DE1617601C3 - Medicines used to treat prostate hypertrophy - Google Patents

Description

The invention relates to an orally, subcutaneously, intramuscularly and intravenously administrable medicament for Treatment of prostate hypertrophy, which is characterized in that it is an extract as an active ingredient from Prunus africana (Hook, f.) C K a 1 k m a η, obtained by extraction of the bark with chloroform or a Methanol / water mixture (80:20) has been obtained.

The above-mentioned plant belongs to the Rosaceae family, in the somewhat older literature it was mentioned for this Plant the name Pygeum africanum Hook. f. preferred (cf. »Flore du Congo Beige et du Rwanda-Urundi «Vol. III, p. 32-33 [1952]), but in the more recent literature C. K a 1 k m a η die Above mentioned designation Prunus africana (Hook, f.) used (cf. C. Kalkman "Old World Species of Prunus ”, published in“ Blumea XIII ”, pages 1 to 115, especially pages 21 to 23 [1965]). To obtain the extract according to the invention, all species of the above-mentioned plant, especially the species found in South Africa and East Africa.

The active ingredient is obtained from the bark of the plant. The bark can also be dried, powdered Shape can be used. For this purpose, the bark can be dried in the shade and then z. Am pulverized in a mill.

To produce the above-mentioned extracts, the bark of the plant is treated with CHCl3 or a methanol / Water mixture 80:20 extracted. For the extraction of the active ingredient in purified, crystalline Form, the extract can be placed on a silica gel column using cyclohexane-benzene as the eluent be chromatographed.

An end product is made from an unidentified mixture of sterols with other ingredients finally obtained as a white, crystalline powder. Both the crude extract and the crystalline product can be used for medical purposes.

The product can be in various pharmaceutical forms, such as tablets, kachets, dragees and as liquid preparation for subcutaneous, intramuscular or intravenous injection.

The following examples illustrate the invention:

Production of the extracts:

a) 100 g of finely ground powder of the Prunus africana bark are mixed three times with 200 ml of a methanol / Extracted water mixture (80:20). The extracts are filtered and placed under vacuum at 30 ° C constricted. A paste-like extract is obtained in a yield of 20%, based on the Weight of the bark powder used.

b) Using chloroform as extraction solvent instead of the methanol / water mixture of example a) a yield of 0.5% of a paste-like extract is obtained, based on the weight of the initial bark powder.

Purification of the extracts: ■

c) 350 g of silica gel are placed in a chromatography column with a diameter of approximately 5 cm with cyclohexane p. a. silted up.

The product obtained according to preparation a) or b) is dissolved, mixed with silica gel, the solvent is drawn off and the powder thus obtained is applied to the column. It is then chromatographed with solvents of increasing polarity, for example pure cyclohexane, cyclohexane-benzene (95: 5),; Cyclohexane-benzene (50:50), pure benzene, methylene _; chloride-benzene (95: 5) etc. ■

The entire sterol fraction is in cyclohexane-ben-; zol mixture (50:50) found. !

The so separated, highly purified, active stock- f | part is approximately 0.05% by weight based on the dry plant initially used. ;

The course of the chromatography and the determination of the separated constituents can after two Procedure to be carried out:

- ₀ 1) - thin layer chromatography:

Carrier: silica gel with plaster of paris as a binding agent,

fluorescent at 254 nm, thickness = 0.25 mm

Activation: 10 minutes at 110 ° C

Mobile phase: cyclohexane-ethyl acetate (85:15)

Development time: 1/2 hour at 20 ° C

Spray solution: 6 N sulfuric acid; gives the Ste

rinfraktion the characteristic purple color when heated.

The ÄrWert is around

0.28.

2) - gas chromatography:

A gas chromatograph is used, e.g. B.

an »Aerograph-204« (registered trademark of Varian) with a flame ionization detector and a stainless steel column which is 60 cm long and 3.2 mm in diameter. As a column filling you can z. B. pumice powder with a particle size of 0.15 to 0.11 mm, which is 5% weak contains polar silicone oil. Any other stainless steel column can be used use, e.g. B. a 1.52 m long with a diameter of 3.2 mm and a column filling from one standardized diatomaceous earth preparation for chromatography, particle size 0.25 to 0.18 mm, the 5% non-polar Contains silicone oil.

Chromatograph either at constant temperature at 250 ⁰ C, while the sterols are shown, or it performs a programmed chromatography between 50 and 250 ° C, while all of the ingredients are shown.

The following pharmacological tests show the effectiveness of the extracts according to the invention.

Attempt a

In the experiments, decrepit male rats weighing over 300 g were used,

which a dose of the extract according to b) was administered. The extract was suspended in olive oil and administered as a suspension at 20 mg / ccm per os. The dose corresponds to 20 to 100 mg of bark powder per trag per kilogram of body weight.

For parenteral administration, rat serum with 10% ethanol was used as a carrier. 3 times weekly i. v. injections made, the dose being 25 mg of bark powder per kilogram Body weight corresponded. At the end of the experiment, the animals were sacrificed and the autopsy immediately performed.

