Non-steroidal anti-inflammatory drugs (NSAIDs) are a major cause of upper gastro-intestinal (GI) ulceration and bleeding as well as cardiovascular (CV) diseases (e.g., myocardial infarction and stroke). A feature common to both these adverse events is a variety of vascular reactions. One approach to overcome these side effects has been the development of nitric-oxide (NO)-donating NSAIDs. The NO is considered to overcome some of these vascular reactions caused by NSAIDs. Unfortunately, the NO-NSAIDs developed so far have not had the expected benefits compared with NSAIDs alone.
Using in vitro preparations it is hoped to gain insight into the vascular and smooth muscle reactions induced by NO-NSAIDs compared with NSAIDs as a basis for improving the protective responses attributed to the NO-donating properties of these drugs.
A range of NO-NSAIDs was synthesized based on the esterification of NSAIDs with the nitro-butoxylate as a prototype of an NO-donor. These compounds, as well as NO-donor agents and NSAIDS, were examined for their possible effects on isolated segments of digital arteries of fallow deer, which provide a robust model for determining the effects of vasodilator and vasoconstrictor activities, in comparison with those of standard pharmacological agents.
The NO-NSAIDs were found to antagonise the smooth muscle contractions produced by 5-hydroxytryptamine (serotonin, 5-HT). However, while almost all their parent NSAIDs had little or no effect, with the exception of the R-(−)-isomers of both ibuprofen and flurbiprofen, which caused vasodilatation, all the NO-NSAIDs tested antagonised the increase in tension produced by 5-HT.
R-(−)-ibuprofen and R-(−)-flurbiprofen, along with the nitro-butoxyl esters of the NSAIDs examined, produce relaxation of segments of deer digital artery smooth muscle in vitro
Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the most widely used drugs for prescription and non-prescription (‘over-the-counter’ or OTC) medications for the treatment of musculo-skeletal and various acute and chronic painful and inflammatory conditions (Rainsford
Current concerns regarding the safe use of NSAIDs have centred on the combined GI and CV risks of these drugs (Lanas et al.
To this end, a range of NSAIDs coupled to an NO-releasing moiety (NO-NSAIDs) has been developed, in the hope that such compounds, by releasing NO in the mucosa, would be less damaging to the GI tract. Some of these NO-NSAIDs have been shown experimentally to cause less gastric injury than the parent NSAIDs (Wallace et al.
Unless otherwise stated, NSAIDs, together with the intermediates used in the synthesis of the nitrobutoxy compounds described below, were obtained from Sigma-Aldrich (Poole, Dorset, UK). 5-Hydroxytryptamine (serotonin, 5-HT), phenylephrine (PHE), the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and other laboratory reagents were also obtained from Sigma-Aldrich (Poole, Dorset, UK).
The propionic acids, ibuprofen and flurbiprofen, are referred to as their racemic mixtures (
The NO-NSAIDs General scheme for the synthesis of NO-NSAIDs Summary of the synthesis of NO-NSAIDs by a method modified from that of Wallace et al (
Melting points are uncorrected and were determined on Stuart Scientific SMP3 apparatus. Infrared spectra were recorded with an ATI Mattson Genesis series FTIR spectrophotometer. 1H NMR and 13C NMR spectra were recorded in CDCl3 using a Brucker AC 250 spectrometer operating at 250 and 62.9 MHz, respectively. Chemical shifts (δ) are recorded in ppm downfield from Me4Si as internal standard and J values are given in Hz. Mass spectra were recorded with EI-VG 7070E mass spectrometer. Accurate masses were determined on VG Autospec EI mass spectrometer with magnetic sector instrument. Optical rotations were measured at 23 °C with a Bellingham and Stanley ADP 440 polarimeter using dichloromethane as the solvent. All solvents were dried and distilled by standard techniques.
The bromobutyl ester (
The bromobutyl ester (
The bromoester (
The bromoester (
The bromoester (
The bromoester (
Nitroxybutyl ester (
Nitroxybutyl ester (
Nitroxybutyl ester (
Nitroxybutyl ester (
Nitroxybutyl ester (
(R)-(-)-Nitroxybutyl ester (
(S)-( +)-Nitroxybutyl ester (
(R/S)-Nitroxybutyl ester (
The methods employed were those previously described (Callingham et al.
