 min after i.v. treatment.
selective EIA (Pradelles et al., 1985), carried out directly on aliquots
Determination of 2NTX-99 and 2NTX-101 in Rat Plasma by
of the incubation media, according to the manufacturer’s instruction HPLC. The choice of the RP-HPLC and the extraction methods were
(Cayman Chemicals, Ann Arbor, MI).
investigated using reference standards of 2NTX-99 and 2NTX-101
Nitrite (NO2Ϫ) was measured using the Griess reaction, which (the primary, denitrated metabolite of 2NTX-99; Fig. 1), with pico-
possesses significant sensitivity limitations but allows measurement tamide as internal standard. Compounds eluted from a LiChrosorb
of the cumulated amount of nitrite, a significant marker of NO
release in the assay sample. 2NTX-99, 2NTX-101, and 120 ␮M stadt, Germany) as well separated peaks at 5.3 Ϯ 0.5, 9.5 Ϯ 0.5, and
isosorbide mononitrate or GTN (40 ␮M) were incubated with rat 34 Ϯ 1.8 min, using solvent A (30% acetonitrile; 70% NaH2PO4
5-␮m, RP-SELECT B, C8, 25-cm ϫ 4-mm i.d. column (Merck, Darm-
ϫ
aortic rings (20 mg wet weight) in 0.2 ml of Krebs-Henseleit buffer, 0.05 M H2O, pH 6) as mobile phase; absorbance was monitored at
pH 7.4, at 37°C for different times (30, 60, 120, 180, and 360 min). 230 nm. Both 2NTX-99 and 2NTX-101 appeared to be pure, and only
Supernatants were allowed to react (1:1, v/v) with the Griess reagent trace amounts of 2NTX-101 were present in 2NTX-99. Extraction
(0.5% sulfanilamide, 0.05% naphtylethylendiammine dihydrochlo- from rat plasma was carried out after alkalinization on C18 Bond
ride, and 2.5% H3PO4) to form a chromophore absorbing at 546 nm. Elut cartridges, followed by elution with ethyl acetate, evaporation,

2NTX-99, an Antithromboxane and NO Donor
833
and reconstitution in 300 ␮l of 0.6 M HCl and 600 ␮l of ethyl acetate
(Fossati et al., 1992). The lower acidic aqueous phase was taken to
dryness and redissolved in 40 ␮l of solvent A before injection into the
HPLC system.
Quantitation was performed using standard curves (200 ng–20 ␮g
of synthetic 2NTX-99 and 2NTX-101, together with the internal
standard) prepared in rat plasma, and extracted, and analyzed as
described. The curve was linear with a correlation coefficient of 0.99
for both compounds.
Data Analysis
The concentration-response curves of platelet aggregation were
analyzed and drawn by means of the computer program ALLFIT,
and evaluation of the statistical significance of the parameter differ-
ence was based on the F test for the extra sum of square principle
(Draper and Smith, 1966). Statistical evaluation of the data was
carried out by analysis of variance (one-way analysis of variance or
repeated measure analysis of variance with one grouping factor, as
indicated); p Ͻ 0.05 was considered statistically significant.
Statistical analysis of ligand-binding data were performed with
the LIGAND program (Munson and Rodbard, 1980). Parameter er-
rors are always expressed in percentage coefficient of variation (CV)
and calculated by simultaneous analysis of at least two different
independent experiments performed in duplicates or triplicates.
Data are presented as means Ϯ mean S.E. of multiple independent
experiments, each performed at least in duplicates. A statistical level
of significance of p Ͻ 0.05 was accepted.
Fig. 2. A, dose-dependent relaxation by 2NTX-99, 2NTX-101, ISMN, and
GTN on norepinephrine-induced rabbit aortic rings contraction. B, dose-
dependent relaxation induced by 2NTX-99 and ISMN on U46619-induced
rabbit aortic ring contraction. Effect of the selective guanylate cyclase
inhibitor ODQ. Data represent mean values. Bars, mean Ϯ S.E. of n
replicates.
Materials
Zoletil 20 (tiletamine and zolazepam) was from Virbac (Milan,
Italy); norepinephrine bitartrate salt, acetylcholine chloride, arachi-
donic acid sodium salt, and OKY-046 were from Sigma Chemical Co.
