DE2630178A1 - NEW MEDICINAL PRODUCTS BASED ON TISSUE AND / OR ORGAN EXTRACTS FROM ANIMALS, METHOD FOR MANUFACTURING THE SAME PHARMACEUTICAL PRODUCTS CONTAINING THESE - Google Patents

Description

New drugs based on tissue and / or organ extracts of animals, processes for the preparation thereof and pharmaceutical compositions containing them

The invention relates to new medicaments based on tissue and / or organ extracts from animals as well as methods for obtaining and purifying the same and pharmaceutical compositions containing them. More particularly medicines with a remarkable antisteatogenen efficacy and a strong inhibitory WIR Vung to the abnormal increase of collagen and, more generally an inhibitory effect on the proliferation of vascular and connective tissues of the liver (antizirrhotischer effectiveness).

• Steatosis and cirrhosis belong - regardless of whether they are caused by poisonous effects, poor nutrition or metabolic disorders or above all caused by alcoholism - still today among the worst plagues of mankind.

S 061 / CAS 2 RPA 6 09 8 80 M Ü30

ORIGINAL INSPECTS

The current therapeutic treatment, which includes numerous biological tests with monitoring of the electrolyte balance, a monitoring of the kidney function and a monitoring of the liver function associated with an extraordinary expensive drug treatment (by antibiotics, plasma, corticoids, human albumin etc.) is included also because of the numerous secondary effects caused by the drugs administered can be quite tricky or dangerous.

The aim of the invention is therefore to create new drugs for combating of steatosis and cirrhosis, which meet the requirements of the practice in particular because of their complete harmlessness and the absence of secondary effects better than the previously known drugs and treatments of a long duration under completely safe conditions * and are also associated with significantly lower production costs.

The medicaments according to the invention with antisteatogenic and Anticirrhotic effects are essentially achieved by tissue and / or organ extracts obtained on enzymatic vege formed by animals.

According to the invention, a method for producing these medicaments is also provided, which is characterized in that the animal tissue and / or organ in a first stage with a proteolytic enzyme in a saline solution with a pH in the region of the neutral point and at treated at a temperature in the region of + 4 ⁰ C, after which the suspension obtained is treated by suitable physical means to obtain a clear supernatant liquid; that the resulting clear supernatant liquid in a second stage with absolute alcohol at a

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pH treated below (inferjeur) 7; and that one from the animal tissues and / or organs extracted substance in a third stage by adding a calcium salt at a pH in the region of 7.5 fails to achieve a product that can be used alone or mixed with others Products used as such or (for example in the salt solution) lyophilized or freeze-dried and then can be sterilized.

According to an advantageous embodiment of the method according to the invention, the enzymatic treatment takes place in the saline solution for two consecutive repetitions.

According to a further embodiment of the method according to the invention, the salt solution contains 6 to 10 g of NaCl, 0.5 to 2 g of NaHCO ₃ , 0.1 to 0.3 g of KCl, 0.04 to 0.08 g of NaHgPO, and 1 to 3 g Glucose per liter of water.

According to yet another embodiment of the method according to the invention, the amount of enzyme used is between 0.1 and 0.5 g per 100 g of tissue and / or organ material.

If desired, the total extract obtained from animal tissues and / or organs can be used to achieve a heat-stable Product and a lipoid fraction are fractionated. The invention is therefore also a Process for the purification of the total extract from animal tissues and / or organs.

According to one of the ways in which it is carried out, the cleaning is carried out by dissolving the extract in a salt solution, heating the liquid to 100 ^° C. for 10 minutes, cooling it to room temperature, filtering off the precipitate.

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ges and sterilizing and freeze-drying the supernatants Liquid.

In accordance with another type of implementation, the cleaning is carried out, by soaking the whole extract for 30 minutes with a Alcohol-ether (2: 1) solution treated at 60 ° C. The "enriched" Solvent is filtered and centrifuged until clear, after which the solvent is evaporated. The dry extract of this fraction, called "lipoid", dissolves easily in propylene glycol.

Further special features will emerge from the following description, which is used to explain the invention in greater detail serves. It includes an example of the implementation of the method according to the invention and a report on analytical examinations and pharmacological tests, which prove the harmlessness of the new drugs as well as their show inhibitory effect on the proliferation of connective tissue such as collagen.