Segments (approximately 3 mm in length), were mounted, in 10 ml, water-jacketed organ baths at 37 °C and attached to Harvard isometric transducers (0–50 g sensitivity), connected, via Harvard amplifiers and A/D converters (PowerLab® 8/35, ADInstruments Ltd, Bishops Mews, Transport Way, Oxford, OX4 6HD, UK) for computer recording of developed tension. Resting tension was adjusted to 3 g, which was maintained during a 45 min period of acclimatisation and beyond. The integrity of the vascular endothelium was tested by measuring the relaxation produced by addition of 10–6 M histamine to segments pre-contracted with either 10–6 M 5-HT or 10–6 M PHE; since acetylcholine, the agent normally employed to detect functional endothelium is without effect in this preparation. In each experiment, the vessel rings were contracted, either with single concentrations or graded concentrations of (5-HT).
Cumulative changes in tension to applied agents were plotted as percentages of maximum responses against log concentrations of the relevant agent and fitted to the Hill equation by use of the non-linear regression facility in Kaleidagraph® (Synergy Software, 2457 Perkiomen Ave., Reading PA, USA 19,606) with n-values referring to the number of animals used. Tests for statistical significance were performed using the unpaired t-test.
In Figs.
Stock solutions of the NSAIDs were made by first dissolving a few milligrams of the compound in 0.25 ml of DMSO (dimethyl sulphoxide) and made up to 10–2 M with an appropriate volume of deionised water. These solutions, together with any dilutions, were kept on ice until used.
This investigation tested four NSAIDs (aspirin, ibuprofen, naproxen, and indomethacin) and four corresponding NO-donating NSAIDs (aspirin nitroxybutyl ester, ibuprofen nitroxybutyl ester, naproxen nitroxybutyl ester and indomethacin nitroxybutyl ester).
Stock solutions of 10−2 M 5-HT), phenylephrine (PHE) and histamine were prepared and kept at 4 °C and diluted with deionised water for use on the day of the experiment and kept on ice. Solutions of methylene blue for use as an inhibitor of nitric oxide synthase (Mayer et al.
Rings of 2–3 mm length were cut from the digital arteries using scissors and mounted in the organ bath by sliding the two hooks into the lumen of the artery. Each water bath was filled with PSS (buffered salt solution) and continuously aerated with 95% O2 5% CO2. The jackets surrounding the water baths had water heated to 37 °C continuously pumped through them to maintain physiological temperature in the water baths. The day’s stock solution flask of aerated PSS was also kept submerged in the water bath so that it was at the correct temperature when it was added to the organ baths. The tension pulled by the rings was adjusted to 3 g before each experiment was begun.
On the morning of each day of experiments, the artery segments were pre-contracted with 10−5 M 5-HT as this concentration was sufficient to achieve the maximum contractile response; previous studies had shown to induce the rings to respond well to subsequent drug additions. When the vessels had reached maximum contraction, 10−6 M histamine was added to the organ baths to test for the presence of a functional endothelium. The organ baths were then washed out and filled with fresh PSS. The rings were left to relax for an hour, with the tension returned to 3 g at intervals and the experiment proper was begun.
The transducers were calibrated by use of the PowerLab® calibration facility and tested for linearity of response by attaching weights from 1 to 20 g. All data were processed by use of LabChart® (ADInstruments) on the recording computer.
The cumulative changes in tension to applied agents were plotted as percentages of maximum responses against log concentrations of the relevant agent and fitted to the Hill equation by non-linear regression, with n-values referring to the number of animals used. Only rings from left feet were used after ensuring, having previously that there were no differences in responses between rings taken from either foot, to ensure that the n-values truly represented individual animals. Tests for statistical significance were performed using the unpaired t-test.
