JPH06507610A - Pharmaceutical compositions containing purprogalin useful as antioxidants and cytoprotective agents - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Pharmaceutical compositions containing purprogalin useful as antioxidants and cytoprotective agents The present invention generally relates to biomedically acceptable antioxidants. Learn more The present invention relates to the free atom of purpurogalin and purpurogalin glucosides. This is related to group-capturing properties.

Approximately 5% of oxygen in the bloodstream is in the form of free oxygen groups such as peroxide group 02 and hydroxyl group OH. It is formed by These free oxygen radicals are highly toxic and can cause irreversible oxidative damage to cells and tissue. For example, during organ transplantation, bypass surgery, etc. When normal blood flow to an organ or tissue is interrupted [known as ischemia] When oxygen is reintroduced into the tissue, peroxide production occurs. secondary hydroxyl groups are formed, and obvious cytotoxicity is exerted. Iskemia Xanthine dehydrogenase is the main source of excess free radicals in the process. . This usually consists of purine bases to nicotinamide, adenine, and dinucleotides. It is an enzyme that transfers electrons to the oxide of hydrogen. During oxygen deprivation, this The enzyme is rapidly and irreversibly converted to xanthine oxidase. This is the An enzyme that generates large amounts of peroxide by transferring electrons directly to oxygen. be.

Free oxygen groups can attack and damage important biomolecules. Inside the cell membrane So, OH can start a chain reaction known as fat peroxidation. can be obtained. In this reaction, highly unsaturated fatty acids are destroyed and become water-soluble n*, resulting in As a result, the membrane morphology is destroyed. *Peroxidation occurs in the cytoplasm of lysosomal hydrolase. This may lead to cell death due to release. Free oxygen groups mutate into DNA may cause depolymerization of hyaluronic acid and other macromolecules. There are times when I can do it.

Living organisms have several anti-msi defenses that minimize oxidative damage. I'm being held back. One of them is the enzyme involved in superoxide dismutase. There is an elementary mechanism that acts catalytically on the combination of 02 free radicals and hydrogen in 2fll. to form hydrogen peroxide. Hydrogen peroxide is a relatively non-toxic molecule. removed by peroxidases such as catalase. another defense The mechanism is provided by natural antioxidants such as vitamin E (1-copherol). glutathione and ascorbic acid in the cell fluid, forming a hydrophobic core in the cell membrane. It is something that causes This type of antioxidant is a compound of peroxide that normally occurs in cells. Suitable for detoxifying most of the substances. But this one after the Isgamia period The large increase in peroxide production that occurs when oxygen is reintroduced to tissues cannot be accommodated. do not have.

Therefore, especially surgeries that involve ischemia of organs such as the heart, liver, and kidneys. to prevent or reduce free oxygen radical damage that can occur with surgical procedures, such as To combat this, therapeutically effective antioxidants are needed.

This has been proposed for use as a free radical scavenger to reduce damage during ischemia-reperfusion. Substances that have been proposed so far include allopurinol, ascorbic acid, l-tocopherol, vitamin E.

and 6-hydroxy-2+ 5+ 7+ 8-tetramethylchroman-2- Carboxylic acids (Trolox) (e.g. U.S. Pat. No. 4.877) ．． 810 am book), Scott et al. J, ^-, Oil, Che-, Soc, C: 200-203 (1974) ) i! , Trolox as an antioxidant for preserving foods and fats The original design is reported.

In the pathogenesis of diseases such as arthritis, inflammation, and some cancers, and in aging. The importance of free oxygen groups has also been recognized, e.g. Cross et al. al,, ^nn, Int, Wed, 107: 526-545) , vitamin E is generally considered to be the best natural antioxidant known to date. It is. However, this has particularly satisfactory therapeutic use, especially under emergency conditions. It cannot be called an antioxidant because it is extremely lipophilic and has no effect on cells. This is because uptake is slow.

Purpurogallin ranks No. 1 in the Merck Index. This is a known compound described in entry number 7963 of the 1st edition. chemically 2, 3. having the following structure. 4. 6-tetrahydroxy-5H-benzosic Rohebuten-5-one.

It is made by oxidation of pyrogallol. Its typical uses are edible and Additive to non-edible fats and oils, hydrocarbon fuels and lubricating oils, preventing oxidation and metal contamination. It is used to prevent.

