DE1252365B - - Google Patents

Description

GERMAN #M PATENT OFFICE Wfltix? German class: 30 h - 6 EDITORIAL «-« ^ File number: J 27798 IV a / 30 h

1252 365 filing date: March 29, 1965

Opened on: October 19, 1967

The present invention relates to a new valuable antibiotic called Neocarzinostatin and processes for its manufacture. In particular, the invention relates on the production by fermentation as well as processes for the extraction and purification of the Substance. Neocarzinostatin inhibits the growth of sarcoma 180, leukemia SN-36 and 1210 tumors in mice. The substance is also effective against various bacteria, e.g. B. Staphylococcus aureus 209 P, Staphylococcus aureus Kishima, Staphylococcus aureus Smith, Staphylococcus aureus 537, Sarcina lutea, Bacillus subtilis and Escherichia coli. The substance is still very valuable for biological research in the separation and classification of mixtures of microorganisms as well as the elimination of microorganisms from laboratory and medical and dental equipment Instruments.

Carzinostatin, a high molecular weight substance, has some effectiveness against malignant tumors and is obtained by fermentation of Streptomyces carzinostaticus. The production has already been described in Japanese Patent Specification 267158. Was carzinostatin but so far only obtained in its raw form and is extremely unstable, especially in Exposure to heat.

If you try to obtain carzinostatin in pure form, the product will break down into two parts A and B, both of which have been shown to be ineffective in fighting tumors. That is, it has not heretofore been possible to manufacture carzinostatin as a preparation. This Problem is already in the journal "The Journal of Antibiotics", Vol. 14 A, p. 27, by Jyun-Chi-Shoji been discussed in detail.

The inventors of the present invention have now discovered that fermentation can produce certain variant strains of Streptomyces carzinostaticus obtained by ultraviolet irradiation and by means of the so-called »single spore isolation« method, produce a relatively stable substance can, which is obtained in pure form. The inventors have this substance "Neocarzinostatin" called.

The new substance has an antibacterial and anti-tumor effect and is resistant against heat and other procedural measures that are necessary for the extraction of the substance in pure form.

The present invention is therefore

Manufacture and extraction of the antibiotic
Neocarcinostatin

Applicant:

Nakao Ishida. Sendai (Japan)
Representative:

Dr. Dr. J. Reitstötter and Dr.-Ing. W. Bünte.
Patent Attorneys, Munich 15, Haydnstr. 5

Named as the inventor. Nakao Ishida, Katsuo Kumagae. Keizo Miyazaki, Mitsuo Rikimaru, Sendai: Masahiko Kuroya, Tokyo (Japan) Claimed priority: Japan of! April 1964 (18 015)

a manufacturing process for an antibiotic that was given the name "Neocarzinostatin". The method consists in applying a variant neocarcinostatin-producing strain of Streptomyces carzinostaticus, called Streptomyces carziaDJlallcus var. Neocarcinostaticus, "to an" aqueous carbohydrate solution containing a nitrogen source under submerged aerobic conditions until the solution is cultured has an actual effectiveness against Sarcina lutea, whereupon the neocarzinostatin can then be obtained from this solution. The present invention also relates to the neocarzinostatin itself obtained by this process.

The F i g. 1 shows the ultraviolet absorption spectrum of neocarcinostatin;

F i g. Fig. 2 shows the infrared absorption spectrum of neocarcinostatin in the form of potassium bromide beads.

Neocarzinostatin is a substance with a high molecular weight, around 6000. Although there are already several means of fighting cancer that have a high molecular weight own, e.g. B. Melanomycin, Peptimycin, Actinogan, Malinaycin, Enolmycin, etc., under-

709 678 Ϊ85

The neocarzinostatin according to the invention is completely different from all these known agents clear in terms of molecular weight, antibiotic effect, etc. In addition, the impact is widespread of neocarzinostatin in the fight against tumors and its high chemotherapeutic Properties that stand out, properties that are not found in any of the known anti-cancer drugs.

