DE1467765A1 - Process for the production of therapeutic masses - Google Patents

Description

PATENT LAWYERS

DR. F. ZUMSTEIN - DR. E. Al DR. R. KOENIQSBERQER - DIPL-PHYS. R. HOLZBAUER

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TEUe «RnMME: ZUMPAT 8MÖNOHENS.

BRÄUHAUSSTRA8ae 4 / in <ONTO: MDNOHEN 911S9

BANK ACCOUNT:
HMUO H. AUFHAUSER

AIKUAM CHEMISfS LIMITED, London E.G. 3 / England

▼ experienced in the production of therapeutic maeeen

Invention relates to new therapeutic measures and methods for their production.

the present invention has been found that • tiaafte Extracts that contain animal species Knoohenaark in the following a valuable activity on the Waohetuwahewaung of Yuaorsellen »own. the New extracts according to the invention and therefore useful for the Treatment of various forms of cancer in aneöhliohen Body and also in the treatment of other pethological conditions, a.B. from Beetrahlangeeohaden »

The new inventive Ifaeaen are aue in Prinsip dear Xnoohenaark of sick animals that have suffered from a sooparaei-

suffer from tary infection «and in particular from bone marrow» the Leucooytosle has «received. Especially need » bare masses can be obtained from the bone marrow of animals somlaeis and allied and davit related conditions are ill. It is particularly advantageous "the new masses according to the invention are derived from animals" who have contracted infections of the stationary type "as occurred in Central, South or East Africa, especially in cattle and doh vines. Infected bone marrow for use in the present invention has been obtained from, for example, Lobatsl, Nairobi and Kitsumu obtain. It is not mentioned that the bone marrow, that is used for the purposes of the present invention often hypertrophic and / or sizeable ante-mortem bacterial infection can and in particular Infections by gram-negative organisms · elneohlieSlioh of various forms of coli species.

The new therapeutic masses are in essence water soluble and are therefore isolated from bone marrow of the stationary type by a process in which an aqueous Phase or an organic, hydroxyl group-containing Solvent is applied.

3He present invention therefore relates to a method for the preparation of a therapeutic composition which is valuable ^v

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ftyiroxylgruppen-twnfclgw »! Msnngssittel is teoataktlert.

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or etnas Sohlelfelttela, see B. Send. As a suitable orsanleefae

Slttel kflnoen ζ · Β · are called monoei hnhole ^ like ethanol and isopropanol "or auob polyalcohols""le QlyomrlM and PropflenglvlGol · Sine worded out-singing

Senlelfialttel in Oegamert von Glycerin, «obel each for

100 Oewlontetelle Knoohenwark about 10 Oewlcfatstelle

SenlellMLttels and about 28 Oewiohfcstelle Olycerln

will. The so obtained olympic extract is mt norsaler pnyelologi «rather Koehealsieetaae w wnd die The solution obtained in this way is filtered through a Seltx filter eterllieiert, so that an Injlderhares preparation is necessary Peutlschen properties and a seed content of solids Sound at least about 0.3 ns / nl is obtained.

ÖAD 809812/1316

In general, it is beneficial to the bone marrow: first of all treatment with a Pett solvent subject to extract fat, and then the defatted material. welter to treat the water-soluble Extract or isolate material su. Suitable degreasing agents for the previous degreasing step are e.g. liquid hydrocarbon and chlorinated hydrocarbon solvents such as carbon tetrahydrofuran, Chloroform, trichlorethylene and especially petroleum fractions such as petroleum ether.

The defatted material is then conveniently subjected to extraction with an aqueous phase which optionally an aqueous one containing hydroxy groups may contain organic solvent. If the degreased material consists mainly of solid matter, which may feel greasy to the touch, treatment with a water-miscible ketone, e.g. acetone, in order to obtain a satisfactory product for the subsequent extraction. However, there is a possibility that the ketone extract may have one Contains part of the oarclnogenic substance, so that carrying out the ketone treatment is not desirable in all cases is. The defatted material is then treated with an organic solvent containing hydroxyl groups extracted and then the extract is mixed with water. In principle, different hydroxyl group

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containing solvents are used, but the Use of a solvent «such as ethanol, isopropanol, Propylene glycol or glycerin preferred. The extract obtained is conveniently diluted with water.

Solutions obtained as described above have usually a total solids content of about 0.3Ji with an ammonia content of 0.04-0.2 mg / ml and generally 0.1 mg / ml.

When the defatted material consists of relatively small amounts of solid suspended in an aqueous medium, as is generally the case when If bone marrow from Nairobi is used, it is advantageous to filter or to filter this aqueous suspension centrifuge to extract the solid residue with glycerol and / or water, taking from the combined extract or extracts and the aqueous filtrate a solution is obtained, which after sterilization for injection suitable 1st. This injection solution can also contain injectable binders, such as glycerine and / or Kresol B.P. contain, generally between about $ 5 and $ 8 based on total solids content are. Preferably this solution is diluted before the injection so that it can be used for therapeutic purposes. injectable solution containing 5 # of total solid material, glycerin and 0.3 µl of cresol.