U.S. Pat. No. 4,181,545 (Anderson) discloses a rubber substrate. Pulp as an additive in vulcanization systems for polymeric binders for solid propellants. The use of logalin and other hydroxy-substituted aromatic compounds has been described. Ru. These compounds simultaneously promote the vulcanization action, and if left unvulcanized or vulcanized, It is reported that it forms a complex with metal ions that promotes the oxidative decomposition of polymeric systems. ing.

One of the objects of the invention is, inter alia, to provide a more effective method than hitherto for use in reperfusion. The objective of the present invention is to provide antioxidants that are effective and biomedically acceptable.

The present invention shows that purpurogalin and its glucosides are unexpectedly effective in biomedical applications. Due to the discovery that it is useful and effective as an antioxidant and cell protectant in the field of application. It is based on The effects of purpurogalin in these fields of use are Compared to the effects of previously used antioxidants such as lox and ascorbic acid. It's large. This thing processes the blood of patients in the Iskemia bunker and Damage caused by oxidative free radicals to tissues and organs by reperfusion of blood into them It is particularly useful in mitigating

Purpurogalin is non-toxic, stable and naturally persists in the bloodstream for several weeks.

One feature of the present invention is that Purpurogar is used as a cell protection antioxidant. and its glucosides.

Another feature of the invention is that it is used as an antioxidant and cell protectant in mammals. The object of the present invention is to provide useful compositions, which compositions contain physiologically acceptable and effective aids. containing an effective amount of purpurogalin or its glucoside in combination with .

Yet another feature of the present invention is that the concentration of oxidative free radicals in mammalian blood is This method provides a method for reducing the level of treatment with an effective amount of purpurogalin or its glucosides.

In the attached drawings, FIG. 1 shows diagrammatically the results obtained according to Example 1 below. .

FIG. 2 shows diagrammatically the results obtained according to Example 2 below. .

A preferred operating procedure of the present invention uses purpurogalin as an antioxidant to The aim is to alleviate ischemia-reperfusion injury. Appropriate for such purposes, in a physiologically acceptable carrier.

A liquid, effective amount of the purpurogalin composition is administered immediately prior to reperfusion of the ischemia organ. first, into the patient's blood at a site in close proximity to the organ to be reperfused. reperfusion If this injection is performed near the organ to be treated, the amount of purpurogalin required will be lower. Purpurogalin can be administered by regular injection into the bloodstream at any convenient location. may give the desired result, but it is wasteful and requires a larger amount of Luprogarin is required. However, in some cases, such as in the case of an injured patient, Injection is unavoidable, and oral administration with a suitable carrier is also possible. .

Physiologically acceptable carriers suitable for use with purpurogalin of the present invention include , water, saline, preferably isotonic saline, or blood. commonly used cardioplegic Contains solutions. As a carrier for injectable Purpurogalin solution for administration to patients Most preferred is a sample of the patient's own blood or blood of the same type as the patient. . These are usually available at the point of surgery involving ischemia. . This makes it an ideal biocompatible medium for patients.

The dose of purpurogalin depends on the patient's weight and blood capacity. In general, Approximately 0.3 mg-15 g per kg of patient body weight, preferably approximately 0.5-10 g 1 g, preferably for adult patients of normal weight and blood capacity. In this case, an amount of purpurogalin of about 3 mg to 100 mg is appropriate. children and children When administering to a patient, these amounts can be adjusted appropriately in proportion to the patient's weight. Wear.

The concentration of purpurogalin in the solution to be administered is not particularly important and is determined by the person administering it. It can be easily devised. Dilute solutions are generally preferred. Purpurogalin solution is preferably administered to the patient slowly, e.g. over 10-20 minutes; Therefore, under these conditions, dilute solutions are easier to administer. 0.1-101 During the administration of a solution of H1, preferably a solution with a concentration of 0.2-5 is suitable. The patient's condition and vital signs should be monitored and the rate of administration should be adjusted if necessary. cormorant.

The invention is further illustrated by the following non-limiting examples. unless otherwise noted All chemicals used were reagent grade and manufactured by Sigma Chem. ical Co, , St, Louts, No, , Ll, S, A,) Supplied. Purpurogalin and Trolox are available from Aldrich (Aldrt). Chemical Co., Ltd.). AAPII C2, 2”- Azo-bis-(2-amidinopropane) hydrochloride] is available from Polyscience Co., Ltd. Sciences Inc.).

111" In this example, experiments were conducted using human red blood cells. i.e. different amounts of pull of AAPH in the reaction mixture in the absence and presence of progalin or Trolox. The free radicals generated by thermal activation are brought into contact with red blood cells and pulled under these conditions. The cytoprotective ability of progalin was determined.

