Link:  Pharm/Biotech Resources

Title:  Synergistic composition of trans-tetracos-15-enoic acid and Apocynin and method of treatment for hepatotoxicity

United States Patent:  6,949,586

Issued:  September 27, 2005

Inventors:  Handa; Sukhdev Swami (Jammu, IN); Suri; Om Parkash (Jammu, IN); Gupta; Vishwa Nath (Jammu, IN); Suri; Krishan Avtar (Jammu, IN); Satti; Naresh Kumar (Jammu, IN); Bhardwaj; Vikram (Jammu, IN); Singh; Bupinder (Jammu, IN); Chandan; Bal Krishan (Jammu, IN)

Assignee:  Council of Scientific Research (IN)

Appl. No.:  102147

Filed:  March 20, 2002

The present invention relates to a synergistic hepatoprotective pharmaceutical composition comprising an effective amount of trans-tetracos-15-enoic acid (TCA) and Apocynin (APO), the present invention also relates to a method of treatment for hepatotoxicity in mammals and humans.

SUMMARY OF THE INVENTION

The present invention relates to a synergistic hepatoprotective composition containing trans-tetracos-15-enoic acid (TCA) and Apocynin (APO). The present invention also relates to a method of treatment for hepatotoxicity in mammals and humans.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a synergistic pharmaceutical composition having enhanced hepatoprotective activity on subjects, obtained from the plant Indigofera tinctoria, said composition comprising an effective amount of:

Still another embodiment of the present invention, wherein said method is used to treat liver disorders caused by Galactosamine, Paracetamol and Carbon tetrachloride.

Yet another embodiment of the present invention, a method wherein the dosage for the treatment of CCl₄ induced hepatotoxicity in mammals is about 50-mg/kg-body weight.

Still another embodiment of the present invention, a method wherein the enhanced hepatoprotective activity in CCl₄ induced hepatotoxic mammals is up to 92%.

Yet another embodiment of the present invention, a method wherein the dosage for the treatment of acetaminophen induced hepatotoxicity in mammals is 50 mg/kg-body weight.

Still another embodiment of the present invention, a method wherein the enhanced hepatoprotective activity in acetaminophen induced hepatotoxicity in mammals is up to 86%.

Yet another embodiment of the present invention, a method wherein the dosage for the treatment of Galactosamine induced hepatotoxicity in mammals is 50 mg/kg of body weight.

Still another embodiment of the present invention, a method wherein the enhanced hepatoprotective activity in Galactosamine induced hepatotoxicity in mammals is up to 75%.

Yet another embodiment of the present invention, a method wherein the dosage for chloretic activity in mammals to control the bile flow and bile solids is 50 mg/kg of body weight.

Still another embodiment of the present invention, a method wherein the enhanced chloretic activity in mammals is up to 39%.

Yet another embodiment of the present invention, a method wherein the composition is used either singly or in combination with pharmaceutically acceptable carriers.

Still another embodiment of the present invention, a method wherein the composition is administered to a subject in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binder or stabilizers.

Yet another embodiment of the present invention, a method wherein the desired dosage is administered for both preventive and curative properties.

Still another embodiment of the present invention, a method wherein the composition is administered systemically, orally or by any clinically/medically accepted methods.

Yet another embodiment of the present invention, a method wherein the subject is selected from animals, mammals, and preferably humans.

Still another embodiment of the present invention, a method wherein the preferred dosage for hepatic disorders in human beings is about 10-15 mg/kg of body weight.

The invention is further explained in the form of preferred embodiments.

i. Animals:

The pharmacological studies are conducted on Wistar albino rats (150-180 g) and Swiss albino mice (25-30 g) of either sex, colony—bred in the Institute's animal house. After procurement, all the animals are divided into different groups and are left for one week for acclimatization to experimentation room and are maintained on standard conditions (23±2° C., 60-70% relative humidity and 12 h photo period). The animals are fed with standard rodents pellet diet and water ad libitum. There are six animals in each group except for general behaviour and acute toxicity studies where ten animals are used in each group.

ii. Hepatotoxins:

It is emphasized that hepatotoxin that causes acute hepatitis should have close resemblance with the viral hepatitis, clinically, biochemically and histologically. Drugs are also causes of chronic hepatic disease as chronic hepatitis, fatty liver, cirrhosis and several vascular lesions of the liver. In many instances drug induced hepatitis proves indistinguishable from viral hepatitis. Chemically induced hepatic injury for experimental studies should be severe enough to cause cell death or to modify hepatic functions. The mechanism of acute hepatic injury depends upon the chemical compound and the species of animals used. Many chemicals produce parenchymal damage, arrest bile flow and cause jaundice (chloretic injury). Hepatoprotective activity against CCl₄, paracetamol and, D-galactosamine induced hepatotoxicity are studied.

Carbon Tetrachloride (CCl₄): CCl₄ is one of the most powerful hepatotoxins (in term of severity of injury). It causes toxic necrosis, which leads to biochemical changes having clinical features similar to those of acute viral hepatitis (Vogel, 1977, Bramanti et. al., 1978, Kumar et. al., 1992). Liver injury is produced by administration of CCl₄ mixed with liquid paraffin. Animals are given single dose of CCl₄ (50 μl.kg^-1, p.o.) in acute single treatment and (0.5 ml.kg^-1, p.o.) in case of multitreatment with drug. It is administered orally (p.o) by gastric intubation. The control animals received the equal volume of liquid paraffin.