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The aqueous extracts obtained by the process described above are fundamental ready for use as soon as they have been sterilized and standardized if necessary.

The new compositions according to the invention are complex mixtures. They contain various amino acids, one or more of the following may be present: Leucine, isoleucine, methionine, cystine, threonine, lysine, Valine and L-phenylalanine. The hates according to the invention can also contain various vitamins, in particular those of the B complex.

The masses also contain serum proteins, including particularly albumins and globulins. Properdln seems to be present as well.

It is also assumed that the invention Containing masses of one or more indole derivatives, possibly of the tryptophane type. Sines or more enzymes may also be present, probably of the proteinase type.

The wet also contain one or more arooatlsohe Primary amine compounds, along with one or more purine bases. Chromatography of the invention new hatred shows the presence of many substances,

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including those of the types mentioned above. The Mac « Sen can also contain a lipopolysacoharide.

Your chemical elements Molybdenum and Elsen can »

if present eein.

Hats also show anti-oleic acid activity.

The compositions according to the invention are preferably formulated as sterile aqueous solutions for injection « preferably in the form of ampoules or other suitable containers, each of which contains e.g. can. Good results have been obtained by administering 0.23-1 ml at 7-14 day intervals.

The masses are preferably administered by subcutaneous or intramuscular injection.

Various forms of cancer can be treated with the new compositions of the invention, including Adenocarcinoma, sarcora, myelomatosls and melanotisohem Careinoma.

To explain the invention is hereinafter referred to as Example given a general representation instruction for one of the novel masses according to the invention.

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The bone marrow from the infected animal is unworthily collected immediately after slaughter and, if it is not treated immediately to extract the fixed tracts, "it is best to preserve it in some way" e.g. by packing it in polyethylene bags and the sealed polyethylene bags in vacuum bottles frozen "or, optionally, by treatment with chloroform and / or storage at relatively low temperatures, e.g. at around 0 - 4 ° C.

Preferably, bone marrow degreasing is carried out next, for example by extensive simple or multiple extraction with a fat solvent, e.g. petroleum ether (4o / 6O) or carbon tetrachloride. 10 - 30 1 fat solvents can be convenient for each Kilo marks are used.

The defatted pulp is then treated with an organic solvent containing hydroxyl groups, which is am most advantageously a polyalcohol and, in particular, glycerine is. This treatment is advantageously carried out by pasting or grinding the marrow with the solvent causes 10-25 ml of solvent per kilo of bone marrow (calculated on non-degreased material) one represent a suitable amount. Then water is added to the mixture and pasting or trituration is continued; the amount of water is advantageously chosen so

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that a mixture is formed that contains 1 part of solvent

Contains 9 parts by volume of water.

The aqueous extract is then separated from the solid material by filtration using paper pulp, to separate colloidal and semi-colloidal materials. The extract is then sterilized, e.g. by aseptic filtration through a sterilization filter.

The Amlnoatickstofl'-Qehalt the aqueous extract is determined conveniently by the method of Pope and Stevens (J.Bioohem. (1959). 22 "1070).

If desired, the preparations can be mixed with buffering agents, e.g. sodium phosphate, and / or preservatives, e.g. cresol B.P. or m-cresol can be formulated. The pH is conveniently in the range of 6.5-3.

The following examples illustrate the invention. example 1

10% by weight of sand (8.98 g) turns into 89.9% white marrow from oetafrlkanisohem cattle suffering from cysticercosis, added and the Oemisoh is made with 20 ml of pure glycerin offset. The mixture is used in a mortar for 5 minutes to marry a pestle. Then it is left to stand for 5 minutes and then mixed with 225 ml of normal saline solution. The Genftsch is stirred for 5 minutes and then through a

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Whatman Ho.! Filter paper filtered. Then the Filfcrafc is filtered under aseptic conditions through a Sterllisations-Seltz-Pilter and the sterile Piltrat obtained in this way is then suitable for injections. The solution has a Oeaantgehalt to Peststoffen of about 0 _f J>%.

Belapiel 2

22.7 X (5 gallons) carbon tetrahedral B.P. were in a vessel brought in «the one with a stirrer and one open the floor tap was fitted. Stirring was started and within an hour to the carbon tetra chloride in small pieces 49T0 g Bone marrow added, which was obtained from wholesaled cattle and obtained from pigs infected with cystioerosis infection and which had been preserved until use by keeping it at a temperature of 0-4 ° C had been kept in the presence of 20 com chloroform per kilo of bone marrow. Stirring was continued for a further 1 j | hours continued and thereafter the liquid was allowed to separate.

