Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/12—Ketones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention generally relates to antioxidants which are biomedically applicable. More particularly, the present invention relates to the oxy ⁇ radical scavenging properties of purpurogallin and purpuro ⁇ gallin glucosides.
• Oxygen free radicals such as the superoxide radical 0 2 and hydroxyl radical OH are formed by approxi ⁇ mately 5% of the oxygen in the bloodstream.
• Oxygen free radicals such as the superoxide radical 0 2 and hydroxyl radical OH are formed by approxi ⁇ mately 5% of the oxygen in the bloodstream.
• Oxygen free radicals such as the superoxide radical 0 2 and hydroxyl radical OH are formed by approxi ⁇ mately 5% of the oxygen in the bloodstream.
• Oxygen free radicals such as the superoxide radical 0 2 and hydroxyl radical OH are formed by approxi ⁇ mately 5% of the oxygen in the bloodstream.
• Such oxyradi- cals are highly toxic and can cause irreversible oxidative damage to cells and tissues.
• ischemia the surgical procedure known as ischemia
• xanthine dehydrogenase an enzyme that normally transfers electrons from purine bases to the oxidized form of nicotinamide adenine dinucleotide.
• this enzyme is rapidly and irreversibly converted to xanthine oxidase, an enzyme that generates large quantities of superoxide by transferring its electrons directly to oxygen.
• Oxygen free radicals can attack and damage important biological molecules. Within cellular membranes, OH can initiate a chain reaction known as lipid peroxida- tion, in which polyunsaturated fatty acids are broken down into water soluble products with consequent disruption of membrane integrity. Peroxidation of membranes may result in cell death due to the release of lysosomal hydrolases into the cytoplasm. Oxygen radicals can produce mutations in DNA and depolymerise hyaluronic acid and related macro molecules.
• the body has several defense mechanisms by which oxidative damage can be minimized.
• One is an enzymatic mechanism which involves superoxide dismutase, which catalyses the combination of two 0 2 free radicals with hydrogen to form hydrogen peroxide, a less toxic molecule which is eliminated by a peroxidase such as a catalase.
• Another defence mechanism is provided by natural ntioxi- dants such as vitamin E (tocopherol) with the hy; ophobic core of cell membranes, and glutathione and ascorbic acid in the cell water.
• Such antioxidants are adequate to detoxify most of the superoxide normally produced within the cell. However they cannot cope with the vastly increased superoxide production which occurs when oxygen is reintroduced into a tissue after a period of ischemia.
• Substances which have previously been proposed for use as free radical scavengers to reduce ischemia- reperfusion damage included allopurinol, ascorbic acid, dl- tocopherol, vitamin E and 6-hydroxy-2,5,7,8-tetramethyl- chroman-2-carboxylic acid (Trolox) - see for example U.S. patent 4,877,810. Scott et al., J. Am. Oil. Che . Soc. 11:200-203 (1974) report the original design of Trolox as an antioxidant for preserving food and fats.
• Vitamin E has generally been considered the best, known, natural antioxidant. However, it is not a particularly satisfactory therapeutic antioxidant, especially under emergency conditions, because it is extremely lipophilic and is taken up by cells only slowly.
• Purpurogallin is a known compound, described at entry number 7963 of the Merck Index, 11th Edition. Chemically, it is 2,3,4,6-tetrahydroxy-5H-benzocycl, tepten- 5-one, of chemical structure:
• It can be prepared by oxidation of pyrogallol. Its stan ⁇ dard uses are as an additive to edible or inedible fats or oils, hydrocarbon fuels or lubricants, to retard oxidation or metal contamination.
• U.S. Patent 4-181,545 Anderson, describes the use of purpurogallin and other hydroxy-substituted aromatic compounds as additives in curing systems for polymeric binders for rubber-based solid propellants. They are reported to accelerate the curing action and to complex with metal ions which will otherwise promote oxidative degradation of uncured and cured polymeric systems.
• the present invention is based upon the discovery that purpurogallin and its glucosides are unexpectedly useful and efficient as antioxidant and cytoprotective agents in biomedical applications.
• the efficiency of purpurogallin in such applications is greater than that of previously used antioxidants such as Trolox and ascorbic acid. It is particularly useful in treating a patient's blood following ischemia, to reduce the damage caused by oxidative free radicals on tissues and organs following reperfusion thereof with blood after ischemia
• Purpuro ⁇ gallin is non-toxic and is persistent, lasting in the bloodstream naturally for several weeks.
• the present invention in one aspect provides purpurogallin and its glucosides for use as cytoprotective antioxidant agents.
• compositions useful as an antioxidant and cytoprotective agent in mammals comprising an effective amount of purpurogallin or a glucoside thereof, in association with a physiologically acceptable adjuvant therefor.
• the invention provides a method of decreasing the oxidative free radical concentra ⁇ tion in mammalian blood, which comprises treating the mammalian blood in vivo with an effective amount of pur ⁇ purogallin or a glucoside thereof.