The microorganism which according to the invention provides the antibiotic neocarzinostatin has been isolated using ultraviolet radiation and the "single spore isolation" method. It is a variant of the species Streptomyces carzinostaticus, which is called Streptomyces carzinostaticus var. neocarzinostaticus. Strains of Streptomyces carzinostaticus var. Neocarzinostaticus received the laboratory designation F-41, F-42 (C-9544-JP 8) and E-793 F 51 (C-9544-JP 9). tribe F-42 (C-9544-JP 8) and E-793 F 51 (C-9544-JP 9) as the most suitable neocarcinostatin-producing strains have been deposited with the American Type Culture Collection, Rockville, Maryland, and placed in the inventory for microorganisms classified under ATCC No. 15945 and ATCC No. 15944.

The following text describes the characteristics of the new neocarzinostatin-producing variant of the Streptomyces carzinostaticus, the process used to separate what is known as neocarzinostatin The substance and its chemical and biological properties are described.

Bacteriological properties of Streptomyces carzinostaticus var. Neocarzinostaticus

1. Morphology

a) Conidial morphology.

The sporosphere of the conidium zigzags from a point on the air mycelium; the length is generally short; closed or open spirals beyond the whorl; no twisted whisk.

b) Conidium.

Under the electron microscope, the surface of the spores appears to be characteristic of the so-called "process type", more precisely, the extensions are more like hair than thorns. The shape of the spores is ovoid; their size is approximately 1.5 x 2.0 μ.

2. Properties in different agar media

medium growth Aerobic mycelium Soluble pigment Tsuabec (glycerin) agar medium Transparent slightly brownish gray no Asparagine agar medium Transparent Brownish gray no Potassium Malate Agar Medium Transparent, dissolves potassium malate slightly brownish gray no Nitrate with peptone water added Transparent, reduced nitrate salt no no Starch agar medium Transparent, releases strength Brownish gray no Tyrosine agar medium Transparent no no Potato slices Cream colored slightly brownish gray Yellowish
Brown Yellow beet slices Cream colored White to tea colored no Egg agar medium Transparent White no Blood agar medium Olive and hemolytic White to yellowish
Gray no Refrel coagulates blood medium Transparent, wrinkled, loosens
medium White no Gelatin medium Transparent, no liquefaction
of the medium White no Milk medium Yellow-brown, milk curdled,
Peptonization White no Ordinary agar medium Transparent, wrinkled White to yellowish
Gray no Cellulose no growth

Comment:

The above color names are based on "The Sumdard ofColor" by the Color Research Institute of Japan, issue 64.

3. Biochemical properties

a) Nitrite formation: occasionally.

b) Melanoid formation: none.

c) sugar consumption.

This strain is processed and decomposed in the agar medium Pridham and Gottlieb as the basic medium Dextrin, maltrose, sucrose, lactose, mannose, galactose, glucose, fructose, xylose, arabinose, Rhamnose, glycerin, mannitol and inositol good,

but only weakly: insulin, dulcitol and sorbitol. The growth in raffinose is somewhat unsatisfactory, but the organism seems to process them.

The properties mentioned differentiate our variant of the »Streptomyces carzinostaticus« fundamentally described by all others in Bergey's Manual of Determinative Bacteriology, 1957 edition Species. Of the species removed with the Streptomyces carzinostaticus var. Neocarzinostaticus can be compared, are only mentioned Streptomyces albus and Streptomyces griseolus. These two species and also Streptomyces pseudogriseolus (by Okami in 1955 as recognized a pseudo-xanthomycin producing species [s. J. of Antibiotics, A, p. 126, Vol. 8, 1955]) be included in the group of closest related species.

Streptomyces pseudogriseolus, however, forms a soluble brown pigment in a gelatin medium and dissolves the gelatin slowly to fairly quickly. These properties are absolutely different, because the variant on which the invention is based does not dissolve gelatin and also does not produce any soluble Pigment. In addition, the substance that Streptomyces pseudo griseolus produces is pseudo-xanthomycin, a low molecular weight anti-cancer agent.

