Pharmacology

We have previously demonstrated that the phenolic compounds catechol, hydroquinone, and phenol increase the prostaglandin (PG) E2/leukotriene (LT) B 4 ratio in human polymorphonuclear leukocytes (PMNs), while resorcinol has the opposite effect. However, in human whole blood phenols have a different effect on the thromboxane (TX) B2/LT ratio than in PMNs on the PGE2/LTB4 ratio. To establish whether the discrepancy between the results of our previous studies is due to different indicators of prostaglandin H synthase activity in PMNs (PGE2) and in whole blood (TXB2), we measured the effect of phenols on PGE 2 synthesis in whole blood. The phenols only inhibited PGE 2 synthesis (ICso values for resorcinol, catechol, hydroquinone, and phenol of 10 gM, 10 gM, 60 gM and 700 gM, respectively). No significant stimulatory activity was seen as earlier in PMNs. Thus, the effect of phenols on PGE2 synthesis in whole blood is different from that in PMNs, although their order of potency to inhibit PGE 2 synthesis is the same.

Ž . Ž . Ž . Ž . Ž . The effects of y -nicotine 0.0005-500 mM , q -nicotine 0.0005-50 mM and y -cotinine 0.0005-500 mM on arachidonic 2q Ž . Ž . acid metabolism were investigated in Ca ionophore A23187 calcimycin -stimulated human whole blood in vitro. y -Nicotine and Ž . y -cotinine stimulated prostaglandin E but inhibited thromboxane B synthesis, as has been observed previously in A23187-stimulated 2 2 w polymorphonuclear leukocytes and platelet-rich plasma Saareks, V., Riutta, A., Mucha, I., Alanko, J., Vapaatalo, H., 1993. Nicotine and

Abstract: The in vitro effects of nicotinic acid (10–1000 μM), pyridoxine (0.1–500 μM) and pyridoxal-5′-phosphate (0.1–500 μM) and the ex vivo effects of nicotinic acid (2500 mg orally during 12 h) and pyridoxine (600 mg orally daily for seven days) on arachidonic acid metabolism were investigated in calcium ionophore A23187 (calcimycin)-stimulated human whole blood. In vitro nicotinic acid stimulated prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis. Pyridoxine at all concentrations and pyridoxal-5′-phosphate at the highest concentration stimulated prostaglandin E2 and thromboxane B2 production, but had no effect on leukotriene E4 synthesis. Nicotinic acid treatment increased ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis to 185%, 165% and 175% of the initial values, respectively. In the pyridoxine-treated subjects, ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis was decreased after seven days to 75%, 65% and 45% of the initial values, respectively. In the present study the effects of nicotinic acid on the 5-lipoxygenase pathway in arachidonic acid metabolism were studied for the first time and the drug was found to stimulate this pathway in vitro and ex vivo. In vitro pyridoxine and pyridoxal-5′-phosphate had no effect on the 5-lipoxygenase pathway. The inhibition of leukotriene synthesis by pyridoxine ex vivo might be of therapeutic importance.

The effects of smoking cessation with and without nicotine substitution on prostaglandin E2, leukotriene B4, leukotriene E4, and thromboxane B2 synthesis ex vivo in man were investigated. Blood samples were obtained from 20 healthy non-smoking controls and from 30 healthy smoking volunteers before and 3, 7 and 14 days after smoking cessation. Half of the smokers used nicotine chewing gum as a substitution therapy. Urinary cotinine and trans-3′-hydroxycotinine as well as thiocyanate excretions were used as indicators for the use of nicotine chewing gum and smoking, respectively. Prostaglandin E2, leukotriene E4, and thromboxane B2 were measured from whole blood after calcium ionophore A23187 stimulation by direct radioimmunoassay and leukotriene B4 by RP-HPLC. Prostaglandin E2 and thromboxane B2 syntheses were about three times and leukotriene B4 and E4 syntheses four times higher in smokers than in controls. Three days after smoking cessation without nicotine substitution, levels were lowered significantly to about 70%, 80%, 45% and 60% of the initial values; and after 14 days to 55%, 80%, 45% and 50%, respectively. In the nicotine substitution group no significant decreases were seen during the two-week follow-up. The increased level of eicosanoid synthesis detected in smokers in this ex vivo study may contribute to the harmful cardiovascular effects of smoking. Long-term nicotine substitution might diminish the beneficial effects of smoking cessation due to the possible stimulatory effects of nicotine and cotinine on eicosanoid synthesis even in vivo.