The prostate is severed and carefully cleaned; the two testicles are also saved. the The effect of the treatment is shown macroscopically by the general appearance and weight of the organ and microscopically during the histological examination of the sections.

The macroscopic examination particularly reveals the color, swelling and the diameter of the gland and especially judged their weight. In order to be able to interpret the results statistically more easily, the "Prostate index" calculated. This is the quotient of the weight of the prostate in mg to the weight of the animals in The testicle index is the quotient of the weight of the two testicles in mg to the weight of the Animal in grams.

During histological examination, the height of the glandular epithelium of the prostate lobes and the middle one Examined surface of the glandular vascular incisions. The spermiogenesis of the testicles is examined.

results

The treated animals experienced a weight loss of 2 to 8%. However, this weight loss stopped in the course of treatment. The weight curve then again reached a shape similar to that of the Comparison animals.

The prostate index of the treated animals was about 20% lower than that of the control animals. Both The organ was visibly thinner, a little darker and significantly softer than in the treated animals Comparison animals. Examination of the histological sections showed a very clear decrease in the height of the Epithelium resembling that of much younger animals in appearance.

The testicle index was essentially the same in the treated animals and the control animals; the Sections showed normal spermogenesis. The examined extract therefore works selectively, it causes the The hypertrophied prostate regresses without affecting the testicles. The effect of the extract increases with concentration without the effect being proportional. The lipophilic fractions are particularly effective. Studies have shown that hydrophilic fractions, e.g. B. water extracts, are less effective.

toxicity

In the rats and mice that i. v. have been treated with a dose ten times greater than that therapeutic dose, which corresponds to 100 mg of bark powder per kg of body weight, did not occur after five weeks Death a.

acute toxicity

The experiments were carried out with rats with a purified chloroform extract c). at one i. v. no death was observed at the administered dose of 2 g / kg body weight.

Chronic oral toxicity in rats
administration

The experiment was carried out for three months with a chloroform extract according to the invention according to c) accomplished.

45 male and 45 female rats were divided into three groups of 30 animals each (15 male and 15 female) shared.

1. A comparison group received only the excipient, that of 0.10 ml of olive oil per 100 g of the rat duration.

2. One group received the extract according to the invention in a dose of 10 mg per kg (1 mg per 100 g diluted in 0.1 ml oil).

3. One group received the extract according to the invention in a dose of 100 mg per kg (10 mg per 100 g when diluted in 0.1 ml of oil).

The animals, which were caged in seven or eight, were weighed twice a week and the Blood counts were taken at regular intervals.

The animals received the drug 5 days a week. After three months there was one animal per cage taken out and brought together with male and female animals to see the possible effect to judge reproduction.

The following effects were tested:

a) - Effect on growth:

The growth curve of the animals showed no noticeable difference between the individual Groups. In the male animals, however, there was greater weight gain in those animals that received the had received extract according to the invention in a dose of 100 mg / kg, found. An analysis of the Differences in weight between the three male groups showed no significant difference.

b) - Effect on the blood count.

Between the control groups and the treated groups, there was no difference in terms of the Number of erythrocytes and leucocytes found.

c) - effect on reproduction:

Those exposed to untreated female rats male rats and those exposed to untreated male rats female rats all reproduced normally. The nests were normal. The appearance of the boys agreed with the appearance of the comparison animals. This confirms, at least for the males, that histological examinations carried out on the testicles in the previous experiment.

Attempt B

Various attempts were made. The first was done in such a way that, apart of the effect of the product on the prostate, the harmlessness to living animals and the Ineffectiveness on organs other than the prostate have been tested.

The experiment was carried out for 52 days. The animals were male rats weighing 266 to 390 g, they received the preparation orally.

Each group initially consisted of 10 animals.

There were the following groups:

1 comparison group

1 group treated with a chloroform extract according to b) a dose of 1 mg / kg of the animal five times in the

Week was treated
1 group with a methanol / water extract according to a) a dose of 30 mg / kg five times in the

Week was treated
1 group treated with the same extract at a dose of 300 mg / kg five times a week.

The doses of 1 mg / kg chloroform extract and 30 mg / kg methanol / water extract correspond to the therapeutic dose.

No death was observed in the animals, apart from incidental causes such as a rhinopharyngitis epidemic, which is not caused by the product.

One animal from the group treated with 300 mg / kg methanol / water extract was removed because the Weight values of the various organs fluctuated, which was obviously due to malformations and was independent of treatment.

The weights of the organs of this animal were taken into account in the various calculations.

The five groups were found to have no toxicity damage caused by the products of the invention exhibited.

1) Checking the weight curves

The weight curves show a normal and continuous increase in the weight of the animals. the statistical comparison of the different groups after the end of treatment shows that no significant There is a difference between the control animals and the treated animals.

2) Examination of the organs

At the end of the experiment, the animals were beheaded. The following organs were removed and weighed: Testicles, adrenal glands, kidneys, seminal vesicles, prostate, pituitary gland. These various organs were subsequently kept for histological examination.

