In vitro pharmacological evaluation of multitarget agents for thromboxane prostanoid receptor antagonism and COX-2 inhibition
Graphical abstract
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) provide analgesic and anti-inflammatory properties by virtue of cyclooxygenase (COX) inhibition. COX is responsible for prostanoid production from arachidonic acid (AA) and can be inhibited reversibly by non-aspirin NSAIDs and irreversibly by aspirin. COX exists in two isoforms, the housekeeping enzyme COX-1 responsible for the gastric cytoprotection and haemostatic integrity, and the inducible isoform COX-2, mostly expressed in response to inflammatory stimuli and constitutively present in some specific tissue such as endothelial cells, brain and kidney [1], [2].
Severe gastric problems such as bleeding, gastric erosion and ulcers are the main side effect of chronic use of conventional NSAIDs and aspirin, mainly due to the inhibition of the COX-1-derived gastroprotective prostaglandin (PG) E2 production [3]. Celecoxib (Celebrex) and rofecoxib (Vioxx) were the first COX-2 selective inhibitors (coxibs) to enter the market as second generation NSAIDs to be used in symptomatic treatments of patients with osteoarthritis and rheumatoid arthritis with the promise of being antiinflammatory while minimizing gastrointestinal (GI) toxicity [4]. However, in 2004 Vioxx was withdrawn from the market and concern over potential cardiovascular (CV) toxicity and risk of myocardial infarction and stroke associated with the extended use of coxibs [5], [6] and traditional NSAIDs in general [7], [8], [9] widespread rapidly, leading the official medicine agencies to issue CV safety warnings for coxibs still on the market and successively also for non-selective NSAIDs.
Early explanation for the potential thrombotic risk included an imbalance in the biosynthesis of thromboxane A2 (TXA2, a potent platelet aggregator and vasoconstrictor) and prostacyclin (PGI2, which has opposing actions) [10] as a result of the observation that urinary excretion of the principal PGI2 metabolite, 2,3-dinor 6-keto PGF1α, was reduced in patients treated with celecoxib and rofecoxib, while TXB2, urinary metabolite of TXA2, was unaltered [11], [12]. Indeed, PGI2 is the major end product of COX-2 in vascular endothelium and reduced prostacyclin receptor signalling has been suggested to contribute to the adverse CV outcomes observed with coxibs [13]. Other explanations have been also proposed to clarify the effect of coxibs (and conventional NSAIDs) that do not involve the isoform of COX present on endothelial cells. For example, it was hypothesized that the hazard could depend upon differences in the levels of lipid peroxides or in the supply of AA substrate between platelets and endothelial cells, such that PGI2 synthesis is inhibited by NSAIDs more readily than platelet-derived TXA2 [14]. Another report suggests that the CV toxicity of rofecoxib could be due to its intrinsic physico-chemical properties and primary metabolism that increase Low Density Lipoproteins and membrane lipids oxidation thus promoting formation of isoprostanes, a characteristic feature of atherogenesis [15]. The involvement of isoprostanes is of particular interest considering that they are nonenzymatic products of fatty acid oxidation, therefore insensitive to the action of aspirin and NSAIDs, they are chemically stable and are produced in vivo in quantities exceeding those of TXA2 and, finally act through the TXA2 prostanoid (TP) receptor [16].
For these reasons, we consider that the addition of a TP antagonist component to a coxib may provide protection against all the harmful activities mediated through the activation of the TP receptor by mediators sensitive and insensitive to aspirin/NSAIDs such as the unopposed platelet-derived TXA2 and the nonenzymatic product isoprostanes.
Recently, an unexpected mechanism of action for diclofenac, a traditional NSAID with a non-selective profile of COX inhibition, and its derivative lumiracoxib was described: the competitive antagonism at the TP receptor [17]. While it is true that increase in CV risk has been reported not only for selective coxibs, but also for conventional NSAIDs [18], [19], [20] including diclofenac [21], its potency as TP receptor antagonist is certainly not sufficient to have an impact in therapy at the prescribed clinical doses [17]. In addition, despite it has been withdrawn from the market due to hepatotoxicity problems (Novartis News (2007) Prexige® receives “not approvable” letter in the US despite being one of the most studied COX-2 inhibitors), a recent meta-analysis of eighteen clinical trials in patients with osteoarthritis taking lumiracoxib concluded that no significant differences in CV outcomes was evident between lumiracoxib and placebo or between lumiracoxib and other NSAIDs [22]. Thus, these findings seems to corroborate our choice to use lumiracoxib as a starting structure for chemical modification and to reinforce the hypothesis that cardiotoxicity associated to the different NSAIDs and coxibs might not depend on COX selectivity per se, but rather on distinctive characteristics of each single molecule, including its pharmacokinetic, that might affect differently the intricate inter-eicosanoid network of biosynthetic and signaling pathways leading to multiple events that may synergize or be functionally opposed, as it is the case for platelet function [23].
In the present study we report the physico-chemical profile and the full pharmacological characterization of four different compounds 18, 20, 7 and 32 (Fig. 1) endowed with dual COX-2 inhibitor activity and TP receptor antagonism out of a large series of compounds previously reported [24]. TP antagonism of these newly synthesized compounds has been evaluated calculating their pA2 for anti-aggregating activity in human platelets as well as for their activity in inhibiting phospholipase C induced inositol phosphate production in Human Embryonic Kidney 293 (HEK293) cells transiently transfected with the TPα receptor in response to the TXA2 stable analogue U46619. Moreover, we determined their COX-1 and COX-2 activity and selectivity in washed platelets and isolated human monocytes, respectively. The same was performed with reference molecules, namely the traditional NSAIDs naproxen, the coxib lumiracoxib, as well as the potent and selective TP antagonist terutroban [25].