(St. Louis, MO). Thromboxane B2 and 6-keto prostaglandin F1␣ EIA
kits, SQ 29,548, and U46619 were from Cayman Chemicals. Gelatin
powder and all inorganic salts were from Merck. Ultrapure water
(MilliQ) was from Millipore Co. (Bedford, MA). Isosorbide mononi-
trate (ISMN) was purchased from Chiesi Farmaceutici S.p.A.
(Parma, Italy). Collagen was from Mascia Brunelli (Milano, Italy).
Transfection reagent ExGen 500 was from MBI Fermentas
(Hanover, MD). Cell culture media, serum, supplements, and molec-
ular biology reagents were purchased from Gibco Invitrogen Co.
(Carlsbad, CA). Inositol-free-DMEM was from ICN Pharmaceuticals
Inc. (Costa Mesa, CA). HEK293 cells were obtained from American
Type Culture Collection (Rockville, MD). Ultima Gold was from
Packard Instruments (Meriden, CT). [5,6-3H]SQ29,548 and myo-[2-
3H]inositol were purchased from Perkin-Elmer (Boston, MA). Stock
solution of these compounds were stored at Ϫ20°C. Anion exchange
resin AG 1X-8 (formate form, 200–400 mesh) and Poly-Prep columns
were from Bio-Rad. All other reagents were of the highest purity
available from Sigma Chemical Co.
EC50 values of the compound varied moderately before (2.5 Ϯ
0.38 ␮M, n ϭ 4) and after (6.2 Ϯ 1.0 ␮M) exposure to 300 ␮M
2NTX-99.
Compound 2NTX-99 (0.1–100 ␮M) caused a concentration-
dependent relaxation (EC50, 17 Ϯ 1.2 ␮M) of aortic rings
precontracted with the stable thromboxane analog U46619,
at a concentration (10 nM) as efficacious as 1 ␮M NE. The
effects of 2NTX-99 (30 ␮M) and ISMN (60 ␮M) were pre-
vented by pretreatment with ODQ (10 ␮M), a selective in-
hibitor of NO-sensitive guanylate cyclase enzyme activity
(Fig. 2B).
Incubation of rat aortic rings with compound 2NTX-99 (120
Ϫ
␮M) triggered a time-dependent formation of NO2 (30 min,
103 Ϯ 42; 60 min, 179 Ϯ 32; 120 min, 213 Ϯ 31; 180 min,
353 Ϯ 97; 360 min, 314 Ϯ 58 ng/ml; n ϭ 4) that plateaued
after 180 min, whereas 2NTX-101 was ineffective. ISMN (120
␮M) also led to nitrite formation (103 Ϯ 33 ng/ml, n ϭ 4), and,
as expected, GTN (40 ␮M, n ϭ 3) led to a significant increase
Results
Ϫ
in NO2 (4.96- Ϯ 0.36-fold over 2NTX-99).
Vascular Response and Modulation of AA Metabolism by
2NTX-99
The ability of compound 2NTX-99 to inhibit TXA2 synthe-
sis and action (and to stimulate PGI2 formation) was inves-
2NTX-99 (0.1–100 ␮M) caused a concentration-dependent tigated in preparations of rabbit aorta incubated with homol-
relaxation of aortic rings precontracted with NE (1 ␮M), ogous platelets, as recently reported (Buccellati et al., 2002).
with a potency approximately 300-fold lower than GTN When intact vessel specimens were incubated with a suspen-
(EC50, 7.9 Ϯ 0.4 and 0.03 Ϯ 0.002 ␮M, respectively) and twice sion of washed platelets (450,000/␮l) and challenged with 12
as potent as ISMN (EC50, 15.9 Ϯ 0.9 ␮M). The relaxation ␮M AA, a strong contraction occurred; pretreatment with
curve of 2NTX-99 was shifted (approximately 12-fold) to the 2NTX-99 (10 and 40 ␮M, 30 min) caused a significant reduc-
right, in a parallel way, by pretreatment with 10 ␮M meth- tion of AA-induced contraction (Ϫ24.5 and Ϫ47%, respec-
ylene blue, an inhibitor of guanylate cyclase (data not tively) (Fig. 3A).
shown). Compound 2NTX-101, the primary denitrated me-
tabolite of 2NTX-99, induced only a modest relaxation at 100 se triggered significant synthesis of TXB2 and 6-keto-PGF1␣
␮M, and no EC50 value could be calculated (Fig. 2A). The which was markedly enhanced following challenge with AA
The coincubation of washed platelets with aortic rings per
,

834
Buccellati et al.
