Activity of dipyrone on intraplatelet arachidonic acid metabolism: An in vitro study

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Pharmacological Research, Vol. 21, No. 1, 1989

ACTIVITY OF DIPYRONE ON INTRAPLATELET ARACHIDONIC ACID METABOLISM : AN IN VITRO STUDY R. ABBATE, S . PINTO, A. M . GORI, R. PANICCIA, M . COPPO and G. G . NERI SERNERI Clinica Medica I, Universita' di Firenze, Viale Morgagni 85, Firenze Received in final form 22 September 1988

SUMMARY The effects of dipyrone on platelet cyclooxygenase and lipoxygenase were investigated in vitro by the study of 1- 14C arachidonic acid (AA) conversion by high performance liquid chromatography (HPLC) on washed platelets at seven different drug concentrations (from 5 to 300 ug/ml) . The effects of dipyrone on thromboxane (TX) B2 generation from endogenous AA were also studied in platelet-rich plasma and in washed platelets by radioimmunoassay . In the study of 1- 14C AA metabolism the inhibitory concentration (IC) 50 for TXB 2 was 40,ug/ml. However, at the lowest drug concentration (5 1ug/ml) a slight but significant inhibition was found (25 . 3%, P< 0 . 001) and a complete one at 300 µg/ml . A relationship between TXB 2 inhibition and log drug concentration was found (r= 0 . 97, P< 0 . 001). Lipoxygenase (LO) activity showed an increase of 45 . 9% at 20,ug/ml and of 251 . 5% at the highest concentration (r= 0 P< 0 . 001) . The inhibition of TXB 2 generation from endogenous AA by washed platelets was of the same order of magnitude of the inhibition of TXB 2 production from exogenous 1- 14C AA. Our results indicate that dipyrone affects intraplatelet AA metabolism at very low concentrations, however its activity, on a molar ratio basis, appears to be lower than that of other non-steroidal anti-inflammatory drugs . KEY WORDS : dipyrone, platelets, arachidonic acid . INTRODUCTION Dipyrone is a pyrazolone derivative, endowed with an analgesic, antipyretic and anti-inflammatory effect . Like other pyrazolone derivatives, dipyrone is an inhibitor of cyclooxygenase (CO), and this inhibitory effect is dependent on the concentration of the drug [1] . The inhibition of prostaglandin production was demonstrated first in microsomal preparations from bovine and ram vesicles [2, 3] . Furthermore, it has been shown that dipyrone inhibits platelet aggregation by decreasing thromboxane (TX) A2 synthesis induced by low concentrations of stimulating agents [1, 4] . However, no investigations have been performed on the effect of dipyrone on platelet lipoxygenase (LO) pathway. 0031-6989/89/010043-08/$03 .00/0

© 1989 The Italian Pharmacological Society



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Platelets do not synthesize leukotrienes and the main product from LO pathway is 12-hydroxyeicosatetraenoic acid (12-HETE). 12-HETE from platelets may be utilized by white cells for the formation of LO metabolites, 5S,12S DiHETE [5] and 12S,20-DiHETE [6], provided with chemotactic property and able to regulate leukotriene synthesis in leucocytes [7] . The aim of this study was to investigate the in vitro effect of different dipyrone concentrations (from 5 to 300 µg/ml) on intraplatelet conversion of 1- 14 C arachidonic acid (AA) both through CO and LO pathways .

