Micronized Purified Flavonoid Fractions and the Treatment of Chronic Venous Insufficiency: Microcirculatory Mechanisms

Contents

Abstract

The standard treatments for venous diseases of the lower limb include compression bandaging and stockings as well as surgical removal of varicose veins. There is a number of conditions in which these conventional treatments are ineffective, particularly in the management of leg ulceration. Drug treatments for healing venous leg ulcers have yet to be developed to the stage of good clinical efficacy, but may assist in the management of patients. Flavonoid drugs have been widely used in the management of the symptoms of venous disease for many years and have recently been studied in some detail to asses their effects on the microcirculation. Work in animal models of the ischaemia-reperfusion show that MPFF (micronised purified flavonoid fraction) modulates leucocyte adhesion and prevents endothelial damage. Similar biochemical effects have been observed in patients with venous disease and may explain the efficacy of this drug in the management of oedema and other symptoms of venous disease. There is some clinical evidence that MPFF promotes venous leg ulcer healing.

Keywords

Varicose ulceration, varicose veins, ambulatory venous hypertension, microcirculation, neutrophils, flavonoid treatment.

Running title:

Pathophysiology and Efficacy of Daflon 500 mg.

Introduction

The management of venous diseases has relied on compression treatments and surgical interventions for many years. The growth of drug treatment in some sectors of medicine has been considerable but the same is not true of venous diseases. In part this is due to an incomplete understanding of the mechanisms involved in the development of venous ulcers. Much recent evidence points to inflammatory mechanisms involving leucocytes which could be the target of drugs. In many countries, drugs remain widely used in patients with vein problems as adjunctive treatments. , One of the most frequently used drug groups are the flavonoids. These have been in widespread use in the management of oedema and other symptoms of venous disease for many years. Recently evidence of the mechanism of action of some of these compounds has been obtained in more detail from both animal and human studies. The aim of this review is to consider the biological mechanisms addressed by flavonoid treatment.