Fig. 3. A, effect of 2NTX-99 on the contraction of isolated whole-rabbit
aorta incubated with homologous platelets (450 ϫ 106/ml) and challenged
with AA. B, effect of 2NTX-99 on TXA2 and PGI2 synthesis in whole-
rabbit aorta incubated with homologous platelets and challenged with
AA. TXA2 and PGI2 were detected as their stable metabolite, TXB2 and
6-keto-PGF1␣, respectively. Data represent mean values. Bars, mean Ϯ
S.E. of n replicates.
Fig. 4. A, dose-dependent inhibition by 2NTX-99 on AA-induced aggre-
gation (1.5–3 ␮M) of rabbit platelets (450 ϫ 106/ml) in the presence or
absence of an aortic ring. B, dose-dependent effect of 2NTX-99 on TXA2
and PGI2 synthesis in rabbit platelets challenged by AA in the absence or
presence of an aortic ring. TXA2 and PGI2 were detected as their stable
metabolite, TXB2 and 6-keto-PGF1␣, respectively. Data represent mean
values. Bars, mean Ϯ S.E. of n replicates.
(data not shown). Pretreatment with 2NTX-99 (10 and 40
␮M) reduced TXB2 synthesis and increased PGI2 formation
significantly (Fig. 3B).
6%, respectively; n ϭ 5) platelet aggregation in human PRP
challenged with threshold collagen concentrations. Com-
pound 2NTX-101 (the primary denitrated metabolite of
2NTX-99) as well as the reference TX synthase inhibitor
OKY-046 (ozagrel, 40 ␮M) were ineffective. In this experi-
mental setting, ozagrel (40 ␮M) inhibited TX formation by
approximately 80%, whereas equimolar concentrations of
2NTX-99 or 2NTX-101 reduced TX formation by 40% (data
not shown).
Effect of 2NTX-99 on Platelet Aggregation
The effect of compound 2NTX-99 on platelet aggregation
was evaluated in washed rabbit platelet suspensions
(450,000/␮l) stimulated with a submaximal aggregating con-
centration of AA (1.5–3 ␮M). 2NTX-99 inhibited platelet ag-
gregation concentration-dependently (IC50 9.83 Ϯ 1.1 ␮M);
its potency was increased in the presence of aortic rings (IC50
1.45 Ϯ 0.15 ␮M) (Fig. 4A). 2NTX-99 inhibited TXB2 forma-
tion either in the absence or presence of aortic rings (Ϫ71 and
Ϫ80%, respectively, at 10 ␮M); conversely, the compound
stimulated 6-keto-PGF1␣ formation in a concentration-de-
pendent way only when vascular rings were present (Fig.
4B). 2NTX-99 (20–40 ␮M) also prevented the aggregation
Binding of 2NTX-99 to Human TP␣ and Total IP
Determination
Mixed-type curves of [3H]SQ29,548 and heterologous
induced by threshold U46619 concentrations (1–4 ␮M) in competition curves of the agonist U46619 clearly display
rabbit PRP (Ϫ74 Ϯ 6.7% and 96.4 Ϯ 2.4%, respectively; n ϭ monophasic binding curves fitting a single-site model by com-
8) thus sharing the behavior of picotamide (Gresele et al., puterized analysis performed with the program LIGAND
1989).
(Fig. 5A). The simultaneous analysis of three independent
2NTX-99 (20–40 ␮M) inhibited (Ϫ48.2 Ϯ 10% and Ϫ79.2 Ϯ experiments indicated typical binding parameters (SQ29,548

2NTX-99, an Antithromboxane and NO Donor
835
below); bleeding time was 4.3 Ϯ 0.2 min (n ϭ 3) in control
conditions and was significantly prolonged (12.8 Ϯ 0.6 min)
by drug treatment.
Determination of 2NTX-99 in Rat Plasma and Inhi-
bition of Thromboxane A2 Synthesis. Intravenous ad-
ministration of 2NTX-99 to rats (25 mg/kg for 30 min) sup-
pressed TXA2 production in clotting blood (94% inhibition
compared with vehicle-treated animals, n ϭ 5); plasma levels
(n ϭ 2) were 25.67 and 27.44 ␮g/ml (mean concentration,
61 ␮M).
