NZ248714A - Oncolytic bacteria and its use in the diagnosis and treatment of cancer - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £48714 2 4 8 7 14 Patents Form 5 Priority Date(s): . JTl*. .'.T.?'. .

Complete Specification Filed: Class: .

Publication Date: P£ P.

P.O. Journal, No: ...

NO DRAWINGS N.Z. No.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION i TREATMENT AND DIAGNOSIS OF CANCER We, ERA-MASIS LTD., an Israel company of c/o CASPI & CO., 33 Yavetz Street, Tel Aviv 65 258, Israel do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- pAT£ft?T OFFICE 2 > SEP 1993 f piSyST - 1 - (Followed by 1A) 24 8 7 1 r TREATMENT AND DIAGNOSIS^ CANCER lOuji uuirtjui 101 10QQ FIELD OF THE INVENTION The present invention relates generally to oncolytic bacteria and more particularly to a bacterial formulation for the treatment of cancer, as well as to a method for the early diagnosis of cancer.

BACKGROUND OF THE INVENTION In spite of the vast research and clinical effort which has been made in the past, in relation to investigation of the occurrence, diagnosis and treatment of cancer, many problems of an etiological and pathogenetic nature, and which bear on the identification, prevention and treatment of cancer, must be regarded as still in need of a solution.

However, it is generally agreed that early diagnosis of the disease is almost always a prerequisite of successful treatment. For example, the WHO Expert Committee's Report on Early Detection of Cancer (1969) stated that over half'of cancer patients could have been cured if the disease had been detected at an early stage and treated soon after detection. In view of the widespread incidence of the disease, mass screening techniques would evidently be of great value, but have not been instituted on a worldwide basis up to the present time, such as is available for example, at least in developed countries, in the field of tuberculosis of the chest by means of mass X-ray examination.

Among previous proposals for the diagnosis of cancer may be mentioned the following. In US 3^7651^. there was described a method of detecting cancer cells by staining test cells with acriflavine-HCl solution, determining indirectly the dye absorbed by the test cells and comparing with * a control. JP 5^143528 proposed a method for diagnosing malignant tumors * Available at the Patent Office . ''J > - > ■> /■ on request. v G;\ 248714 which utilized an injectable composition containing an endotoxin extracted from cultured bacteria. In GB 1587244, there was described inter alia, the use in a serum agglutination test on the sera of patients, for the detection of neoplasms, of an antigen produced by a species of the genus Streptococcus.

Bodily health is known to be affected by the nature of the intestinal flora, which apparently influences, for example, metabolic processes and both local and general body immune response. It has also been known for some time that certain of the intestinal flora bacteria of normal humans have oncolytic activity, and that there exists a relationship between intestinal microfloral composition and cancer morbidity, see e.g., Oleynik, S.F. and Panchishina, M.V., "About Coliflora and Cancerolycity and Carcinogenicity of the Intestine", Vrachebnoye-delo, 1968, 5: 13-17-However, mere knowledge of a relationship between the intestinal microflora, the immune system and cancer has not resulted up to now in the development of a reliable method either for the early diagnosis of cancer, or for treatment.

As regards the treatment of cancer, the three methods in widespread clinical use at the present time are radiation therapy, chemotherapy and surgical excision of tumors, which may be employed in combination. However, use of these methods in order to eliminate cancer cells in the body will not necessarily be complete or ultimately successful, in part because there is a failure rate associated with any therapeutic or surgical method, but more particularly because if a patient has a tendency to cancer because of a defect in the immune system, or more precisely because of an insufficiency or instability in inherent nonspecific antitumor protection in the body, cancer cells will be again generated. Thus, an agent to eliminate any defect in the body's nonspecific antitumor protection would seem to be indicated, but the present inventor could find no evidence of any such agents in the literature. 2 24 8 / A small number of patents assert that particular bacteria can be used for the treatment of cancer, although the fact remains that these published patents (or the work on which they were based, or any similar work) have not resulted effectively in the introduction of a bacterial method of treatment into clinical practice. In US 3192116, spores of a particular strain Clostridium butyricum, isolated from humus soil and cultured, were # said to show activity against various tumors. In FR 2088231, a suspension of inactivated Brucella bacteria in which 10-50# bacterial cells are whole and the remainder a lysate, was said to be useful in increasing the non-specific immunity of an organism, and for the treatment of tumors.

GB 1587244 discloses a particular strain of Streptococcus faecalis ■ isolated from the air in Italy, which is said to be useful for combatting and diagnosing neoplasms in humans and animals. However, from animal tests which are described in GB 1587244 (no in vivo tests on humans are described), it is clear that the effectiveness of this bacterium in treating neoplasms depends on the number of days for which it is cultured and whether the culture is oxygenated or grown under hypoxic conditions. Even at the level of animal tests the results do not present a consistent picture, and it is apparent, for these reasons, why it is unlikely that the strain described in this patent would ever be used as suggested therein, for clinical treatment in practical terms.