Prostacyclin (PGI2) and thromboxane A2 (TXA2) play an important role in the pathophysiology of various cardiovascular diseases. The balance between PGI2 and TXA2 regulates the interaction between platelets and the vessel wall in vivo. In this study we measured PGI 2 and TXA 2 synthesis by analysing their urinary index metabolites 2,3-dinor-6-keto-PGFl~ and 11-dehydro-TXB2, respectively, in acute (10 patients) and chronic (10 patients) lower limb ischaemia. Both PGI2 and TXA2 synthesis were increased about two-fold in patients with acute lower limb ischaemia compared to chronic lower limb ischaemia. However, the PGIJTXA2 ratio was more or less the same in acute and chronic lower limb ischaemia. In patients with acute lower limb ischaemia caused by thrombotic occlusion, PGI 2 and TXA2 formation were about two times higher than in patients with acute lower limb ischaemia caused by embolic occlusion. Elevation of PGI2 and TXA2 synthesis in acute lower limb ischaemia may reflect increased platelet-vascular wall interactions without changing the PGI2/TXA2 ratio.

We have previously demonstrated that adrenaline infusion increases the thromboxane/leukotriene (TX/LT) ratio in whole blood in healthy volunteers. The aim of the present study was to see whether other catecholamines -noradrenaline and dopamine -are also capable of modulating arachidonic acid (AA) metabolism in man. Low doses of noradrenaline (0.025 gg/kg/min) and dopamine (3.0 g/kg/min), which did not change hemodynamics, were infused for 60m in into healthy male volunteers. Both dopamine and noradrenaline decreased TX synthesis stimulated by spontaneous clotting, but no remarkable effect was seen when calcium ionophore A23187 was used as a stimulus. Dopamine but not noradrenaline increased prostaglandin E 2 (PGE2) synthesis in A23187-stimulated whole blood. They both marginally decreased LTB4 formation in A23187-stimulated whole blood. The findings indicate that not only adrenaline but also noradrenaline and dopamine modulate AA metabolism in man.

8-Isoprostaglandin F 2a is one of a series of isoprostanes formed by free radical catalysed peroxidation of arachidonic acid. Urinary 8-isoprostaglandin F 2a is a new marker which reflects oxidative stress in vivo and can be utilized as a diagnostic tool to assess the extent of oxidative stress in various disease states associated with lipid peroxidation.

The effects of nicotinic acid (2500 mg orally during 12 hr) and pyridoxine (300 mg orally twice daily for seven days) on the excretion of urinary 2,3-dinor-6-ketoprostaglandin F 1a , 11-dehydrothromboxane B 2 and leukotriene E 4 , the markers of systemic prostacyclin, thromboxane A 2 and cysteinyl leukotriene production, respectively, were investigated in healthy male volunteers (nΩ6-8). Nicotinic acid increased 11-dehydrothromboxane B 2 and leukotriene E 4 excretions to 2.6-and 2.0 times the initial values (PϽ0.05), respectively. In the volunteers treated with pyridoxine, 11-dehydrothromboxane B 2 and leukotriene E 4 excretions were decreased to 70% (PϽ0.05) and 65% (PϽ0.01) of the initial values, respectively, but the excretion of 2,3-dinor-6-ketoprostaglandin F 1a was increased 1.7 times (PϽ0.01). The results suggest that nicotinic acid increases thromboxane and leukotriene synthesis which may not be beneficial for patients with cardiovascular diseases or asthma. In contrast, the increase in prostacyclin production and the inhibition in thromboxane and leukotriene synthesis by pyridoxine might be beneficial in disorders where the production of prostacyclin is decreased and the formation of thromboxane and cysteinyl leukotrienes is enhanced.