Fresh human red blood cells were centrifuged at 1500 g x 10 μl in saline. Washed at least 3 times in phosphate-buffered saline (pH 7,4) then 20 %RBC! ! A suspension was prepared. (Miki et al.

^rch, Biochem, Biophys, 258: 373-380. 1 987).

Azo initiator, 2,2'-azo-bis(2-amidinopropane) hydrochloride (^^PF The 0 reaction mixture (0.5 μL volume) in which free radicals were generated by thermal activation of I) was 101 G in phosphate saline: 10% red blood cell (RBC) suspension, AAP H (final concentration level 100-A) and various concentration levels of purbrogarin. It was. This mixture was incubated at 37°C for 180 minutes while slowly incubating. Remove 35 minutes of the reaction mixture, dilute with 1.5 mL of saline, and centrifuge. This supernatant was incubated separately (1500g, 10win) at 525mM. The absorbance was compared to the PB^ control. Similarly, distill the reaction mixture into 1.5 mL. Complete hemolysis was obtained by treatment with water and sonication for 2 minutes.

The hemolysis rate was calculated according to the aforementioned Miki et al.

Figure 1 shows the results graphically. The rate of inhibition of red blood cell lysis increases with increasing concentration of It is shown that. An important index characterizing the antioxidant efficiency of cytoprotective agents is This is the drug concentration that achieves a 50% inhibition of dissolution (IC% (1). Purpurogalin So, IC,, is at 0.12 position, and for Trolox, it is about 0.74 ■N. Ru.

That is, purpurogalin requires only one-sixth of the amount needed for Trolox. A concentration of 50% protects against cell lysis. Therefore, Purpu support 11 units In this example, experiments were conducted using rat hepatocytes. That is, various concentration levels Free radicals were applied to hepatocytes in the presence and absence of Bell's purpurogalin.

Hepatocytes were prepared according to Princen et al. Invest.

78: 10B4-1071 (1986)). Dori type rats (400g - 450g) were prepared. The flow is first 250 sL f) 0.5 sM EGTA and 142 mN 10 MM Hepes Slow Positive Solution (pH 7.4) containing NaCl Toro, 7 mN KCI ) at 20 sL/mln.

gravity dropping at a rate of l.

Re@, 82: 391-398 (1973)) G: Jutte, 5 sM 100μL of magnesium-depleted water containing CaC1z and O,OS% collagenase It was performed using Hanks' balanced salt solution. The details of the metal have been previously reported in the literature. As stated in (wu et al,, Biochem, Ce1lBio1.68.1189-1194. (1990)).

For free radical studies, the cell media was removed and the cells were exposed to 66.7 IU/L of oxidase <<XOO>> and 2 sM of hypoxanthine (these two 3 mL of 0.05 N phosphate soda containing Buffered saline (PBS) (pH 7.4) was added. Each concentration of purpurogalin The criterion for comparing the effects in level was 105 cells of the same generation in each culture dish. This was the time required to cause necrosis (necrosis) in 95% of the cells. Antioxidant If present, add it to cells along with xOD and hypoxanthine and evaluate A blind study was conducted on 3 groups randomly selected by a person.

Purpurogalin solution was prepared using PBS that had been degassed for at least 30 minutes beforehand. It was prepared using Purpurogalin is partially water soluble, so the 4 sM soaking intensity level Lupurprogalin solution was prepared by brief sonication. solid pull pro Once galin was almost completely dissolved, the pH was adjusted to 7.40±0.05.

The control group is PBS containing cells only, PBS and XOD, or PBS and hypoxanthine cultured together with cells.

The results are shown schematically in Figure 2. In Figure 2, the vertical axis represents 100,000 hepatocytes. The time required for necrosis to occur is expressed in minutes. The horizontal axis is The concentration of purpurogalin used in the experiment is expressed in IM. How many of the same concentration level The average value of the experiment was shown, and statistical analysis was performed using the Student's t-test. It is a bond.

Data were expressed as SD (standard deviation) above the mean. As a comparison with the control, the data Statistical significance was indicated by a P value < 0.05.

Figure 2 shows the dose-dependent effect of purpurogalin on rat hepatocytes. As the concentration of brogalin increases, the liver tissue exposed to free oxygen groups tm too, oo The time (minutes) required for om to undergo necrosis is prolonged. Purpurogalin The best cytoprotective effect is obtained at the 3-M concentration level; At certain concentration levels, the time required for liver cells to undergo necrosis can be reduced to 30 minutes. Extended. Control group (i.e. exposed to hypoxanthine and XOD without additives) In approximately 6 minutes, 100,000 cells showed clear necrosis. occured.