The entire disclosures of the above-mentioned patents are explicitly incorporated herein by reference. The published literature (including patents) thus supports a relationship between bacteria, nonspecific immunity and potential for the treatment of cancer, but suggests to the skilled person that a considerable research effort including a high level * Available at the Patent Office . - v on request. _ r , 3 w -\ ' 'y • , _ t "8 MAR 1994 ^: a y H/?- ■ ' 2 4 8 7 14 of inventive ingenuity, would be necessary in order to attain a situation where a bacterial method for the treatment of cancer would (at least) constitute an important feature of clinical practice, in addition to the presently recognized methods of radiation therapy, chemotherapy and surgery.

It is an object of the present invention to provide a bacterial formulation for the treatment of cancer, as well as a method for the early diagnosis of cancer. Other objects of the invention will appear from the description which follows.

SUMMARY OF THE INVENTION The present invention provides in one aspect an isolated and biologically pure strain of oncolytic bacteria derivable from human intestinal microflora, which is selected from the species Escherichia coli, Streptococcus faecalis and other bacterial species occurring in" human intestinal microflora.

In another aspect, the present invention provides a bacterial formulation, for use in the treatment or prevention of cancer, comprises, together with at least one pharmaceutical^ acceptable diluent, carrier or adjuvant, an active ingredient selected from (i) and (ii), namely: (i) at least three oncolytic strains of bacteria from at least two bacterial species, wherein said strains are obtainable or derivable from human intestinal microflora; and (ii) at least one oncolytic strain of bacteria selected from Escherichia coli A.T.C.C. 55373. Escherichia coli A.T.C.C. 55374, Escherichia coli A.T.C.C. 55375 and Streptococcus faecalis G35 A.T.C.C. 55376. The specified strains are also the subject of the invention. 4 2 4 8 7 14 In yet another aspect, the present invention provides a method for the early diagnosis of cancer in a human, wherein a human feces-derived sample of bacteria selected from Escherichia coli and Streptococcus faecalis, is subjected to incubation in vitro with a standard culture of cancer cells containing a predetermined number of such cells, for a period of time sufficient to enable the extent of interaction between the bacteria and the standard culture of cancer cells to be determined, and effecting the determination based on a count of the number of the interacted and/or non-interacted cancer cells present at the end of the period of time, the extent of interaction being preferably calibrated against the extent of interaction between a control preparation of the oncolytic bacteria which are the subject of the invention, incubated under the same conditions with the standard culture of cancer cells.

The expression "early diagnosis of cancer" in the present specification and claims, is intended to convey such diagnosis, whether or not the cancer has reached the stage in which it is detectable by other methods presently available to the clinician.

DETAILED DESCRIPTION OF THE INVENTION In a preferred embodiment of the formulation aspect of the invention, this comprises as active ingredient at least four such oncolytic bacterial strains. In another embodiment, the at least two bacterial species comprise Escherichia coli and Streptococcus faecalis, and may comprise, e.g., three strains of Escherichia coli and one strain of Streptococcus faecalis. More specifically the formulation may comprise the four specific bacterial strains mentioned above, samples of which were deposited (1) at the L.A. Tarasevitch Research Institute for Standardization and Control of Medico-Biological Preparations, Moscow, and in respect of which certificates nos. 2 4 Q 7 •• *■ - 4 01-07/208, /209» /210 and /211 have been issued, dated June 8, 1983, and (2) with the American Type Culture Collection (A.T.C..C.) under the Budapest Treaty, on November 13. 1992. Details of these four strains are as follows.

Escherichia coli G35 strain no. 1-59, A.T.C.C. Designation 55373.

Cultural and morphological features indicate that the strain belongs to the Escherichia genus. The straight rods of 1.1 - 1.5 x 2.0 - 6.0 mm (alive) occur separately or in pairs, and are gram-negative. They are mobile, and move using peritracheal cilia. They form pink-colored colonies in endomedium; nutritional agar colonies are smooth, moist, greyish with regular edges; gelatin colonies are turbid, greyish-white, moist. When grown on potato they give an extensive yellowish diffused coating. The strain is preserved in 2% plain agar, in a refrigerator at 4-8°C, with weekly reincubation. Reproduction is by incubation at 37°C (thermostat) for 24 hours. Oncolytic activity is shown under incubation at 37°C (thermostat) for 2-6 hours in contact with tumor cells, in vitro, as well as under in vivo conditions by administration per os.

The culture generates indole but not hydrogen sulfide, gives negative Voges-Proskauer reaction and positive reaction with methyl red. It does not eliminate urea and does not utilize citrate. It does not ferment rhamnose, raffinose, and salicin. Glucose and other carbohydrates are fermented producing pyruvate, which is then converted to lactic, acetic and formic acids. Part of the formic acid is broken down by the complex hydrogen lyase enzymatic system into equal quantities of carbon dioxide and hydrogen. The culture has weak hemolytic properties. The strain is resistant to kanamycin, oxacillin, erythromycin, methicillin, polymyxin, ampicillin, penicillin, oleandomycin, neomycin, streptomycin, rheotomycin and 6 2 4 8 7 1 4 tetracycline, and has low sensitivity to carbenicillin, gentamycin and levomycetin.