Neocarzinostatin is obtained by cultivating the strain Streptomyces carzinostaticus var. Neocarzinostaticus under suitable culture conditions. All breeding methods customary for breeding other actinomycetes are also used in breeding des Streptomyces carzinostaticus var. neocarzinostaticus. Neocarzinostatin is obtained preferably by converting the neocarcinostatin-producing organism into a suitable Inoculated medium and then grown under aerobic conditions. Although different material than Nutrient can be used in the cultures, the medium is said to be, preferably as a nitrogen source an organic material such as B. peptone, meat extract, yeast extract, corn softened water, soybean meal, Peanut meal, hydrolyzed ^ protein substance, cottonseed meal, fish meal, soluble distillation preparations, and possibly inorganic stick- sources of material such as nitrates and ammonium salts, e.g. B. ammonium sulfate contain; as a carbon source serve dextrose, lactose, maltose, glycerin, molasses, sucrose, starch and other inorganic Salts such as B. sodium chloride, potassium chloride and magnesium sulfate; as a buffering agent Calcium carbonate or phosphate and traces of heavy metal salts can be used. Such Nutrient media ingredients are enumerated in Canadian Patent 513,324, British Patents 730 341 and 736 325 as well as in the USA patents 2,691,618, 2,658,018, 2,653,899, 2,586,762, 2,516,080, 2,483,892, 2,609,329 and finally 2,709,672. In the case of aerated, submerged fermentation, one uses an anti-foam agent, e.g. B. liquid paraffin, fatty oils or silicones. Also can be used for making of neocarzinostatin several carbon sources, nitrogen sources or antifoam agents are used will.

For a large-scale culture, the sub- ⁶ S merse, aerobic process has proven to be particularly suitable. The fermentation temperatures are usually between 25 and 30 ° C., but are preferred

at 27 to 28 ° C. The pH of the medium fluctuates between 6 and 9 and is optimally around 7. The neocarzinostatin-producing strains were grown with stirring for 2 to 5 days at 28 ° C and in sterile media (pH 7.2), the meat extracts, peptone, sodium chloride and calcium carbonate, but which have different carbon sources, e.g. B. glycerine, dextrose, sucrose, maltose, Starch, etc., it was found that the medium containing dextrose as a carbon source was the most stable medium and also gave the greatest yield of neocarcinostatin.

Adding yeast to the medium increased productivity. It also turned out that the Culture medium containing starch, sodium chloride, inorganic salts, yeast extract and soybean powder contained, greatly accelerated the growth of organisms.

Fermented at 28 ⁰ C with constant stirring and using the optimal nutrient medium (pH = 7.2), ie 3% textrose, 0.5% meat extract, 0.5% peptone, 0.5% sodium chloride and 0, 2% calcium carbonate, the pH of the medium fell to 6.6 to 6.0 within 24 hours and then rose again to 6.8 to 7.2 48 to 72 hours later. The filtrate obtained inhibited the growth of Sarcina lutea at a dilution of 1: 120. When added to normal cells, cell degeneration was found at a dilution of 1: 1200.

To obtain the neocarcinostatin, the culture medium is made using known methods, such as Filtration, centrifugation or similar processes, into a liquid part and a solid part, which is the Contains mycelium, divided. The water-soluble neocarcinostatin is mainly contained in the liquid part.

After separating the mycelium, it was difficult to remove the neocarzinostatin from the liquid part - and at all pH values - with organic solvents that are not mixed with water, z. B. ethyl acetate, butyl acetate, butanol and the like. To extract.

In some cases the neocarcinostatin in the liquid was destroyed. Although neocarzinostatin on When activated carbon is adsorbed, it is usually difficult to elute. However, it is possible to use neocarzinostatin on weak adsorbents, e.g. B. acid kaolin clay, phlorzyl, magnesol, etc., adjusted to pH 2 are acidified to adsorb; it is then poured into neutral water. It is also possible to adsorb or eliminate foreign constituents by having a suitable pH value the solution adjusts, e.g. B. pH = 7, with neocarzinostatin not being adsorbed. This procedure represents is a very advantageous and convenient cleaning method.

It is difficult to get neocarzinostatin in the nutrient solution using the usual ion exchange resins to clean. Neocarzinostatin is namely in a medium or in weakly alkaline aqueous Solution quite unstable. For this reason, it does not adsorb on the resin when it is acidified and with an anion exchanger, such as. B. Amberlite IRA-400®, IR 4 B or IRC-50®, brought into contact will. However, neocarzinostatin adsorbs on some strong cation exchangers, such as B. Amberlite IR 120. Even so, it's hard to neocarzinostatin with ammonia, water or a

Pour combination of water and organic solvents from the resin. In this context it may be said that Dowex-50 Na and H ion exchange resins are partly neocarcinostatin adsorb if you try the casting methods just mentioned, but only here inactive components can be poured off. It can be seen from this that when this Purification process the neocarzinostatin is not stable.