Objective. To determine the dose of acetylsalicylic acid (ASA), that inhibits the production of the vasoconstrictive, aggregatory thromboxane A 2 while sparing the production of the vasodilatory antiaggregatory prostacyclin. Design. A controlled study comparing the effects of three doses of ASA on the production of thromboxane A 2 and prostacyclin. Methods. Seven pregnant hypertensive patients and five non-pregnant healthy women received 0.5, 1.0 and 2.0 mg/kg/day of ASA, each dose for 10-12 days, the treatment periods following each other immediately. Seven normotensive pregnant women served as controls and were given no ASA. Blood and urine samples were taken at baseline and after the treatment periods to determine serum thromboxane B 2 and the urinary 2.3-dinor-6-ketoprostaglandin F 1a and 11-dehydrothromboxaneB 2 , the major stable metabolites of prostacyclin and thromboxane A 2 , respectively. Results. The urinary excretion of 11-dehydrothromboxaneB 2 was significantly higher in both hypertensive (34.9∫18.3 pg/mmol creatinine) and normotensive (39.3∫14.4 pg/mmol creatinine) pregnant women than in non-pregnant women (14.8∫6.4 pg/mmol creatinine). The urinary excretion of 2.3-dinor-6-ketoprostaglandinF 1a was also higher in normotensive pregnant women (93.9∫50.9 pg/mmol creatinine) than in non-pregnant women (18.2∫11.3 pg/mmol creatinine). The excretion rate of 2.3-dinor-6-ketoprostaglandinF 1a in hypertensive patients was lower than in normotensive pregnant women (44.7∫24.2 pg/mmol creatinine). At baseline the urinary 2.3-dinor-6-ketoprostaglandin F 1a /11-dehydrothromboxaneB 2 ratio was almost the same in the hypertensive patients (1.6) and in the non-pregnant women (1.2). The ratio was 2.6 in normotensive pregnant women. In the hypertensive group, already the lowest dose of ASA inhibited urinary 11-dehydrothromboxaneB 2 excretion significantly. Because none of the doses of ASA inhibited 2.3-dinor-6-ketoprostaglandinF 1a production, the 2.3-dinor-6-ketoprostaglandinF 1a /11-dehydrothromboxaneB 2 ratio was shifted in the favor of prostacyclin at all dose levels. In the non-pregnant women, even the highest dose level of ASA failed to affect the ratio. Conclusion. In the dose range of 0.5-2.0 mg/kg/day, ASA has a favorable effect on the ratio of prostacyclin to thromboxane A 2 in hypertensive pregnancies.

Percutaneous transluminal angioplasty is an acute, local stimulus to platelets which activation is regarded as an important factor for a later restenosis. The balance between the production of prostacyclin and thromboxane A2 is of (patho)physiological importance due to their opposite actions on vascular tone and platelet reactivity. In this study we investigated the influence of percutaneous transluminal angioplasty of the peripheral arteries on prostacyclin and thromboxane A2 productions in vivo by measuring the excretions of their urinary index metabolites, 2,3-dinor-6ketoprostaglandin FI~ and 11-dehydrothromboxane B2, respectively, in 10 patients. We found a twofold increase in thromboxane A2, but no significant change in prostacyclin, production after peripheral transluminal angioplasty which shifted prostacyclin/thromboxane A2 balance to the direction of thromboxane A 2 formation. This gives theoretical support to the use of thromboxane A2 synthase inhibitors and receptor antagonists as well as prostacyclin analogues in combination with peripheral percutaneous transluminal angioplasty to prevent thrombosis and restenosis.

Although theophylline has been used in the treatment of asthma for decades, it is not a first line choice any more. It is a well-known bronchodilator, but was recently discovered also to be an anti-inflammatory, immunomodulatory and bronchoprotective agent. Therefore we wanted to establish the role of theophylline on prostaglandin and leukotriene production, which plays a part in the pathogenesis of asthma. Theophylline was infused (bolus 5 mg/kg in 15 min and infusion 0.4 mg/kg/h for 1 h 45 min) into healthy volunteers. Thromboxane B2, prostaglandin E 2 and leukotriene E4 were measured from the A23187-stimulated whole blood samples and stable metabolites of thromboxane A2; prostacyclin and leukotriene E4 were measured from urine. Theophylline increased prostaglandin E a production and decreased leukotriene E4 production ex vivo in whole blood, thus increasing the prostanoid/leukotriene ratio. It did not change thromboxane B2 production stimulated by either spontaneous clotting or A23187 in the whole blood. Theophylline had hardly any effect on in vivo thromboxane, prostacyclin and leukotriene E, production measured as urinary metabolites, 11-dehydro-thromboxane B2, 2,3-dinor-6-keto-prostaglandin FI~ and leukotriene E4, respectively. Serum theophylline concentrations were at the lower level of normal therapeutic range during the infusion. The increase in PGE2 and the decrease in LTE4 synthesis ex vivo may offer a new explanation for the mode of antiasthmatic action of theophylline. It is notable that this phenomenon occurs at low serum theophylline concentrations. These results confirm the idea that theophylline has an anti-inflammatory and bronchoprotective action and support the use of theophylline as a therapeutic agent in asthmatic patients.

The effects of substituted catechols (3-methylcatechol, 4-methylcatechol, 4-nitrocatechol, and guaiacol) and trihydroxybenzenes (pyrogallol, propyl gallate, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) on the synthesis of prostaglandin (PG)E 2 and leukotriene (LT)B 4 were tested in human A23187-stimulated polymorphonuclear leukocytes. The effects were related to their peroxyl-radical-scavenging (antioxidant), superoxide-scavenging (antioxidant), and superoxide-generating (prooxidant) properties. In general, compounds with hydroxyl groups in the ortho position increased PGE 2 /LTB 4 ratio, and compounds with hydroxyl groups in the meta position decreased PGE 2 /LTB 4 ratio. Catechols, which have hydroxyl groups in the ortho position, were the most potent peroxyl radical and superoxide anion scavengers. Trihydroxybenzenes (pyrogallol, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) generated superoxide, whereas dihydroxybenzenes did not. Thus, the positions and number of hydroxyl groups seem to be the most important properties determining the action of phenolic compounds on PGE 2 /LTB 4 ratio and their antioxidant/ prooxidant activities.