Chronic venous insufficiency (CVI) resulting in skin changes and leg ulceration.
Venous ulceration and the skin changes which precede ulceration are best managed by careful objective evaluation of the venous system of the lower limb followed by compression bandaging or stockings and surgical treatment where appropriate. Surgical intervention is appropriate where leg ulceration is attributable mainly to superficial venous incompetence alone in a patient fit enough for this procedure. In some studies this would apply to as many as half the patients presenting with venous ulceration. , A number of studies show that healing usually progresses well in such patients and recurrent ulceration is not a common problem . In general patients with damage to the deep veins patients are best managed by compression treatments alone. Unfortunately, whilst compression treatment can usually achieve healing if high enough levels of compression are used, recurrence is common problem with an annual recurrence rate of 25% per year. This is clearly a place in which drug treatments may have a role.
Pathogenesis of CVI
Many authors have attempted to explain the mechanism by which leg ulcers form. In recent years the simple concept that tissue hypoxia leads to leg ulceration has been largely abandoned. The causes of leg ulceration are very much more complex than simple failure to deliver oxygen or other tissue nutrients to the location in which they are required. In 1988 I suggested that leucocyte sequestration in the lower limb, which was observed during venous hypertension, was part of the mechanism that eventually results in leg ulceration. Subsequently a considerable amount of work has been done by a number of authors to evaluate this proposal by investigating several inflammatory mechanisms in CVI.
The role of leucocytes in chronic venous insufficiency
During ischaemia in any tissue the white blood cells, which are normally thought to be protective for the body, may cause damage to the tissues. Neutrophils have the ability to become ‘activated’ which would normally occur in destroying bacteria or ‘foreign’ material. In this state they release enzyme granules from within their cytoplasm and make oxygen free-radicals , both of which are very poisonous to all biological molecules and cause destruction of bacteria. Unfortunately, neutrophil activation occurs during tissue ischaemia, , but this time it is the normal tissues which are attacked. In acute ischaemia the endothelium is damaged and neutrophils block many capillaries . Some of these mechanisms may be at work in venous disease, occurring in response to venous hypertension.
Serial measurement of white and red cell counts in blood taken from the foot showed that during venous hypertension there was a fall in the leucocyte count relative to the red cell count. This was especially marked in patients with venous disease. Subsequently the same response to venous hypertension was reported in the upper limb, with more leucocyte sequestration in the limbs of patients with venous disease than with control subjects. There was clearly a systemic inflammatory response in these patients. The extent of this response has been evaluated both immuno-histochemically and by blood sampling. It has been found that leucocytes retrieved from the lower limbs of volunteers and patients with venous disease following venous hypertension undergo activation. , Patients with venous disease have increased neutrophil degranulation shown by ELISA testing for plasma neutrophil elastase and lactoferrin. However, these particular indices of inflammation showed similar values in patients with uncomplicated varicose veins and those with venous ulceration. It is clear that all clinical stages of venous disease result in a systemic inflammatory response.
Endothelial cells shed their surface adhesion molecules into the blood and the plasma levels of these can be used to assess injury to endothelium. Plasma levels of several endothelial adhesion molecules have been shown to be elevated in patients with venous disease. In one paper it was found that plasma levels of soluble ICAM-1 and VCAM-1 increased in patients with venous disease (superficial and deep venous incompetence). In those patients with deep venous disease, who presumably had the more severe symptoms, the plasma levels of these adhesion molecules remained raised after resting supine. This observation confirms the suggestion that there is circulating evidence of damage to the vascular endothelium in patients with chronic venous disease in the form of soluble adhesion molecules. The exact origin of these is unclear at present, whether it is from damaged skin, damaged veins or some other source.
Micronised purified flavonoid fraction (MPFF)
Diosmin is a member of the flavonoid family. MPFF as a commercial product which combines diosmin with another flavonoid, herperidine (Daflon 500 mg, Detralex, Arvenum 500, Capiven, Venitol, Variton, Ardium, Servier, Courbevoie, France). Laurent examined the efficacy of MPFF in 200 patients in a double blind, placebo-controlled trial lasting 2 months. Outcome measures assessed the symptoms of venous disease using visual analogue scales. MPFF improved functional and organic venous insufficiency (symptoms of venous disease not associated with major venous valvular incompetence) by 71% and 66% respectively compared to 36% and 38% for placebo. The effect on oedema was assessed by measuring ankle circumference. The mean difference in the reduction of supra-malleolar circumference was 6.7 mm (right limb) and 6.1 mm (left limb) in favour of micronized diosmin in patients with functional symptoms. In patients with varicose veins or post thrombotic syndrome the reduction was 6.8 mm (right limb) and 6.5 mm (left limb). The supra-malleolar circumference remained unchanged in the placebo group.
The mode of efficacy of this drug is incompletely understood. A number of investigations have been undertaken in animals to assess some possible modes of action. MPFF has been found to increase venous tone , and lymphatic flow , , . It also decreases hyperpermeability and increases capillary resistance . These findings may explain why MPFF reduces tissue oedema, but do not indicate the mode of action at a molecular level.
A recent paper has shown that it modifies the interaction of leucocytes with endothelium. A hamster dorsal skin fold model was used to investigate the effect of MPFF on the microcirculation following ischaemia-reperfusion. The group of animals pre-treated with MPFF exhibited less neutrophil adhesion in the post-capillary venules at 30 mins, 2 hours and 24 hours after reperfusion following ischaemia, compared to the control group . This observation well may be linked to the protective effect of flavonoids in the treatment of oedema. Decreased leucocyte activation is also associated with a decreased platelet and complement system activation, leading to a lowered release of histamine and decreased leucocyte-dependent endothelial damage .
The studies referred to above reflect observations in ischaemia-reperfusion models. These subject the microcirculation to a standardised challenge, allowing objective assessment of an intervention, such as pre-treatment with MPFF. However, they do not reflect what happens in venous hypertension. This cannot easily be replicated in an animal model, since venous hypertension in human subjects requires a vertical distance of 1.2 m – 1.5 m between the leg and the heart to produce a large enough venous pressure to cause injury to the microcirculation. This level of pressure (60 – 70 mm Hg) can be achieved over short periods of a few days using arterio-venous fistulae. However, this results in a high-flow state which again is the situation in human patients. Takase et al have developed a venous hypertension model in the rat mesentery by occluding a vein draining part of the gut. This results in a pressure of about 30 mm Hg in the mesenteric venules, and results in increased leucocyte adhesion to post-capillary venules, seen through a capillary microscope. Pre-treatment with MPFF for seven days significantly decreased the number of rolling, adherent and migrating leucocytes in the rat mesentery. MPFF reduced neutrophil expression of CD62L even though CD18 was not affected.
Clinical use of MPFF
The effect of MPFF in a placebo-controlled venous ulcer healing study has been reported . Patients were randomised to receive MPFF or placebo combined with standard compression bandaging during an 8 week follow-up period. In 91 patients with an ulcer diameter of 10 cm or less, 14 of 44 (32%) patients receiving MPFF compared to 6 of 47 (13%) receiving placebo healed their ulcers (p=0.028, chi squared) after 8 weeks treatment. The time to achieve healing was shorter in the MPFF group than in the placebo group (p=0.037). This is the only member of the ‘oedema protective’ drug group which has been shown to modify ulcer healing. Despite the fact that the study was relatively small and the duration of treatment was short (8 weeks) the results are encouraging. A further open study with a placebo control has also been published in 140 patients with leg ulcers due to CVI. Patients were randomly assigned to receive MPFF or placebo and the outcome assessed after 24 weeks’ treatment. 47% of ulcers in the MPFF treatment group and 28% of patients in the control group healed their ulcers. The healing rate in the control group is low, suggesting that low levels of compression were used. The data suggest that MPFF treatment speeds ulcer healing, although the results of open studies should be interpreted with caution.
The possible mechanism of action of this drug is not yet clear, although several of its effects have been described above in the section on treatment of varicose veins. The effects of MPFF on systemic inflammatory mediators have been studied in a recent pilot study. , Twenty patients with chronic venous disease (CEAP clinical stage 2-4) were treated for 60 days with MPFF 500 mg twice daily taken orally. There was no placebo control group in this pilot study.
The expression of the leucocyte adhesion molecule CD62L (L-selectin) was substantially decreased on monocytes and neutrophils by MPFF treatment, however, CD11b expression was not modified (fig 1). This finding suggests that leucocyte L-selectin interaction with endothelial selectins responsible for the initial stages of adhesion to endothelium may be modulated by MPFF treatment, reducing the likelihood of leucocyte adhesion and presumably acting as an anti-inflammatory mechanism. Significant down regulation of plasma levels of sVCAM-1 and sICAM-1 activity following therapy was also observed (data not shown) indicating that endothelial damage which ensues in venous disease from chronic venous
hypertension was mitigated by MPFF treatment.