Example of the production of whole extract

100 g of striated bovine muscle was finely cut and chopped into pieces not more than 1 mm thick. These pieces were placed in 147 ml of an aqueous solution containing 1.2 g NaCl, 0.3 g glucose, 0.15 g NaHCO ₃ , 0.15 g trypsin, 0.03 g KCl and 0.007 g NaH ₂ PO ₄ . The pH of the suspension was adjusted to 7.2 with sodium hydroxide solution and the suspension was then ^{left at 4 °} _{C. for 24 hours (the pH was checked at 7 f} 2 during the first 3 hours to stabilize it). After this time, the suspension was filtered and the liquid was centrifuged in a centrifuge cooled to + 2 ° C. for 1 hour at 3000 rpm. The supernatant "enriched" liquid was collected and the centrifuged material

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rial extracted again with 150 ml of saline solution, which was again ^{centrifuged for 1 hour at 4 0} C with 3CX) 0 rpm.

The combined supernatant liquids were treated with 75 ml of absolute ethanol. The liquid was left at 4 ° C for 24 hours and the pH was adjusted back to values below 7 if necessary. A white precipitate formed which was filtered through cheesecloth and then centrifuged at 3,000 rpm at 2 ° C for 1 hour. 0.02% CaClp (5%) and a sufficient amount of sodium hydroxide solution to bring the pH to 7.6 were then added to the supernatant liquid obtained in this way. The extract showed slow excretion. To complete the precipitation, the liquid was left at 4 ° C for 24 hours. The precipitate was then separated off by centrifugation, dissolved in the saline solution, sterile filtered through a membrane with a pore diameter of about 0.45 / U and then lyophilized.

Analytical testing

A) fractionation

1 g of freeze-dried total extract gives (in dry weight):

by the purification leading to a thermostable fraction 666.5 mg

by becoming a lipoid fraction

leading purification: 67.7 mg

a thermolabile fraction of: 82.8 mg

mineral salts 183.0 mg

B) Determination of proteins

This determination can only be carried out if the total extract or the thermostable fraction of the Whole extract with a polyacrylamide gel (for example

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wise "LYPHOGEL" by Gelman) as a precautionary measure, the Water absorbs up to 5.5 times its own weight as well as molecules whose molecular weight is lower than 20,000, while substances whose molecular weight is over 20,000 - i.e. all of the proteins in Solution remain.

For such a determination, 250 mg of total extract were dissolved in 25 ml of water and one for 10 ml of this solution sufficient amount of "LYPHOGEL" to achieve final volume of 1.5 ml was added. The determination of the total proteins was carried out according to the Folin method 0.1 ml of the concentrated extract. It was found that 1 g of total extract contains 50 to 70 mg of proteins.

C) electrophoresis

Electrophoresis of the total extract shows essentially two fractions, one that migrates in accordance with albumin and a second that migrates in accordance with β-globulins. The electrophoresis was carried out in a Gelman cell on cellulose polyacetate in 0.05 M Tris-barbital buffer of pH 8.8 at a voltage of 350 V over a period of 35 minutes; the acetate bands were stained for 10 minutes with a solution containing 500 mg of Ponceau S red in 100 ml of 5% trichloroacetic acid; the bands were decolorized with 5% acetic acid and it was cleared with a 12% solution of acetic acid in methanol; then it was ^{dried at 90 °} C. for 30 minutes. The optical density and the integration areas were determined, for example, with a "Digiscreen-R" device. Was found:

Albumin about 13 % ß-globulins about 87 %.

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D) Determination of the anti-collagen activity in vitro

The following procedure was used: normal rat liver became with a sharp knife 10 to 15 pieces about 0.5 mm thick, corresponding to 120 to 150 mg fresh weight, cut out Then incubated in small dishes with 3 ml of bicarbonate-containing Krebs-Ringer solution, the 6 ^ C radioactive Proline and precisely weighed and increasing amounts of the total extracts contained.

The gas phase was formed by 95 % oxygen and 5 % CO «.

It was incubated for 60 to 120 minutes in a Dubnoff apparatus with a stirrer. After the end of the incubation, the pieces were removed, dried on filter paper and homogenized in 1 ml of Ringer's solution. Then 1 ml of 30% trichloroacetic acid (TCA) was added and everything was centrifuged. The sediment or precipitate was washed twice with 2 ml and 1 ml of 10% TCA. The supernatant liquids were discarded and the sediment resuspended in 1 ml of 5% TCA and then ^{brought to 90 °} C. for 30 minutes, after which it was centrifuged. The fraction soluble in the TCA contained the collagen. This was first hydrolyzed for 16 hours at 105 ^° C. in centrifuge tubes closed with screw stoppers with 6N HCl, treated with charcoal to remove the nucleic acids and neutralized with sodium hydroxide solution, after which the nitrogen was finally determined by the ninhydrin method to determine the collagen content.