Escherichia coli G35 strain no. 2-60, A.T.C.C. Designation 55374.

Cultural and morphological features indicate that the strain belongs to the Escherichia genus. The straight rods of 1.1 - 1.5 x 2.0 - 6.0 mm (alive) occur separately or in pairs, and are gram-negative. They are mobile, and move using peritracheal cilia. They form pink-colored colonies in endomedium; nutritional agar colonies are smooth, moist, greyish with regular edges; gelatin colonies are turbid, greyish-white, moist. When grown on potato they give an extensive yellowish diffused coating. The strain is preserved in 2% plain agar, in a refrigerator at 4-8°C, with weekly reincubation. Reproduction is by incubation at 37(thermostat) for 24 hours. Oncolytic activity is shown under incubation at 37 °C (thermostat) for 2-6 hours in contact with tumor cells, in vitro. as well as under in vivo conditions by administration per os.

The culture generates indole but not hydrogen sulfide, gives negative Voges-Proskauer reaction and positive reaction with methyl red. It does not eliminate urea and does.not utilize citrate. It does not ferment dulcite, salicin or cellobiose, but weakly ferments lactose and saccharose (by 6-10 days). Glucose and other carbohydrates are fermented producing pyruvate, which is then converted to lactic, acetic and formic acids. Part of the formic acid is broken down by the complex hydrogen lyase enzymatic system into equal quantities of carbon dioxide and hydrogen. The culture has weak hemolytic properties. The strain is resistant to kanamycin, oxacillin, erythromycin, methicillin, polymyxin, ampicillin, penicillin, oleandomycin, neomycin, streptomycin, rheotomycin and tetracycline, and has low sensitivity to carbenicillin, gentamycin and levomycetin. 7 24 8 7 1 4 Escherichia coli G35 strain no. 3"6l, A.T.C.C. Designation 55375- Cultural and morphological features indicate that the strain belongs to the Escherichia genus. The straight rods of 1.1 - 1.5 x 2.0 - 6.0 mm (alive) occur separately or in pairs, and are gram-negative. They are mobile, and move using peritracheal cilia. They form pink-colored colonies in endomedium; nutritional agar colonies are smooth, moist, greyish with regular edges; gelatin colonies are turbid, greyish-white, moist. When grown on potato they give an extensive yellowish diffused coating. The strain is preserved in 2% plain agar, in a refrigerator at 4-8°C, with weekly reincubation. Reproduction is by incubation at 37°C (thermostat) for 2k hours. Oncolytic activity is shown under incubation at 37 °C (thermostat) for 2-6 hours in contact with tumor cells, in vitro, as well as under in vivo conditions by administration per os.

The culture generates indole but not hydrogen sulfide, gives negative Voges-Proskauer reaction and positive reaction with methyl red. It does not eliminate urea and does not utilize citrate. It does not ferment rhamnose, raffinose and salicin. Glucose and other carbohydrates are fermented producing pyruvate, which is then converted to lactic, acetic and formic acids. Part of the formic acid is broken down by the complex hydrogen lyase enzymatic system into equal quantities of carbon dioxide and hydrogen. The culture has weak hemolytic properties. The strain is resistant to kanamycin, oxacillin, erythromycin, methicillin, polymyxin, ampicillin, penicillin, oleandomycin, neomycin, streptomycin, rheotomycin and tetracycline, and has low sensitivity to carbenicillin, gentamycin and levomycetin. 8 24 87 14 Streptococcus faecalis G35 strain no. 4-62, A.T.C.C. Designation 55376.

Cultural and morphological features indicate that the strain belongs to the Streptococcus faecalis genus. The cells sire spherical or oval, in pairs or diversified chains, diameter less than 2 mm. They are stable, do not form endospores and are gram-positive. The strain is preserved in 2% plain agar, in a refrigerator at 4-8°C, with weekly reincubation. Reproduction is by incubation at 37°C (thermostat) for 24 hours. Oncolytic activity is shown under incubation at 37°C (thermostat) for 2-6 hours in contact with tumor cells, in vitro, as well as under in vivo conditions by administration per os.

The culture grows in 6.5% NaCl broth at pH 9-6, or in milk with 0.1% methylene blue. For growth in plain media, folic acid is required. It ferments arabinose and sorbite, but does not produce ammonia from arginine. It contains Lancefield 0 and D group antigens. The strain is resistant to kanamycin, oxacillin, erythromycin, methicillin, polymyxin, ampicillin, penicillin, oleandomycin, neomycin, streptomycin, rheotomycin and tetracycline, and has low sensitivity to carbenicillin, gentamycin and levomycetin.