After oral administration (250 mg/kg), plasma levels of
2NTX-99 and TXA2-synthesis inhibition were assessed up to
16 h postdosing, showing sustained plasma concentrations
and long-lasting pharmacological activity. In a few selected
experiments, 2NTX-99 was administered at a lower dose (50
mg/kg), and plasma levels were followed up to 3 h; inhibition
of TXA2 production peaked at 90 min (Ϫ71%), and plasma
levels (n ϭ 2) were 9.29 and 6.95 ␮g/ml (mean concentration
19 ␮M) (Fig. 6, A and B).
An area under the curve, 0 to 16 h, of 149.5 h/␮g/ml was
calculated for the 250 mg/kg dose, with a Cmax value of 31.8 Ϯ
8.2 ␮g/ml and an estimated half-life of 6 h, even if the spread
(from 0.29–9.69 ␮g/ml) of plasma values at 16 h was broad.
The results of the 250 mg/kg dose at 16 h were compared with
the more limited data of the 50 mg/kg dose. Areas under the
curve, 0 to 3 h, were, respectively, calculated as 66 and 18.3
h/␮g/ml, showing, when adjusted for the dose, a higher
(ϩ38%) value for the lower dose. The difference might be
attributable to slower dissolution and absorption of the in-
soluble compound from the administered suspension of the
higher dose. An excellent correlation between the plasma
concentrations of 2NTX-99 and the inhibition of TXA2 syn-
thesis was observed (r2 ϭ 0.72; EC50, 11.8 ␮M).
Fig. 5. A, binding of [3H]SQ29,548 in HEK293 transiently expressing
human TP␣ receptor. For the sake of clarity, only curves from a repre-
sentative experiment are shown. B, total IP formation in HEK293 tran-
siently expressing human TP␣ receptor; accumulation was measured
after incubation in the absence (basal) and presence of U46619 for 30
min. SQ29,548 and 2NTX-99 were added 30 min before U46619. Data
represent mean values. Bars, mean Ϯ S.E. of n replicates.
Kd ϭ 3.48 Ϯ 36% CV; U46619 Ki ϭ 64.2 Ϯ 83% CV), as
previously reported (Capra et al., 2004). On the contrary,
compound 2NTX-99 did not compete for the labeled antago-
nist (Fig. 5A).
Signaling of TP␣ receptor was also investigated by mea-
suring the capacity of 2NTX-99 to inhibit agonist-induced
total IP production (Fig. 5B). HEK293 cells expressing the
human TP␣ responded to 1 ␮M U46619 stimulation with a
marked elevation of total IPs (3.2-fold increase), an effect
that was specifically and significantly (p Ͻ 0.01) prevented
by 30-min pretreatment with 1 ␮M SQ29,548. In contrast,
30-min pretreatment with 2NTX-99 up to 40 ␮M was totally
ineffective, suggesting that the compound does not inhibit
TP␣-induced phospholipase-C activation.
Animal Studies
Effect of 2NTX-99 on Systemic Blood Pressure and
Bleeding Time in the Rat. Administration of 2NTX-99 to
anesthetized rats (25 ␮g/kg i.v. over a 3-min period) led to a
transient drop of systemic blood pressure from control values
of 117.3 Ϯ 1.3 to 109.3 Ϯ 1.3 mm Hg (n ϭ 3); the decrease in
blood pressure peaked 3 to 4 min after administration and
fully recovered thereafter, reaching control values (118.7 Ϯ
1.3 mm Hg) between 30 and 60 min. No significant change in
heart rate was observed. The effect of 2NTX-99 on bleeding
time was also investigated 30 min after i.v administration of
Fig. 6. Time course of inhibition of rat serum TXB2 (A) and 2NTX-99
plasma levels (B) after oral (250 mg/kg) administration of 2NTX-99;
comparison with i.v. (25 mg/kg, 0.5 h). In selected experiments (0.5–3 h)
2NTX-99 was administered p.o. at 50 mg/kg. Columns represent mean
25 mg/kg, a level that fully inhibited platelet function (see values. Bars, mean Ϯ S.E. of n replicates.

836
Buccellati et al.