One of the most effective methods of separating neocarcinostatin is to obtain neocarcinostatin salted out from the aqueous solution, better still from a concentrated solution, with the addition of Ammonium sulfate, sodium sulfate, etc. or by adding zinc chloride or by precipitation appropriate pH and addition of methanol, ethanol, acetone.

Probably the best method of cleaning is to precipitate the neocarcinostatin, after adding an appropriate amount of a saturated aqueous solution or ammonium sulfate powder in slightly acidic water, and then by means of a semi-permeable film, such as. B. Cellophan®, dialyzed to remove ammonium sulfate and low molecular weight foreign matter will. With the same goal you can also use gel filter media, e.g. B. Sephadex G-25, G-50 and G-75; the neocarzinostatin is then isolated from the fractions. These filter media are commercially available crosslinked dextran polymers which form gels with water and act as molecular sieves act, d. that is, they absorb polyglucose molecules with molecular weights below 3000 and 7000, respectively or 10,000. Another useful way of purifying the neocarzinostatin is to use it a DEAE-Sephadex column (diethylaminoethyl-Scphadex).

Neocarzinostatin, which can be explained in more detail by the above-mentioned and later in examples The various procedures explained above have the following properties:

1. Neocarzinostatin is an acidic white powder; Analysis (free acid): C = 41.34%; H = 9.43%; N = 11.13%.

2. The melting point of neocarcinostatin is 260 ° C

3. The ultraviolet absorption spectrum of neocarzinostatin in aqueous solution is shown in Fig.l shown. It shows a weak absorption at 278 to 280 πΐμ with a peak at 290 ΙΤΙμ.

4. The infrared absorption spectrum is from FIG. 2 can be seen.

5. The measurement of the sedimentation constant of an aqueous solution of neocarzinostatin with a concentration of 10 mcg / ml gave the following value: S - ^ - - · 1.3, giving a molecular weight of 7000 is displayed (assuming that it is a spherical particle). One was used Spinco centrifuge model E (number of revolutions 52,640 rev./min.).

6. If the dispersion constant of an aqueous solution of neocarcinostatin with a concentration of 7.5 mcg / ml was also investigated using a Neurath apparatus at 20 ° C., the following value was obtained: Dr = 1.4; If the coefficient of friction was replaced by the value 5 - ^ - just mentioned, the molecular weight was 6000.

7. Necarzinostatin is very easily soluble in water; well soluble in hydrogenated methanol and hydrogenated Ethanol; slightly soluble, however, in methanol, ethanol, butanol; practically insoluble in Acetone, ethyl acetate, butyl acetate, ether, chloroform and petroleum ether.

8. Neocarzinostatin is more stable at acidic pH than at neutral pH and unstable at alkaline pH Area.

9. Neocarzinostatin is biuret positive and orcinol negative. It is hydrolyzed in a sealed flask for 18 hours at IOO ⁰ C with concentrated nitric acid, it is found amino acids, eg. B. aspartic acid, glycine, alanine, serine, glutamic acid, proline, leucine, phenyl-alanine, threonine, arginine, lysine, valine, cysteine, isoleucine, etc. 10. Neocarzinostatin can be obtained from an aqueous solution by means of precipitants, e.g. B. ammonium sulfate, phosphotungstic acid, zinc chloride and the like., Are precipitated. However, this cannot be achieved with pure ammonium, picric acid, flavic acid, methyl orange etc.

The most important biological properties of the substance are shown below:

1. Antibiotic activity

The antibiotic activity of the new substance can be seen in the table below.

Test bacteria Staphylococcus aureus 209 P. Staphylococcus aureus Kishima Staphylococcus aureus Smith ... Staphylococcus aureus 537 Sarcina lutea PCI 1001 Sarcina lutea Bacillus subtilis 219 Escherichia coli NHIJ Shigella flexneri 2a Shigella sonnei Salmonella typhosa Vibrio comma Proteus rettgeri Xanthomonas oryze Mycobacterium tuberculosis 607 Candida albicans

Minimal inhibitory concentration

mcg / ml

30 60 15 30 4 4 60

> 1,000> 1 OOO

1,000> 1,000> 1,000> 1,000

1,000> 1,000> 1,000

1

2. Action against tumors

The characteristic of neocarcinostatin is that besides its growth-inhibiting properties for microorganisms Concentrations also have a degenerative effect on various tumor cells possesses, even at an extremely low concentration, i. H. the product that at 2.0 mcg / ml the growth of Sarcina lutea is blocked, already shows at a concentration of 0.2 to 0.1 mcg / ml have a degenerative effect on cells grown in a test tube had been.

