JPH0316326B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to hepatic DNA synthesis stimulators. The liver has a great ability to self-repair, and is an organ that can be expected to be repaired by administering drugs when dysfunction occurs. The present inventor previously reported that when papain was injected intraperitoneally into rats and mice, disappearance of acidic glycans on the surface of hepatocytes and increases in DNA synthesis and mitosis of hepatocytes were observed. Similar changes in the surface layer of hepatocytes also occur in hepatocytes left after partial liver resection, but if protease inhibitors (leupeptin or pepstatin) are administered at this time,
Since the above change did not occur, it was predicted that there was a factor in the serum that activated liver protease. The present inventor has also previously reported that the serum of various animals contains low molecular weight components that activate cathepsin D release from lysosomes and high molecular weight components that inhibit it as well. In view of these circumstances, the present inventor conducted a more detailed study and found that the fraction that activates cathepsin D release promotes hepatic DNA synthesis, and that further purification of the above fraction revealed that its active ingredient The present invention was achieved based on the discovery that the following exists. That is, the gist of the present invention is to perform thin layer chromatography (developing solvent n-
R _f of butanol:acetic acid:water=12:3:5v/v)
It is a hepatic DNA synthesis stimulant whose active ingredient is a fraction with a value of 0.25 to 0.55. The present invention will be explained in detail below. Examples of mammals include humans, rabbits, cows,
Examples include horses, pigs, and sheep. From these animals,
The active ingredient of the present invention is present in plasma or serum obtained by blood collection using conventional methods. The above-mentioned fraction (hereinafter referred to as this fraction), which is the active ingredient of the hepatic DNA synthesis stimulating agent according to the present invention, can be obtained by, for example, converting mammalian plasma or serum into dialysis, ultrafiltration,
By appropriately using a well-known method such as gel filtration, a portion with a molecular weight of 1000 or less is fractionated, and then by appropriately using a well-known method such as column chromatography, thin layer chromatography, ion exchange chromatography, etc. It can be manufactured by separating a portion corresponding to the above R _f value. When producing this fraction, before performing dialysis, ultrafiltration, gel filtration, etc., a step of heating plasma or serum to remove coagulating proteins and a step of removing lipids with an organic solvent are provided. It gives favorable results because the fractionation becomes easy. In addition, the active ingredient is gel-filtered (Sephadex G)
According to
When 400 is re-chromatographed, it is separated into 400 and 200 and the activity is eluted, so there is an active ingredient in the fraction with a molecular weight of 1000 or less, and no active ingredient in the fraction with a molecular weight of 1000 or more. I can understand.
In any case, the active ingredient is a heat-resistant low molecular weight substance and is not a polymeric substance. In addition, when thin layer chromatography using cellulose is performed, the activity is R _f value of 0.20 to 0.25, 0.32 to 0.38,
It is found in three places with R f value of 0.43 to 0.55, and cannot be found in other places, and among these, R _f value is 0.32 to 0.32.
0.38 seems to be the main component. The details of whether the three spots mentioned above are molecularly distinct, or whether they are caused by some kind of artefact as seen in the case of gel filtration, remain to be investigated. It is. This fraction exhibits physical properties such as those described in Examples. When this fraction is used as a hepatic DNA synthesis stimulant, it is administered alone or in combination with a pharmaceutically acceptable carrier. Its composition is determined by the route of administration, administration schedule, etc. The dosage is determined depending on the patient's age, health condition, weight, severity of symptoms, type of concurrent treatment, if any, frequency of treatment, nature of desired effect, etc. Therapeutic doses generally range from 0.05 to 50 mg per parenteral dose.