During the separation period it accumulated on the surface a brownish, greasy foam of the liquid, while the bone chips sank to the bottom of the vessel. The lower one Schioht was removed through the drain cock on the bottom, leaving the bones and foam in the jar.

The liquid was yellow in color and rather cloudy, obwoal no significant residue was left on the paper, when a sample of the liquid was filtered through filter paper.

Ss became about 11.4 liters (2-4 gallons) of raw carbon tetrachloride B.P. added to the residue in the vessel and the mixture was stirred for about 20 minutes. Then it was Left overnight. The next morning the solution was stirred for an additional 15 minutes and then allowed to settle. Eventually a clear, pale yellow solution was obtained, together with a brownish layer of sohweed and the more seiger ones Bone particles. The liquid was removed through the floor drain cock. " with the unochen the fibers, etc. held back. The bones were then removed from the jar along with the light material and held for three hours vigorously with 5 1 acetone B.P. touched. The slurry was then decanted into a suction filter (Buohner funnel), whereby In this procedure, the main amount of bone in the Bottle remained. The filter cake was brownish in color and was again In 2 | 1 suspended acetone and more Stirred for 2 hours and then filtered. The filter cake was washed on the suction filter with 1 liter of acetone ur "! left to dry at room temperature.

Weight of discarded bones 110 g. Weight of air-dry product 92.5 g corresponding to 1.86% by weight of bone marrow used.

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The air-dry residual material was then in triturated in a mortar, the glycerine being added in an amount of 20 mg / kg of original starting material, that is, 100 ml of glycerin was used since approximately 5 kg of starting material was used. The glycerin and the remaining material was ground in the mortar for 30 minutes and afterwards 900 ml of double-glass distilled were added Water was added to make a final solution of $ 10 (v / v) glycerin to obtain. The resulting sludge was then transferred to an appropriately sized vessel and stirred for one hour and then filtered through a cloth. The volume of the filtrate was measured and the residue on the cloth was measured with so much Double-glass distilled water washed that the filtrate volume was filled to 1 1.

The piltrat was very cloudy; it was stirred and with about 20 g pilter porridge (Pord No. 428) mixed in small pieces and when the addition of the filter slurry was complete, stirring was continued for an hour. The mixture was then into a 13 cm suction filter, which is applied to a 2! suction bottle was, decanted. Once the pulp had formed a thick bed at the bottom of the funnel, but not earlier, the pump was gradually turned on and the mash compressed into a firm bed. The filtrate was returned to the filter funnel two or three times and filtered again until the final filtrate was only sloppy. The sloughy filtrate was then filtered through a sterilization

«Η q R 19/1 3 ι κ

Filter pad (Ford 2k "steraraat") filtered under atmospheric pressure. The filtration was fairly rapid at first, but slowed down; as soon as the filtration became very slow, the sterilization filter pad was replaced. The resulting piltrate was suitable for use in the manufacture of injectable preparations.

Example 3

A preferred embodiment consists of the following Process steps:

a) degreasing the bone marrow of East African cattle, suffering from cystioercosis by stirring with carbon tetrachloride or preferably petroleum ether in the cold,

b) separating the residue, which is aqueous in nature, but contains solids,

o) Clarification of this aqueous residue by filtration or centrifugation (filtrate ^B A ⁿ ) and vigorous shaking of the separated solid with a small amount of water and filtration (filtrate ^Β Β ^η ) and combining the filtrates "A ⁿ and" B ",

d) keeping this solution under vacuum in order to dissolve Remove carbon tetrachloride or petroleum ether, and Filtration to separate precipitated fat,

e) Examination of this solution for total solids content (usually between 5 and 8 pounds),

f) Adding glycerin and cresol B.P. so that the final concentration at these connections are 2 or * 0, j5 #, and an-

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then dilute with water for injection so that the preparation contains $ 5 total solids, and g) Sterilize the final solution by aseptic filtration and aseptic filling into sterile vessels.

9 kg of freshly soled bone marrow from Nairobi Cattle suffering from cystioeroosls «are in 22.7 1 (5 gallons) of carbon cetranium chloride and a Stirred for an hour, during which time most of the fat is dissolved. The mixture is left to stand overnight and the lower Carbon tetrachloride layer then peeled off. Another 11.4 liters (2 s gallons) of carbon tetrachloride will be added added, the mixture is stirred for two hours and then left to settle for 1.5 hours. The lower carbon tetrahedral layer is drawn off and the reddish-brown sludge is poured into a 10 l oven with 2 l carbon tetra chloride transferred and the mixture is stirred for one hour and then left to stand overnight.