Formation of Denitrated Metabolite 2NTX-101. In all we performed classic competition experiments in a recombi-
plasma samples from treated animals, a prominent chro- nant system expressing the TP␣ receptor (it would have been
matographic peak corresponding to intact 2NTX-99 was ob- the same using the TP␤ because the two isoforms are iden-
served, together with less relevant peaks at the retention tical for the first 328 residues and differ only in the C-
time of 2NTX-101, the primary, denitrated metabolite of terminal tail of no relevance for ligand binding). The receptor
2NTX-99; quantitative analysis indicated amounts ranging was labeled with the competitive antagonist [3H]SQ29,548
between 3 and 6% of the concentrations of 2NTX-99. Incuba- and competed with the unlabeled 2NTX-99. Furthermore, in
tion of 2NTX-99 (40 ␮M) in rat plasma in the presence of the same system, we also demonstrated that 2NTX-99 does
aortic rings (at 37°C for 30 min) resulted in amounts of not antagonize the U46619-induced IP production, clearly
2NTX-101 equal to 0.41 Ϯ 0.26% (mean Ϯ S.D.) of the parent demonstrating that 2NTX-99 is not a TP receptor antagonist.
compound, whereas 2NTX-101 was not detected in incuba- Rather, inhibition of U46619-induced platelet aggregation
tion of 2NTX-99 in plasma alone.
indicates that 2NTX-99 behaves as a functional antagonist,
possibly with the contribution of the NO-releasing compo-
nent of 2NTX-99. The exact molecular mechanisms behind
this somehow unexpected finding are presently unclear and
Discussion
In the present article, we describe the pharmacology of will be addressed specifically in separate investigations.
2NTX-99, an orally active, innovative chemical entity with In addition, 2NTX-99 attenuated platelet aggregation in
plural actions on TXA2 synthesis, PGI2 formation, and NO human PRP stimulated with threshold concentrations of col-
availability, resulting in functional effects that span from lagen (0.5–1 ␮g/ml) and reduced partially (Ϫ40%) TX forma-
vascular relaxation to inhibition of platelet aggregation and tion. Ozagrel did not affect collagen-induced platelet aggre-
their interactions with the vessel wall. 2NTX-99 is a struc- gation despite a marked reduction of TX formation (80%), in
tural analog of picotamide, a dual thromboxane synthase line with the existence of a nonlinear relationship linking TX
inhibitor/TXA2 receptor antagonist (Gresele et al., 1989). synthesis and platelet aggregation (Reilly and FitzGerald,
2NTX-99, although retaining the thromboxane synthase in- 1987; Buccellati et al., 2002). These findings are of particular
hibitory activity, did not bind to the TP␣, nor did it affect interest since exposed collagen represents a primer of plate-
TP␣-induced signal transduction. Moreover, the contribution let adhesion, activation, and release of inflammatory and
of the NO donor properties of 2NTX-99 has been well docu- prothrombotic mediators (Farndale et al., 2004). As expected,
mented and confirmed by the lack of activity of the deni- a similar degree of TX synthesis inhibition was shared by the
trated derivative 2NTX-101.
denitrated derivative 2NTX-101, which failed to affect plate-
In NE-precontracted rings, 2NTX-99 showed NO-depen- let aggregation. These results, altogether, are suggestive of
dent vasorelaxant potency (EC50, 7.9 ␮M) markedly lower an NO-mediated antiplatelet effect of 2NTX-99.
than GTN (EC50, 0.03 ␮M) but higher than ISMN (EC50, 15.9
The in vitro antiaggregatory activity of organic nitrates is
␮M). GTN is considered as an organic nitrate with high well recognized and shown to depend on structure and on
vasorelaxant potency, whereas our data clearly indicate that steric orientation of the nitrate function (Weber et al., 1993);
2NTX-99 (and ISMN) belong to the group of organic nitrates ISMN possesses ex vivo antiaggregatory effect in patients
with lower vasorelaxant potency but less prone to tolerance (De Caterina et al., 1990), comparable with those of ISDN
development (Daiber et al., 2004).
(De Caterina et al., 1984). The actions of organic nitrates on
ISMN, the main metabolite of isosorbide dinitrate, is an vessels and platelets, at difference from spontaneous NO
orally active, clinically proven antianginal agent with sus- donors, are dependent upon enzymatic mechanisms for acti-
tained effects. ISMN is considered a more reliable reference vation and release of NO (Ahlner et al., 1991). These mech-
for in vitro pharmacological comparison than its parent com- anisms are generally more prominent in vascular cells than
pound, whose pharmacokinetic and pharmacodynamic be- in platelets. 2NTX-99, combining the structural features for
havior is governed by biphasic NO release from two nitrate antithromboxane activity with the insertion of an NO donor
functions with very different rates of activation (Ahlner et moiety, i.e., a nitrate ester of secondary hydroxy group, equa-
al., 1991). 2NTX-99 fully relaxed aortic rings precontracted torially oriented on a cyclohexane ring, was predicted to show
with U46619 (EC50, 17 ␮M), whereas ISMN caused only a slow rate of activation. Indeed, this NO donor moiety af-
partial relaxation. The effect of 2NTX-99 was abolished by forded adequate stability and metabolic resistance, as needed
ODQ, an inhibitor of guanylate cyclase, and shifted to the for absorption and for sustained effect, at variance with other
right by the guanylate cyclase inhibitor methylene blue (data NO donors, mostly characterized by scarce stability, short
not shown).