Nitroxybutyl-aspirin (NO-aspirin) effectively reduced the contractile responses of digital artery segments produced by increasing concentrations of 5-HT, whereas aspirin was without effect (Fig. Effect of aspirin and NO-aspirin on the cumulative log[concentration]—vasoconstrictor responses of fallow deer isolated arterial rings to 5-HT. A comparison of the contractile responses of arterial rings to 5-HT in the presence and absence of aspirin (10–4 M) and NO-aspirin (10–4 M), showed that aspirin had no significant effect on the responses of the arterial rings to 5-HT (
When the maximum tension that could be developed by the segments, in response to applied 5-HT, was examined, the relaxation in tension produced by NO-aspirin alone was reduced from 50 to 30% in the presence of methylene blue (Fig. Effect of methylene blue on the cumulative log[concentration]—vasoconstrictor responses of fallow deer isolated arterial rings to 5-HT. In the presence of 10–4 M methylene blue, a direct nitric oxide synthase and guanylyl cyclase inhibitor (Mayer et al. Effect of indomethacin and NO-indomethacin on the cumulative log[concentration]—vasoconstrictor responses of fallow deer isolated arterial rings to 5-HT. A comparison of the contractile responses of arterial rings to 5-HT in the presence and absence of indomethacin (10–4 M) and NO-indomethacin (10–4 M,) showed that while indomethacin had no significant effect on the responses of the arterial rings to 5-HT ( Effect of naproxen and NO-naproxen on the cumulative log[concentration]—vasoconstrictor responses of fallow deer isolated arterial rings to 5-HT. A comparison of the contractile responses of arterial rings to 5-HT in the presence and absence of naproxen (10–4 M) and NO-naproxen (10–4 M), showed that naproxen had no significant effect on the responses of the arterial rings to 5-HT, while NO- naproxen, significantly reduced the maximum tension produced ( Effect of ibuprofen and NO-ibuprofen on the cumulative log[concentration]—vasoconstrictor responses of fallow deer isolated arterial rings to 5-HT. A comparison of the contractile responses of arterial rings to 5-HT in the presence and absence of ibuprofen and NO-ibuprofen show no significant difference between the effects of the two drugs on the rings’ responses to 5-HT (
However, when the effects produced by
Furthermore, since ibuprofen and flurbiprofen are diastereo-isomeric Effect of increasing concentrations of S-( +)-ibuprofen and R-(-)-ibuprofen on the tension produced in fallow deer isolated arterial rings by a constant concentration of 3 × 10–6 M 5-HT. S-( +)-ibuprofen, in concentrations up to 10–4 M had no significant effect on the maintained tension ( Effect of increasing concentrations of S-( +)-flurbiprofen and R-(−)-flurbiprofen on the tension produced in fallow deer isolated arterial rings by a constant concentration of 3 × 10–6 M 5-HT. S-( +)-flurbiprofen, in concentrations up to 10–4 M had no significant effect on the maintained tension (
Removal of the vascular endothelium (a source of NO) reduced ( Effect of increasing concentrations of R-(−)-ibuprofen on the tension produced in fallow deer isolated arterial rings by a constant concentration of 3 × 10–6 M 5-HT, in the presence and absence of vascular endothelium. While there appears to be no significant difference (
These relaxant effects were reduced to near control values by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (Feelisch et al. Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on the relaxation of tension by increasing concentrations of R-(−)-ibuprofen on the tension produced in fallow deer isolated arterial rings by a constant concentration of 3 × 10–6 M 5-HT. In contrast to the highly significant relaxation of tension produced by R-(−)-ibuprofen alone of the maintained control tension (
These results demonstrate that the NO-donating analogues of aspirin, indomethacin, etc., significantly reduced the contractile responses of vascular smooth muscle to electrical stimulation and to applied 5-HT and PHE (results not shown), while, with the exception of ibuprofen and flurbiprofen, the parent NSAIDs were without effect. It was also shown that methylene blue (an inhibitor of NO action) significantly reduced the effect of NO-aspirin (Fig.
The fact that R-(−)-ibuprofen produced a relaxation of a similar magnitude to racemic NO-ibuprofen suggests that either R-(−)-ibuprofen released NO on a similar scale to NO-ibuprofen, or that it caused relaxation by some other means. There are several other means possible, including the induction of iNOS or direct activation of soluble guanylate cyclase. Some previous work has been done on the possible involvement of ibuprofen with iNOS. One study suggests that the concentration of NO in cells can be raised by the presence of ibuprofen, through the induction of iNOS (Menzel and Kolarz
The reduction in the relaxation caused by
By comparison, another diastereoisomeric propionic acid,
The results overall suggest that R-(–)-ibuprofen directly activates sGC. They also suggest that NO-ibuprofen does not work in the same fashion. If it did then it would be likely to produce greater relaxation given its coupling with a nitric oxide-releasing moiety. The combination of the release of NO and direct activation of sGC by ibuprofen should produce a greater relaxation than just the activation of sGC alone but it does not, suggesting that the change in the chemical composition by esterification causes sufficient change in structure to prevent the compound working in the same way as R-(−)-ibuprofen. As S-( +)-ibuprofen is much less effective than the R(−)-isomer, the activation must be very specific. Due to the similarity between flurbiprofen and ibuprofen, it is no surprise that the former causes relaxation. There is also the possibility that other heme proteins are involved. It has been suggested that ODQ is non-selective and may inhibit enzymes other than sGC (Feelisch et al.
We thank the following Cambridge Natural and Medical Science Tripos undergraduates for their valuable contributions to the research in this manuscript —Samantha Benedict, Ian Gregory, Falak Masood, Alexander Maini, Maleeha Munnawwar and Richard Turner. Our grateful thanks are due to Dr Lisa Fagg for her expert help and support. We are also most grateful to the owners and staff of the Denham Park Estate, Bury St Edmunds (UK) for the generous donation of deer feet and for the provision of facilities and help in the removal of their digital arteries at the abattoir.
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