The effect of neocarcinostatin can also best be demonstrated by tumor tests' 5 with mice.

If you vaccinate z. B. Mice 4,000,000 Sarcoma 180 ascites cancer cells in the abdomen, then gives daily and 6 days for neocarzinostatin, 24 to 28 hours after tumor vaccination with Doses of 3.2 to 0.1 mg / kg (body weight) begin, so the formation of ascites markedly suppressed, with no toxicity occurring in the mouse and the life of the test animals at the same time is greatly extended. At doses of 3.2, 1.6 and 0.8 mg / kg, all were treated Mice recovered and lived on. This fact proves that the chemotherapeutic effect of the neocarcinostat is very large. Because the effective dose is between 0.1 and 3.2 mg / kg and the acute toxicity of the product in the mouse is 30 mg / kg, the therapeutic ratio is the minimum dose sufficient to prolong the life of the cancerous mouse at 300.

The product, applied at doses of 3.2 to 0.2 mg / kg, was able to save the life of the test mice significantly prolong when applied to mouse leukemia SN-36, i.e. a tumor, for for which there was no effective antidote at all.

The effect on the two types of cancer mentioned was found in the culture filtrate, confirmed during of the entire cleaning process, and finally it was found that the implementation of the cleaning process, with continuous testing of the inhibitory effect in Sarcina lutea, the same importance is to be attached as the extraction of the active active substance.

Even when using a raw substance obtained from ammonium sulphate deposition could activity even in Ehrlich ascites, ascitic mouse liver cancer MH 134, mouse leukemia L-1210, Ehrlich subcut. solid cancer, Sarcoma 180 subcut. solid cancer and Bashford's subcut. solid cancer can be detected. In order to have one with subcutaneous cancers (solid form) In order to achieve effective success it was preferably injected intravenously.

These excellent properties of Neocarzinostatins show that the invention Product is a new and very valuable substance.

Practical examples for the production and purification of the neocarcinostat are given below Text brought. From these examples it can be seen that by combining the various extraction methods a neocarzinostatin of very high purity can be obtained. It is clear to those skilled in the art that the insulation can also be achieved by 365

other or modified methods not specifically described can be used. These procedures too are claimed by the patent.

example 1

An aqueous culture medium with the following ingredients was prepared:

Starch 2.0%

De-oiled soybean powder 2.0 ° / o Dry yeast 0.5% Sodium chloride 0.25% Manganese chloride 0.0005% Copper sulfate 0.0005% Zinc sulfate 0.0005% Calcium carbonate 0.2%

After sterilizing and adjusting the pH to 7.0, 100 ml of the medium were poured into different Test flasks with a capacity of 500 ecm are introduced and sterilized. The strain Streptomyces carzinostaticus var. neocarzinostaticus F 41 was inoculated and then at a temperature of 27 ° C for 24 hours fermented for a long time with stirring.

The culture thus obtained was used as the mother culture for further work.

The following aqueous medium was then prepared for production:

Glucose 3.0%

Peptone 0.5%

Meat extract 0.5% Sodium chloride 0.5% Calcium carbonate 0.2%

After the sterilization had taken place, the medium was adjusted to pH 7.0. 100 ml each of this medium were placed in 70 sample flasks (500 ecm) and sterilized. Then you set 5 percent by volume of the above-mentioned mother culture to the medium in each of the test flasks and fermented, under Stirring, at a temperature - of 27 ° C. After an incubation time of 36 hours, the pH 6.6, after 48 hours 6.8 to 7.2. Later there were no more changes in the pH value. How to estimate the amount of neocarcinostat in the liquid according to its effect on Sarcina lutea examined, it was 40 mcg / ml in the 24-hour culture, 73 mcg / ml after 36 hours, 100 mcg / ml after 48 hours and 130 mcg / ml after 64 hours. The fermentation was interrupted after 64 hours, and the solids containing the mycelium were separated by filtration. One used Filter paper. 51 culture liquids were obtained with a content of 130 mcg / ml active ingredient.