In a particular embodiment of the invention, the numbers of bacteria present in the formulation may be in the ratio of 100-390 Escherichia coli to 100 Streptococcus faecalis, e.g., when three species of Escherichia coli and one of Streptococcus faecalis are present in the formulation, then the numbers of bacteria present in the formulation may be in the ratio of 35-130 (such as 35"70 or 70-130) for each strain of Escherichia coli to 100 Streptococcus faecalis. In a presently preferred embodiment, the bacterial formulation of the invention is in unit dosage form and comprises 25-30 billion bacteria of each of the four strains (i.e. three 9 2 4 8 7 14 of Escherichia and one of Streptococcus) , or a sub-multiple of such formulation where the bacteria are present in the same ratio.

In an alternative presently preferred embodiment, the bacterial formulation is in unit dosage form and comprises 15~30 billion bacteria of each of three strains of Escherichia coli and 25~30 billion of one strain of Streptococcus faecalis, or a sub-multiple of such formulation where the bacteria are present in the same ratio.

Insofar as an objective of the invention is to enable the bacterial formulation to act internally in- the body, the formulation may be adapted for the usual means of administration known in the art, e.g. for oral, parenteral, rectal or transdermal administration. The use of diluents, carriers and adjuvants is well known in the pharmaceutical art, and is elaborated e.g. in publications such as above mentioned GB 1587244.

As regards the early diagnosis aspect of the invention, in a particular embodiment by which the present invention may be practiced, determination of the extent of interaction between the test sample of bacteria and the standard culture of cancer cells is made by counting the number of cancer cells remaining in the visual field of a microscope of a smear on a microscope slide, after fixing and dyeing as necessary. The operative methods for counting cells on a microscope slide are per se known to persons in the field.

In order to avoid errors and for the sake of improved accuracy in the determination, it is preferred that determination of the extent of interaction between the bacteria and the standard culture of cancer cells be calibrated against the extent of interaction between a control preparation of 1G 24 87 14 bacteria, in particular the oncolytic bacteria which are the subject of the present invention and are described herein, incubated under the same conditions, and the standard culture of cancer cells.

Thus, for example, when carrying out a control determination in addition to the test determination, there may be calculated a Tumor Cell Necrosis Index (TCNI) from the following formula c{%) = 100(a-b)/a, where a » the number of standard cancer cells destroyed in the control experiment, b = the number of standard cancer cells not destroyed in the experiment on the test sample, and c = the TCNI. Qualitatively, it will be apparent that in a healthy patient in which the intestinal Escherichia coli and Streptococcus faecalis will have a similar activity to the control bacteria, b will' be low (and a in any case should be approaching 100%), giving a relatively high TCNI, whereas in a relatively less healthy patient b will have a relatively high value and the TCNI will be lower. More precisely, the inventors have found the TCNI values indicated in Table A in relation to healthy patients and patients having various classes of diseases.

Table A Diseases No. of patients c(average) Sigma c(range) (Normal subjects) 1200 86 1.1 68- 100 Gastrointestinal 1340 62 1.1 50- 98 Tuberculosis 141 70 2.3 67- 100 Communicable 123 62 1.1 60- 89 Radiation 157 52 1.1 - 56 Oncological 1531 29 1.1 - 49 Oncological post- operative 682 53 3.4 21- 82 11 24 8 7 14 It should be particularly noted that the c range of 68-100 for normal human subjects does not overlap the c range of 10-49 found for oncological subjects. These facts underline the precision and thus the utility of the present method for early diagnosis of cancer. Based on their overall experience to date, the inventors believe that c values within these ranges indicate the following in human subjects under test: 492 and below: malignant tumors present in the body; >492 and <6l%: the subjects have a possible predisposition to malignant tumors, i.e. a potential risk group; 6l-100%: absence of malignant tumors in the body.

Without prejudice to' the broad concept of the early diagnosis aspect of the invention as set forth herein, suitable strains of bacteria for use as control, in a preferred embodiment of the method of the invention, are the following four strains, isolated from healthy human feces, which have been deposited with the American Type Culture Collection (A.T.C.C.) under the Budapest Treaty, on November 13, 1992.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The following non-limitative exemplification is intended to illustrate the present invention, including its utility.