duration of action, or lack of oral bioavailability (Megson,
2NTX-99 inhibited aggregation of washed rabbit platelets 2000). The cumulated release of NO from 2NTX-99 and
stimulated by AA, and its potency was increased approxi- ISMN following incubation with rat aortic rings at 3 h goes
mately 7-fold in the presence of aortic rings with intact en- hand in hand with the potency of the compounds in relaxing
dothelium. 2NXT-99 inhibited TXA2 synthesis, either in the rabbit aorta, i.e., 5.4 and 2.4% of the amount released by
absence or presence of aortic rings (70–80% at 10 ␮M), and in GTN, respectively, indicating for both compounds the re-
the latter situation increased PGI2 synthesis (approximately quirement of concentrations over 200 times higher than GTN
3-fold at 10 ␮M), as shown by other TX synthase inhibitors to equal its rate of release (7.9 and 15.9 ␮M, respectively,
(Gresele et al., 1991; Buccellati et al., 2002). 2NTX-99 inhib- versus 30 nM GTN), as well as the relaxing effect of nano-
ited U46619-induced platelet aggregation, thus showing a molar concentrations of the endothelial flow of endogenous
profile similar to dual TX synthase inhibitors-TP antagonists NO (Moncada et al., 1991). 2NTX-99 did not spontaneously
(Gresele et al., 1989; Hanson et al., 2005). To assess the release NO in phosphate buffer nor generate detectable
thromboxane A2 receptor antagonist properties of 2NTX-99, amounts of 2NTX-101 when incubated in rat plasma,

2NTX-99, an Antithromboxane and NO Donor
837
CAPRIE (1996) SCCAPRIE. Lancet 348:1329–1339.
whereas 2NTX-101 formation was observed by incubation in
rat plasma in the presence of aortic rings.
Coto V, Oliviero U, Cocozza M, and Milani M (1998) Long-term safety and efficacy of
picotamide, a dual-action antithromboxane agent, in diabetic patients with carotid
atherosclerosis: a 6-year follow-up study. J Int Med Res 26:200–205.
Daiber A, Oelze M, Coldewey M, Bachschmid M, Wenzel P, Sydow K, Wendt M,
Kleschyov AL, Stalleicken D, Ullrich V, et al. (2004) Oxidative stress and mito-
chondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tet-
ranitrate with other organic nitrates. Mol Pharmacol 66:1372–1382.
De Caterina R, Giannessi D, Crea F, Chierchia S, Bernini W, Gazzetti P, and
L’Abbate A (1984) Inhibition of platelet function by injectable isosorbide dinitrate.
Am J Cardiol 53:1683–1687.
De Caterina R, Lombardi M, Bernini W, Mazzone A, Giannessi D, Moscarelli E,
Weiss M, and Lazzerini G (1990) Inhibition of platelet function during in vivo
infusion of isosorbide mononitrates: relationship between plasma drug concentra-
tion and hemodynamic effects. Am Heart J 119:855–862.
Dogne JM, de Leval X, Delarge J, David JL, and Masereel B (2000) New trends in
thromboxane and prostacyclin modulators. Curr Med Chem 7:609–628.
Draper NR and Smith H (1966) Applied Regression Analysis. Wiley, New York.
Eikelboom JW and Hankey GJ (2004) Failure of aspirin to prevent atherothrombo-
sis: potential mechanisms and implications for clinical practice. Am J Cardiovasc
Drugs 4:57–67.
Farndale RW, Sixma JJ, Barnes MJ, and de Groot PG (2004) The role of collagen in
thrombosis and hemostasis. J Thromb Haemost 2:561–573.
Forbes CD (1998) Secondary stroke prevention with low-dose aspirin, sustained
release dipyridamole alone and in combination: ESPS Investigators: European
Stroke Prevention Study. Thromb Res 92:S1–S6.
Fossati T, Parisi S, Abbiati G, and Castiglioni C (1992) Determination of picotamide
in human plasma and urine by high-performance liquid chromatography. J Chro-
matogr 577:382–386.
