FR2534271A1 - PYRIDINE SOLUBLE MICROORGANISM EXTRACT AND PROCESS FOR THE PRODUCTION THEREOF - Google Patents

Abstract

PROCESS FOR PRODUCING A PURIFIED PYRIDINE SOLUBLE EXTRACT FROM A MICROORGANISM CONTAINING BETWEEN APPROXIMATELY 7 AND 20 BY WEIGHT OF PROTEIN, BETWEEN APPROXIMATELY 10 AND 16 BY WEIGHT OF SUGAR, AND BETWEEN APPROXIMATELY 35 AND 55 BY WEIGHT OF FATTY ACIDS . THE EXTRACT COMBINED WITH A CELLULAR WALL SKELETON IN A PHARMACEUTICALLY ACCEPTABLE MEDIUM IS USEFUL AS ANTI-TUMOR AGENT IN THE TREATMENT OF ANIMALS AND HUMANS.

Description

Microorganism extract soluble in pyridine and

process for obtaining it.

  The present invention relates to an extract soluble in pyri dine from a microorganism which, combined with a skeleton

  cell wall (SPC), provides a pharmaceutical composition

  ceutique possessing anti-tumor properties.

  Bacteria such as Corynebacterium parvum have made

  the object of experimental work to isolate them and character-

  the component to which inhibition of growth is due

  tumor growth (see for example Anti Tumor Activity and Lymphoreticular Stimulation Properties of Fractions Isolated from C parvum; Cantrell, et al, Cancer Research 39, pp 3554-3563 (September 1979)) Apart from its anti-tumor activity, C parvum has been shown to be a powerful stimulator of the lymphoreticular system, leading to an undesirable increase in the weights of the spleen and liver and to blastogenesis The Applicant has discovered that an extract of microorganism soluble in pyridine has

  powerful anti-tumor properties without the toxic effects

  that undesirable associated with the technical products

anterior.

  The cell wall skeleton is essentially a cell wall from which much of the protein and lipids normally found there have been eliminated. It is a polymeric acid mucopeptide mycoliquearabinogalactane containing remnants of trehalose mycolates ("P 3"). and undigested tuberculoproteins We get the skeleton

  cell wall from any micro-

  organism including, but not limited to, M smegmatis,

  M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacte-

  rium diphteria, Corynebacterium parvum, M kansasii, M tuber-

  culosis (strains H 37 RV and Ayoma B), and M bovis strain BCG.

  The cell wall skeleton can also be obtained from other microorganisms such as E coli, B abortus

and Coxiella burnettii.

  We can produce the cell wall skeleton by first cultivating and harvesting bacteria such as M bovis strain BCG (Bacille Calmette Guérin) We pass the residue

  whole cell resulting in a cell fractionator

  the (Ribi Cell Fractionator (Sorvall, Model RF-A)) which breaks

  cells, separating the outer envelope or cell wall

  the protoplasmic impurities layer The resulting cell walls are then subjected to a series of solvent extracts and enzyme treatments (for example trypsin and / or chymotrypsin) to arrive at the wall skeleton

purified cell.

  The object of the invention is therefore to provide a pharmaceutical composition containing an extract soluble in pyridine of a microorganism in combination with a skeleton of

cell membran.

  Another object of the invention is to provide a method for obtaining

  retention of the pyridine-soluble extract of a micro-

  organization. Yet another object of the invention is to provide a method of treating tumors in warm-blooded animals and humans, using the composition containing the pyridine-soluble extract of a microorganism and the skeleton

cell wall.

  The present invention relates to com-

  pharmaceutical positions comprising a pyridine-soluble extract of a microorganism, containing between approximately 7 and 20% by weight of protein, approximately 10 to 16% by weight of sugar and approximately 35 to 55% by weight of fatty acids

  combination with the cell wall skeleton (SPC).

  The extract preferably contains about 12% by weight of protein, as much sugar and about 45% by weight of fatty acids. Any microorganism can be used to obtain the pyridine-soluble extract, including for example

  M bovis BCG, M, phlei, M smegmatis, M kansasii, Nocardia-

  rubra, Corynebacterium'diphteriae and Corynebacterium 'parvum.

  Corynebacterium parvum is especially preferred.

  Whole micro-cells are mixed with pyridine

  organism, preferably in the form of a paste The resulting mixture is separated to obtain a supernatant fraction which contains the extract soluble in pyridine and a residue

  'Optionally, you can submit the residue -

  to repeated separation procedures as described

  above using pyridine to remove amounts

  additional tities of the desired extract.

