DE3715990C2 - Use of coumarin and 7-hydroxy-coumarin - Google Patents

Description

The invention relates to the use of coumarin and / or 7-hydroxy-coumarin for the prevention and treatment of oxidative Blood vessel damage and prevention necrotization of tissues or blood vessels.

Numerous metabolic processes depend on the Redox function of enzymes or their cofactors, Substrates or coenzymes. On the other hand grab Medicines, poisons and xenobiotics in these processes and change the redox layer. Unless at these processes also the oxygen is involved, arise frequently Situations that are called "oxygen stress" summarizes.

In the cellular process of aerobic "normal metabolism" is the process of oxygen activation and the Oxygen detoxification controlled precisely. Oxygen activating and deactivating systems are finely tuned. Come this balanced System by external interference in imbalance, so can arise pathological situations, starting from a barely measurable ORP shift to the Tissue death (necrosis). The body's antioxidants, like z. As the ascorbic acid, tocopherol or glutathione must therefore constantly fed to the aerobic organism or in regenerated continuously in a cyclic process become. However, there are also reactions in which the regeneration of a substrate of such processes not is possible. In these cases, the respective substrate be replaced. Within the scope of all possibilities oxidative There are of course a great variety of processes  Regeneration, repair, replacement or substitution reactions, which partly mesh or overlap.

Furthermore, it is known that the damage to blood vessels often due to oxidative processes. The DE-A-36 23 255 describes a pharmaceutical agent which the use of a xanthine Oxidaseblockers in combination with an iron chelator. This remedy can be found in further an OH radical scavenger can be added. As OH Radical scavengers are suitable in addition to those already mentioned above Tocopherol the S-methyl-alpha-ketonbutyric acid, Formate, mannitol and sorbitol.

N. B. Piller, Brit. J. Exp. Path. 56 (1), 1975, pages 83-91 describes coumarin with proteolytic effects.

N. B. Piller, Clinic. Exp. Pharmacol. Physiol. 3 (2), 1976, pages 127 to 139 describes investigations of Effect of benzopyrones, e.g. Coumarin, on endothelium. Cells.

J.R. Casley-Smith et al., Am. J. Clin. Nutr. 28 (11), 1975, pages 1242 to 1254 describe the vitamin P Effect of Benzopyrone Coumarin and Troxerutin in Rats.

K. Sekiya et al., Biochim. Biophys. Acta, 713 (1), 1982, Pages 68 to 72 describe the inhibition of epoxygenase and activation of cyclooxygenase by esculetin (6,7-dihydroxy-coumarin), this effect being the radical trapping property attributed by Exculetin.

The object of the present invention is the provision a drug for the prophylaxis and treatment of oxidative  Blood vessel damage and prevention the necrosis of tissues and blood vessels.

It has now been found that one this task by using coumarin and / or 7-hydroxy-coumarin as an active ingredient can solve.

The subject of the present invention is therefore the use of coumarin and / or 7-hydroxy-coumarin for prevention and treatment of oxidative blood vessel damage and to prevent necrosis of tissues or blood vessels.

Coumarin is a lactone of o-hydroxycinnamic acid; it comes in numerous grass and clover species as well as in woodruff and many other plants.

In the inventive use of coumarin and / or 7-Hydroxy-coumarin will be the active ingredients together with appropriate Administered carriers or excipients.  

In the use according to the invention coumarin and / or 7-hydroxy-cumane is preferably used in the concentration range of 5 × 10 ^-5 to 2.5 × 10 ^-4 molar. Concentrations outside this range may also be appropriate, depending on the mode of administration and the severity of the disease.

For administration are suitable for the invention Use the usual pharmaceutical Dosage forms, z. As ointments, solutions, dragees, Capsules, tablets, injectable or infusion solutions, oral or parenteral, e.g. Intramuscular, intraarterial or intravenously, or topically, e.g. In the form of percutaneously acting patches. Usual daily doses are between 5 and 400 mg.

For the application are z. B. sterile aqueous solutions, which contain the active ingredient provided according to the invention. These solutions may, if necessary, in a suitable Be buffered; the liquid diluent can also with sufficient saline or glucose be adjusted isotonic. The used sterile, By the way, aqueous media are slightly by themselves available methods.  