3) Results

on the other hand, none of the fractions shows pathological changes in the other, distant organs.

Thin layer chromatography for characterization
of the starting material

a) Using a methanol / water extract of the bark powder and n-butanol / 2N HCl (98: 2) mixture as the mobile phase and a thin layer of silica gel as the carrier, a number of stains are obtained, the clearest of which is 0,1,0,4,0,7 and 0,8 respectively.
If the same extract and the same mobile phase are used, but paper is used as the chromatographic support, a very clear spot with a value of 0.36 is observed.

b) Using a chloroform extract of the bark powder, a methylene chloride / benzene (1: 1) mixture as solvent and paper as carrier, two spots with values of 0.3 are obtained and 0.7. The spot with the /? R value of 0.7 becomes cut out and eluted with chloroform. A few drops of acetic anhydride are added to the eluate, and 1 cm of concentrated sulfuric acid is slowly added to the wall of the test tube run down the eluate. At the level of the interface between the two phases, an image is formed pink ring, while the chloroform phase is colored light green.

If the same extract and chromatographic carrier are used, but a mixture of methanol with 1% glacial acetic acid and water (30:70) as the mobile phase, the component that had a value of 0.3 in the previous chromatogram has a value this time A value of 0.6; the other component does not migrate at all.
All of these chromatograms are developed in UV light or with iodine vapor or concentrated sulfuric acid.

The medicament according to the invention can be administered orally, subcutaneously, intramuscularly and intravenously will; the following preparations are suitable for this:

Oral preparations:

The weights of the animals and the weights of the various organs were given in each Groups compared statistically. The result of this comparison shows a significant difference in the Weights of the prostate. The histological examination confirms the previously established fact and gives even a more accurate picture. It was found that the four extracts used in the following decreasing Order were effective:

a) - methanol / water extract according to a) in one dose

from 300 mg / kg;

b) - methanol / water extract according to a) in one dose

of 30 mg / kg and chloroform extract at a dose of 1 mg / kg;

c) - chloroform extract at a dose of 1 mg / kg.

The effect can be described as follows:
The cells of the prostate epithelium of the comparison animals are cylindrical-cubic. The epithelial wall is thin. The cells of the treated animals develop into cylindrical, tall, secreting papilla-shaped structures that penetrate the interior. On the

a) Soft capsules, one capsule contains: 25 mg Extract made according to 100 mg Manufacture b) Peanut oil to fill up Gelatine shell, consisting of Gelatin, glycerin and potassium sorbate b) beads, one bead contains: 25 mg Extract made according to 35 mg Manufacture b) 33 mg Magnesia 2 mg Lactose 5 mg Silicon dioxide Magnesium stearate Gelatine shell. c) Soft capsules, one capsule contains: 5 mg Purified extract fraction, 100 mg manufactured according to c) Peanut oil to fill up

Gelatin shell, consisting of
Gelatin, glycerin and potassium sorbate.

d) beads, one bead contains:

Purified extract fraction, 5 mg manufactured according to c) 45 mg Magnesia 43 mg Lactose 2 mg Silicon dioxide 5 mg Magnesium stearate The shell is made of gelatin.

ok

Injectable preparations, subcutaneously, intramuscularly or intravenously injectable preparation:

a) Ampoules, one 5 ml ampoule contains:

Purified extract fraction,

produced according to c) 5 mg

Polyoxyethylene sorbitan monooleate 1 ml

Physiological serum

to fill up to 5 ml

«5

20th

b) Ampoule for subcutaneous and intramuscular injection, one ampoule of 5 ml contains:

Purified extract fraction,

produced according to c) 5 mg 25

Neutralized olive oil
to fill up

5 ml

The medicament according to the invention has already been successfully clinically tested. B. on three works in "Medecine Interne, March 1970, Vol. 5, No. 3 Bis" by A. G r e ν y and J. P. F a ν r e; by Y. Guillaud-Vallee and referenced by G. Grein er and B a 11 ο f .. The first-named author examined the effect of the drug according to the invention on 26 patients, the second author administered that Medicines according to the invention on 25 patients and the third author evaluated 16 cases. The patients received 4 capsules containing 25 mg of active ingredient each day. Treatment generally lasted from 15 to 45 days. from 21 cases with a prostate adenoma were identified as 9 patients after the specified treatment time Discharge completely cured, with 6 patients substantial partial successes were achieved. From 4 patients with a disease of the bladder neck three could be discharged as completely cured, the fourth Patients have achieved significant partial successes. The drug is used today with great success Used to treat prostatic hypertrophy in humans.

709518/354

Claims (1)

Claim: Orally, subcutaneously, intramuscularly and intravenously for the treatment of the Prostate hypertrophy, characterized by that it contains as active ingredient an extract from Prunus africana (Hook, f.) C. K a 1 k m a n, obtained by extracting the bark with chloroform or a methanol / water mixture (80:20) has been.