Effects of five alkyl hydroxytyrosol ether derivatives on platelets function

JJ Reyes de la Vega1, MI Ruiz-Moreno1, A Benitez-Guerrero3, R Guzmán-Moscoso1, A Guerrero2, J Muñoz-Marin2, JA Gonzalez-Correa2,1, JP De la Cruz2. 1Faculty of Health Sciences. University of Malaga, Research Group LIAIT. Department of Pharmacology, Spain, 2School of Medicine, Research Group LIAIT. Department of Pharmacology, Spain, 3University Hospital Carlos Haya, UGC Physical Medicine and Rehabilitation, Spain

Introduction: Platelet activation is the main cause of arterial thrombus formation. Hydroxytyrosol (HT) inhibits platelet aggregation both in vitro and ex vivo, and modifies the synthesis of prostanoids, nitric oxide and some leukocyte inflammatory mediators. However, this compound has a low lipophilicity, for that reason efforts have been made to synthesize HT derivatives with a better hydrophile/lipophile balance and increase its bioavailability. In this connection a series of hydrophobic derivatives (alkyl HT ethers) of HT have been synthesized.

Aims: To investigate the “in vitro” antiplatelet and anti-inflammatory effects of five alkyl hydroxytyrosol (HT) ether derivatives in human whole blood and to evaluate the effects of the oral administration of five alkyl HT ethers on platelet aggregation comparing with HT.

Methods: “in vitro” study was carried out in blood samples from 36 healthy volunteers. HT and HT alkyl ether derivatives (ethyl (E), butyl (B), hexyl (H), octyl (O) and dodecyl D)) were incubated. Maximum intensity of platelet aggregation induced with collagen, arachidonic acid or ADP, calcium-induced thromboxane B2 and nitric oxide production (constitutive activities of cyclooxygenase and nitric oxide synthase), LPS-induced prostaglandin E2 and nitric oxide production (inducible activities of cyclooxygenase and nitric oxide synthase) and LPS-induced interleukin 1ß production were measured. The “ex vivo” experiments were carried on using 78 wistars rats (six animals per group) which were administrated by gavage 20 or 50 mg/Kg/day of saline, HT or the different HT alkyl ethers derivatives during 7 days. All animals were housed at the University of Malaga Centro de Experimentación Animal under standard temperature and light/dark conditions, with access to food and water ad libitum. At the end of the treatment period, all rats were anesthetized with 40 mg/kg of sodium pentobarbital i.p., blood was obtained via puncture of the abdominal aorta and finally killed by decapitation. Statistical analysis was performed using the t student test and ANOVA.Results: All the compounds inhibited in a concentration-dependent manner platelet aggregation, thromboxane B2 and inflammatory mediators. The concentrations of each compound that inhibited 50% the corresponding variable with respect to control sample (IC50) were in the range 10-7-10-6 M for HT hexyl ether, while for the other compounds these values were in the range 10-5 M. For thromboxane B2 production this was in the range 10-4 M. HT alkyl ether derivatives showed a higher effect than HT. Maximum effect was observed in the range C4-C6. In “ex vivo” experiments maximum intensity of collagen-induced aggregation was the 12,00±1,50/9,08±0,74 ohmios (20 and 50 mg/Kg/day) for HT vs. 12,00±1,60/10,25±2,75 (E), 6,40±0,70/10,00±0,99 (B), 10,50±0,50/6,25±0,75 (H), 10,20±1,70/6,50±0,50 (O) and 15,75±2,30/8,00±0,50 (D). On the other hand serum TxB2 (pg/mL) levels were: 60,42±2,05/54,82±3,27 (HT), 15±8,07/72,78±5,69 (E), 39,39±4,29/50,15±2,81 (B), 22,21±5,01/28,78±4,43 (H), 43,29±7,25/69,77±5,81 (O) and 66,31±7,86/73,4±1,27 (D).

Conclusion: HT alkyl ether derivatives exert antiplatelet and antiinflammatory effects that are greater than HT. This effect involves a slight decrease in platelet thromboxane.