MATERIALS AND METHODS Venous blood for platelet study was withdrawn by a clean venepuncture without venous stasis into polypropylene syringes using a 19 G needle from 10 overnight fasting healthy non-smoker subjects (5 males, 5 females, mean age 32 . 6 ± 4 . 8 years) . All subjects were free from any drug for at least 2 weeks . Blood was immediately transferred into tubes containing acid-citrate-dextrose (ACD) (NIH, formula A) in a ratio of 8 . 5 :1 . 5 (v/v) and sodium citrate (0 . 11 mol/1) in a ratio of 9:1 (v/v) . Platelet-rich plasma (PRP) was obtained by centrifuging at 160g for 6 min at 20°C . The platelet suspensions were prepared by centrifuging PRP (adjusted to pH 6 . 5 with additional acid-citrate-dextrose) at 1800 g for 30 min at 20°C and washed twice in buffer containing 8 mmol/l Na 2HPO4, 2 mmol/l NaH 2PO4, 10 mmol/l ethylenediaminetetra-acetic acid (EDTA), 5 mmol/l KC1, 135 mmol/l NaCl, pH 7 . 2 and recentrifuged at 1800 g for 30 min at 20°C . The washed platelets were resuspended in the same buffer. If necessary, additional assay buffer was added to obtain a platelet concentration of 3 x 10 5 platelets/µl . One hundred ul dipyrone (dissolved in distilled water and diluted to 1, 2 . 5, 5, 10, 20, 40, 100, 200 and 300 tg/ml final concentration (f.c .) corresponding to 15 mm, 30 mm, 60 mm, 120 mm, 300 mM, 900 mm) were added to 900 ul of PRP and to 900,ul of platelet suspension and incubated for 30 min at 37 °C; controls were set up, adding the same volume of distilled water to PRP and to platelet suspension. In order to investigate TXA2 production, PRP (adjusted to 3 x 10 5 platelets/,Cl by diluting with platelet-poor plasma) and washed platelets were incubated with dipyrone and stirred with human thrombin (5 NIH U/ml f.c .) for 10 min at 37 °C. TXA 2 was measured as its stable metabolite TXB 2 by radioimmunoassay according to Granstrom et al. [8] by using a commercial kit [9]. The detection limit was 10 pg/ ml of TXB2; the variation coefficients (CV) of intra-assay and interassay were 8 . 5% and 11 . 58% . One ml of platelet suspension pretreated with dipyrone was incubated with 1- 14C AA (specific activity 52 . 9 mCi/mmol) at 1 umo 1 / 1 f .c . in 50 mmol/l Tris buffer pH 7 . 4 for 40 min at 37°C . Subsequently CaCl, (4 . 05 mmol/l f.c.) and human albumin (0 . 5 mg/ml f.c .) were added to labelled platelets before stimulating with human purified thrombin (5 NIH U/ml f.c .) for 30 min at 37 °C with magnetic stirring . Three hundred ul of 0 . 12 mol/l EDTA and 22,ul of undiluted dimethylsulphoxide were added to stop the reaction . After centrifuging at 3500 g



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for 20 min at 20°C the platelet suspensions were immediately separated and the supernatant was stored at - 80°C before subsequent HPLC processing . Separation of AA metabolites was performed using a Waters HPLC Model ALC GPC 244 (Waters Associated, Milford, Massachusetts), containing a Model 6000 A pump and a U6K injector with a 2 ml sample loop . The stationary phase was a Waters reverse phase uBondapak phenyl column, 3 . 9 mm x 30 cm . The mobile phase and the technique injection were performed according to Russell and Deykin [10]. The AA metabolites were identified by the elution time using labelled standards in a continuous flow with a radiochemical computer assisted detector (Trace 7140, Packard Instrument Company, Downers Grove, Illinois) which provided chromatograms, areas and relative radioactivity percentage of each peak . The CV was 3 . 9% for TXB 2, 5 . 6% for prostaglandin (PG) F 2a , 6 . 8% for PGE 2, 7 . 2% for PGD 2, 4 . 6% for hydroxyheptadecatrienoic acid (HHT) and 4 . 9% for 12-HETE . The recovery of the sample injected in this system was greater than 98% . The CV of the overall procedure was 6 . 3% for TXB 2, 7 . 5% for PGF2,,, 7 . 7% for PGE 2, 7 . 8% for PGD2, 6 . 4% for HHT, 7% for 12-HETE . Statistical analysis of the results was carried out by the Student's t-test for paired data and polynomial fitting . Results are reported as mean ± SEM . 1-14 C AA (52 . 9 mCi/mmol), 3HTXB2 (115 . 9 Ci/mmol), 1-14C PGF,, (52 . 9 mCi/mmol), 1- 14C PGE 2 (52 . 9 mCi/mmol), 3H-PGD 2 (100 Ci/mmol), 3H-HHT (80 Ci/mmol), 3H-12-HETE (60 Ci/mmol) and Riafluor were purchased from New England Nuclear, Dreieich, FRG . 12-HETE was purchased from Hilran Biochemicals, Ltd, Tel Aviv, Israel . TXB 2, PGF2a, PGE 2, PGD2 were purchased from Upjohn, Kalamazoo, Michigan . Tetrahydrofuran and acetonitrile, HPLC quality, were obtained from Riedel-de Haen Ag Seelze-Hannover, FRG . Human purified thrombin (homogeneity greater than 95% by isoelectrofocusing on polyacrylamide gel) was obtained from Boehringer Biochemia, Mannheim, FRG . Reagents for TXB 2 determinations were purchased from American Biological Technologies, Esslingen, FRG . Human albumin was obtained from Behringwerke AG, Marburg, FRG . Dipyrone was supplied from Hoechst AG, Frankfurt, FRG (purity 99 . 9%) . RESULTS Arachidonic acid metabolism by labelled platelets