• FIGURE 1 is a graphical presentation of the results obtained according to Example 1 below;
• FIGURE 2 is a graphical presentation of the results obtained according to Example 2 below.
• the preferred process of the present invention is the use of purpurogallin as an antioxidant to reduce organ ischemia - reperfusion injury.
• an effective amount of the purpurogallin composition in a suitable physiologically acceptable carrier, in liquid form, is injected into the patient's blood immediately prior to reperfusion of the organ following ischemia, and at a location adjacent to the organ to be reperfused. If such injection takes place adjacent to the organ to be reperfused, lesser amounts of purpurogallin are necessary.
• Beneficial results can also be obtained by a general injection into the bloodstream of the purpurogallin, at any convenient location, but this is wasteful, and larger quantities of purpurogallin are then necessary. Sometimes, however, in the case of injured patients, injection at other locations is inevitable. Oral administration with a suitable carrier is also possible.
• Suitable physiologically acceptable carriers for use with purpurogallin in the present invention include water and saline solution, preferably isotonic saline solution, or any commonly used cardioplegic solution, for ready mixing and compatibility with the blood.
• Most preferred as the carrier for an injectable purpurogallin solution for administration to a patient is a sample of the patient's own blood, or blood of the patient's type. Such is normally available at the site of the ischemia-involving surgery. It provides ideally biocompatible medium for the patient.
• the quantities of purpurogallin to be adminis ⁇ tered vary based upon the body weight and blood capacity of the patient. In general, it is preferred to provide a patient with from about 0.3 mg - 15 mg of purpurogallin per kilogram body weight of the patient, preferably about 0.5 - 10 mg per kg. For a human adult patient of normal body weight and blood capacity, an amount from about 3 mg - 100 mg of the purpurogallin is suitable. Appropriate adjust ⁇ ments can be made to these quantities in proportion to a patient's weight, when administering to children, animals, etc.
• the concentration of purpurogallin in the sol ⁇ ution to be administered is not critical, and can readily be devised by the administrator. Dilute solutions are usually preferred. It is preferred that the purpurogallin solution be administered to the patient slowly, e.g. over a 10 - 20 minute period, so that a dilute solution is more easily administered under such conditions. Solutions of concentration 0.1 - 10 mM, preferably 0.2 - 5mM, are suitable. The patient's condition and vital signs should be monitored as the solution is administered, and the rate of administration adjusted if necessary.
• Freshly obtained human erythrocytes were washed at least three times in saline with centrifugation at 1500 g x 10 min. Then, a 20% suspension of RBC was prepared in the phosphate buffered saline (pH 7.4). (Miki et al.. Arch. Biochem. Biophys. 258: 373-380, 1987).
• the free radicals were generated by thermal activation of the azo-initiator 2,2'-azo-bis(2-amidinop- ropane)HCl(AAPH) .
• the reaction mixture (0.5 mL volumes) contained 10% red blood cell (RBC) suspension, AAPH (100 mM final level) and various levels of purpurogallin in 10 mM phosphate buffered saline.
• the above mixture was incubated at 37°C for 180 in, while shaking gently.
• The, 35 ⁇ l aliquot of reaction mixture was taken out, diluted in 1.5 mL of saline, and centrifuged (1500 g for 10 min). The absorption of the supernatant at 525 mM was read against a PBA blank.
• the reaction mixture was treated with 1.5 mL of distilled water with sonication for 2 min to obtain complete hemolysis. Percent hemolysis was calcu ⁇ lated according to Miki et al., op. cit.
• Figure 1 which is a graphical presentation of these results, shows that increasing concentrations of antioxidants (purpurogallin and Trolox) raised the percen ⁇ tage inhibition of red cell lysis.
• An important index for characterizing the antioxidant efficacy of a cytoprotective agent is the concentration of the agent that elicits 50% inhibition of cells lysis (IC so ).
• IC so concentration of the agent that elicits 50% inhibition of cells lysis
• Trolox is about 0.74 mM.
• purpurogallin can protect 50% of the cells from lysis at a concentration only one-sixth of that required with Trolox, so that purpurogallin can be said to be six times as effective as Trolox.
• Hepatocytes were prepared from male Sprague- Dawley rats (400 g - 450 g) according the method of Princen et al.. J. Clin. Invest.
• Free radical studies were done by removing the cell medium and adding to the cells 3 mL of 0.05 M sodium phosphate-buffered saline (PBS) (pH 7.4) containing 66.7 IU/L of xanthine oxidase (XOD) and 2 mM hypoxanthine, the latter two being used to generate oxyradicals.
• PBS sodium phosphate-buffered saline
• XOD xanthine oxidase
• hypoxanthine 2 mM hypoxanthine
• Purpurogallin solutions were made up with PBS that had been degassed for at least 30 min. Since purpurogallin is partly water-soluble, sol ⁇ utions with 4 mM level of purpurogallin was prepared with brief sonication. The pH was adjusted to 7.40 ⁇ 0.05 after almost complete dissolution of the solid purpurogallin.