  The pyridine is then removed from the extract and the dried extract is dialyzed against a suitable liquid such as distilled water The absence of whole cells or of fragments of cells constituting contaminants is confirmed by electron microscopy The purified extract resulting can then be lyophilized by known methods to obtain a stable product, The pyridine-soluble extract produced in accordance with the invention can be combined with SPC to provide a composition having a powerful anti-tumor activity without stimulating hypertrophy of the spleen and liver Cancers which can be treated with this composition include animal tumors such as bovine squamous cell carcinoma, bovine fibrosarcoma, equine sarcoldus, equine melanoma, equine squamous cell carcinoma, mammary tumors canines, canine adenoma and canine melanoma, and human tumors such as breast tumors, lung tumors, tumors of the colon, malignant melanoma, squamous cell carcinomas and

ovarian tumors.

  The composition is preferably administered by injection, in a pharmaceutically acceptable medium, such as an emulsion of oil droplets, directly in the tumor,

  under conditions described more particularly below.

  The above composition can be stabilized, for example by a lyophilization procedure and then reconstituted

without loss of activity.

  The amount of pyridine-soluble extract in a single injection for the treatment of animals is between about 375 and 2500 micrograms / milliliter The amount of SPC is between about 125 and 375 micrograms / milliliter. The number of milliliters injected into the tumor is determined by the size of the tumor according to the following table

  Animal dosage depending on the size of the tumor.

  t 30 Tumor diameter (cm) Quantity injected (ml) 0-1 to 0.5

1-2 0.5 to 2.5

2-3 2.5 to 5

3-5 5 to 10

-8 10 to 15

  greater than 8 15 to 20 The maximum dose per injection is approximately 40 milligrams

  both for the pyridine-soluble extract and for the SPC.

  The course of treatment includes up to six injections

  given about two weeks apart.

  The present composition in a suitable injection medium such as an emulsion of oil droplets is administered directly to human tumors The amount of extract

  soluble in pyridine in a single injection is included

  is between about 200 and 5,000 micrograms, preferably

  between about 800 and 1,200 micrograms, while the quan-

  tity of SPC is between about 50 and 2,000 micro-

  grams The preferred unit dosage for $ PC is between

  about 475 and 525 micrograms All the dosages men-

  above are based on an adult patient represented

  sentative weighing 70 kilograms The injections are administered

  approximately once a week and up to a total of

fifteen injections.

  As described above, the composition for the treatment of warm-blooded animals and humans can be used in the form of an emulsion of oil droplets. The amount of oil used is in the range from about 0.5 at

  3.0% by volume based on the total volume of the composition.

  It is preferred to use between about 0.75 and 1.5% by volume of oil. Examples of such oils include mineral oil, squalane, squalene, 7-n-hexyloctadecane, superior oil Conoco and Drakeol 6 VR mineral oil

  (produced by Penreco Company, Butler, Pennsylvania).

  The mixture containing the oil, homogenized, is then

  washed with a detergent which can optionally be dissolved in saline before mixing The amount of

  detergent is typically between about

  0.02 and 0.25% by volume and preferably between approx.

  0.10 and 0.20% by volume based on the total volume of the composition Any common detergent can be used, including Tween-80 and Arlacel (produced by Atlas Chemical Company), The resulting mixture is then homogenized adding detergent to form a slurry with a percentage

  high level of oil droplets coated by the components

  active health as observed

under the microscope.

  The following examples are given for illustration only.

  and are not intended to limit or redefine in any way the invention as claimed in the

appended claims.

  Example 1 Preparation of an extract soluble in pyridine

  from Corynebacterium Parvum.

  Corynebacterium Parvum (P acnes, sou-

  che 4182) at 370 ° C. in NIH thioglycolate broth for 48 to 72 hours to obtain a whole cell paste. The paste was then washed with 500 milliliters of distilled water. 90 grams (wet weight) of washed paste were mixed. with 200 milliliters of dry pyridine and centrifuged at 1,700 g for one hour at 40 ° C. A pyridine-soluble extract was removed in the form of a supernatant fraction The residue which remained was extracted again with pyridine under conditions identical to those described above After filtration using Whatman No. 1 paper, the pyridine extracts were combined and the solvent was removed by evaporation at 500 ° C. in a Buchi Rotavapor (Brinkmann Instruments, Westbury, New York). dried pyridine was dialyzed thoroughly against distilled water and then lyopohilised The resulting purified pyridine extract contained about 12% by weight of protein, about 12% by weight of sugar and about 45% by weight of fatty acids On examined the extract under an electron microscope and found it to be free from contamination with whole cells and cell wall fragments.

  the pyridine-soluble extract was 9% (8.1 grams).