After incubation with dipyrone (5, 10, 20, 40 ug/ml) no labelled AA was found in the supernatant ; at concentrations higher than 40,ug/ml a little amount (14-16% of total radioactivity) was found unmetabolized . After incubation with dipyrone a decrease of 1- 14 C AA conversion by platelets into CO metabolites was observed . The inhibition of PGF 2 , PGE 2 and PGD2 could be observed at 5 and 10 ,ug/ml dipyrone concentratrion . After incubation with concentrations higher than 10 ug/ml the amount of PGs became undetectable and so the inhibition could not be evaluated . A 50% inhibition of TXA2 was obtained at 40 ug/ml dipyrone concentration and a complete one at 300,ug/ml (Fig . 1) . However, already at 5 ug/ml dipyrone concentration a significant (P<0-001) inhibition (25 . 3%) was found . The polynominal regression curve of the inhibition of TXB2 formation on log dipyrone concentration was represented by the equation :



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Pharmacological Research, Vol. 21, No. 1, 1989 y= -

35 . 5 + 186 . 6x- 204x2 + 100 . 4x3 -16x4

(r= 0

P< 0 . 001)

The HHT production at different concentrations is shown in Fig . 1 . The concentration of dipyrone able to induce an inhibition of 50% of HHT production was 100 ug/ml and at the highest concentration tested the inhibition was 93 . 3%. The equation of the polynomial curve of the inhibition of HHT formation on log drug concentration was : y=-49 . 1+164 . 7x-144 . 9x2 +61 . 1x3 -8 . 1X'

(r=0 . 97,P<0 . 001)

In contrast to the effects of dipyrone on CO pathway, the formation of LO pathway metabolites was enhanced by dipyrone .

A 100

0 50 a

.

r=0 97 0

• I 1 1 5 10 20 40 Dose

(µg/ml)

BAS

.

.

P<0 001

r=0 97

P<0 .001

I I l I 1 I I I I 40 100 200 300 100 200 300 5 10 20 Dose

(µg/ml )

5

10

20

40

100 200 300

Dose ()ug/ml)

Fig. 1 . Relationship between TXB 2 (A) and HHT (B) at seven dipyrone concentrations and in vitro 1- 14 C arachidonic acid conversion into TXB, (empty columns) and HHT (filled columns). The conversion of 1- 14 C AA into the main LO metabolite, 12-HETE, was significantly affected even at 5 ug/m1 (Fig . 2) . The 12-HETE production at 20 µg/ ml increased by 45 . 9%. By enhancing dipyrone concentration the amount of 12HETE produced by washed platelets further increased ; at the highest concentration the percentage of increase was 251 •5 % . Log drug concentration correlated with the increase of 12-HETE production : y=140 . 7 - 444 . 3x+ 560 . 3x2 - 273 . 7x3 + 51 . 3x4

(r= 0 . 97, P< 0 . 001)



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Pharmacological Research, Vol . 21, No. 1, 1989

300

r=0 .97

1

P<0 .001

//

15 000

0

200

1 i/

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0

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,

V

100

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J

10

20 40 100 Dose (µg/m l)

200 300 T

5000 3000 0f

10

5

BAS

20

40

100

200

300

Dose (,ug/ml)

Fig. 2. In vitro 1- 14 C arachidonic acid conversion into 12-HETE at seven dipyrone concentrations and relationship between 12-HETE increase and log drug concentration .

100 90 80 70 0 60 50 40 30 20 10

r

0

- A

100- B 90-

-

so7060504030 7201 10 1

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I

1

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20

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Fig. 3 . Relationship between TXB, production inhibition in PRP (A) and washed platelets (B) and log drug concentration .



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Pharmacological Research, Vol. 21, No. 1 . 1989

TXB2 production by platelet rich plasma and by washed platelets

In order to verify whether the inhibitory activity of dipyrone on TXA 2 formation by exogenous AA also occurs on TXA 2 generation by endogenous AA in the presence of plasma, TXB, formation by PRP and washed platelets was measured . The inhibitory concentration 50 (IC50) of TXB 2 generation by PRP stimulated with thrombin was 2 . 5 µg/ml (from 73 . 2 ± 13 . 3 to 34 . 4 ± 5 . 3 ng/10 8 plt); at 40 ,ug/ml dipyrone TXB 2 production was found almost completely inhibited (94 . 7% inhibition) and at 100,ug/ml TXB2 was detectable only in trace amounts (99 . 1% inhibition) . The relationship between log drug concentration and TXB 2 inhibition was found to be linear (r=0 . 98, P< 0 . 001, Fig . 3A) . TXB, production by washed platelets was found slightly but significantly decreased after incubation with 5 and 10 ug/ml dipyrone concentration (from 102-1±12-4 to 82-8±9-5 and to 79-8± 10 . 02 ng/10 8 plt, P<0 .001) . At these drug concentrations the percentages of inhibition were 18 . 7 and 21 . 1% respectively. The IC50 was 20,ug/ml and at the highest concentration tested (200,ug/ml) the inhibition was nearly complete (94 . 6%). Log drug concentration and TXB 2 inhibition showed a linear correlation (r= 0 P< 0 . 001, Fig. 3B). Inhibition of TXB 2 production does not relate to the amounts of TXB, production in the absence of the drug either in PRP or in washed platelets .

