Clinical and Experimental Pharmacology and Physiology (2004) 31, 159–162 MICRONIZATION ENHANCES THE PROTECTIVE EFFECT OF PURIFIED FLAVONOID FRACTION AGAINST POSTISCHAEMIC MICROVASCULAR INJURY IN THE HAMSTER CHEEK POUCH Fátima ZGA Cyrino,* Daniel A Bottino,* Laurence Lerond † and Eliete Bouskela* *Laboratório de Pesquisas em Microcirculação, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil and † Institut de Recherches Internationales Servier, Courbevoie, France SUMMARY 1. The present study was designed to evaluate the effect of micronization on the protective effect of the purified flavonoid fraction (MPFF) on increases in macromolecular permeability induced by ischaemia–reperfusion in the hamster cheek pouch microcirculation. 2. Male hamsters (Mesocricetus auratus) were treated orally, twice a day, with vehicle (lactose), MPFF and non-micronized purified flavonoid fraction (PFF) at 5, 20, 80 and 320 mg/kg per day for 10 consecutive days. On the 11th day, cheek pouches of anaesthetized animals were prepared for intravital microscopy. 3. Local ischaemia was obtained by clamping the neck of the everted pouch and the increase in microvascular permeability was quantified as leakage (leaks) of intravenously injected fluorescein isothiocyanate-labelled dextran (FITC–dextran 150; MW = 150 000). 4. Reperfusion, after 30 min ischaemia, resulted in an immediate but reversible increase in post-capillary leakage. The MPFF induced a significant dose-related reduction in the increased permeability, with 83.4% inhibition compared with control at 320 mg/kg per day (19.2 ± 1.9 vs 115.7 ± 4.1 leaks/cm 2 ; P < 0.0001). Non-micronized PFF was signifi- cantly less effective: only 47.9% inhibition compared with control was observed at 320 mg/kg per day (60.3 ± 1.0 vs 115.7 ± 4.1 leaks/cm 2 ; P < 0.0001) and there was no dose–effect relationship. 5. In conclusion, micronization significantly enhances the protective effects of the purified flavonoid fraction on reper- fusion injury in the hamster cheek pouch. This improvement is likely to be related to the better absorption of the micronized formulation, which could explain the superior clinical efficacy shown in previous studies. Key words: cheek pouch, hamster, macromolecular perme- ability, micronization, micronized purified flavonoid fraction. INTRODUCTION The micronized purified flavonoid fraction (MPFF), consisting of 90% micronized diosmin (diosmetin-7-rhamnoglucoside) and 10% flavonoids expressed as hesperidin (hesperitin-7-rhamnogluco- side), is used clinically to treat chronic venous insufficiency and haemorrhoidal disease because of its anti-inflammatory proper- ties. 1,2 The mechanism underlying the development of chronic venous insufficiency seems to involve cyclical periods of ischae- mia–reperfusion. 3,4 Early restoration of blood flow to the ischaemic tissue is essential to stop the progression of cellular injury associ- ated with the decrease in oxygen and nutrient delivery. However, it is now clear that reperfusion to an ischaemic area initiates a complex cascade of pathological events that could potentially lead to the same end result as prolonged hypoxia (i.e. cellular dysfunc- tion and necrosis). Reactive oxygen species promote the formation of pro-inflammatory stimuli (such as platelet-activating factor, leukotriene B4 and activated complement components), modify the expression of adhesion molecules on the surface of leucocytes and endothelial cells (like CD11b/CD18, P-selectin and intercellular adhesion molecule (ICAM)-1) and reduce the levels of nitric oxide (NO) in the muscle, a potent anti-adhesive substance. 5 This latter effect is exacerbated by a postischaemic decline in tissue levels of NO synthase. As a consequence, the leucocytes start ‘rolling’ at the endothelial surface, which is followed by adherence to endothelial cells of the post-capillary venules. The activated leucocytes migrate into the interstitial tissues, inducing microvascular barrier dysfunction via release of oxidants and hydrolytic enzymes. 6 Flavonoids inhibit phosphodiesterases involved in cell activation. Much of this effect is upon the biosynthesis of protein cytokines that mediate adhesion of circulating leucocytes to sites of injury. Thus, enhanced vascular macromolecular leakage is one of the earliest signs of microvascular dysfunction elicited by ischaemia– reperfusion. The increased permeability leads to an enhanced fluid filtration, with a consequent increase in interstitial fluid pressure, which could physically compress the capillary and lead to the development of the ‘no-reflow’ phenomenon. 7,8 The MPFF has been shown to exert a protective effect on microvascular barrier function under experimental conditions such as ischaemia–reperfusion, 9–11 oxidative stress, 12 inflammation 9,13 or venous hypertension. 14 In the cheek pouch microcirculation, 10 day oral treatment with MPFF has been shown to inhibit the increase in macromolecular permeability induced by histamine, bradykinin, leukotriene B4 and ischaemia–reperfusion. 9,13 The present study was designed to evaluate the influence of micronization on the protective effects of the purified flavonoid Correspondence: Professor Eliete Bouskela, Laboratório de Pesquisas em Microcirculação, Pavilhão Reitor Haroldo Lisboa da Cunha, térreo, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier 524, 20550-013 Rio de Janeiro RJ, Brazil. Email: eliete@uerj.br Received 17 March 2003; revision 1 October 2003; accepted 16 November 2003.