Conclusions.

A series of inflammatory processes involving leucocyte-endothelial interaction are involved in the development of CVI. The precise mechanisms which culminate in the development of leg ulceration are not fully understood. Therapeutic modulation of these processes may provide of means of speeding ulcer healing and preventing recurrence. MPFF is widely used to treat the symptoms of venous diseases. There is clinical evidence of efficacy in the management of symptoms as well as in oedema reduction. The molecular mechanisms which are addressed by flavonoid treatment include those which influence leucocyte adhesion to endothelium. This is a fundamental biological process and central to many aspects of the inflammatory response. In animal models of ischaemia, venous hypertension and inflammation MPFF reduces leucocyte adhesion and mitigates the consequences of these noxious stimuli. The result appears to be to prevent damage to the microcirculatory endothelium. In clinical practice MPFF treatment results in reduced L-selectin expression by leucocytes and reduces plasma levels of soluble endothelial adhesion molecules as well as VEGF. These probably reflect the anti-inflammatory effects of this drug. Reduced leg ulcer healing times have been observed in two placebo-controlled trials but the role of MPFF has yet to be fully established in promoting venous ulcer healing.
References

Figure legends.

Figure 1.

Changes in neutrophil (N) and monocyte (M) surface fluorescence (arbitrary units A.U.) for adhesion molecules CD62L and CD11B before & after flavonoid therapy for sixty days (n=20). All statistics employ the Wilcoxon test for paired data. These results show a decrease CD62L (L-selectin) following therapy.

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