GB2123006A - Pyridine soluble extract of a microorganism - Google Patents

Abstract

A method of producing a purified pyridine-soluble extract of a microorganism is disclosed which contains between 7 and 20% by weight of protein, between 10 and 16% by weight of sugar, and between 35 and 55% by weight of fatty acids. The extract when combined with cell wall skeleton in a pharmaceutically acceptable medium is useful as an anti-tumor agent in the treatment of animals and humans.

Description

SPECIFICATION Pyridine soluble extract of a microorganism The present invention is directed to a pyridinesoluble extract of a microorganism which, when combined with cell wall skeleton (CWS), provides a pharmaceutical composition possessing antitumor properties, and to a process for its production.

Bacteria such as Corynebacterium parvum have been the subject of experimental work to isolate and characterise the component responsible for inducing inhibition of tumor growth (see, for example, Anti-tumorActivity and Lymphoreticular Stimulation Properties of Fractions Isolated from C. parvum; Cantrell, et al, Cancer Research 39, pgs. 3554-3563 (September, 1979). Apart from anti-tumor activity, C. parvum has shown to be a potent stimulator of the lymphoreticular system resulting in undesirable increases in spleen and liver weights and blasto-genesis. Applicant has discovered that a pyridine-soluble extract of microorganism possesses potent anti-tumor properties without the undesirable toxic effects associated with the prior art products.

Cell wall skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid arabinogalactan mucopeptide containing remnants of trehalose mycolates ("P3") and undigested tuberculoprotines. Cell wall skeleton is obtained from any microorganism including, but not limited to, M.smegmatis, M.phlei, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheria, Corynebacterium parvum, M.kansasii, M.tuberculosis (Strain H 37 RV and Ayoma B), and M.bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such other microorganisms as E.coli, B.abortus and Coxiella burnettii.

Cell wall skeleton may be produced by first growing and harvesting bacteria such as M.bovis Strain BCG (Bacillus Calmette-Guerin). The resulting whole cell residue is processed through a cell fractionator (Ribi Cell Fractionator (Sorvall, Model RF-1)) which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g., trypsin and/or chymotryspin) to give purified cell wall skeleton.

According to one aspect of the invention there is provided a process for producing a purified pyridine-solubie extract of microorganism comprising: (a) preparing a whole cell paste of said bacterium; (b) washing said paste; (c) reacting said paste with pyridine to produce an extract and a residue; (d) removing pyridine from said extract; and (e) dialyzing said dried extract to obtain purified pyridine-soluble extract.

According to another aspect of the invention there is provided a pyridine-soluble extract product obtained from a microorganism comprising between about 7 and 20% by weight of protein, between about 10 and 16% by weight of sugar and between about 35 and 55% by weight of fatty acids.

The extract may contain cell wall skeleton (CWS). It preferably contains about 12% by weight of each of protein and sugar and about 45% by weight of fatty acids.

Any microorganism may be used to obtain the pyridine-soluble extract including, for example, M.bovis BCG, M.phlei, M.smegmatis, M.kansasii, Nocardia rubra, Corynebacterium diphtheriae and Corynebacterium parvum. Corynebacterium parvum is especially preferred.

Whole cells of the microorganism, preferably in the form of a paste, are mixed with pyridine. The resulting mixture is separated to obtain a supernatant fraction which contains the pyridinesoluble extract and pyridine residue. Optionally, the pyridine residue may be subjected to repeated separation procedures as described above using pyridine to remove further quantities 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 cell fragment contaminants is confirmed by electron microscopy. The resulting purified extract may then be lyophilized by known methods to obtain a stable product.

The pyridine-soluble extract produced in accordance with this invention may be combined with CWS to produce a composition having potent anti-tumor activity without stimulating the induction of spleen and liver enlargements. The cancers which may be treated by this composition include animal tumors such as bovine squamos cell carcinoma, bovine fibrosarcoma, equine sarcoid, equine melanoma, equine squamous cell carcinoma, canine mammary tumors, canine adenoma and canine melanoma and human tumors such as breast tumors, lung tumors, colon tumors, malignant melanoma, squamous cell carcinomas and ovarian tumors.

The composition is preferably administered by injection in a pharmaceutically acceptable medium such as an oil-droplet emulsion directly into the tumor under conditions more particularly desribed below. The aforesaid composition may be stabilised as for example, by a lyophilization procedure and then reconstituted without loss of potency.

The amount of the pyridine-soluble extract in a single injection for the treatment of animals is between about 375 and 2500 micrograms/milliliter. The amount of CWS is between about 125 and 375 micrograms/milliliter.

The number of milliliters of the biologic injected into the tumor is determined by the size of the tumor in accordance with the following table: Animal dosage according to tumor size Diameter of Amount of biologic tumor (cm) injected (ml) 0--1 up to 0.5 1-2 0.5to2.5 2-3 2.5to5 3-5 5 to 10 5-8 10 to 15 greater than 8 15to20 The maximum dose per injection'is about 40 milligrams for each of the pyridine-soluble extract and CWS. The course of treatment comprises up to six injections administered at about two week intervals.

The present compostiion in a suitable injection medium such as an oil-droplet emulsion is administered directly into human tumors. The amount of the pyridine-soluble extract in a single injection is between about 200 and 5000 micrograms, preferably between about 800 and 1200 micrograms, while the amount of CWS is between about 50 and 2000 micrograms. The preferred single dosage level for CWS is between about 475 and 525 micrograms. All of the abovementioned dosage levels are based on a typical 70 kilogram adult patient. The injections are administered about once every week for up to a total of 1 5 injections.