• the controls included PBS containing either cells alone, or PBS with either XOD or hypoxanthine incubated with cells.
• Fig. 2 The results are shown graphically in Fig. 2.
• the vertical axis represents the time, in minutes, taken to necrose 100,000 hepatocytes
• the horizontal axis represents the concentration, in mM, of purpurogallin used in the various tests.
• Mean values of several experiments at the same level are shown, the statistical analysis having been done using the Student's t test.
• the data are expressed as means ⁇ SD (standard deviation). In comparisons with the control, statistical significance of the data was indicated by a p value ⁇ 0.05.
• Figure 2 illustrates the dose-dependent effect of purpurogallin on rat hepatocytes.
• the increasing concen ⁇ trations of purpurogallin prolonged the time (min) taken to necrose 100,000 hepatocytes that were exposed to oxyradi- cals.
• the best cytoprotective effect given by purpuro ⁇ gallin was at the level of 3mM, which prolonged the time taken to necrose hepatocytes up to 30 min.
• the control i.e. hepatocytes exposed to hypoxanthine and XOD without additive
• Rat kidney mesangial cells were isolated and tested with purpurogallin versus Trolox over the same range of concentrations, for protection against oxyradical damage using methods and procedures described by Wu et al., op cit. Clearly, purpurogallin protects the kidney cells substantially better than Trolox over all the levels examined. The results are given below in Table III.
• the model involves the occlusion of blood vessels supplying to the left lateral, median, and Spigelian lobes of the rate liver by clamping the left portal vein, left hepatic artery and left bile duct.
• the right portal vein, hepatic artery and bile duct were left intact as an interal shunt.
• the occlusion was done in Sprague-Dawley rats (0.3 - 0.4 kg) which had fasted overnight.
• a p value ⁇ 0.05 is statistically significant.
• New Zealand white rabbits (3.0 - 3.5 kg by weight) were anaesthetized with intramuscular injection of Keta ine (35 g/kg) and Atravet (0.4 rag/kg), and intra ⁇ venous injection of atropine (0.1 mg/kg).
• anaesthesia was maintained by performing tracheotomy and ventilating the animal with positive pressure respiration using a Harvard small animal respirator and a gas mixture of 2.5% ethrane (or enflurane) in oxygen. ' A midline sternotomy was done. The pericardium was opened and the heart was exposed. The main branch of the anterior ventricular coronary artery that supplies blood to a great
• part of the left ventricle and apex in rabbits (also referred to as the left circumflex coronary artery) was temporarily ligated with a 5-0 silk thread for 1 hour at the site between 1/2 to 2/3 from apex to the atrio- ventricular groove.
• a 30-ml bolus of 1 mM test solution was injected through the right external jugular vein.
• a 30-mL bolus of normal saline was given instead of the test solution.
• a 3-hour reperfusion followed. After this period, the heart was harvested, stained for enzyme activity with a tetrazolium dye and the areas of necrosis determined by planimetry.
• the original ligature in the heart was tightened, and an 22G angiocath was inserted into the aorta for injection of a 30-mL bolus of Evans' Blue solution.
• the heart was then sliced transversely into 2 mm thick slices and stained with 1.25% nitro red tetrazolium dye for 30 min.
• the negative nitro- red tetrazolium staining pattern on each slice was traced on a transparent acetate sheet for calculating the necrotic area by computerized planimetry.
• the 75% mean organ salvage achieved with the relatively low dosage here is statistically and clinically highly significant. This may be the highest myocardial salvage demonstrated with a simple compound under the conditions of this heart model.
• a diglucoside of purpurogallin (C 23 H 28 0 18 ) was used according to the present invention in methods and pro ⁇ cedures as described above, to protect erythrocytes, hepatocytes and myocytes, as well as in the liver and heart models as described above. It performed with similar effectiveness to purpurogallin itself. By high performance liquid chromatography, another (presumably mono-) glucoside was isolated and tested. This compound also performed similarly to purpurogallin. Thus, either purpurogallin or its mono- and diglucosides are superior cytoprotectants, both in cells and in vivo.

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• 1992
  • 1992-05-15 WO PCT/CA1992/000204 patent/WO1992020332A1/en not_active Application Discontinuation
  • 1992-05-15 CA CA002103040A patent/CA2103040A1/en not_active Abandoned
  • 1992-05-15 EP EP92909999A patent/EP0584148A1/en not_active Withdrawn
  • 1992-05-15 HU HU9303246A patent/HUT66375A/hu unknown
  • 1992-05-15 JP JP4509614A patent/JPH06507610A/ja active Pending
  • 1992-05-15 AU AU17455/92A patent/AU1745592A/en not_active Abandoned

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