Use of Zn-Protoporphyrin for Hepatitis Treatment

Male ICR mice aged 4 weeks were used in groups of 10 animals. The test substance was suspended in 4% aqueous gum arabic solution and the suspension was administered by oral gavage once a day for 7 consecutive days. Concurrently with the last administration, carbon tetrachloride in 25% olive oil was administered subcutaneously in a volume of 2 ml/kg (0.5 ml/kg as carbon tetrachloride). The animals were then deprived of food for 24 hours, at the end of which time the blood was drawn from the abdominal aorta under ether anesthesia. The blood was centrifuged (3,000 r.p.m., 15 minutes) to separate the serum and GPT and GOT activities in the serum were determined (Reitmann-Frankel's method). In addition, the liver was excised and weighed.

To the control group, 4% aqueous gum arabic solution was orally administered for 7 days and carbon tetrachloride-olive oil was subcutaneously administered. To the normal group, 4% aqueous gum arabic solution was orally administered for 7 days and olive oil alone was subcutaneously administered. As the control drug, malotilate was used.

The results are shown below in the table.

According to the above results, protoporphyrin IX-Zn(II) complex of the present invention dose-dependently inhibited the elevation of serum GPT and GOT and also inhibited the increase of liver weight. Furthermore, the administration of the drug at 100 mg/kg caused significant decreases in GPT, GOT and liver weight. It was thus made clear that the drug prevents carbon tetrachloride-induced hepatic disorder.

The test procedure of Example 1 was repeated except that protoporphyrin IX-Sn(IV) complex was used as the test substance. The results are shown in the following table.

The above results indicate that protoporphyrin IX-Sn(IV) complex has no prophylactic effect on carbon tetrachloride-induced hepatic disorder.

The test procedure of Example 1 was repeated except that protoporphyn disodium was used as the test substance. The results are shown in the following table.

The above results indicate that protoporphyn disodium has little prophylactic effect on carbon tetrachloride-induced hepatic disorder.

The protoporphyrin compounds employed in the above examples are prepared as shown in the following:

Protoporphyrin IX-Zn(II) complex:

Protoporphyrin IX dimethyl ester (1.1g) was dissolved in methylene chloride (200 ml). The solution was refluxed gently, a saturated solution of zinc acetate in methanol (50 ml) was added thereto and refluxed for further 2 hours. After it was allowed to come to room temperature, the mixture was washed three times each with 200 ml of water and dried over anhydrous magnesium sulfate, and then solvent was removed from the mixture by distillation. The residue was subjected to alumina column chromatography employing chloroform as an eluant to obtain red fraction. Solvent was removed from the fraction by distillation and the remaining residue was recrystallized from the mixed solvent of methylene chloride and methanol to obtain 800 mg of protoporphyrin IX dimethylester-Zn(II) complex as reddish purple crystals.

The crystals were dissolved in pyridine (250 ml), 1 N aqueous sodium hydroxide solution (16 ml) was added thereto, and the mixture was refluxed for 2 hours. After the mixture was allowed to come to room temperature, solvent was removed from the mixture by distillation and the resulting residue was dissolved by adding water (50 ml) thereto. To the solution, a suitable amount of acetic acid was added, and resulting precipitates were collected by filtration, washed with water and dried to obtain 560 mg of protoporphyrin IX-Zn(II) complex as dark red crystals.

Electronic spectrum (.gamma..sub.max.sup.nm, DMF): 419.8, 547, 584.2

IR (KBr, cm.sup.-1): 3450, 2910, 1710.

NMR (DMF-d.sub.7, 8 ppm): 3.31 (t, 3H), 3.64 (s, 3H), 3.65 (s, 3H), 3.78 (s, 6H), 4.44 (t, 3H), 6.25 (dd, 2H), 8.53 (dd, 1H), 10.22 (s, 1H), 10.25 (s, 2H), 10.33 (s, 1H).

Protoporphyrin IX-Sn(IV) complex:

Protoporphyrin IX dimethyl ester (100 mg) was dissolved in methylene chloride (100 ml) and the solution was refluxed gently. To the solution, a saturated solution of tin(II) actate in methanol (20 ml) was added gradually and the mixture was refluxed for further 2 hours. After it was allowed to come to room temperature, the mixture was washed three times with each 100 ml of water and fried over anhydrous magnesium sulfate. Then, solvent was removed from the mixture by distillation and the residue was purified by alumina (Merck, active grade II-III) column chromatography to obtain 80 mg of protoporphyrin dimethyl ester-Sn(IV) complex.

The complex was dissolved in a solution of potassium hydroxide (100 mg) in methanol, a small amount of water was added thereto and the mixed solution was refluxed for 2 hours. The resulting residue was dissolved by the addition of water (10 ml) and acetic acid (0.1 ml) was added to the solution.

Resultant precipitates were collected by filtration, washed with water and dried to obtain 65 mg of protoporphyrin IX-Sn(IV) complex.

Electronic spectrum (.gamma..sub.max.sup.nm, DMF): 416.5, 546, 584.5.

IR (KBr, cm.sup.-1): 3450, 2910, 2850, 1705.

NMR (.delta.ppm, CF.sub.3 COOD): 12.3 (s, 1H), 12.1 (s, 1H), 11.7 (s, 2H), 9.33 (dd, 2H), 7.23 (dd, 4H), 5.30 (br.t, 4H), 4.33 (br.s, 12H), 4.03 (br.t, 4H).