kg/day, orally 0.1 to 100 mg/Kg/day. When this fraction is administered orally, it is used in the form of tablets, capsules, powders, granules, solutions, elixirs, etc., and when administered parenterally, it is used in the form of sterile liquids such as liquids or suspensions. It will be done. When used in the forms described above, solid or liquid non-toxic pharmaceutical carriers can be included in the composition. As an example of a solid carrier, conventional gelatin-type capsules are used. The active ingredient may also be tabletted, granulated, or packaged as a powder with or without adjuvants. Excipients used in combination include water: gelatin: types of lactose, glucose, etc.: starches such as corn, wheat, rice, arrowroot starch, fatty acids such as stearic acid, calcium stearate, magnesium stearate, etc. Fatty acid salts: Talc: Vegetable oil: Alcohols such as stearyl alcohol and benzyl alcohol: Gum,
Examples include polyalkylene glycol. These capsules, tablets, granules and powders generally contain 5 to 100% by weight of active ingredient, preferably 25 to 100% by weight. Liquid carriers include water, petroleum, soybean oil,
Oils of animal or plant origin or synthetic oils such as peanut oil, sesame oil, mineral oil, etc. are used. Generally, physiological saline, textulose or similar saccharide solutions, and glycols such as ethylene glycol, propylene glycol, polyethylene glycol, and the like are preferred as liquid carriers. When administered parenterally by intramuscular, intravenous, or subcutaneous injection, the fraction is used as a sterile solution with the addition of other solutes such as saline or glucose to make the solution isotonic. Suitable vehicles for injection include sterile water, lidocaine hydrochloride solution (for intramuscular injection), physiological saline, dextrose, intravenous fluids, electrolyte solutions (for intravenous injection), and the like. These injection solutions usually contain 0.5 to 20% by weight, preferably 1 to 10% by weight of the active ingredient. In the case of liquid preparations for oral administration, suspensions or syrups containing 0.5 to 10% by weight of the active ingredient are preferred. In this case, aqueous excipients such as fragrances, syrups, and pharmaceutical micelles are used as carriers. This fraction has hepatic DNA synthesis stimulating action. Due to this action, this fraction promotes the regeneration or new generation of liver parenchymal cells when their number or function decreases due to liver diseases such as acute hepatitis and chronic hepatitis, or drug addiction.
It is useful as a drug for restoring the original liver function. The present invention will be explained in more detail with reference to production examples and examples below, but the present invention is not limited by the following examples unless it exceeds the gist thereof. Test Example Activity Measurement Method From 10% homogenate of rat liver in 0.25M sucrose-20mM Tris-HCl buffer (PH7.2),
Differential centrifugation was performed first at 750 x g for 10 minutes, second at 3300 x g for 10 minutes, and third at 16500 x g for 20 minutes.
Gently wash 16,500×g of pellets with 0.25M sucrose-1mM ethylenediaminetetraacetic acid solution (PH7.2), and suspend in the same solution so that each 20ml is equivalent to 10g of liver-derived pellets to produce lysosomes. did. Lysosomal suspensions (2 ml) were incubated with or without sample at 37°C for 60 min and centrifuged at 16500 xg for 20 min. Pellets are 0.25M sucrose-0.2% Triton X-100 (trademark of nonionic surfactant manufactured by Rohm and Haas)
Suspend in 4.4 ml of solution, leave at 4℃ overnight, and incubate at 16500×
Centrifuged at g for 20 minutes. The supernatant was used to measure the activity of residual cathepsin D by the method described below. To measure the activity of total cathepsin D, a lysosomal suspension was solubilized and used in the same manner as described above, except that incubation was not used. The percentage of cathepsin D release (100×(difference in activity)/total cathepsin D activity) was calculated from the difference in activity between total cathepsin D and residual cathepsin D. Cathepsin D activity was measured by the method described below. 0.1 ml of the supernatant obtained by centrifugation of the lysosomal suspension solubilized with the nonionic surfactant was added to a freshly prepared 0.18 M acetate buffer (PH3.2) of acid-denatured hemoglobin. solution
Mixed with 1.9ml. The mixture was incubated at 37°C for 60 min and then diluted with ice-cold 10% trichloroacetic acid-water.
1.0 ml was added to stop the reaction. After centrifugation, supernatant
0.5 ml each was reacted with 2.75 ml of Lowry's reagent (alkaline copper reagent + fluorin reagent), and the absorbance at 750 nm was measured. The % stimulation of cathepsin D release from lysosomes by the active ingredient was defined as follows. (% release in the presence of active ingredient) - (% release in the absence of active ingredient)/(% release in the absence of active ingredient) One unit of activity was defined as the activity providing 10% stimulation. The activity of the active ingredient was measured over a concentration range that provided a linear relationship between concentration and activity (below 100% stimulation). This serum fraction (factor) has the effect of promoting the release of cathepsin D and L from lysosomes (a type of organelle containing various hydrolytic enzymes) prepared from the liver. A quantitative method has been constructed based on its activity. One unit corresponds to an activity that promotes the release of cathepsin D from lysosomes by 10%. Cathepsin D from lysosomes, as mentioned above.