We infused noradrenaline (0.025 pg.kg/min for 60 min. n=7) and dopamine (3.0 lq'I@nin for 60 n-tin, n==6) into healthy male volunteers to study the effects of these catecholamines on in vivo thromboxane A,, prostacyclin and leukotriene E4 production measured as urinary excretions of 1 1-dehydro-thromboxane (TX) I!$, 2,3dinor-6-keto-prostaglandin (PG) F,, and leukotriene (LT) E4, respectively. Plasma noradrenaline and dopamine concentrations were 2.910.3 and 233+17 nrnol/l at the end of the noradrenaline and dopamine infusions, respectively. Noradrenaline decreased thromboxane production and increased leukotriene production almost twofold. It had hardly any effect on prostacyclin production. Dopamine had no significant effects on any of the variables, however it had a tendency to increase prostacyclin and leukotriene production. The results indicate that noradrenaline is a more important modulator of arachidonic acid metabolism than dopamine in vivo.

Objective. To determine the dose of acetylsalicylic acid (ASA), that inhibits the production of the vasoconstrictive, aggregatory thromboxane A 2 while sparing the production of the vasodilatory antiaggregatory prostacyclin. Design. A controlled study comparing the effects of three doses of ASA on the production of thromboxane A 2 and prostacyclin. Methods. Seven pregnant hypertensive patients and five non-pregnant healthy women received 0.5, 1.0 and 2.0 mg/kg/day of ASA, each dose for 10-12 days, the treatment periods following each other immediately. Seven normotensive pregnant women served as controls and were given no ASA. Blood and urine samples were taken at baseline and after the treatment periods to determine serum thromboxane B 2 and the urinary 2.3-dinor-6-ketoprostaglandin F 1a and 11-dehydrothromboxaneB 2 , the major stable metabolites of prostacyclin and thromboxane A 2 , respectively. Results. The urinary excretion of 11-dehydrothromboxaneB 2 was significantly higher in both hypertensive (34.9∫18.3 pg/mmol creatinine) and normotensive (39.3∫14.4 pg/mmol creatinine) pregnant women than in non-pregnant women (14.8∫6.4 pg/mmol creatinine). The urinary excretion of 2.3-dinor-6-ketoprostaglandinF 1a was also higher in normotensive pregnant women (93.9∫50.9 pg/mmol creatinine) than in non-pregnant women (18.2∫11.3 pg/mmol creatinine). The excretion rate of 2.3-dinor-6-ketoprostaglandinF 1a in hypertensive patients was lower than in normotensive pregnant women (44.7∫24.2 pg/mmol creatinine). At baseline the urinary 2.3-dinor-6-ketoprostaglandin F 1a /11-dehydrothromboxaneB 2 ratio was almost the same in the hypertensive patients (1.6) and in the non-pregnant women (1.2). The ratio was 2.6 in normotensive pregnant women. In the hypertensive group, already the lowest dose of ASA inhibited urinary 11-dehydrothromboxaneB 2 excretion significantly. Because none of the doses of ASA inhibited 2.3-dinor-6-ketoprostaglandinF 1a production, the 2.3-dinor-6-ketoprostaglandinF 1a /11-dehydrothromboxaneB 2 ratio was shifted in the favor of prostacyclin at all dose levels. In the non-pregnant women, even the highest dose level of ASA failed to affect the ratio. Conclusion. In the dose range of 0.5-2.0 mg/kg/day, ASA has a favorable effect on the ratio of prostacyclin to thromboxane A 2 in hypertensive pregnancies.

The metabolism of melatonin to 6-sulphatoxymelatonin (aMT6S) and N-acetylserotonin (NAS) is catalyzed by cytochrome-P450 (CYP) isozymes CYP1A2 and CYP2C19, respectively. We studied the in vivo effect of CYP2C19 substrate (citalopram, omepratzole, or lansopratzole) on the metabolism of endogenous and exogenous melatonin by measuring the excretion of urinary aMT6S, the main metabolite of melatonin and a reliable estimate of plasma melatonin in 15 insomniac psychogeriatric inpatients. The effect of melatonin treatment on sleep parameters was also assessed. The patients with or without CYP2C19 substrate were treated for 21 days randomly in a double-blind way with placebo or 2 mg exogenous melatonin orally. aMT6S excretions were measured radioimmunologically from night urine at baseline (day 0), on day 21, and one day after the treatment was discontinued (day 22).