| Another study on the permeability of blood vessels in general (not just the BBB) using rats showed "that previous treatment of animals with procyanidolic oligomers prevented the permeability increase produced by collagenase injection." ref 4 abstract "In animals pretreated with OPC the collagenase induced permeability increase is abolished. OPC protects the microvasuclar wall against the proteolytic attack." ref 4 fig. 1 caption This is important, because this is the very effect we want to achieve in our proposed treatment. I.e., by keeping the blood-brain barrier supplied with appropriate flavonoids, it is hoped that when something comes along that would normally have caused a breach and subsequent lesion, that instead the flavonoids will prevent damage from occurring. (Incidentally, OPC is the French equivalent of PCO). Before discussing the manner in which flavonoids may potentially assert their effectiveness, we will first review some fundamentals. The BBB is a barrier which is caused by the capillary cells within the central nervous system (CNS) being bound together much more tightly than they are in the rest of the body. Because of the tightness, most substances are prevented from passing out of the capillary and into the CNS tissue proper. An extracellular matrix consisting of a number of proteins such as collagen and fibrin make up the matrix and function as a glue to hold the cells together. Certain cells in the immune system produce enzymes which can dissolve the extracellular matrix. For most parts of the body this is useful in allowing white blood cells greater access to an area of infection. However, when these cells release their chemicals in the capillaries within the CNS, there is a resulting breakdown of the BBB, leading to an MS lesion, at least in certain individuals. One example of this process is with matrix metalloproteinases (MMPs), which, depending on their specific kind, can act on collagen, gelatin, fibronectin, and other components of the extracellular matrix. Of particular significance as we shall discuss shortly is that MMPs are not active in the form in which they are released, but must be converted to an active form by other enzymes. ref 5 There is another process at work which also has the potential to attack BBB integrity. During phagocytosis of target cells by phagocytes, free radicals are released. These free radicals are extremely active and can destroy many of the tissues they contact. This can be a useful feature when a phagocyte is trying to destroy an invading organism: "The superoxide ion and the derived oxygen free radicals are highly reactive and capable of destroying a variety of molecules. Their liberation during phagocytosis as potent bactericidal agents as well as their action in inflammatory events have been clearly demonstrated." ref 6 So, the generation of super oxides and free radicals are useful against infections. However, when the process takes place at the blood-brain barrier, such as takes place at the beginning of an MS lesion, it can be just one more attack on the integrity of the barrier. I would now like to discuss three proposed mechanisms by which flavonoids might be effective in MS treatment. 1). Flavonoids have a tremendous affinity for joining themselves to enzymes. Thus, if they were to join themselves to either the MMPs or their activators, they might be able to block this process. This would then be a specific mechanism by which the BBB would be preserved and an impending MS lesion thwarted. The following excerpts from an article on the flavonoids are relevant: "More recent studies indicate that certain flavonoids have rather extraordinary capacities to affect the activity of many enzyme systems.... It is also obvious that some of the enzyme systems affected by flavonoids are important in the regulation of secretory, contractile and motility processes, such as the release of inflammatory chemical mediators from mast cells, basophils, neutrophils, and macrophages." ref 7 In this article the author states that studies have clearly shown that certain flavonoids have an extraordinary capacity to affect the activity of the enzyme systems related to the ones we want to inhibit. The test results quoted above in ref 2 and ref 3 demonstrate these ideas on a practical basis. The mechanism we envision here is that when MMPs are released as discussed earlier, that the flavonoids would "disable" them before they joined with their activator enzyme. Thus, they would be rendered ineffective. 2) Flavonoids tend to become part of the cellular matrix, or at least adhere to it. This is particularly true of the PCOs. While in the matrix, they have receptor sites exposed which tend to grab onto the enzymes which would otherwise attack the matrix. Thus, they become "sacrificial lambs" so to speak in maintaining the integrity of the matrix. In an experiment it was "conclusively evidenced that PCOs bind to skin elastic fibres when injected intradermally into young rabbits. As a result, these elastic fibres were found more resistant to the hydrolytic action of porcine pancreatic elastase when injected to the same site. These in vivo studies further emphasized the potential effect of these compounds in preventing elastin degradation by elastase(s) as occurred in inflammatory processes." In the same article an in vito experiment is discussed where elastin is pretreated with PCO and as a result elastin becomes completely inhibited from dissolving the elastin: "The inhibitory effect of procyanidolic oligomers is even more pronounced with human leukocyte elastase. Pretreatment of elastin with this flavonoid completely inhibits the action of 10 micrograms of purified enzyme." "In the present work, we evidenced that flavonoids could efficiently and selectively bind to elastic fibres....Our results indicated that procyanidolic compounds bound on to insoluble elastin increased the non-productive binding sites of elastases(s)" ref 8 Although the medium here was elastin and not collagen or any of the other components of the BBB matrix, it is reasonable to anticipate that the effects would have been similar if collagen had been tested instead. Indeed, that is what was done in an experiment studied in the next paragraph. 3) Flavonoids are potent anti-oxidants. Free flavonoids (i.e., those not bound in the matrix) will rapidly destroy free radicals before the radicals do their damage. Although this is only a secondary issue, it could still be significant. In one experiment collagen taken from calf skin was exposed to super oxidants. It was found that the oxidants readily split the collagen into peptides of a relatively small size: "by use of a system generating oxygen free radicals in vitro, we were able to cleave fibrillar preparations of acid-soluble collagen into peptides of a relatively small size... We find here that the action of superoxide is even more complete and drastic than expected." ref 6 Thus, once inflammation starts in the blood brain barrier, any oxygen free radicals produced may be assumed to compound the attack on the BBB matrix, reinforcing the breakdown process. However, the authors showed that if the collagen were first soaked in anthocyanosides, allowing them to bind with the collagen, and next the collagen were rinsed so that there were no free anthocyanosides available, that the oxygen free radicals still had decreased ability to dissolve the collagen: "It was found that the anthocyanoside-treated fibrils do not contain any more free anthocyanoside and that the bound anthocyanosides do not exert a scavenging effect.... When the collagen fibrils have been pretreated by anthocyanosides prior to the exposure of the superoxide ion, the amount of solubilized peptides decreases..." Table 3 of the article discloses the degree of protection offered by various anthocyanosides and in various concentrations. Thus, the protection offered the collagen by pretreatment was due to action by anthocyanosides which had become bound to the matrix and were not simply floating freely. This confirms the process in step 2 above as well as showing the importance of anti-oxidants such as the flavonoids in bolstering the integrity of the collagen before it was attacked. Again, this is what we would like to accomplish in our efforts to prevent or reduce the number of new MS lesions by flavonoid treatment. It should also be pointed out that efforts to test the effects of super-oxide ions on the collagen but in the presence of free anthocyanosides were fruitless. The anthocyanosides neutralized the free radicals so quickly that the radicals never had any effect on the collagen: "It was not worthwhile to incubate collagen directly with both the superoxide ion generating system and the anthocyanoside preparations because the latter alone were found to directly inhibit the super-oxide forming system (Table 4)." An extremely interesting experiment was performed with mice. Although MS does not naturally occur except with human beings, there is a disease called Experimental Autoimmune Encephalomyelitus (EAE) which behaves like MS. This diseased is induced experimentally in an animal such as a rat, mouse, or rabbit by artificially sensitizing the animal to proteins such as are found in its central nervous system. Once sensitized, the disease will progress in a fashion very similar to the naturally occurring human disease MS. In the experiment of interest, EAE was induced in mice. The progress of the disease was stopped by treating the mice with a particular drug. The following quotes are taken from the journal article recounting the experiment: |
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