Section snippets
Reagents
Animal serum, antibiotics, other supplements, Lipofectamine 2000, Opti-MEM I and molecular biology reagents were purchased from Invitrogen (Carlsbad, CA). Inositol-free Dulbecco’s modified Eagle’s medium (DMEM) was obtained from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Ultima Gold was from PerkinElmer Life and Analytical Sciences (Boston, MA), as were myo-[2-3H]inositol. U46619 ([1R-[1ααβ(Z)α(1E, 3S*)][1α,4α,5β(Z), 6α(1E, 3S*)][1αα,5β(Z), 6α(1E, 3S*)][1α,4α,5β(Z), 6α(1E, 3S*)][1ααβ(Z), 6α(1E,
Chemistry
For the synthesis of compounds 7 (2-[(2-chloro-6-fluorophenyl) amino]-5-methyl-benzoic acid) and 32 (2-(((4-chlorophenyl) sulfonyl) amino)-5-methyl-benzoic acid) the procedure reported in Scheme 1 (see Supplementary materials) was used. The synthesis of lumiracoxib analogue 7 was accomplished as previously described [24]. Briefly, the Chan–Lam coupling was used by reacting 2-amino-5-methylbenzoic acid with 2-chloro-6-fluorophenylboronic acid in the presence of 1,8-diazabicylo-[5,4,0]undec-7-ene
Discussion
In this contribution we report a full in vitro pharmacological characterization of selected molecules from a series of compounds recently synthetized [24] and two additional molecules with dual COX-2 inhibitor activity and TP receptor antagonism. As reference compounds we have considered the non selective first generation NSAID naproxen, the most selective COX-2 inhibitor lumiracoxib and the TP antagonist terutroban. This work directs to a new generation of multitarget compounds among which two
Conclusions
We show here that structure-activity analysis allowed us to obtain prototypes of new chemical entities endowed with higher TP antagonist potencies and a more balanced COX-2 selectivity. The modification of existing drugs targeted against COX-2 (lumiracoxib) can lead to new bivalent molecules with obvious advantage in the pharmacological properties. The main goal of a multitarget drug is to have both activities at the same concentration range with a consistent pharmacodynamic and single
Acknowledgements
We gratefully thank Dr. Alain Rupin (Institut de Recherches Servier, France) for generously providing us with terutroban. This work was supported in part by a grant from Regione Lombardia (SAL-02) and Fondazione Banca del Monte di Lombardia (GER, VC) and by University of Turin (Ricerca locale 2013 to MB).
References (59)
- et al.
Gastric mucosal defense and cytoprotection: bench to bedside
Gastroenterology
(2008) - et al.
COX-2 selectivity alone does not define the cardiovascular risks associated with non-steroidal anti-inflammatory drugs
Lancet
(2008) - et al.
COXIB/TP antagonists: a possible new twist in NSAID pharmacology and cardiovascular risk
Trends Pharmacol. Sci.
(2010) - et al.
Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2
FEBS Lett.
(2006) - et al.
EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin
Thromb. Res.
(2012) - et al.
The specific thromboxane receptor antagonist S18886: pharmacokinetic and pharmacodynamic studies
J. Thromb. Haemost.
(2005) - et al.
Thromboxane antagonism with terutroban in peripheral arterial disease: the TAIPAD study
J. Thromb. Haemost.
(2010) - et al.
Terutroban versus aspirin in patients with cerebral ischaemic events (PERFORM): a randomised, double-blind, parallel-group trial
Lancet
(2011) - et al.
Vascular events after stroke: terutroban fails to PERFORM
Lancet
(2011) - et al.
Dimerization of the human receptors for prostacyclin and thromboxane facilitates thromboxane receptor-mediated cAMP generation
J. Biol. Chem.
(2004)
Molecular determinants for the selective inhibition of cyclooxygenase-2 by lumiracoxib
J. Biol. Chem.
A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385
J. Biol. Chem.
Cyclooxygenases: structural, cellular, and molecular biology
Annu. Rev. Biochem.
The coxibs, selective inhibitors of cyclooxygenase-2
N. Engl. J. Med.
Adenomatous polyp prevention on vioxx trial I. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial
N. Engl. J. Med.
Adenoma prevention with celecoxib study I. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention
N. Engl. J. Med.
Risk of myocardial infarction in patients taking cyclo-oxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: population based nested case-control analysis
BMJ
Do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials
BMJ
Coxibs and cardiovascular disease
N. Engl. J. Med.
Effects of specific inhibition of cyclooxygenase-2 on sodium balance, hemodynamics, and vasoactive eicosanoids
J. Pharmacol. Exp. Ther.
Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2
Proc. Natl. Acad. Sci. U. S. A.
Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition
Circ. Res.
Stronger inhibition by nonsteroid anti-inflammatory drugs of cyclooxygenase-1 in endothelial cells than platelets offers an explanation for increased risk of thrombotic events
FASEB J.
A biological rationale for the cardiotoxic effects of rofecoxib: comparative analysis with other COX-2 selective agents and NSAIDS
Subcell. Biochem.
Cardiovascular responses to the isoprostanes iPF(2alpha)-III and iPE(2)-III are mediated via the thromboxane A(2) receptor in vivo
Circulation
Antagonism of thromboxane receptors by diclofenac and lumiracoxib
Br. J. Pharmacol.
Increased mortality and cardiovascular morbidity associated with use of nonsteroidal anti-inflammatory drugs in chronic heart failure
Arch. Intern. Med.
Risk of myocardial infarction and death associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) among healthy individuals: a nationwide cohort study
Clin. Pharmacol. Ther.
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These authors equally contributed to the work.