For further investigation, rat hepatocytes were cultured, and Wu et al. (mentioned above) and C. l1n, Ches+, 36: 1172-1173 (abstract), 199 Release using purpurogalin and Trolox as additives according to the procedure of The damage caused by oxygen groups was tested and the results shown in the following table were obtained.

2nd quantity free oxygen base layer 105 rat hepatocytes In control experiments without additives, cells showed approximately 6.5 Survived for a minute. This means that at equivalent concentrations, purpurogalin is derived from vitamin E. Trolox, a potent anti-oxidant, is more effective against rat hepatocytes. This shows that it has an excellent protective effect on crabs.

Example 3 Human ventricular myocytes were removed and subjected to the methods and procedures described in Wu et al. (cited above). The protective action of purpurogalin and Trolox against free oxygen radical damage was determined using the following procedure. The use was tested. The results are shown in Table 1I below.

No. L Todoroki after free oxygen radical damage 1 (Is habitual muscle cells In control experiments without additives, cells survived for 2 minutes of exposure to free oxygen radicals. .

Each result shown in Table 1 is the average of 5-7 repeated experiments with closely aligned conditions. It is. This result shows that purpurogalin, especially at concentration levels above 0°5- In this study, the protective effect on muscle cells was at least an order of magnitude better than Trolox. Which indicates that.

1151 Rat kidney vascular membrane cells were removed and treated using the method and procedure described in Wu et al. (cited above). , regarding the protective effect against free oxygen radical damage, purpurogar in the same concentration range A phosphorus vs. Trolox test was conducted. across all concentration levels tested. Luprogalin showed substantially better renal cytoprotection than Trolox.

The results are shown in table m below. LL true XOD (66,7IU/L) and Hypoxanthine (0.5mW) exposed to 0 (control) 5.7 5.7 0.13 11.5 6.4 G, 25 13.4 6.8 0.50 16.0? , 4 1.00 19.4 7.8 E1 Wataru1 An in-vivo study was conducted on rat liver, and the pull-up during partial liver ischemia-reperfusion was The cytoprotective effect of progalin was determined.

Wu et al. (wu et al., Hepatology 13: 5) 5- 580 (1991)) was used. the main points In other words, the model was created by placing the left portal vein, left hepatic artery, and left bile duct with forceps. Closing off the blood vessels that supply blood to the left side of the liver, the middle lobe, and the collection of Subigerius. Consisting of The right portal vein, hepatic artery, and bile duct were kept naturally as internal shunt flow. − Sprague-Dawley rats (0.3-0.4 kg) that had been deprived of night food were used to test blood. Tube closure was performed. Approximately 45 seconds before Iskemia bond completion, 3-L saline solution (control), or containing purpurogalin or trolox (at the concentration levels indicated below) Saline was injected into the pedicle vessels over a period of 3 minutes. This was followed by 24 hours of reperfusion. 0 Next, the degree of necrosis (based on liver wet weight) is determined by the currently widely accepted histochemical methods (Fred-Eriks et al., Exptl. Mo1. Path, 41: 119-125.1984) Analyzed.

The results are shown in Table ■ below. When the number of oles increased from 4.4 to 18μoles per kg of body weight, liver necrosis It can be seen that the degree of cis is significantly reduced. This is an injectable Purpurogalin administration It is also shown that organ recovery increases markedly as the dose increases. All of Organ recovery was statistically significant relative to the control at the concentration levels used. It is noted that all effective doses are in μ-ole units per kg body weight. .

1ヱ1 Effect of purpurogalin on rats subjected to partial ischemia-reperfusion Control Purpurogalin Salt water only 4.4 8.8 18 Injection dose (no antioxidant) (μmoles/kg body weight) Average necrosis ( Weight%＞30.3 17.7 7.3 4.4±SD 11.9 4.6 4 ．． 9 1.1 Liver recovery % O427686 Number of rat population 6 6 6 6 P bond vs. control 0.05 G, 005 0.001 P aspiration < O, OS is statistically significant It is meaningful.

1151 In this example, we demonstrated the in vivo effects of purpurogalin during cardiac ischemia-reperfusion in rabbits. The cytoprotective effect was tested.

New Zealand white rabbits (body weight 3.0-3.5 kg) were treated with ketamine (35-g /kg) and atravet (0.4% g/kg), and atravet (0.4% g/kg); The animals were anesthetized by intravenous injection of phthalate (0.1% g/kg).