Example 2

The liquid medium obtained according to Example 1 was, with the aid of a saturated oxalic acid solution adjusted to pH = 3.0 and then the resulting precipitate was taken up by filtration. 50 mg each of kaolin and Celite 545 in powder form (diatomaceous earth) were added to the filtrate, followed by 15 hours at 4 ° C stirred, and then chromoprotein was allowed to act in an optimally absorbent manner and then filtered. The obtained filtrate was divided and placed in cellophane bags; one blew on these sacks Dry air at a temperature of 27 ° C for 24 hours, about 600 ml of the contents condensing

709 -678 385

Claims (1)

sated. This now concentrated solution was at 4 ° C by means of cellophane dialysis for 24 hours in desalinated distilled water. The yield of desalted concentrated solution from the culture liquid was approximately 80% (867mcg / ml, 600 ml). Example 3 A concentrated solution, obtained according to Example 2, was stirred vigorously at 4 ° C, then was up to 25 percent by volume (150 mg) of solid ammonium sulfate added; the resulting brown precipitate was collected by centrifugation and washed thoroughly. Then another 150 mg of ammonium sulfate were added added and allowed to stand at 4 ° C for 15 hours. An evolving one gray-white precipitate was isolated by freeze centrifugation. The precipitate was with a cold aqueous ammonium sulfate solution washed several times, dissolved in 20 ml of distilled water and Dialized with distilled water overnight at 4 ° C. After desalting was done, the liquid became passed through a Sephadex G-25 column. The solution passed through was then lyophilized. Man received 660 mg of neocarzinostatin in the form of a slightly yellow, crude powder. By measuring the antibiotic potency against Sarcina lutea was found to be 330 meg / mg. The yield from the culture liquid of Example 1 was 56%. Example 4 30th 500 mg (330 mcg / ml) of the crude neocarzinostatin powder, obtained according to Example 3, were in 4.0 ml (pH 6.0, O.OOlmolar) phosphoric acid buffer solution dissolved, then to remove all insoluble Fabrics for 20 minutes at 10,000 rev / min. centrifuged and then chromatographed, d. H. 32 ml of DEAE-Sephadex A-25 was packed in a 2 cm column and with 0.001 molar Phosphoric acid buffer solution (pH 6.0) vigorously pretreated; then 4.0 ml of neocarcinostatin solution were added (pH 7.0) passed through the column. The adsorbed neocarzinostatin was determined by means of a Salt solution of increased sodium chloride concentration poured off. When performing the chromatography the active ingredient was poured off, a point showing for the first time as the transition to a sodium concentration of 0.15 mole was achieved. Which contain the active material Tending fractions were pooled and treated with distilled water for 24 hours at 4 ° C dialyzed, then lyophilized. The yield was 138 mg as a white powder. 44% Raw powders with a purity of 535 mcg / mg were obtained. The chromatographic was repeated Treatment, neocarzinostatin with a purity of 1000 mcg / mg was obtained. Example 5 Streptomyces carzinostaticus var. Neocarzinostaticus ATCC No. 15945 was prepared according to Example 1 fermented. After 64 hours, the culture fluid had actual activity against Sarcina lutea, and the fermentation stopped. The mycelium was separated, and one Liquid containing neocarzinostatin as an effective ingredient was obtained. After the Methods as described in Examples 2, 3 and 4 were then obtained neocarzinostatin with a purity of 1000 mcg / mg. Example 6 Streptomyces carzinostaticus var. Neocarzinostaticus ATCC No. 15944 was prepared according to Example 1 fermented. After 64 hours, the culture fluid had actual activity against Sarcina lutea, and the fermentation stopped. The mycelium was separated, and one Liquid containing neocarzinostatin as an effective ingredient was obtained. According to the methods as described in Examples 2, 3 and 4, neocarzinostatin was then obtained with a purity of 1000 mcg / mg. Claim: Process for the production of the antibiotic neocarzinostatin, characterized in that that Streptomyces carzinostaticus var. neocarzinostaticus ATTC 15944 and ATCC 15945 under submerged, aerobic conditions grown in a common nutrient solution and the antibiotic from the culture liquid through Precipitation or adsorption is obtained. Considered publications:
Japanese Patent Publication No. 267 158. 1 sheet of drawings 709 678 385 10.67 ® Bundesdruckerei Berlin