The optical density of the various samples was at

570 τψ read and the radioactivity determined with the aid of scintillation fluid, such as "Omnlfluor" from Nen Chemicals in a "tri-carb" apparatus. By plotting the curve of the nitrogen content e of the various samples obtained with increasing total extract content, it was found that the amount of extract, based on the protein content, which inhibits collagen synthesis by 50% , was 136 g . Since the protein content of the total extract was about 60 mg / g, it can be seen that the standard preparation of the extracted complex contained 441 anti-collagen units (LFS).

REPORT ON PHARMACOLOGICAL EXAMINATIONS AND PROPERTIES

Materials and methods

The test animals were albino Wistar rats of both sexes weighing in the region of 100, 150 and 200 g and castrated male pigs weighing 20 to 30 kg with chronic hepatopathy were used.

1) Treatment of the animals

The cirrhosis in the rat was caused by subcutaneous injection of 0.25 ml of a 1: 1 solution of CCl ^ cereal oil induced per 100 g body weight of the animals. The injections were given three times a week on one of two days Performed for three to four months. The total extract (E.T.) was either made at the same time as the carbon tetrachloride or the next day in amounts of 25 mg of lyophilisate dissolved in 0.5 ml of water per 100 g of body weight administered to the rat. The comparison animals were given subcutaneous amounts of grain oil or Saline solution administered.

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The E.T. in drinking water Dissolved in amounts of 800 mg lyopbilisate per 300 ml of water, which amount is available for 10 rats for 24 hours was standing.

The steatogenic diet was composed as follows:

Casein 5 bacon 20th glucose 15th tapioca 48.3 1-cystine 0.50 Cod liver oil 2 Vegetable oil 3 Salt mixture 4th Vitamin mix 2.2

This diet was given to rats starting with treatment with CCl ^ as a substitute for the control animals given normal diet or food given. The E.T. in amounts of 1 g lyopbilisate injected into the subcutaneous cervical area per 20 kg of body weight dissolved in 5 ml of water. The comparison animals received equivalent amounts of saline under the same circumstances.

2) analytical methods

a) Collagen determination

The determination was based on the content of oxyproline. This amino acid is known to be 13 % present in collagen. The extraction of the hepatic collagen was carried out using the method described by PATEC and others, and the oxyproline was determined using the LOGAN method. The following working method was used: Approx

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3 g of liver were ground in a mortar and, after successive treatments in acetone and ether to remove water and lipoids, the powder obtained was dried in a drying cabinet to constant weight. About 50 mg of this powder were weighed into a centrifuge tube with a screw cap and washed twice with 20 mg urea to remove the soluble proteins. The powder was then hydrolyzed for 16 hours at 11O ⁰ C. The samples obtained were neutralized with sodium hydroxide solution, diluted to 10 ml with water and filtered. The oxyproline content was determined in 1 or 2 ml of the filtrate using the above-mentioned LOGAN method. The oxyproline content was calculated on the basis of a standard sample obtained on the basis of a solution of pure, commercially available oxyproline. The collagen content was obtained by multiplying the oxyproline content by 7.46.

b) Determination of the triglycerides

The liver triglycerides were extracted using the FOLCH method and the VAN HANDEL method modified method determined by ZILVERSMIT.

c) Determination of the proteins

This determination was carried out using the biuret method described by COLOWICK and KAPLAN.

d) collagen synthesis

This was done according to the recommended by HIRAYAMA and Mitt Method.

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e) Collagen Solvent Activity

The collagenolytic activity was tested in vitro on a liver homogenate; following procedure was applied: 1.5 ml of sodium acetate buffer from pH 5.5 and 1.0 ml of 15% liver homogenate in one Acetate buffer as well as increasing amounts of E.T. were placed in nitrocellulose centrifuge tubes. the final amount of mixture was 2.5 ml and by this amount for each E.T. Keep addition constant, an equivalent amount of acetate buffer was withdrawn.