Preparation of the Bacterial Formulation containing four strains Fresh milk (11) is boiled gently in a clean vessel for 10-15 minutes, covered, cooled to 36-38°C, and 30~35 g of a mixture (stored at -5 to +5°C) of equal volumes of Escherichia coli G35 strain no. 1-59 A.T.C.C. 553731 Escherichia coli G35 strain no. 2-60 A.T.C.C. 55374, Escherichia coli G35 strain no. 3"6l A.T.C.C. 55375 and Streptococcus faecalis G35 strain no. 4-62 A.T.C.C. 55376 (the concentration of each strain being 1.5-2.0 billion bacteria/ml) was added, the whole being stirred with a sterile spoon. The 12 ? 4 3 7 f 4 vessel was tightly covered, insulated and allowed to stand for 3~4 hours at external ambient temperature, when the internal temperature was about 35 °C, and was then stored at -5 to +5°C. A slightly acidic taste, non-coagulation of the milk and fluidity of the mixture confirm that the formulation is ready for use. Further quantities may be prepared similarly. (Alternatively an aq. NaCl solution can be similarly prepared.) The 11 of formulation is taken orally in 100 g portions morning and evening for 5 days. An exemplary total period of administration may be 35 days, 100 g being taken morning and evening. (Formulations containing the single strains were prepared similarly.) Utility of the Bacterial Formulation 1. The above formulation containing four bacterial strains was tested in volunteers, each of whom received a single dose of about 100 billion bacteria, per os. No pathological deviations were found. Results showing a significant subsequent increase in intestine microflora oncolytic activity are shown in Table I, below. (Oncolytic activity in Table I and TCNI in Table II mean the Tumor Cell Necrosis Index.) 2. 22 children who suffered from the Chernobyl event were diagnosed according to IL 101409 and were found to have a TCNI in the range 40-522. They were subjected to clinical, endoscopic, X-ray and morphological examination, and were found to have inter alia intestine microflora disturbances; 9 of the patients had intestinal dysbacteriosis. They took the four-strain formulation of the invention for 1 month along the lines indicated at the end of the formulation example (above), the actual dosage rate being tailored to each patient's circumstances including size and weight of the patients. Results are indicated in Table II below, showing a 13 24 8 7 1 4 «■ i significant increase in the TCNI value and elimination of dysbacteriosis, after taking the formulation. Additionally, all the children showed a general improvement in health and appetite, absence of discomfort, absence of negative responses and good restoration of the gastric function.

TABLE I Group Amount Oncolytic X activity y Validity Relatively healthy 6 51.5±2.7 6l.5i2.l 95 Cancer patients 4 • 40.510.57 52.512.0 99-9 x = prior to treatment, y = following treatment 14 24 8 7 1 TABLE II TCNI(2) Intestinal No.

Patient Age (years) X y dysbacteriosis x y 1 Sirenok Natasha 8 40 48 + 2 Shevchenko Tamara 40 50 + - 3 Potapenko Sasha 13 40 . 60 - 4 Polishchuk Lena 8 42 48 - Vasilenko Tanya 14 42 48 - 6 Sidorenko Anya 9 42 50 + 7 Matushenko Sasha 12 42 50 - 8 Churikova Ira 7 42 50 - 9 Churikova Sasha 42 48 + Matushenko Sergei 44 56 - 11 Avgrimenko Tolya 8 44 60 - 12 Polishchuk Denis 44 50 + 13 Kalmaz Anton 12 44 52 + 14 Sirenok Sasha 13 44 56 - Krutko Andrei 9 46 50 + 16 Sidorenko Sveta 11 46 80 + - 17 Ushchapovsky Denis 12 48 54 . + - 18 Polishchuk Sergei 48 54 - 19 Vaks Anya 7 50 70 - Polishchuk Nina 17 50 70 - 21 Kachur Oxana . 11 52 80 - 22 Kachur Sasha 7 54 80 - x = prior to treatment y = following treatment 24 87 14 3. A four-strain bacterial formulation prepared as above was fed to separate groups of rats having different types of cancer, and using different modes and/or media for administration, with the results referred to relative weight of cancer nodes in the test animals on the 10th day following administration, shown in Tables III below. There were 10 rats in each group. It may readily be seen that there is significant improvement in the treated rats as compared with the control groups.

TABLE III: ANTITUMOR EFFECT OF BACTERIAL FORMULATION Cancer type Administration Average cancer Efficiency (J!) node weight (g) MELANOSARCOMA Control 32.8 Per os (milk) 12.7 61 Per os (0.92 NaCl) 14.9 55 SARCOMA 45 Control 13.8 Intramuscular 4.3 69 (0.92 HC1) Per os (8.92 NaCl) 4.7 65 WALKER Control 27.9 ADENOCARCINOMA Per os (milk) 7.3 74 Per os (0.92 NaCl) 8.5 70 PLIES Control 27-1 LYMPHOSARCOMA Per os (milk) 16.1 40 4. Single-strain bacterial formulations in milk, prepared as above, were administered to rats per os, as described in the preceding paragraph. The results are shown in Table IV, which compares also the results of using the four-strain formulations under similar conditions. 16 24 87 14 TABLE IV: ANTITUMOR EFFECT OF SINGLE-STRAIN AND FOUR-STRAIN FORMULATIONS Cancer type Efficiency{%) in reduction of weight of tumor nodes Single strains (A.T.C.C. designations) 55373 55374 55375 55376 Four-strain . formulation MELANOSARCOMA 52% 48% 65% 50% 61% SARCOMA 45 76% 80% 82% 51% 98% WALKER ADENOCARCINOMA 68% 60% 12% % 74* PLIES LYMPHOSARCOMA % 38% 36% 32% 40* SCHWEIZ LEUCOSIS 50% 56% 5Q% 61% . Case history A patient, 46, was hospitalized November 30. 1990 with diagnosis of ovarian cystadenoma. Histology showed right ovarian and omental metastatic tumors, granular cell cancer with thecomatosis and metastases into the omentum and the intestinal peritoneum. Disturbance of the intestine microflora (dysbacteriosis) was found, and ovarian cancer with metastases was detected. The patient took the four-strain formulation of the invention, as prepared above, for one month as indicated at the end of the preparative example. Following treatment, dysbacteriosis had been eliminated. General improvements in the patients clinical state were noted, nausea, vomiting and diarrhea had ceased, and the stools had become normal. Bacterial oncolytic activity (TCNI) had changed from 36% prior to treatment, to 58# thereafter. 17 24 87 14 an increase of 22% on the TCNI scale.