DISCUSSION Our results indicate that dipyrone, in concentration of 5 ug/ml, is able to modify significantly the exogenous 1- 14 C arachidonic acid conversion by washed platelets . TXB 2 , HHT and the other CO metabolite production decreased with increase in log concentration, reaching a complete inhibition at the maximum concentration studied (300,ug/ml) . However, the inhibitory potency of dipyrone seems to be lower than other non-steroidal anti-inflammatory drugs. In fact the dipyrone IC50 of TXA 2 generation is sixfold higher than that of acetylsalicylic acid [11], as molarity ratio, and is notably higher 15-fold, if compared to indobufen [12], an anti-inflammatory drug which, like dipyrone, reversibly binds to platelet membrane . Conversely, LO product, 12-HElE, formation increased with the log concentration of dipyrone and the magnitude of the increase of 12-HETE is more marked than the decrease of CO metabolites . This suggests that in the presence of dipyrone higher amounts of 1- 14C arachidonic acid were shifted from CO pathway to LO pathway, and that the increased availability of the substrate enhanced LO activity. A similar pattern has been reported in the presence of other CO inhibitors, such as aspirin [13, 14] or indobufen [12] . The fact that dipyrone enhances LO metabolite formation by stimulated platelets might explain the rare occurrence of asthma episodes during dipyrone treatment [2], similar to those observed after administration of other CO inhibitors [15] . Indeed, platelet-derived LO products have been reported to be able to induce in vitro the activation of C-5 lipoxygenase in human blood leucocytes and so to promote leukotriene formation [7] . On the other hand, dipyrone does not inhibit LO activity in in vitro leucocyte suspensions . The inhibitory activity of dipyrone on the



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conversion of exogenous 1- 14 C arachidonic acid into TXB 2 by washed platelets was of the same order of magnitude of the inhibition of TXB 2 generation from endogenous arachidonic acid by washed platelets . However, a higher inhibition of TXA 2 formation by dipyrone was observed when platelet-rich plasma, instead of washed platelets, was incubated with the drug . In our experimental conditions the amounts of TXB 2 assayed in PRP, before the addition of the drug, were lower than those assayed in washed platelets . This difference could depend on the presence of proteins, such as albumin, in PRP, which aspecifically binds TXA 2 [16] and decreases the conversion of arachidonic acid into CO and LO metabolites [17, 18]. Moreover, the presence of plasma proteins may potentiate the inhibitory effect of the drug by increasing its stability in the incubation medium [3] . In conclusion, dipyrone at therapeutic concentrations was found to be able to affect arachidonic acid metabolism through CO pathway and to decrease TXA, production by platelets . However, its potency, on a molar ratio basis, appears to be lower than that of other non-steroidal anti-inflammatory drugs . The degree of the inhibitory effect of dipyrone on platelets cannot be considered indicative of its inhibition extent on CO pathways of other tissues : in fact the inhibitory potency of dipyrone is different in relation to the different target cells [19] as it is also shown in relation to other CO inhibitors, which exert unequal CO inhibitory effect on different tissues [3, 20] .

ACKNOWLEDGEMENTS This investigation was supported by a grant from Fondazione Hoechst Milano .

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15 . Morris HR, Piper PJ, Taylor GW, Tippins JR . BrJ Pharmac 1979; 66 :452P. 16 . Granstrom E, Kindahl H. In: Frolich JC, ed. Methods in prostaglandin research . New York: Raven Press, 5 :119-210 . 17 . Deykin D, Russel FA, Vaillancourt R . Prostaglandins 1979; 18 : 17-25 . 18 . Stuart MJ, Gerrard JM, White JG . Blood 1980 ; 55 : 418-23 . 19 . Luthy C, Multhaupt M, Oetliker 0, Perisic M . BrJ Pharmac 1983 ; 79 : 849-54 . 20 . Weksler BB, Pett SB, Alonso D, et al. New Engl J Med 1983 ; 308 : 800-5 .