For the preparation of coumarin-containing medicines can use the usual carriers and additives become. Usual carriers are, for example, water, physiological saline, alcohols, Polyethylene glycols, glycerol esters, gelatin, Carbohydrates, such as lactose and starch, magnesium stearate, Talcum. Usual additives are, for example Preservatives, sterilants, lubricants, Wetting agents and emulsifiers, dyes, Taste Correctives and Flavorings. The selection of Carrier and additives depends on whether the preparations according to the invention enteral, parenteral or should be applied locally.

The following examples are intended to illustrate the invention in more detail explain without limiting it.

Examples 1. tablets with coumarin

For the preparation of tablets with 200 mg each Individual weight, containing 50 mg of coumarin needed you

Coumarin | 50 mg Avicel pH 102 129 mg Polyplasdone XL 20 mg magnesium stearate 1 mg

2. Capsules with coumarin to 300 mg

Coumarin | 50 mg Yellow beeswax 10 mg soy 10 mg vegetable oil 130 mg capsule shell 100 mg

3. Injection ampoules with coumarin

For the preparation of injection vials with 2 each ml of content containing 4 mg of coumarin is needed

Coumarin | 4 mg Phys. NaCl, ad 2.0 ml

4. Infusion solutions with coumarin

Coumarin | 2 mg phys. NaCl, ad 500 ml

5. Liquid oral dosage form with coumarin

Coumarin | 1 g sorbitol 10 g sodium saccharine 0.050 g Ethanol 20% (v / v), ad 100 ml Aroma essence in sufficient quantity

6. Ointment with coumarin

Coumarin | 1 g Emulgade F 17 g Cetiol V 20 g Nipasorbin 0.5 g H₂O 61.5 g

7. Transdermal system with coumarin

Coumarin | 150 mg propanediol 600 mg Polymerisationsreagenz 300 mg Silastic TDX 4-4210 1950 mg

The following experiments serve the mode of action of Coumarin and 7-hydroxy-coumarin as "antioxidants" To illustrate model reactions:

test series

Oxygen in the ground state of its atmospheric existence is very inert: since he is in the "diradical" Triplet state, it must be in order to react with atoms in the First of all, to be able to react to a singlet ground state to be activated; that requires the so-called "spin ban" the chemical reactivity. In principle, the Oxygen can be physically or chemically activated. Dye-catalyzed, light-driven processes The extremely reactive singlet oxygen can arise; reductions can convert oxygen from superoxide anion, Hydrogen peroxide and the very reactive OH radical be formed, the dismutation of the superoxide is spontaneous, but by the enzyme superoxide dismutase can be accelerated up to 10,000 times. The critical So oxygen activation step is the One electron reduction to superoxide. The formation of Peroxide is in contrast to the endergonic reduction exergonic and uncatalyzed; the others, thermodynamically very favorable reactions to the OH radical and more to water in biological systems or less uncontrolled, with the peroxide reduction transition metal catalyzed and the further reaction of the OH radical to the water with any H donors in Microseconds goes on.

The reaction of the OH radical with biomolecules is considered Key reaction of oxygen toxicity.  

1. xanthine oxidase system

The oxygen reduction under hydrogen peroxide formation can be traced quite easily in the oxygraph. The formation of H₂O₂ as a reduction product of xanthine oxidase is a popular model system for tracking oxygen activation and deactivation reactions. In Fig. 1, a scheme of this reaction is shown. The xanthine oxidase reaction forms intermediate superoxide; This can be proved by adding hydroxylamine to a buffered mixture with xanthine and xanthine oxidase and detecting the nitrite formed as a dye after the reaction with the aid of an azo coupling. Hydroxylamine nitrite formation is inhibited in a concentration-dependent manner by superoxide dismutase (SOD). The bound in the xanthine oxidase iron is formed in this reaction, not only superoxide and H₂O₂, but also on the so-called "Haber-white" reaction, the reactive OH⁻ radical. This can be detected with the aid of a methionine derivative, the S-methyl-alpha-ketobutyric acid (KMB), by gas chromatography, the fragmentation of the molecule under ethene formation.

In reperfusion of ischemic areas, this seems to be Xanthine oxidase system play a significant role. The oxygen radicals from reperfusion of the OH type or modifications thereof cause tissue necrosis.