The frontal region of the animal was pricked, the tracheotomy was performed, and the small animal respiratory tract and acid 2.5% ethrane (or enflurane) ventilate the animal by positive pressure breathing using a gas mixture containing anesthesia was maintained. A midline sternal incision was made. 6 membranes were opened to expose the heart.

Main division of the anterior ventricular coronary artery that supplies blood to the left ventricle and most of the apex of the heart (left The circumflex coronary artery (also called the circumflex coronary artery) is located between 172 and 2/3 of the way from the apex of the heart to the atrioventricular groove. , and was temporarily ligated using 5-0 silk for 1 hour. Approximately 1 minute before releasing the closure, press the right 30-ml of 1mM test solution was injected at once through the external jugular vein. In control animals, Instead of the test solution, 30-1 ml of normal saline was given at once. In either case, Reperfusion was then performed for 3 hours.

The moat and heart were collected and stained with tetrazolium dye to examine enzyme activity. The extent of necrosis was determined by planimetry.

To determine the risk area, the primary ligation of the heart is strengthened and a 22G angiocarcinoma is placed in the aorta. Insert the angiocath and pour 30 mL of Evans Blue solution. The heart was sliced transversely into 21-thick slices and treated with 1.25% nitro. Stained with red tetrazolium for 30 minutes. Nitro Red Tetrazolium Non The dye bales were traced onto transparent acetate paper and determined by computerized planimetry. The area of necrotic field was calculated.

The results are shown in Table V below.

1 brother 1 Purpurogalin Control saline solution (8,6-10u moles/kgL sl, 11u l) in the field of risk organ necrosis Average % (±SD) 46.6± 10 11.8± 6.2 Average organ recovery rate %　075 Rabbit population: 66 P value vs. control <0.001 The 75% average organ recovery vehicle achieved here with relatively low doses is consistent with the This is extremely meaningful both quantitatively and clinically. This is the condition of this heart model. This may be the best myocardial recovery demonstrated using a single compound in this case.

11■Yu According to the present invention, the diglucoside of purpurogalin ( C23H2801@), red blood cells, hepatocytes, and muscle cells, as well as the aforementioned Protective effects were confirmed in liver and heart models. This one is purbro It showed similar effects as galin itself. Separate by high performance liquid chromatography The glucoside (probably mono) was isolated and tested. This compound also produces plump It showed similar behavior to Rogalin. In this way, purpurogalin, its monoglyceride Both lucosides and diglucosides are excellent cytoprotective agents in cells and in vivo. Become.

Interdiction rate [〃] Tomisaroen adventure international search report Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), 0A (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG. ), AU, BB, BG, BR, CA, C3, FI, GB, HU.

JP, KP, KR, LK, MG, MN, MW, No, PL, R O, R.U., S.D.

Claims (12)

[Claims] 1. an effective amount of purpurogalin or A mammalian antioxidant and cell preserver characterized by containing the glucoside. Compositions useful as protective agents. 2. A composition according to claim 1, wherein the active ingredient is purpurogalin. 3. 3. The composition of claim 2, wherein the adjuvant is an aqueous liquid. 4. The adjuvant is water, 4. The composition of claim 3, which is physiological saline or whole blood. 5. Treating mammalian blood in vivo with an effective amount of the composition of claim 1. A method for reducing the concentration of oxidative free radicals in the blood of mammals, characterized by Law. 6. Treating mammalian blood in vivo with an effective amount of the composition of claim 4. A method for reducing the concentration of oxidative free radicals in the blood of mammals, characterized by Law. 7. Adding an effective amount of the composition according to claim 1 to blood in vivo. during reperfusion of mammalian organs after ischemia. How to reduce tissue damage caused by 8. Adding an effective amount of the composition according to claim 4 to blood in vivo. during reperfusion of mammalian organs after ischemia. How to reduce tissue damage caused by 9. Purpurogalin is administered in an amount of about 0.3-15 mg per body weight of the mammal. 5. The method of claim 4. 10. Purpurogalin is administered in an amount of about 0.5-10 mg per body weight of the mammal. 6. The method of claim 5. 11. an effective amount of purprogalin or a glucoside thereof that is physiologically acceptable; acid in mammalian blood by treating blood in vivo with a composition containing Method of using purpurogalin or its glucosides to reduce the concentration of oxidizing free radicals . 12. In order to reduce the concentration of oxidative free radicals in mammalian blood, to prepare physiologically acceptable compositions for processing the blood within the How to use Purpurogalin.