In addition to the effect of promoting withdrawal, this serum factor is also known to have the effect of increasing the level of cathepsin D (more precisely, cathepsin D-like acid protease) in the blood in vivo. Research into where cathepsin D, which is increased in the blood by this serum factor, comes from has revealed that red blood cells are the main source of cathepsin D. That is, it was demonstrated that cathepsin D (like acidic protease) bound to the red blood cell membrane (also called ghost) is released from the membrane by this serum factor. Therefore, this active ingredient inhibits the release of cathepsin D from lysosomes.
It also has the activity of promoting the release of cathepsin D-like acidic protease from the red blood cell membrane, increasing the amount of acidic protease in the blood in vivo, and stimulating DNA synthesis in the liver. Test Example Toxicity Test This serum factor is not a synthetic product, but is a substance that actually exists in the blood of animals, and in a broad sense can be called a natural physiologically active substance.As a result of a series of studies, It has been found that it is a new hormone secreted by the parathyroid glands that is dependent on glucagon (pancreatic hormone). This serum factor stimulates DNA synthesis specifically in the liver in vivo.
From experiments using mice, the effective amount is 1,000 to 10,000 units/mouse.

[Table] Three mice were used in each group in the above experiment, and the specific activity of serum factors was 25,000 units/mg.
There were no deaths at 36 or 48 hours after administration. This serum factor has been purified almost to a pure product, and the specific activity of this purified sample reaches 10 ⁸ to 10 ⁹ units/mg. The effective amount of the hepatic DNA synthesis stimulant is less than 10 ⁴ units/mouse, and the weight is less than 4 μg/Kg body weight, so acute toxicity is not expected at all. Production example (step) Mix 30% of bovine serum with an equal amount of water and adjust to PH4.5 with hydrochloric acid.
and heated to 100°C for 20 minutes. After cooling, the coagulated protein was filtered off using a Teflon cloth (a trademark of Dupont's fluorocarbon resin), and the residue was washed twice with water. The filtrate and washing solution were combined and filtered through filter paper. The heated serum filtrate (at this stage is referred to as heated serum filtrate) was neutralized with sodium hydroxide and then evaporated to dryness on a rotary evaporator. (Step) The dried product obtained in step was dissolved in an appropriate amount of water, and lipids were extracted with an organic solvent [ethanol-ethyl ether (1:3 v/v) and ethyl ether]. The organic solvent layer was washed with water. The aqueous layer and aqueous washings were combined and concentrated on a rotary evaporator. (The material at this stage is referred to as the delipidated material.) (Step) The delipidated material obtained in step was diluted with water so that the sodium chloride concentration was 1.5M or less. The solution was subjected to ultrafiltration using an ultrafiltration membrane made from dextran (trademark: Diafilter G-05T, manufactured by Bioengineering Co., Ltd., hereinafter referred to as G-05T membrane). Filtrate (at this stage, G-
The 05T filtrate) contains components with a molecular weight of less than 5000, which is filtered using a dextran ultrafiltration membrane (Bio Engineering Co., Ltd. trade name Diafilter G-
05H (hereinafter referred to as G-05H membrane) was subjected to ultrafiltration. Most of the activity is caused by the G-05H membrane filtrate (at this stage, which contains components with a molecular weight of less than 500).
-05H filtrate), but the residue of G-05H membrane (this stage is called G-05H residue)
Some of it remained. (Step) Adjust the G-05H filtrate to pH 5.0 and equilibrate with Na ⁺ type weakly acidic cation exchange resin (Rohm and Haas trademark Amberlite CG-50-10 ^-5 M hydrochloric acid).
column chromatography. After putting the filtrate into the column, add A _{260 on} of the filtrate with 10 ^-5 M hydrochloric acid.
Washed until absorption was negligible. The active moieties adsorbed on the resin were flushed out with 1M aqueous ammonia.
The active part near the tip of the ammonia water was neutralized with hydrochloric acid and evaporated to dryness using a rotary evaporator.
(The product at this stage is referred to as CG-50 ammonia effluent.) (Step) The CG-50 ammonia effluent was quickly washed twice with ethanol and methanol, the residue was dissolved in water, and impurities were removed by centrifugation. . The supernatant was subjected to gel filtration using dextran gel (Sephadex G-10, a trademark of Pharmacia Fine Chemicals) and monitoring activity and absorbance at 280 nm and 260 nm. The activity was divided into two bands. One is the molecular weight of approx.