Paracetamol (APAP, Acetaminophen): It is a therapeutic agent widely used as analgesic/antipyretic drug. When taken in large doses it causes hepatic necrosis which leads to biochemical changes having clinical features similar to those of acute viral hepatitis in humans (Proudfoot and Wright, 1970). The similar effect is observed in animals. The toxic effect can be potentiated if it is given several hours after the anesthetic ether inhalation (Wells et. al., 1986).

Liver injury is induced by injecting paracetamol (200 mg.kg^-1) interaperitoneally in normal saline (pH 9.4) six hour after inhalation of anesthetic ether (4 ml/4 min/6 animals) in a closed chamber. The control animals received the equal volume of vehicle.

D-Galactosamine: It is one of the toxins that induce hepatic inflammatory conditions in the rat liver that clinically resembles to viral hepatitis. The mechanism of GalN induced liver injury has been extensively examined and this model is now accepted as one of the authentic systems of liver damage (Bauer et. al., 1974, A1-Tuwaijiri et. al., 1981)

Hepatic damage is produced by injecting GalN (300 mg.kg^-1) subcutaneously in normal saline. The control animals received the equal volume of vehicle.

iii. Treatment with Bio-Active Compound and Silymarin:

Freshly prepared suspension (1%, w/v) in 0.2% gum acacia in normal saline is used for all the experiments except for toxicity studies where (10%, w/v) suspension is used. Silymarin suspension (1%, w/v) in 0.2% gum acacia is used as a reference standard (positive control).

iv. Experimental Models:

Effect on Serum and Hepatic Biochemical Parameters:

CCl₄ Induced Hepatotoxicity:

Treatment of Test Material Before and After Hepatotoxin: The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of rats at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (CCl₄, 0.5 ml.kg^-1, p.o.) intoxication. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations. All the animals are then killed by decapitation, their livers are quickly excised, cleaned of adhering tissue, weighed and homogenised in phosphate buffer saline for the analysis of hepatic parameters (Agarwal and Mehendale, 1983, Klingensmith and Mehendale, 1982, Zimmerman, 1973, Edmondson and Peter, 1985, Mitchell, et al, 1973).

Paracetamol Induced Hepatotoxicity:

Treatment of Test Material Before and After Hepatotoxin:

The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 72 hours, 48 hours and 24 hours, 1 hour before diethyl ether inhalation and 1 hour after hepatotoxin (paracetamol, 200 mg.kg^-1, i.p.) given 6 hours after exposure to diethyl- ether. Blood is collected from orbital sinus in all the animals 18 hours after last treatment and serum separated for different estimations. A portion of the liver is processed for histopathological studies.

D-Galactosamine Induced Hepatotoxicity:

(a) Treatment of Test Material before and after Hepatotoxin:

The doses of TCA and APO individually and in mixture, silymarin (50 mg/kg, p.o. each) and vehicle (normal saline) are fed to different groups of mice at 48 hours, 24 hours and 2 hours before and 6 hours after hepatotoxin (GalN, 300 mg.kg^-1, s.c.) intoxication. Blood is collected from orbital sinus in all the animals 18 h after last treatment and serum separated for different estimations. All the animals are then killed by decapitation, their livers are quickly excised, cleaned of adhering tissue, weighed and homogenised in phosphate buffer saline for the analysis of hepatic parameters. A portion of the liver is processed for histopathological studies.

Parameters Studied:

GPT and GOT: Pyruvate formed by transamination reaction is determined spectrophotometrically after reaction with 2,4-dinitrophenylhydrazine (Reitman and Frankel, 1957).

ALP: p-nitrophenol formed in alkaline medium is measured spectrophotometrically using p-nitrophenyl phosphate as substrate (Walter and Schutt, 1974).

Bilirubin : Total bilirubin is measured by diazotization reaction with NaNO₂ (Malloy and Evelyn, 1937)

Triglycerides: Triglycerides from serum are extracted with isopropanol and sopanified with KOH. The liberated glycerol is converted to formaldehyde by periodate and determined after reaction with acetyl acetone. Triolein is used as standard (Neri and Firings, 1973).

Glutathione: It is determined after deproteination by reaction with DTNB (Ellman 1959 as modified by David 1987).

Lipid peroxidation: Thiobarbituric acid reacting substances are determined spectrophotometrically at 535 nm. Buege. and Aust. (1978).

Hepatoprotective Activity:

Hepatoprotective activity (H) is calculated by the following equation:


Where TC, VC, and V are drug+toxin, vehicle+toxin and vehicle treated groups of animals respectively.

Human Dose:

Doses for human being can be calculated by equivalent surface area doses conversion factor (equivalency on the basis of mg/sq. m)

Effect on Bile Flow and Bile Solids

The liver, by producing bile, plays an important role in digestion. The presence of bile in the intestine is necessary to accomplish the digestion and absorption of fats as well as absorption of the fat-soluble vitamins—A, D, E & K. Bile is also an important vehicle of excretion. It removes many drugs, toxins, bile pigments and various inorganic substances either derived from the diet or synthesized by the body as cholesterol or as cholic acid. Increase in the bile flow is suggestive of stimulating action of liver microsomal enzymes.
 

Claim 1 of 36 Claims

1. A pharmaceutical composition having hepatoprotective activity on subjects, said composition comprising an effective amount of:

(a) trans-tetracos-15-enoic acid (TCA) obtained from the plant Indigofera tinctoria;

(b) Apocynin (APO) obtained from the plains Apocyanum cannabium and A. androsaemifolium; and

the ratio of APO and TCA is in the range of 3:1 to 1:3.

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