Gresele P, Deckmyn H, Arnout J, Nenci GG, and Vermylen J (1989) Characterization
of N,NЈ-bis(3-picolyl)-4-methoxy-isophtalamide (picotamide) as a dual thrombox-
ane synthase inhibitor/thromboxane A2 receptor antagonist in human platelets.
Thromb Haemost 61:479–484.
The i.v. administration of 2NTX-99 caused a moderate and
transient drop in systemic blood pressure, without affecting
heart rate, and significantly prolonged bleeding time. These
findings were largely expected, given the capacity of the
compound to cause vascular relaxation, and are in line with
its ability to markedly inhibit platelet function and TX syn-
thase enzyme activity. Moreover, the metabolic fate of
2NTX-99 was addressed in vivo in a preliminary study of oral
administration in rats. Sustained plasma levels of the intact
molecule were observed, along with low concentrations of the
denitrated metabolite (3–6% with respect to the parent com-
pound). These amounts may well represent the kinetic bal-
ance between the formation of the metabolite in vascular and
other tissues and its elimination and appear compatible with
the amount generated in vitro and the moderate, but phar-
macologically significant, release of NO measured in arterial
tissues.
The pharmacokinetic study, within the limits of the small
number of animals and the degree of variability, allowed an
estimate of the half-life of 2NTX-99 of 6 h. Importantly, an
excellent correlation (r2 ϭ 0.724) between the plasma levels
of 2NTX-99 and the inhibition of TXA2 synthesis in clotting
blood was observed. The in vivo experiments indicate that
2NTX-99 represents a novel chemical entity, not a prodrug or
a mutual prodrug of two active molecules (Bolla et al., 2005),
that is absorbed and exerts its sustained action in intact
form. As a consequence, the multiplicity of its diverse effects,
observed in vitro in a balanced fashion, can be elicited in vivo
consistently with a sole pharmacokinetic pathway of absorp-
tion and distribution.
Gresele P, Deckmyn H, Nenci GG, and Vermylen J (1991) Thromboxane synthase
inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic
disorders. Trends Pharmacol Sci 12:158–163.
Gurbel PA and Bliden KP (2003) Interpretation of platelet inhibition by clopidogrel
and the effect of non-responders. J Thromb Haemost 1:1318–1319.
Habib A, Vezza R, Creminon C, Maclouf J, and FitzGerald GA (1997) Rapid, agonist-
dependent phosphorylation in vivo of human thromboxane receptor isoforms.
Minimal involvement of protein kinase C. J Biol Chem 272:7191–7200.
Hanson J, Rolin S, Reynaud D, Qiao N, Kelley LP, Reid HM, Valentin F, Tippins J,
Kinsella BT, Masereel B, et al. (2005) In vitro and in vivo pharmacological char-
acterization of BM-613 [N-n-pentyl-NЈ-[2-(4Ј-methylphenylamino)-5-nitrobenzene-
sulfonyl]urea], a novel dual thromboxane synthase inhibitor and thromboxane
receptor antagonist. J Pharmacol Exp Ther 313:293–301.
In conclusion, 2NTX-99 offers an innovative profile of plu-
ral actions on platelet activation and interaction with the
vascular wall, inhibiting the synthesis of thromboxane, in-
creasing that of prostacyclin, and providing a pharmacologi-
cally relevant supply of NO. NO, in turn, may stimulate PGI2
formation and suppress TXA2 synthase activity; NO also
activates guanylyl cyclase to increase cGMP and acts syner-
gistically with PGI2 to increase cAMP levels in, e.g., platelets
and vascular smooth muscle cells (Antman et al., 2005).
Taken together, the net effect of these actions is to provide
optimal control of platelet and vessel function in athero-
thrombosis.
Maclouf J, Folco G, and Patrono C (1998) Eicosanoids and iso-eicosanoids: constitu-
tive, inducible and transcellular biosynthesis in cardiovascular disease. Thromb
Haemost 79:691–705.
Megson IL (2000) Nitric oxide donor drugs. Drugs Future 25:701–715.
Mehta SR, Yusuf S, Peters RJG, Bertrand ME, Lewis BS, Natarajan MK, Malmberg
K, Rupprecht HJ, Zhao F, Chrolavicius S, et al. (2001) Cure Study Investigators.
Lancet 358:527–533.
Modesti P (1995) Picotamide: an inhibitor of the formation and effects of TXA2.
Cardiovasc Drug Rev 13:353–364.