1A-

15 ml of collagen with C -labelled were added to this mixture Added proline. In addition, comparative samples with 1 ml acetate buffer instead of 1 ml Liver homogenate prepared to check for spontaneous collagen hydrolysis during incubation he follows.

The samples produced in this way were ^{incubated in air at 37 °} C. for 3 hours. After this incubation, the samples were centrifuged at 35,000 rpm for 30 minutes and the radioactivity was determined on aliquots of the supernatant fluid. The collagenolysis was determined from the difference between the activity in the presence and absence of liver homogenate and based on 1 g of fresh liver.

The effect of the E.T. was tested on the liver of rats, which it was injected subcutaneously simultaneously with (also subcutaneously administered) CCl ^ and the were given either a normal diet or a steatogenic diet.

A) NORMAL DIET ;. SUBcutaneous injection of CCl, (Duration of treatment 105 days)

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Results:

a) mortality

The quotient of the mortality Qm was determined according to the following equation:

2m sqm =

N + S

in which m = the number of dead animals;

N = the number of the initial population; and S = the number of surviving animals.

The result was found:

Group of male rats:

Comparison animals: Qm = 0.033 treated with CCl ^: Qm = 0.666 CCl ₄ + ET: Qm - 0.142

Group of female rats:

Comparison animals: Qm = »0 treated with CCl ^: Qm * 0.500 CCl + E.T. : Sqm ~ 0.162

b) Ratio of the weight of the liver (in g) to the body weight of the rat (in g)

The weight ratio of (liver: total body weight) the comparison animals was chosen as the reference value and set equal to 1.00.

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The following values were then found for the other groups:

Group of animals treated with CCl ^: 1.14 Group of CCl + E.T. treated animals: 1.00

c) collagen content of the liver

If the collagen content in the comparison animals is set equal to 100, this is obtained in the other groups the following values:

Group of male rats:

treated with CCl. :

treated with CCl ^ + E.T ":

Group of female rats;

treated with CCl ₄ : treated with CCl ₄ + ET:

d) content of the liver

If the content of triglycerides in the comparison animals is set equal to 1Q0, one obtains for the other groups the following values:

Group of male rats:

treated with CCl ₄ : treated with CCl ₄ + ET:

Group of female rats;

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treated with CCl ^:
treated with CCl ₄ + ET: 101

B) STEATOGENIC DIET

a) mortality

Qm of the comparison animals: 0.162 Qni for diet: 0.500

Qm at Diet + E.T. : 0.285

b) Triglyceride content of the liver

If one considers the content of triglycerides in animals of the same kind equals 100, the following values are obtained for the other groups:

Diet treated rats only: 800 diet + E.T. treated rats: 300.

COMPARISON OF EFFECTS OF TOTAL EXTRACT (E. T.). THE THERMOSTABLE FRACTION OF THE TOTAL EXTRACT (F.T.) and OF THE TOTAL EXTRACT LIPOID FRACTION (F.L.)

Table I provides comparative values for the antisteatogenic efficacies of the E.T., the F.T. and the F.L.

6 0 9 8 8 6/1 Q 3 Q

TABLE I.

In 5 groups (20 rats each), the following values were obtained after treatment for 45 days:

Rat group
treated with Collagen content
the liver Triglyceride content
the liver I.
I.
i (comparison animals)
I. 100 100 j ^CC1 4 176 284 : cci ₄ + ε.τ. 101 146 CCl ₄ + PT 108 130 I.
; CCl ₄ + FL 108 107

C) EFFECT OF TOTAL EXTRACT IN PIGS WITH CHRONIC LIVER DISEASE

To prove that the total extract is also not opposite experimentally produced hepatopathy is effective, some ten young pigs of the same breed and of the same age, who suffered from chronic hepatopathy, divided into two groups: The animals of the first group served as comparison animals, while the others were treated with whole extract. At the end After the treatment, which lasted 60 days, the animals were sacrificed and the following parameters were checked:

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Mortality and weight development;

Ratio of liver weight to body weight of the pig;

macroscopic evaluation of the liver; Collagen content of the liver; Glyceride content of the liver.

The results obtained clearly agreed with the rat results reported above.

D) ACUTE AND CHRONIC TOXICITY TEST

a) Acute toxicity

The rats were divided into four groups of 10 animals each (5 male and 5 female) who received the following injections subcutaneously:

2 ml solution with 150 mg E.T. 2.5 ml solution containing 200 mg E.T.

3 ml solution with 300 mg E.T.