In experiments it could be shown that coumarin does not inhibit the xanthine oxidase reaction. In contrast, however, the xanthine oxidase-driven KMB cleavage was inhibited by about 100 μM to about 35%, and by about 500 μM to about 55%. These findings indicate that coumarin in the range of about 5 x 10 ^-5 M to about 5 x 10 ^-4 M is not a xanthine oxidase blocker but appears to be a fairly reactive partner of an OH⁻-type oxidant.

2. Diaphorase system

Flavoproteins are cellular redox catalysts that ubiquitous in the living world. mitochondria, Microsomes, plasmatic membranes, leukocytes and Chloroplasts contain Flavinenzyme in their Electron transport systems. There are a lot of Electron acceptors known for such "diaphorases" couple nonspecifically and by auto-oxidation Decompose oxygen radicals. On it goes the toxicity many xenobiotics and drugs back.

If anthraquinone sulfonic acid is chosen as such an auto-oxidizable electron acceptor, it is also possible to use superoxide, peroxide and an OH⁻-type oxidant, similar to the xanthine oxidase system but via a completely different reaction pathway. The pathway of diaphorase-catalyzed oxygen reduction is shown in FIG .

Experiments have shown that coumarin has no effect on the flavin catalysis of NADH-driven oxygen uptake. In contrast, the diaphorase-catalyzed KMB cleavage with ethene release is clearly inhibited as an indicator reaction for OHat-type oxidants at 50 μM, where I₅₀ is about 5 × 10 ^-4 .

This finding is in accordance with the above demonstrated results from the xanthine oxidase system.  

3. Autooxidation of dihydroxyfumaric acid

For some time it has been known that among diabetic Conditions can autooxidize sugar, with oxidants of the OH⁻ type arise. A simplified model molecule such enolized sugar provides the dihydroxyfumaric acid The auto-oxidation of dihydroxyfumaric acid proceeds at neutral pH concentration dependent, with KBM too Ethen is split.

Typical OH scavengers, such as ethanol, formate or mannitol, inhibit the KMB cleavage.

In this chemical system coumarin behaves like a typical OH scavenger: An inhibition is already at 50 μM recognizable.

4. Iron-Catalyzed OH⁻ Formation - Perferry Oxidations

There is ample evidence that most "derailed" oxidation damage is iron or copper catalyzed, with iron playing the more significant role. Here the catalysis of the "Haber-Weiss" reaction is attributed the main role. In a xanthine oxidase-catalyzed reaction, the methionine cleavage, which indeed proceeds at far lower reaction rates than the KMB cleavage, is considerably stimulated by Fe-EDTA in concentrations of about 100 μM. The underlying reaction mechanism explains the inhibition of this sequence by both SOD and catalase, as both OH and peroxide must be available to produce OH⁻ ( Figure 3). The importance of such reactions may be estimated, bearing in mind that the side effects of many drugs may be due to similar reaction sequences.

The KMB cleavage by Fe-EDTA-H₂O₂ ("Fentons Reagent ") was in the experiment by coumarin in a Inhibited "step process". A threshold is 100 to 500 μM. An increase in inhibition occurs from 0.5 mM.

By the biological system "ascorbate iron", the sure is of high relevance, both KMB and with lower rate of methionine to ethene fragmented. Both systems are coumarin with initially inhibited steeper kinetics. Also here lets recognize a bifunctional character of the inhibitory effect.

5. Riboflavin-catalyzed, photodynamic reactions

It became riboflavin catalysis as a test system selected. Riboflavin - without further additives - in one buffered system fragmented KMB in the light (30 Klux). This reaction, which is a mixture of photodynamic Type I (charge separation) and Type II reactions (Exiton transfer to oxygen to form Singlet oxygen) is characterized by coumarin in the Concentration range of about 100 uM inhibited to 50%.

All results of the above model experiments show that coumarin is an OH⁻ radical scavenger which does not have any negative side effects in the sense shows an unspecific enzyme blockade.

Claims (1)

Use of coumarin and / or 7-hydroxy-coumarin for Prevention and treatment of oxidative blood vessel damage and to prevent necrosis of tissues or blood vessels.