As described above, the composition for treatment of warm blooded animals and humans may be used in the form of an oil droplet emulsion. The amount of oil used is in the range between about 0.5 and 3.0 percent by volume based on the total volume of the composition. It is preferred to use between about 0.75 and 1.5 percent by volume of the oil. Examples of such oils include light mineral oil, squalane, squalene, 7-n-hexyloctadecane, Conoco superoil and Drakeol 6 VR mineral oil (produced by the Penreco Company, Butler, Pennsylvania).

The homogenized oil containing mixture is then combined with a detergent which may optionally be dissolved in a saline solution prior to mixing.

The amount of detergent is typically between about 0.02 and 0.25 percent by volume and preferably between about 0.10 and 0.20 percent by volume based on the total amount of the composition. Any common detergent material may be used including Tween-80 and Arlacel (produced by the Atlas Chemical Company)l The mixture resulting from the addition of detergent is then homogenised to form a suspension which has a high percentage of oil droplets coated with active components as determined by observation under a microscope.

The following examples are for illustrative purposes only and are not intended to limit or in any way redefine the invention as claimed in the claims appended hereto.

Example 1 Preparation of pyridine-soluble extract from Corynebacterium parvum Corynebacterium parvum (P.acnes, Strain 4182) was grown and harvested at 370C in NIH thioglycolate broth for between 48 and 72 hours to obtain a whole cell paste. The paste was then washed with 500 ml of distilled water. 90 grams (wet weight) of the washed paste was mixed with 200 ml of neat pyridine and centrifuged at 1700xg for one hour at 40C. A pyridine-soluble extract was removed as a supernatant fraction.

The remaining residue was extracted with additional pyridine under identical conditions as desribed above. Following filtration, using Whatman No. 1 paper, the pyridine extracts were pooled and the solvent was removed by evaporation at 500C in a Buchi Rotavapor (Brinkmann Instruments, Westbury, New York).

The dried pyridine extract was extensively dialyzed against distilled water and then lyophilized. 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. The extract was examined under an electron microscope and found to be free of contaminating whole cells and cell wall fragments. The yield of the pyridine-soluble extract was 9% (8.1 g).

Example 2 Preparation of pyridine-soluble extract from M.bovis strain BCG M.bovis strain BCG was grown and harvested in Sautons medium at 370C for between 3-4 weeks to obtained a washed whole cell paste. 50 grams (wet weight) of the washed paste was then treated in the same manner as Example 1 to produce a yield of the pyridine-soluble extract at 7% (3.5g). The extract contained 15% by weight of protein, 1 0% by weight of sugar and 52% by weight of fatty acids.

Example 3 Guinea-pig line-i 0 tumor tests Seven strain 2 guinea pigs having Line-lO tumor growths of about 9mm in diameter were injected once with 0.4 ml of a sterile oil droplet emulsion, i.e., Drakeol 6 VR mineral oil (Pennsylvania Refining Company, Butler, Pennsylvania), containing 300 micrograms of the pyridine-soluble extract prepared in accordance with Example 1 and 50 micrograms of cell wail skeleton, directly into the tumor tissue.

After three months, the animals were examined and in 6 of the 7 animals, total regression had occurred.

In a control experiment, six strain 2 guinea pigs having Line-lO tumor growths of about 9mm in diameter were injected once with 0.4 ml of the sterile oil droplet emulsion described above without the pyridine extract or cell wall skeleton.

The injections were made directly into the tumor tissue. None of the six tumors showed any signs of regression after three months.

Claims (13)

Claims

1. A process for producing a purified pyridinesoluble extract of microorganism comprising: (a) preparing a whole cell paste of said bacterium; (b) washing said paste; (c) reacting said paste with pyridine to produce an extract and a residue; (d) removing pyridine from said extract; and (e) dialyzing said dried extract to obtain purified pyridine-soluble extract.

2.The process of claim 1 further comprising lyophilizing said purified pyridine-soluble extract.

3. The process of claim 1 or 2, wherein the microorganism is M.bovis BCG, M.phlei, M.smegmatis, M.Kansasii, Nocardia rubra, Corynebacterium diphtheriae or Corynebacterium parvum and preferably Corynebacterium parvum.

4. The process of any of the preceding claims characterised by reacting the residue with pyridine to obtain additional amounts of the pyridine-soluble extract.

5. A pyridine-soluble extract product obtained from a microorganism comprising between about 7 and 20% by weight of protein, between about 10 and 1 6% by weight of sugar and between about 35 and 55% by weight of fatty acids.

6. The product of claim 5 wherein the amount of protein and sugar is each about 12% by weight and the amount of fatty acids is about 45% by weight.

7. A pharmaceutical composition comprising a therapeutically effective amount of the product of claim 5 in combination with cell wall skeleton and a pharmaceutically acceptable carrier.

8. The composition of claim 7 in lyophilized form and wherein the carrier is an oil droplet emulsion, the oil being present in amount between 0.5 and 3.0% by volume based on the total volume of the composition.

9. The composition of claim 7 or 8, wherein the oil is squalane, light mineral oil, squalene, 7-n- hexyl-octadecane, Conoco superoil or Drakeol 6VR mineral oil and there is also present in the composition a detergent in an amount between about 0.02 and 0.25% by volume based on the total volume of the composition.

10. The composition of any of claims 7 to 9 wherein the amount of each of said pyridinesoluble extract product and cell wall skeleton is up to about 40 milligrams and suitably, the amount of the pyridine-soluble extract product is between about 200 and 5000 micrograms and the amount of cell wall skeleton is between about 50 and 2000 micrograms.

11. A process according to claim 1 substantially as described herein with reference to any one of the Examples.

12. A composition according to claim 5 substantially as described herein with reference to any one of the Examples.

13. A composition according to any one of claims 5 to 10 or claim 12 when prepared by a process according to any of claims 1 to 4 or claim 11.