4 ml solution with 400 mg E.T.

No mortality was observed.

b) Chronic toxicity

The effect of the medicament according to the invention was found in healthy male and female rats long-term (311 days) subcutaneous and oral administration. There was no abnormality whatsoever of the parameters examined, including:

the weight development; at urinalysis;

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the number of red and white blood cells and amount of hemoglobin;

of urea nitrogen and blood sugar; the GPT and GOT transaminase; at free and esterified cholesterol and the triglycerides of plasma; at the weight of the main organs and their histological Verification.

c) Teratogenic activity

No abnormality or deviation was compared with either subcutaneous or oral administration found to comparison animals.

E) CONCLUSIONS

The results obtained in the various investigations show that the medicaments according to the invention are a remarkable antisteatogenic and anticirrhotic efficacy against both experimentally generated and spontaneous ones Have liver changes. The medicaments of the present invention penetrate the abnormal accumulation of collagen Treatment with CCl ^ back, and not just at the same time Administration with the toxic substance, but also when an accumulation of collagen has already been detected has been. In addition, the medicaments according to the invention have proven themselves as effective to prevent or reduce the further damage, the seat of which is the liver and which are caused by CCl, proved, as compared to the accumulation of triglycerides, the changes in the content of Proteins and water as well as the necrotic districts or tablets, which are observed histologically. It was also proven that the total extract as well as the thermostable and fat-soluble fractions have an effect on the damage caused by protein malnutrition

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be how in terms of a decrease in hepatic Steatosis.

Viewed overall, the medicaments according to the invention therefore show a wide variety of direct effects the prevention or suppression of various changes, regardless of whether they were triggered experimentally or are of a spontaneous nature.

A certain number of already in humans with diverse Forms of chronic liver disease (generally associated with alcohol) obtained show the anticirrhotic effectiveness of the new drugs. The administration to the test subjects took place in the form of gastro-resistant tablets with 0.300 g of active ingredient in quantities of 6 tablets per day.

The new drugs can be used alone or in conjunction with other drugs and in different appropriate ways Administration forms are used.

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Claims (9)

Claims 1. Process for the preparation of drugs with antisteatogenic and anticirrhotic effectiveness, characterized in that one in one first stage a tissue and / or organ from the animal with a proteolytic enzyme in a saline solution at a pH value treated in the region of neutrality and at a temperature in the region of + 4 ° C and the suspension obtained subject to appropriate physical treatment resulting in a clear supernatant liquid; that one this clear liquid treated in a second stage with absolute alcohol at a pH value below 7 and that from the animal tissues and / or organs extracted substance in a third stage by adding calcium salt to a pH value in the region of 7 »5 fails to achieve a product that alone or mixed with other products than such can be used or lyophilized and then sterilized. 2. The method according to claim 1, characterized in that the enzymatic treatment in the salt solution in two successive Repetitions or painting takes place. 3. The method according to claim 1, characterized in that the product is lyophilized in a salt solution containing 6 to 10 g NaCl, 0.5 to 2 g NaHCO ₃ , 0.1 to 0.3 g KCl, 0.04-0 .08 g NaHpPO, and 1 to 3 g glucose per liter of water. 4. The method according to claim 1, characterized in that the amount of enzyme used is between 0.1 and 0.5 g per 100 g Tissue and / or organ material lies. 609886/1030 5. The method according to any one of claims 1 to 4, characterized in that the total extract of animal tissue and / or organs fractionated to obtain a thermostable product and a lipoidal fraction will. 6. The method according to any one of claims 1 to 5, characterized in that the cleaning is carried out by dissolving the extract in a salt solution, heating to 100 ⁰ C for 10 minutes, cooling to room temperature, filtering off the precipitate and sterilizing and lyophilizing the supernatant liquid. 7. The method according to any one of claims 1 to 6, characterized in that the purification by treating the total extract with an alcohol-ether solution at 60 ⁰ C, filtering off the "enriched" solvent, centrifuging and evaporating the liquid and subsequent dissolution of the obtained Product in propylene glycol. 8. New drugs with antisteatogenic and anticirrhotic effectiveness, characterized by extracted components from animal tissues and / or organs which can be obtained by enzymatic means according to any one of claims 1 to 7 are· 9. Pharmaceutical agents with anti-steatogenic and anti-cirrhotic Effectiveness, characterized by a drug content according to Claim 8. 609886/1030