  Example 2 Preparation of pyridine-soluble extract from M bovis strain BCG M bovis strain BCG was cultivated and harvested in Sautons broth at 370 ° C. for approximately three to four weeks to obtain a washed whole cell paste. then treated 50 grams (wet weight) of washed dough in the same way as in Example 1 to obtain a yield of

  7% pyridine-soluble extract (3.5 grams).

  The extract contained 15% by weight of protein, 10% by weight

  sugar and 52% by weight of fatty acids.

  Example 3 Test on guinea pig rfnl 10 tumors.

  Seven strain 2 guinea pigs with row 10 tumor sizes of approximately 9 millimeters were injected once

  diameter, 0.4 milliliter of a sterile drop emulsion-

  oil scraps, namely Drakeol 6 VR mineral oil

  (Pennsylvania Refining Company, Butler, Pennsylvania), story-

  300 micrograms of the pyridine-soluble extract prepared according to Example 1 and 50 micrograms of cell wall skeleton directly into the tumor tissue. The animals were examined after three months, and in six of the

  seven animals total regression had occurred.

  In a control experiment, six strain 2 guinea pigs with rank 10 tumor sizes were injected once

  about 9 millimeters in diameter 0.4 milliliter of the emulsion

  sterile oil droplet described above without pyridine extract or cell wall skeleton

  injections were made directly into the tumor tissue.

  None of the six tumors showed signs of regression

after three months.

Claims (11)

Claims.   1 Process for producing a pyridine-soluble extract, purified from a microorganism, comprising:   (a) the preparation of a paste of whole cells of the-   said bacteria; (b) washing said paste;   (c) reacting said paste with pyridine to pro-   down extract and residue; (d) removing pyridine from said extract; and (e) dialysis of said dried extract to obtain the extract soluble in purified pyridine.   2 Method according to claim 1, characterized in that said lyophilized extract soluble in purified pyridine.   3 Method according to one of claims 1 and 2, characterized   in that the microorganism is M bovis BCG, M phlei, M smegmatis, M Kansasii, Nocardia rubra, Corynebacterium diphtheriae or Corynebacteriumparvum and, preferably, Corynebacterium parvum.   4 Method according to one of the preceding claims, charac-   r 30 terized in that the residue is reacted with pyridine to obtain additional quantities of soluble extract in pyridine.   Pyridine-soluble extraction product obtained from a microorganism, comprising between approximately 7 and% by weight of protein, between approximately 10 and 16% by weight   sugar and between about 35 and 55% by weight of fatty acids.   6, Product according to claim 5, characterized in that each of the quantities of protein and sugar is approximately 12% by weight and that the quantity of fatty acids is approximately % in weight.   7. A pharmaceutical composition comprising a therapeutically effective amount of the product according to claim in combination with the cell wall skeleton and   a pharmaceutically acceptable carrier.   8 Composition according to Claim 7, characterized in that it is in lyophilized form and that the support is an emulsion of oil droplets, the amount of oil present being between 0.5 and 3.0% by volume on the   base of the total volume of the composition.   9 Composition according to one of claims 7 and 8,   characterized in that the oil is squalane, light mineral oil, squalene, 7-n-hexyloctadecane, superior oil from Conoco or mineral oil Drakeol   6 VR and a detergent is also present in the compound   sition for approximately 0.02 to 0.25% by volume based on the total volume of the composition.   10 Composition according to Claim 7, characterized in that the quantities of said pyridine-soluble extract and of the cell wall skeleton can each reach approximately 40 milligrams, the quantity of   Pyridine-soluble extractant being before-   suitably between about 200 and 5000 micrograms   and the amount of cell wall skeleton between approxi- ron 50 and 2000 micrograms.   11 A method of treating tumors in warm-blooded animals, characterized in that the composition according to claim 7 is administered to warm-blooded animals by carrying out up to six injections, at intervals of approximately two weeks, of the composition directly in the tumor, the composition advantageously containing between approximately 375 and 2500 micrograms per milliliter of the extraction product   soluble in pyridine and between about 125 and 375 milli-   grams per milliliter of cell wall skeleton. 12, A method according to claim 11, characterized in that the composition containing between   about 200 and 5000 micrograms, preferably between approx.   ron 800 and 1200 micrograms, of the extraction product soluble in pyridine and between approximately 50 and 2000 micrograms of cell wall skeleton, preferably between approximately 475 and 525 micrograms.