The carbon tetrachloride layer is siphoned off and the remaining aqueous slurry was centrifuged at 2400 rpm for one hour. The separating red liquid is saved and the remaining solid is vigorously stirred with 130 ml of water for a few minutes using of a high speed a MIsoh / Deeintegratore with sharp Knives. The suspension obtained is centrifuged and the separated liquid to the previously obtained red

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Liquid added. The combined liquids are » The placed in a desiccator, which is kept under vacuum and provided with suitable solvent traps is, and there will be traces of carbon tetrachloride removed within two hours.

The cloudy liquid is filtered until it is clear, a total volume of 700 ml with a total solids content of 5 # 7 # being obtained. The total volume is adjusted to 798 ml with glycerine »cresol BP and water and the binding solution (2% glycerine, 0.3 # cresol BP, total solids content $ 5) is sterilized by filtration through a sterilization pad into a sterile container.

Example 4

18OO g bone marrow from Kenya suffering from Cystlcercosls » since it was essentially free of bone and contained only small amounts of blood, it was broken into rather small pieces and extracted for 24 hours with 15 1 petroleum ether (40/60) with gentle stirring. The bone marrow was then Allowed to settle and the resulting clear solution decanted from the residue. The residue was further degreased by a second and third extraction using 15 liters and 3 liters of petroleum ether, respectively. After the third extraction the petroleum ether and the residue were filtered better than decanted. Petroleum ether is known according to the usual methods

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to be recovered.

The obtained defatted rake was air-dried to give a coarse brown powder, the smell of which is reminiscent of gland extracts such as pituitary powder. The yield of defatted Mark was 86.5 g ($ 4.26).

The air-dry product was ground to a thin paste with 56 ecm glycerine 10 minutes, which was then gradually diluted with 560 com n-3 salt solution. The resulting suspension was clarified by filtration through a bed of filter slurry and then through a sterilization pad. 0.1% cresol was added to the yellow-brown solution obtained and the volume was adjusted to 360 ecm. After a further filtration through a Seitz sterilization filter, the product was aseptically filled into 10 ccm bottles. The product was sterile, pyrogen-free and had no detectable toxicity. 1 ecm of injectable material was produced from each 5 g of the original, non-defatted cysticercosis diseased pulp.

Example 5

Following the procedure of Example 4, 2430 g of the bone marrow were defatted, with 120 g of product (Yield 4.9 #). 86.5 g of the air dry Product resulted in 490 ecm of an injectable solution, so that 1 ecm of injectable material from each 5 g of the original

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lichen, non-defatted, cysticercosis-afflicted marrow was obtained. The product was sterile, pyrogen-free and showed no detectable toxicity.

Example 6

30 g of air-dry intermediate product according to Example 5 were extracted with glycerol according to the method of the catfish examples wherein the extraction was carried out 5 ~ ₂ minutes instead of 10 Hinuten. 120 .mu. of injectable solution were obtained, corresponding to 1 ecm of injectable material from 5 g each of the original, non-defatted, oysticeroosis-diseased pulp. The product was sterile, pyrogen-free and had no detectable toxicity.

Example 7

5000 g oystioeroosls-diseased bone marrow were defatted according to the general production method of Example 4, 200 g air-dry material (yield 6.65ε) were obtained. This product yielded 600 ecm of an injectable solution, so that 1 ecm of injectable material from each 5 g of the original, non-defatted, cysticercesis sufferers Bone marrow was obtained. The product was sterile, pyrogen-free and had no detectable toxicity on.

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Example 8

According to the procedure of Example h , 3000 g of cysticercosis-diseased bone marrow were defatted, whereby Il6 g of air-dry material (yield 3 * 7 $) were obtained. This product yielded 500 ecm of an injectable solution, so that 1 com of injectable material was obtained from 10 g of the original, non-defatted, cysticceroosis-diseased pulp. The product was sterile, pyrogen-free and showed no detectable toxicity.

Example 9

According to Example h , 4000 g of the cysticercosis-diseased bone marrow were defatted, 223 S of air-dry product (yield 5 # 6 #) being obtained. This product gave 400 ecm of an injectable material, so that in each case 1 ecm of injectable material was obtained from 10 g of the original, non-defatted, cvaticeroosis-diseased pulp. The product was sterile, pyrogen-free and showed no detectable toxicity.

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Claims (2)

Claims ) Process for the preparation of a therapeutic mass for use in controlling the growth of tumor cells «characterized in that it consists of bone marrow of animals «such as cattle passing through a zoo-parakeet Infection like cysticeroosis »trypanosoniasis or one Hydatid disease, essentially water-soluble Substances extracted by contacting the bone marrow with water or an organic * solvent containing hydroxyl groups. 2. The method according to claim 1, characterized in that The bone marrow can be treated beforehand with a fat solvent, such as a liquid hydrocarbon or chlorohydrocarbon solvent., degreased. 8 0 9 8 12/1316