Although the utility of the formulation aspect of the invention is not limited by any theory, it is believed that because of deterioration in the intestinal flora associated with cancer, the bacterial formulation in accordance with an embodiment of the invention which contains a multiplicity of bacterial strains is effective because it restores normal intestinal flora and eliminates dysbacteriosis. Given that the normal intestinal flora itself contains a spectrum of bacterial strains, it is hardly to be expected that administration of a single bacterial strain would be as effective as the formulation of the invention containing a multiplicity of strains. Nevertheless, there may well be cases where the administration of one or more. strains will effect an improvement in the patient's condition. However, it is believed that the present preferred formulation of a multiplicity of "strong" oncolytically active bacterial strains of intestinal origin enables the intestinal flora to remain oncolytically active for a long period of time and to promote elimination of incipient cancerous activity in the subject's organism.

The following non-limitative Examples, of which Examples 2-4 are case studies, illustrate the diagnosis aspect of the present invention.

Example 1: Isolation and Use of Feces-derived Bacteria.

A faeces inoculation of 1.0 g/10 ml saline was made in Endo agar, and a portion of this mixture was added to an agar-agar medium to isolate Streptococcus faecalis, which were placed in an incubator for 24 hours at 37 °C. The inoculation was then transferred from Endo agar to 2% plain agar to isolate Escherichia coli, and from special medium to blood bile salt agar (BBSA) to further isolate Streptococcus faecalis; the isolated bacteria were 18 24 87 14 placed in an incubator for 24 hours at 37°C. The cultures were checked by Gram staining, biochemical tests and the requirements of the Bergi specifications (1980).

The isolated bacteria (20 ml containing 2 billion microbes/ml) were smeared with G.02 g of a suspension of standard human or animal malignant cancer cells (e.g. sarcoma 45), then the mixture was incubated at 37°C for 2-6 hours (e.g. 6 hours in the case of sarcoma 45), depending on the strain of cancer cells. In the control experiment 0.4 ml saline solution was mixed with 0.02 g cancer cells; smears were prepared, fixed by the Mai-Grunwald technique and dyed by the Romanovsky-Gimza technique. Thereafter, destroyed and non-destroyed cancer cells were counted in 5 or 10 (H.P. or L.P.) visual fields, the average index was taken and the TCNI was calculated, as explained above, from the equation c(%) = 100(a-b)/a.

Example 2 Patient K.R.S. was first examined on December 27, 1979 where she complained of irregular menses; initial uterine myoma was diagnosed. The patient was hospitalized, the uterine cavity was curretted and the myomatous node was excised for identification purposes. On histology, endometrial fragments with fibrosis were identified. According to the technique of the invention described herein, the TCNI was found to be 40%. On January 4, 1979, the patient was discharged in satisfactory condition.

The patient subsequently complained (June 19, 1979) of prolonged menstrual hemorrhage and was again hospitalized. Hysterography was performed and polypoid-cystic neoplasm was found in the mucosa of the uterine cervix. An operation was carried out on September 13, 1979, and supravaginal amputation of the uterus without adnexal uteri was performed due to the presence of a submucous myomatous node. Histology confirmed uterine myoma. 19 24 87 14 The patient was discharged September 25, 1979 in satisfactory condition. On March 10, 1982, due to the symptoms indicated above, the patient was operated on with high vaginal cervicectomy with dissection of the cervical canal mucosa and resection of ligation granulation polyps. Surgical intervention was complicated by internal hemorrhage which resulted in laparotomy with abdominal cavity drainage.