One is the peak at the molecular weight position of 200 (this is referred to as the MW-200 component), and the other is the peak at the molecular weight approximately 400 (this is referred to as the MW-400 component).
Each component was evaporated to dryness. (Step) Wash the MW-200 component obtained in step three times with ethanol and methanol to remove the residue.
Dissolved in 76% methanol-water. The methanolic solution was placed in a silica gel column and the column was washed with 76% methanol-water and water. Activity is 76%
It was divided into two parts: methanol-water effluent and water effluent. The specific activity of the latter is greater than that of the former. The aqueous effluent was evaporated to dryness and the residue was dissolved in 87% methanol-water and column chromatographed again on silica gel. Column is 87% methanol
Washed with water, 76% methanol-water and water. Activity was recovered in the 87% methanol-water and water effluents, but the specific activity of the water effluent was higher than the specific activity of the 87% methanol-water effluent. (Step) The water effluent from the step was evaporated to dryness and subjected to thin layer chromatography using cellulose (manufactured by Merck & Co., Ltd., non-fluorescent), and was subjected to n-butanol:acetic acid:water (12:3:5v/v). ). Fluorescent band (R _f 0.33−) within the active band (R _f 0.25−0.55)
0.44) Extracted with water, subjected the extract to paper chromatography using Watzman 3MM paper, developed with the above solvent system, extracted the active portion with water and rechromatographed in the same manner. (Step) Collect the parts of Step with a specific activity of 20×10 ³ units/mg or more, and add the dried product to a small amount of 80% ethanol-water,
It was quickly washed with methanol. Dissolve the residue in a small amount of water and store in a vacuum desiccator.
(This is referred to as the purified serum factor) (Step) When the purified serum factor was sequentially differentially extracted with aqueous ethanol, activity was observed in the 90-80% ethanol and 15-0% ethanol portions. The results for the products manufactured in the manufacturing example are shown in Table 1.
It was shown to.

[Table] Data in parentheses in the table is reference data that is not used in the next purification step. The physical properties of the purified serum factor are as follows. colorless. Soluble in water. Relatively sparingly soluble in methanol and other organic solvents. Aqueous solutions are usually 260nm and
Does not show absorption maximum at 280nm. Detection of sugar by Moritshu reaction Detection of imidazole group by negative Pauly reaction Detection of sulfur by negative iodoplatinate reaction Detection test of indole (yellow color) or N-carbamyl group by positive Ehrrich reagent (p-dimethylaminobenzaldehyde + hydrochloric acid) Example Effect on DNA synthesis in the liver in living organisms The heated serum filtrate produced in the production example (activity approximately 1.2 units/mg) is dissolved in water, and the activity is 200 to 400 units/mg.
ml samples were prepared. In addition, partially purified serum factors (activity approximately 265 units/mg) were prepared from the heated serum filtrate by chromatography using the weakly acidic cation exchange resin and paper chromatography (R _f 0.52 to 0.76). The one prepared in the production example was used, and both were dissolved in physiological saline. Each sample was injected intraperitoneally into 2 to 3 mice weighing 19 to 24 g according to the method shown in Table 2.
1.5 hours before killing mice, ^14C -thymidine (5.0~
50.7 Ci/mol) was injected intraperitoneally at 1 μCi. Liver DNA was collected using the Schmitt-Tannhauser method.
The incorporated radioactivity was counted using a scintillation spectrometer using a 0.6% toluene solution of 2,5-diphenyloxazole as the scintillation liquid. DNA was measured using the calf's thorax DNA as a standard using Deitshu's method. The results are shown in Table 2. Note that as a control, physiological saline was injected intraperitoneally.

【table】

Claims (1)

[Claims] 1 Molecular weight 200-400 obtained from mammalian plasma or serum, thin layer chromatography using cellulose (developing solvent: n-butanol: acetic acid: water =
12:3:5v/v) Rf value is 0.20~0.25, 0.32~
0.38, 0.43-0.55, slightly soluble in non-polar solvents, soluble in aqueous methanol, aqueous ethanol and water, adsorbed on cation exchange resin and eluted with ammonia, silica gel in 76%-87% methanol by silica gel chromatography A hepatic DNA synthesis stimulant whose active ingredients are the above fractions that are adsorbed to and eluted with water.