Inhibition of cyclooxygenases by dipyrone

Abstract

Background and purpose: Dipyrone is a potent analgesic drug that has been demonstrated to inhibit cyclooxygenase (COX). In contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low gastrointestinal toxicity, indicating a different mode of action. Here, we aimed to investigate the effects of dipyrone on COX.

Experimental approach: The four major metabolites of dipyrone, including the two pharmacologically active metabolites, 4-methyl-amino-antipyrine (MAA) and amino-antipyrine (AA), were used to characterise their binding to COX and haem as well as their effects on the biochemical properties of COX. Mass spectrometry, UV and visible photometry were used to study binding and prostaglandin production. Levels of anti-oxidant enzymes were assessed by Western blotting.

Key results: The pharmacologically active metabolites of dipyrone, MAA and AA, did not inhibit COX activity in vitro like classical COX inhibitors, but instead redirected the prostaglandin synthesis, ruling out inhibition of COX through binding to its active site. We found that MAA and AA formed stable complexes with haem and reacted with hydrogen peroxide in presence of haem, ferrous ions (Fe(2+)) or COX. Moreover, MAA reduced Fe(3+) to Fe(2+) and accordingly increased lipid peroxidation and the expression of anti-oxidant enzymes in cultured cells and in vivo.

Conclusions and implications: Our data suggest that the pharmacologically active metabolites of dipyrone inhibit COX activity by sequestering radicals which initiate the catalytic activity of this enzyme or through the reduction of the oxidative states of the COX protein.

Figure 1

MAA inhibits PGE₂ and TXB₂ synthesis. (a) Structures of the four dipyrone metabolites. (b) LPS-stimulated RAW 264.7 were treated with the indicated MAA concentrations for 16 h. Human platelets were incubated with varying amounts of MAA for 30 min before induction of the release of intracellular arachidonic acid with A23187 for 10 min. PGE₂ and TXB₂ synthesis was determined as described in the ‘Materials and methods' section. The mean±s.e.m. of at least three experiments are shown. The control values (100%) correspond to 6 ng ml⁻¹ for PGE₂ and 70 ng ml⁻¹ for TXB₂. (c) Conditions as in (b) except that all dipyrone metabolites were used at 100 μM. The mean±s.e.m. of at least three experiments, each in triplicate, are shown. Students t-test ^*P<0.01, ^**P<0.001 metabolite vs control.

Figure 2

MAA does not inhibit COX activity by competitive binding to the active site. (a) LPS-stimulated RAW 264.7 were lysed and prostaglandin synthesis was measured in the presence of 100 μM MAA, AA, FAA, AAA or 1.25 mM aspirin, as described in the ‘Methods' section. Asterisks indicate the prostaglandins that were found only in MAA and AA treated samples. (b) Purified COX-1 (20 U) was incubated with [1–¹⁴C]-arachidonic acid (a.a.) in the presence or absence of 100 μM MAA and 1 μM haem and the products analysed by TLC. (c) 1.3 μM PGG₂ was incubated with the indicated concentrations of MAA for 30 min and analysed by LC-MS/MS as described in the ‘Materials and methods' section. (d–h) 13 μM PGG₂ alone (d) or incubated with 100 μM MAA (e), AA (f), AAA (g) or FAA (g) were incubated at room temperature for 30 min prior to analysis with LC-MS/MS. The arrows indicate the positions of the PGG₂-specific signals. (i) RAW 264.7 cells were metabolically labelled as described in the ‘Methods' section. Cells were stimulated with 5 μg/ml⁻¹ LPS 100 μM MAA and the prostaglandins in the medium (m) or in the cells (c) were analysed by TLC. (j) RAW 264.7 cells were incubated with LPS for 16 h in presence and absence of 100 μM MAA. PGF_2α levels were analysed with LC-MS/MS as described under the ‘Methods' section. Students t-test ^*P<0.001 MAA vs LPS-treatment without MAA.

Figure 3

Dipyrone metabolites form stable complexes with haem. (a) Full scan mass spectrometric analysis (ESI-MS) in the negative ion mode of haem alone. The inset shows a magnification of the indicated signals. (b) Full scan mass spectrometric analysis (ESI-MS) in the positive ion mode of haem and MAA. The inset shows a magnification of the indicated signals. (c) Fragmentation of the suspected haem–MAA complex with the m/z of 831.2 in the negative ion mode. (d) Full scan mass spectrometric analysis in the positive ion mode of haem with FAA. Arrows indicate the position of the proposed haem–metabolite adduct.

Figure 4

MAA and AA react with hydrogen peroxide in presence of an iron source. (a) UV-visible absorption spectra of MAA in absence or presence of haem or haem and peroxide. The inset shows absorption changes of the metabolite–haem complexes after addition of hydrogen peroxide. The arrows indicate MAA specific changes. (b) As in (a) except that AA, AAA or FAA were used instead of MAA. (c–e) Changes in the absorption spectrum of MAA after addition of 100 μM hydrogen peroxide in presence of 10 μM, Fe³⁺ (c), Fe²⁺ (d) or 65 nM reconstituted COX-1. (e) For better comparison, the absorption changes in presence of haem (dotted line) were included in (c and d).

Figure 5

MAA and AA reduce Fe³⁺ to Fe²⁺. (a and b) Determination of Fe²⁺ formation after addition of dipyrone metabolites or ascorbate to FeCl₃ by potassium ferricyanide (a) or bathophenanthroline (b) as described in the ‘Materials and methods' section. The mean±s.e.m. of at least three experiments each carried out in duplicate are shown. Student's t-test ^**P<0.001 MAA or AA vs control. (c) Lipid oxidation in RAW 264.7 cells after incubation with 100 μM Fe²⁺ or MAA in presence or absence of 100 μM deferoxamine (DFO) as determined by levels of malondialdehyde (MDA). The mean±s.e.m. of at least four experiments is shown. Students t-test ^*P<0.05 MAA or Fe²⁺ vs control. (d) Western blot analysis of the expression of superoxide dismutase and peroxiredoxin V in spinal cords of adult rats. The spinal cords were excised 4 or 24 h after i.p. application of 1 g kg⁻¹ dipyrone. Hsp 70 expression was used to control for even loading.