Moncada S, Palmer RM, and Higgs EA (1991) Nitric oxide: physiology, pathophysi-
ology and pharmacology. Pharmacol Rev 43:109–142.
Munson PJ and Rodbard D (1980) Ligand: a versatile computerized approach for
characterization of ligand-binding systems. Anal Biochem 107:220–239.
Napoli C, Aldini G, Wallace JL, de Nigris F, Maffei R, Abete P, Bonaduce D,
Condorelli G, Rengo F, Sica V, et al. (2002) Efficacy and age-related effects of nitric
oxide-releasing aspirin on experimental restenosis. Proc Natl Acad Sci USA 99:
1689–1694.
Napoli C and Ignarro LJ (2001) Nitric oxide and atherosclerosis. Nitric Oxide
5:88–97.
Neri Serneri GG, Coccheri S, Marubini E, and Violi F (2004) Picotamide, a combined
inhibitor of thromboxane A2 synthase and receptor, reduces 2-year mortality in
diabetics with peripheral arterial disease: the DAVID study. Eur Heart J 25:1845–
1852.
Nowak J and FitzGerald GA (1989) Redirection of prostaglandin endoperoxide me-
tabolism at the platelet-vascular interface in man. J Clin Investig 83:380–385.
Pradelles P, Grassi J, and Maclouf J (1985) Enzyme immunoassays of eicosanoids
using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal
Chem 57:1170–1173.
Reilly IA and FitzGerald GA (1987) Inhibition of thromboxane formation in vivo and
ex vivo: implications for therapy with platelet inhibitory drugs. Blood 69:180–186.
Tremoli E, Maderna P, Colli S, Morazzoni G, Sirtori M, and Sirtori CR (1984)
Increased platelet sensitivity and thromboxane B2 formation in type-II hyperli-
poproteinaemic patients. Eur J Clin Investig 14:329–333.
References
Ahlner J, Andersson RG, Torfgard K, and Axelsson KL (1991) Organic nitrate esters:
clinical use and mechanisms of actions. Pharmacol Rev 43:351–423.
Antithrombotic Trialist Collaboration (2002) Collaborative meta-analysis of random-
ised trials of antiplatelet therapy for prevention of death, myocardial infarction
and stroke in high risk patients. BMJ 324:71–86.
Antman EM, DeMets D, and Loscalzo J (2005) Cyclooxygenase inhibition and car-
diovascular risk. Circulation 112:759–770.
Bhatt DL and Topol EJ (2003) Scientific and therapeutic advances in antiplatelet
therapy. Nat Rev Drug Discov 2:15–28.
Bolla M, Almirante N, and Benedini F (2005) Therapeutic potential of nitrate esters
of commonly used drugs. Curr Top Med Chem 5:707–720.
Bossant MJ, Ninio E, Delautier D, and Benveniste J (1990) Bioassay of paf-acether
by rabbit platelet aggregation, in Methods in Enzymology (Murphy RC and Fitz-
patrick FA eds) pp 125–130, Academic Press Inc., San Diego, CA.
Buccellati C, Ciceri P, Ballerio R, Casagrande C, Folco G, and Nicosia S (2002)
Evaluation of the effects of anti-thromboxane agents in platelet-vessel wall inter-
action. Eur J Pharmacol 443:133–141.
Capra V, Habib A, Accomazzo MR, Ravasi S, Citro S, Levy-Toledano S, Nicosia S,
and Rovati GE (2003) Thromboxane prostanoid receptor in human airway smooth
muscle cells: a relevant role in proliferation. Eur J Pharmacol 474:149–159.
Capra V, Veltri A, Foglia C, Crimaldi L, Habib A, Parenti M, and Rovati GE (2004)
Mutational analysis of the highly conserved ERY motif of the thromboxane A2
receptor: alternative role in G protein-coupled receptor signaling. Mol Pharmacol
66:880–889.
Walford G and Loscalzo J (2003) Nitric oxide in vascular biology. J Thromb Haemost
1:2112–2118.
Weber AA, Strobach H, and Schror K (1993) Direct inhibition of platelet function by
organic nitrates via nitric oxide formation. Eur J Pharmacol 247:29–37.
Address correspondence to: Dr. Giancarlo Folco, Department of Pharma-
cological Sciences, Center for Cardiopulmonary Pharmacology, School of Phar-
macy, Via Balzaretti 9, 20133 Milano, Italy. E-mail: giancarlo.folco@unimi.it