On December 2, 1983, due to the occurrence of an ovarian tumor with 7.5 month pregnancy dimensions, laparotomy was performed. When the abdominal cavity was opened, a 14 x 16 cm right ovarian tumor was found, with numerous adhesions to the parietal peritoneum. After adhesion lysis the tumor was removed. A 1G x 10 cm left ovarian tumor was also found, with adhesions and infiltrative growth into adjacent organs (bladder, sigmoid colon); the left ovarian tumor was deemed inoperable. Histology of the right ovarian tumor and omental biopsies showed granular cell carcinoma with thecomatosis and metastases involving the omentum and enteric peritoneum. This case study demonstrates the usefulness of the method for early diagnosis of malignant tumors, in accordance with the present invention.

Example 2.

A.A.K., 53, was hospitalized May 27, 1988 and discharged June 8, 1988; clinical diagnosis indicated systemic disease of left lung complicated by exudative pleurisy, as well as osteoma of the left frontal bone. From previous hospital history (May 1988), biopsy of the pleural cavity followed by cytology showed metastasis of adenocarcinoma with effect on the mucosa. The patient first felt dyspnea in May 1988. TCNI as set forth herein was found to be 36%. In the period May 17"26 1988 the patient had been examined with a resulting diagnosis of exudative pleurisy of ambiguous etiology; pleural punctures were performed with the evacuation of about 1.6 1 of 24 8 7 n hemorrhagic fluid and cytology, as stated above. This case study demonstrates the correlation of a low TCNI, determined in accordance with the present invention, with the existence of malignant tumor in a patient.

Example 4 P.C.G., 56, was hospitalized on April 3. 1980. with diagnosis of left crural melanoma. The 2 x 2 cm tumor was located in the lower posterior portion of the left crus, of soft consistency, mobile on a narrow pedicle, the inguinal lymph nodes not being enlarged. The TCNI as set forth herein was found to be 30% • On April 9. 1988, electro-dissection of the tumor was effected under local anesthesia, with a normal post-operation period; the sutures were removed on the 10th day. The patient was discharged on April 24, 1980, apparently in good condition. Histology showed cancer. There was no TCNI rise (determined in accordance with the present invention) following surgical intervention. On July 20, 1980, TCNI as determined in accordance with the present invention was found to be 3&%- On September 2, 198I, the patient was readmitted to hospital and metastases in the inguinal lymph nodes and in the peritoneum were diagnosed. This case study demonstrates the correlation of a low TCNI, determined in accordance with the present invention, with the existence of malignant tumor in a patient, and further indicated that the persistence of malignant cancer after the patient had been discharged from initial surgery in apparent good condition.

Discussion The present early diagnosis method according to a particular aspect of " the present invention has been shown not only to correlate with clinical findings of cancer, but has demonstrated the presence of cancer not found by conventional clinical procedures. The method of the invention is simple to 21 248714 operate, accurate, and involves no discomfort of the human subject under examination. The method of the invention is potentially readily adaptable to mass screening techniques, in order to identify persons with cancer as well as risk groups. Persons skilled in the art will be aware that the counting of destroyed and/or non-destroyed bacteria which forms part of the inventive method is capable of computerization. As appears from the above case studies, the method of the invention can be used to confirm the efficiency of clinical treatment of cancer, and for detection of recurrence of malignant disease.

The skilled addressee will appreciate that oncolytic strains of bacteria which occur in human intestinal microflora, and which in biologically pure form constitute a particular aspect of the present invention, and form the basis for the presently claimed bacterial formulations for the treatment of cancer, are also the basis for the diagnostic method according to another aspect of the present invention, since it is the activity in practice of a particular patient's oncoloytic intestinal bacteria which is responsible for the in vitro extent of interaction with the standard culture of cancer cells, such extent of interaction being in effect determined in accordance with the present diagnostic method.

While particular embodiments of the invention have been particularly shown and/or described hereinabove, it will be appreciated that the present invention is not limited thereto, since, as will be readily apparent to skilled persons, many variations and modifications can be made. Accordingly, the essential concept, spirit and scope of the present invention will be better understood in the light of the claims which follow. 22 248714

Claims (5)

WHAT WE CLAIM IS:

1. An isolated and biologically pure strain of oncolytic bacteria derived from human intestinal microflora, which is selected from the species Escherichia coli and Streptococcus faecalis.

2. A bacterial formulation, suitable for use in the treatment of cancer, which comprises, together with at least one pharmaceutical^ acceptable diluent, carrier or adjuvant, active ingredient (i) or (ii) as defined below, namely, (i) at least three oncolytic streiins of bacteria as defined in claim 1, or (ii) at least one oncolytic strain of bacteria as defined in claim 1 and selected from Escherichia coli G35 strain no. 1-59 A.T.C.C. 55373. Escherichia coli G^5 strain no. 2-60 A.T.C.C. 55374. Escherichia coli G35 strain no. 3~6l A.T.C.C. 55375, and Streptococcus faecalis G35 strain no. 4-62 A.T.C.C. 55376.

3- A bacterial formulation according to claim 2, which comprises as active ingredient four different oncolytic strains of bacteria. 4. A bacterial formulation according to claim 2, which comprises three strains of Escherichia coli and one strain of Streptococcus faecalis. 5. A bacterial formulation according to claim 4, which comprises Escherichia coli G35 strain no. 1-59 A.T.C.C. 55373. Escherichia coli G35 strain no. 2-60 A.T.C.C. 55374, Escherichia coli G35 strain no. 3~6l A.T.C.C. 55375 and Streptococcus faecalis G35 strain no.

4-62 A.T.C.C. 55376. 23 248714 6. A bacterial formulation according to claim 4 or claim 5, wherein the numbers of bacteria present in the formulation are in the ratio of 100-390 Escherichia coli to 100 Streptococcus faecalis. 7. A bacterial formulation according to claim 4, wherein the numbers of bacteria present in the formulation are in the ratio of 3

5-130 for each strain of Escherichia coli to 100 Streptococcus faecal •>* q. 8. A bacterial formulation according to claim 7, wherein the numbers of bacteria present in the formulation are in the ratio of 70-130 for each strain of Escherichia coli to 100 Streptococcus faecalis.. 9. a bacterial formulation according to claim 4 or claim 5, which is in unit dosage form, and in which the number of bacteria of each of the four strains to one another is in the ratio 1.0-1.2:1.0-1.2: 1.0-1.2: 1.0-1.2. 10. A bacterial formulation according to claim 9, which comprises 25-30 billion bacteria of each of the four species. H A bacterial formulation according to claim 7, wherein the numbers of bacteria present in the formulation are in the ratio of 35"70 for each ' strain of Escherichia coli to 100 Streptococcus faecalis. 248714 12. A bacterial formulation according to claim 4 or claim 5, which is in unit dosage form, and in which the number of bacterial of each of the four strains to one another is in the ratio 3.0-6.0: 3.0-6.0: 3.0-6.0: 5.0-6.0, where said 5.0-6.0 refers to Streptococcus faecalis. 13. A bacterial formulation according to claim 12, which comprises 15-30 billion bacteria of each of the three strains of Escherichia coli and 25-30 billion bacteria of the one strain of Streptococcus faecalis. 14. A bacterial formulation according to any one of claims 2-5 and 7-13, which is adapted for oral, parenteral, rectal or transdermal administration. 15. A bacterial formulation according to claim 6, which is adapted for oral, parenteral, rectal or transdermal administration. 16. A bacterial strain according to claim 1, which is Escherichia coli G35 strain no. 1-59 A.T.C.C. 55373. Escherichia coli G35 strain no. 2-60 A.T.C.C. 55374, Escherichia coli G35 strain no. 3~6l A.T.C.C. 55375 or Streptococcus faecalis G35 strain no. 4-62 A.T.C.C. 55376. 17. Use of an active ingredient as defined in claim 2 suitable for the manufacture of a medicament for the treatment of cancer in a human or non-human mammal. 18. Use of an active ingredient as defined in claim 2 suitable for the manufacture of a medicament for the prevention of cancer in a human or non-human mammal. 248714 19. A method for the early diagnosis of cancer in a human, wherein a human feces-derived sample of bacteria selected from Escherichia coli and Streptococcus faecalis, is subjected to incubation in vitro with a standard culture of cancer cells containing a predetermined number of such cells, for a period of time sufficient to enable the extent of interaction between said bacteria and said standard culture of cancer cells to be determined, and effecting said determination baaed on a count of the number of the interacted and/or non-interacted cancer cells present at the end of the period of time, 20. A method according to claim 19, wherein said extent of interaction is calibrated against the extent of interaction between an isolated and biologically pure strain of oncolytic bacteria as defined in claim 1, or against a combination of such strains, incubated under the same conditions, and said standard culture of cancer cells. 21. A method according to claim 19 or claim 20, wherein said period of time lies within the range of substantially 2 to 6 hours. 22. A method according to claim 19 or claim 20, wherein said determination is made by counting the number of cancer cells remaining in the visual field of a microscope of a smear on a microscope slide, after fixing and dyeing as necessary. 23. A method according to claim 20, wherein said determination is made by counting the number of cancer cells remaining in the visual field of a microscope of a smear on a microscope slide, after fixing and dyeing as necessary. 24. A method according to any one of claims 19-23, wherein in said control preparation of bacteria, there is used at least one strain selected from Escherichia coli A.T.C.C. 55373. Escherichia coli A.T.C.C. 55374, Escherichia coli A.T.C.C. 55375 and Streptococcus faecalis A.T.C.C. 55376. 26 248714 25. An isolated and biologically pure strain of oncolytic bacteria according to claim 1 substantially as herein described or exemplified. 26. A bacterial formulation according to claim 2 substantially as herein described or exemplified. 27. A method according to claim 19 substantially as herein described or exemplified. ERA-MASIS LTD. By Their Attorneys HENRY HUGHES Per: 27